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WO2016189881A1 - Pharmaceutical composition for treating cerebral infarction - Google Patents

Pharmaceutical composition for treating cerebral infarction Download PDF

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Publication number
WO2016189881A1
WO2016189881A1 PCT/JP2016/002598 JP2016002598W WO2016189881A1 WO 2016189881 A1 WO2016189881 A1 WO 2016189881A1 JP 2016002598 W JP2016002598 W JP 2016002598W WO 2016189881 A1 WO2016189881 A1 WO 2016189881A1
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Prior art keywords
compound
pharmaceutical composition
group
cerebral infarction
integer
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PCT/JP2016/002598
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French (fr)
Inventor
Yukihiko Mashima
Tatsuya Matsukawa
Akiko Kawasaki
Satoru Kitani
Yuichiro Tanaka
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R Tech Ueno Ltd
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R Tech Ueno Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/56—Amides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/74—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to a pharmaceutical composition for treating cerebral infarction comprising a VAP-1 inhibitor, which is a benzene derivative having a certain structure.
  • Cerebral stroke has been one of the major therapeutic targets. In Japan, the number of cerebral stroke patients has increased and is estimated to reach three million in 2020. Further, such patients are highly likely to suffer from aftereffects of the strokes. This also gives major challenges from the perspective of welfare and medical economics.
  • Cerebral stroke broadly classified into two subtypes.
  • One subtype is ischemic stroke such as cerebral infarction, which is caused by blockage in the blood vessels in the brain; and the other subtype is hemorrhage stoke such as cerebral hemorrhage or subarachnoid hemorrhage, which is caused by bleeding due to rupture of an artery in the brain.
  • cerebral infarction occurs when blood supply to the brain is interrupted due to e.g., occlusion or stenosis of cerebral arteries.
  • cerebral ischemia leads to insufficient oxygen or nutrients supply, causing brain tissues to necrotize completely or partially.
  • Cerebral infarction includes, for example, lacunar infarction, which is caused by lesions in intracerebral small artery; atherothrombotic cerebral infarction, which is caused by atherosclerosis of relatively large artery in cervical region or skull; and cardiogenic embolism, which is caused by a cardiac disease.
  • thrombolytic treatment using tissue plasminogen activator is the first-line choice.
  • Plasminogen is an enzyme that strongly dissolves thrombi formed in blood vessels.
  • tPA is administered to dissolve the thrombi and make the blood flow reperfuse.
  • the recovery rate representing the rate of the patients who has recovered from the disease to the feasible level of living an independent life, with tPA treatment has been reported 40 to 50%.
  • Administration of tPA is limited for the patients within 4.5 hours from the onset of acute stroke, considering the risk of cerebral hemorrhage when administered after a lapse of prolonged time from the onset.
  • An intravascular therapy which removes thrombi from the blood vessels with mechanical method, has gradually used as a therapeutic option which can be applied within six hours from the onset of the acute stroke. Nevertheless further enhancement of the recovery rate and expansion of the time window for the treatment are strongly desired in clinical practice. Developing a novel therapy for cerebral infarction that decreases the risk of brain hemorrhage and enlarges the patient population to be treated will be highly appreciated.
  • Vascular adhesion protein-1 is a protein which is synonymously referred to as semicarbazide-sensitive amine oxidase (SSAO).
  • SSAO semicarbazide-sensitive amine oxidase
  • Membrane-bound VAP-1 is present at the surfaces of vascular endothelium and circulating SSAO is present in serum.
  • the former is mainly involved in inflammation and works as an adhesive molecule to leukocytes such as granulocytes related to inflammation, and lymphocytes or monocytes related to inflammation and immunity. The latter is responsible for detoxicating amines through the amine oxidase activity.
  • VAP-1/SSAO activity increases in plasma and various tissues of patients of diseases such as diabetes, atopic dermatitis, psoriasis, obesity, arteriosclerosis, and cardiac diseases. Therefore, VAP-1/SSAO inhibitors are expected to treat the diseases through suppressing and normalizing the increased VAP-1/SSAO activity.
  • Non Patent Literature 1 VAP-1 inhibitors were reported to produce a neuroprotective effect in rat models of cerebral infarction in which cerebral ischemia was caused by autologous thrombi or a plug (Non Patent Literatures 1 and 2).
  • Patent Literatures 1 and 2 Benzene, thiophene, and thiazole derivatives having VAP-1 inhibitory effect have been known and their use in prevention or treatment of VAP-1 associated diseases has been suggested (Patent Literatures 1 and 2).
  • An object of the present invention is to provide a pharmaceutical composition for treating cerebral infarction. Further object of the present invention is to provide a novel benzene derivative.
  • the present invention provides a pharmaceutical composition for treating cerebral infarction comprising a compound of formula (I): wherein m is an integer of 1 or 2, n is an integer of 2 or 3, and A is selected from the group consisting of wherein R and R' are independently selected from C1-C6 alkyl, or a pharmaceutically acceptable salt thereof.
  • the present invention provides a compound of formula (I'): wherein m is an integer of 1 or 2, n is an integer of 2 or 3, and A' is selected from the group consisting of or a pharmaceutically acceptable salt thereof. All compounds represented by formula (I') are also covered by formula (I).
  • Fig. 1 shows effects of Compound 1 and Synthesized Compound 1 on the cerebral infarct volume in a rat model of transient focal cerebral ischemia.
  • Each value represents the mean ⁇ standard deviation (S.D.) of 10 rats.
  • *P ⁇ 0.05 and **P ⁇ 0.01 represent significant differences between the vehicle group and the Compound 1, Compound 2, or Synthesized Compound 1 group (Dunnett's multiple comparison test).
  • ##P ⁇ 0.01 represents significant difference between the vehicle group and the positive control group (Student's t-test).
  • Fig. 2 shows effect of Compound 1 on the cerebral infarct volume in a rat model of transient focal cerebral ischemia. Each value represents the mean ⁇ S.D. of 10 rats. **P ⁇ 0.01 represents significant difference between the vehicle group and the Compound 1 group (Dunnett's multiple comparison test). ##P ⁇ 0.01 represents significant difference between the vehicle group and the positive control group (Student's t-test).
  • Fig. 3 shows effect of Compound 1 on the neurological symptoms score in a rat model of transient focal cerebral ischemia. Each value represents the mean ⁇ S.D. of 10 or 11 rats.
  • *P ⁇ 0.05 represents significant difference between the vehicle group and the Compound 1 group (Steel-Dwass test).
  • #P ⁇ 0.05 represents significant difference between the vehicle group and the positive control group (Steel-Dwass test).
  • Fig. 4 shows effect of Compound 1 in the rota-rod test in a rat model of transient focal cerebral ischemia. Each value represents the mean ⁇ S.D. of 10 or 11 rats.
  • *P ⁇ 0.05 represents significant difference between the vehicle group and the Compound 1 group (Tukey-Kramer test).
  • #P ⁇ 0.05 represents significant difference between the vehicle group and the positive control group (Tukey-Kramer test).
  • Fig. 5 shows effect of Compound 1 and effect of the combination of rt-PA and Compound 1 on the neurological symptoms score in a rat model of cerebral ischemia caused by autologous thrombus.
  • *P ⁇ 0.05 represents significant difference between the vehicle group and the Compound 1 group or the combination group (Wilcoxon test).
  • substituents A and A' may be attached to any suitable position on the pyridine ring.
  • alkyl means a straight chain or branched chain alkyl having 1 to 6 carbon atoms, for example 1 to 4 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, tert-pentyl, and hexyl.
  • the pharmaceutical composition may comprise a compound represented by the following formula or or a pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable salt means any nontoxic pharmaceutically acceptable salt, including salts with an inorganic or organic base and acid addition salts.
  • examples of the salt with an inorganic or organic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, an ammonium salt, and amine salts such as triethylamine salt and N-benzyl-N-methylamine salt.
  • the acid addition salt examples include salts derived from mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and salts derived from organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, maleic acid, benzoic acid, glycol acid, gluconic acid, succinic acid, and arylsulfonic acids such as p-toluenesulfonic acid.
  • mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid
  • organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, maleic acid, benzoic acid, glycol acid, gluconic acid, succinic acid, and arylsulfonic acids such as p-to
  • the compounds of formula (I) and the pharmaceutically acceptable salts thereof can be prepared with reference to known literatures such as WO2009/096609 or WO2009/145360, or in accordance with synthesis examples shown in the specification.
  • Cerebral infarction means damages in the brain caused by cerebral ischemia and related symptoms, including, but not limited to, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic embolism, and also including sequelae of cerebral infarction such as neurologic symptoms, higher brain dysfunction, and affective disorder.
  • the cause of cerebral ischemia includes, but not limited to, cerebral thrombosis, cerebral embolism, vasospasm, hypotension, and hypoxemia.
  • pharmaceutical composition for treating cerebral infarction means a pharmaceutical composition that is administered to a subject for the purpose of treating cerebral infarction in the subject.
  • treating cerebral infarction means alleviating, relieving, and/or healing cerebral infarction, or delaying or suppressing the progression of cerebral infarction.
  • the subject to be treated by the pharmaceutical composition is a mammal such as a human, mouse, rat, pig, dog, cat, horse, and cow, especially a human.
  • the pharmaceutical composition may be administered via any route.
  • it may be administered systemically or locally via an administration route such as oral, subcutaneous, percutaneous, transmucosal, intravenous, intra-arterial, intramuscular, intraperitoneal, intranasal, subdural, rectal, or gastrointestinal route, or by inhalation.
  • Spinal injection, epidural injection, or administration into cerebral ventricle is also possible.
  • a physician who treats the disease may properly determine the administration route of the pharmaceutical composition.
  • the pharmaceutical composition may be administered via two or more routes, wherein one route may provide more rapid and effective activity than the other.
  • the administration route is not limited to the routes exemplified above.
  • the pharmaceutical composition may be administered for treating cerebral infarction in the acute phase.
  • acute phase means a period of 14 days from the onset of cerebral infarction.
  • the pharmaceutical composition may be administered shortly after the onset of cerebral infarction in a mammalian subject, especially a human, for example, within 3, 4.5, 6, 12, or 24 hours or longer during the acute phase after the onset of cerebral infarction.
  • the pharmaceutical composition When administered to a mammalian subject, especially a human, the pharmaceutical composition should provide the compound of formula (I) or a pharmaceutical acceptable salt thereof at a dose that is sufficient to effect a desired response in the subject for a reasonable period of time. Such dose is also called as a therapeutically effective amount.
  • the dose is appropriately determined according to various factors including the activity of the used compound, the species, age, sex, and body weight of the subject, and the symptom and severity of cerebral infarction. The dose also depends on the route, timing and frequency of the administration, the nature and degree of the possible adverse effect caused by the used compound itself or the desired physiological activity of the compound.
  • Those of ordinary skill in the art understand that plural times of administration may be necessary in accordance with the symptom or severity of cerebral infarction. Suitable dose and administration schedule may be determined within the common technical knowledge of those of ordinary skill in the art.
  • the pharmaceutical composition may be administered to provide the compound of formula (I) or a pharmaceutical salt thereof, for example, at a dose of about 0.001 to about 100 mg/kg body weight/day, about 0.005 to about 50 mg/kg body weight/day, about 0.01 to about 25 mg/kg body weight/day, or about 0.1 to about 10 mg/kg body weight/day.
  • the pharmaceutical composition may be administered one, two, three, four or more times per day, or in a sustained manner.
  • the pharmaceutical composition may comprise the compound of formula (I) or a pharmaceutically acceptable salt thereof as the active ingredient in an amount sufficient for treating cerebral infarction together with a pharmaceutically acceptable carrier.
  • the carrier is not particularly limited and may be any carrier which is generally used in pharmaceutical compositions, unless it has unsuitable physicochemical property, for example unfavorable solubility or reactivity with the compound, or is not suitable for the administration route.
  • the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical composition may vary in accordance with the formulation of the composition, for example, being 0.00001 to 10.0% by weight, 0.001 to 5% by weight, or 0.001 to 1% by weight.
  • the pharmaceutical composition may be formulated in various forms without particular limitation to achieve the treatment of cerebral infarction.
  • the composition may be formulated in a form suitable for oral administration or parenteral administration and may comprise the compound of formula (I) or a pharmaceutically acceptable salt thereof alone or two or more thereof in combination. Characteristics and properties of the composition may be determined in view of chemical properties of the active ingredient including solubility and the selected administration route according to standard pharmaceutical practice.
  • Examples of the composition to be used for oral administration include solid dosage forms such as a capsule, tablet, or powder, or liquid forms such as a solution or suspension.
  • Examples of the composition to be used for parenteral administration include an injection and infusion in the form of an aseptic solution or suspension.
  • the solid oral composition may contain an excipient generally known in the art.
  • the liquid oral composition may contain various additives such as flavoring agent, colorant, preservative, stabilizer, solubilizer, and suspending agent.
  • the composition may contain various isotonicity agents.
  • the pharmaceutical composition may also contain another active ingredient unless the effect of the invention is inhibited.
  • the present invention provides a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutical salt thereof and another active ingredient.
  • the present invention provides a kit or package comprising the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutical salt thereof and a composition comprising another active ingredient.
  • the kit or package may be associated with a written instruction for the administration.
  • the pharmaceutical composition may be administered to a subject in combination with another active ingredient concomitantly, sequentially, or at different times unless the effect of the invention is inhibited.
  • the compound of formula (I) and another active ingredient may be provided in a single composition or in separate compositions.
  • the administration route of the separate compositions may be same or different.
  • the another active ingredient or active compound examples include, but not limited to, thrombolytic agents such as tPA, rt-PA (alteplase), desmoteplase, urokinase, ozagrel sodium, and aspirin, selective thrombin inhibitors such as argatroban, heparin, low molecular weight heparin, heparinoid, cerebroprotective agents such as edaravone, hypertonic glycerol, and hypertonic mannitol.
  • thrombolytic agents such as tPA, rt-PA (alteplase), desmoteplase, urokinase, ozagrel sodium, and aspirin
  • selective thrombin inhibitors such as argatroban, heparin, low molecular weight heparin, heparinoid
  • cerebroprotective agents such as edaravone, hypertonic glycerol, and hypertonic mannitol
  • administration of the pharmaceutical composition may be combined with a surgical therapy such as mechanical thrombectomy, decompressive craniectomy, emergency carotid endarterectomy, acute recanalization therapy, and acute carotid revascularization (angioplasty/stent implantation).
  • a surgical therapy such as mechanical thrombectomy, decompressive craniectomy, emergency carotid endarterectomy, acute recanalization therapy, and acute carotid revascularization (angioplasty/stent implantation).
  • the present invention provides use of the compound of formula (I) or a pharmaceutically acceptable salt thereof for manufacturing a pharmaceutical composition for treating cerebral infarction.
  • the compound of formula (I) or a pharmaceutically acceptable salt thereof, which is a VAP-1 inhibitor, is useful for manufacturing a pharmaceutical composition for preventing or treating a disease associated with VAP-1, especially cerebral infarction.
  • the present invention provides a method for treating cerebral infarction comprising administering a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • the present invention provides the compound of formula (I) or a pharmaceutically acceptable salt thereof for use in treating cerebral infarction.
  • a pharmaceutical composition for treating cerebral infarction comprising a compound of formula (I): wherein m is an integer of 1 or 2, n is an integer of 2 or 3, and A is selected from the group consisting of in which R and R' are independently selected from C1-C6 alkyl, or a pharmaceutically acceptable salt thereof.
  • a method for treating cerebral infarction comprising administering a therapeutically acceptable amount of a compound of formula (I): wherein m is an integer of 1 or 2, n is an integer of 2 or 3, and A is selected from the group consisting of in which R and R' are independently selected from C1-C6 alkyl, or a pharmaceutically acceptable salt thereof.
  • the present invention provides pharmaceutical composition comprising the compound of formula (I), which is a benzene derivative, or a pharmaceutically acceptable salt thereof for use in treating cerebral infarction.
  • the compound or salt is a VAP-1 inhibitor and thus can treat cerebral infarction through a mechanism different from that of the known thrombolytic agent, which may cause brain hemorrhage, and can be used without limitation of the timing at which it is applied.
  • Synthesis Example 1 Synthesis of 2- ⁇ 4-[2-(3,4'-bipyridine-6-yl)ethyl]phenyl ⁇ acetohydrazide Step 1 To a solution of 5-bromopyridine-2-carbaldehyde (4.97 g, 26.9 mmol) in toluene (50 mL), ethylene glycol (8.34 g, 134 mmol) and p-toluenesulfonic acid monohydrate (51.2 mg, 0.27 mmol) were added. The mixture was heated to reflux for 13 hours while the generated water was separated by a water-measuring tube. The reaction mixture was cooled to room temperature.
  • Step 2 A mixture of bromo-2-(1,3-dioxolane-2-yl)pyridine (2.08 g, 9.00 mmol), pyridine-4-yl boronic acid (1.28 g, 10.4 mmol), palladium acetate (101 mg, 1.3 ⁇ mol), triphenylphosphine (708 mg, 2.70 mmol), sodium carbonate (1.91 g, 18.0 mmol), dioxane (50 mL) and water (50 mL) was heated to reflux for 25 hours. The mixture was cooled to room temperature and water (50 mL) was added. The mixture was extracted with ethyl acetate (150 mL) three times.
  • Step 3 6-(1,3-dioxolane-2-yl)-3,4'-bipyridine (961 mg, 4.21 mmol), p-toluenesulfonic acid monohydrate (640 mg, 3.37 mmol), acetonitrile (25 mL) and water (5 mL) were mixed and stirred for 8 hours at 80°C. The mixture was cooled to room temperature. A saturated aqueous solution of sodium hydrogen carbonate (25 mL) and water (25 mL) was added. The mixture was extracted with dichloromethane three times. The organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure.
  • Step 4 To a suspension of [4-(2-methoxy-2-oxoethyl)benzyl](triphenyl)phosphonium bromide (906 mg, 1.79 mmol) in tetrahydrofuran (7 mL), t-butoxy potassium (216 mg, 1.79 mmol) was added at 0°C and the mixture was stirred for 15 minutes. 3,4'-bipyridine-6-carbaldehyde (300 mg, 1.63 mmol) was added. The mixture was stirred for 30 minutes and water (10 mL) was added. The mixture was extracted with ethyl acetate three times.
  • Step 5 The mixture of methyl ⁇ 4-[2-(3,4'-bipyridine-6-yl)ethenyl]phenyl ⁇ acetate and triphenylphosphine oxide obtained in Step 4 was dissolved in a solvent consisting of ethyl acetate and ethanol (1:1, 30 mL). 10% palladium on carbon (wetted with 50% water, 400 mg) was added and the solution was hydrogenated at room temperature under atmospheric pressure. The reaction mixture was filtered through Celite (registered trademark) and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (FUJI SILYSIA CHEMICAL LTD.
  • Synthesis Example 2 Synthesis of 2- ⁇ 4-[2-(3,3'-bipyridine-6-yl)ethyl]phenyl ⁇ acetohydrazide Step 1 In a similar manner as in Step 2 of Synthesis Example 1, from 5-bromo-2-(1,3-dioxolane-2-yl)pyridine (2.03 mg, 8.82 mmol) and pyridine-3-yl boronic acid (1.25 g, 10.2 mmol), 6-(1,3-dioxolane-2-yl)-3,3'-bipyridine (1.78 g, 7.81 mmol, yield 89%) was obtained as a pale yellow solid.
  • Step 2 In a similar manner as in Step 3 of Synthesis Example 1, from 6-(1,3-dioxolane-2-yl)-3,3'-bipyridine (1.77 g, 7.75 mmol), 3,4'-bipyridine-6-carbaldehyde (797 mg, 4.32 mmol, yield 56%) was obtained as a yellow solid.
  • Step 3 to Step 5 In a similar manner as in Steps 4 to 6 of Synthesis Example 1, from [4-(2-methoxy-2-oxoethyl)benzyl](triphenyl)phosphonium bromide (778 mg, 1.54 mmol) and 3,4'-bipyridine-6-carbaldehyde (258 mg, 1.54 mmol), the title compound (335 mg, 0.94 mmol, yield 61%) was obtained as a white solid.
  • Synthesis Example 3 Synthesis of 2- ⁇ 4-[2-(5-pyrimidine-5-ylpyridine-2-yl)ethyl]phenyl ⁇ acetohydrazide Step 1 In a similar manner as in Step 2 of Synthesis Example 1, from 5-bromo-2-(1,3-dioxolane-2-yl)pyridine (2.06 g, 8.95 mmol) and pyrimidine-5-yl boronic acid (1.28 g, 10.3 mmol), 5-[6-(1,3-dioxolane-2-yl)pyridine-3-yl]pyrimidine (1.24 g, 5.41 mmol, yield 60%) was obtained as a pale yellow solid.
  • Step 2 In a similar manner as in Step 3 of Synthesis Example 1, from 5-[6-(1,3-dioxolane-2-yl)pyridine-3-yl]pyrimidine (1.24 g, 5.41 mmol), 5-pyrimidine-5-ylpyridine-2-carbaldehyde (453 mg, 2.45 mmol, yield 45%) was obtained as a yellow solid.
  • Step 3 to Step 5 In a similar manner as in Steps 4 to 6 of Synthesis Example 1, from [4-(2-methoxy-2-oxoethyl)benzyl](triphenyl)phosphonium bromide (900 mg, 1.78 mmol) and 5-pyrimidine-5-ylpyridine-2-carbaldehyde (300 mg, 1.62 mmol), the title compound (162 mg, 0.49 mmol, yield 52%) was obtained as a white solid.
  • Test Example 1 Analysis of SSAO and MAO-A/B inhibitory activities Using the method described in WO2009/145360 (Patent Literature 2), we measured SSAO (VAP-1) and human monoamineoxydase (MAO)-A/B inhibitory activities of the compounds synthesized in Synthesis Examples 1 to 3 (hereinafter referred to as Synthesized Compounds 1 to 3, respectively) and Compounds 1 and 2 as shown in Table 1 below. Results are shown in the table below.
  • Test Example 2 Evaluation of pharmacological activity in rat model of transient focal cerebral ischemia Test substance
  • test substance Compound 1, Compound 2, and Synthesized Compound 1 in solution (the concentration of each compound : 0.2 mg/mL).
  • Physiological saline (pH 4.0) and Edaravone Mitsubishi Tanabe Pharma Corporation, commercial name: RADICUT (registered trademark) injection, 30 mg was used as a vehicle and a positive control, respectively.
  • transient focal cerebral ischemia model in rats Eight weeks old male rats [strain: Crl:CD(SD), Charles River Laboratories Japan, Inc.] having 260 to 350 g of body weight were used for preparing a model of transient focal cerebral ischemia.
  • the rats were anesthetized by inhalation of 2% isoflurane (Mylan Seiyaku Ltd.) and were restrained in the supine position.
  • the right common carotid artery, external carotid artery, and internal carotid artery were exposed.
  • the right common carotid artery and the external carotid artery were ligated to occlude the middle cerebral artery (MCA).
  • a 19 mm-long 4-0 nylon suture (plug) precoated with silicone (Heraeus Kulzer Japan Co., Ltd.) was inserted into the bifurcation of the external and internal carotid arteries to occlude the MCA.
  • the plug was removed 2 hours after the MCA occlusion to restore the blood flow to the MCA.
  • benzylpenicillin potassium (Meiji Seika Pharma Co., Ltd.) at a dose of 20,000 units/kg was intramuscularly injected. Flexion of the forelimb opposite to the MCA occlusive site was confirmed 30 minutes after the surgery.
  • test substances The test substances or vehicle was administered 3 minutes before and 2 hours after the MCA reperfusion on the day of the surgery.
  • physiological saline pH 4.0
  • test substance groups i.e. Compound 1, Compound 2, and Synthesized Compound 1 groups
  • Edaravone at a dose of 3 mg/2 mL/kg in each injection was administered intravenously via the caudal vein, 3 minutes before and 30 minutes after the MCA reperfusion on the day of the surgery.
  • the rats were decapitated 24 hours after the MCA occlusion, and their brains were cut into 2-mm thick brain coronal sections.
  • the brain coronal sections were stained in 1% (W/V) 2,3,5-Triphenyltetrazolium chloride (Nacalai Tesque, Inc.) and photographed.
  • the cerebral infarct areas in the photographs were measured by graphical analysis. Based on the measured areas, the cerebral infarct volume was calculated.
  • Results Study results are shown in Fig. 1 and Table 3.
  • the cerebral infarct volumes in the vehicle, Compound 1, Compound 2, Synthesized Compound 1, and positive control groups were 344.0 ⁇ 36.3 mm 3 , 287.7 ⁇ 42.9 mm 3 , 305.2 ⁇ 41.2 mm 3 , 273.2 ⁇ 57.9 mm 3 , and 273.7 ⁇ 56.6 mm 3 , respectively; in which each value represents the mean ⁇ S.D.
  • the cerebral infarct volumes in the Compound 1 and Synthesized Compound 1 groups were statistically reduced as compared with that in the vehicle group.
  • the cerebral infarct volumes in Compound 1, Compound 2, and Synthesized Compound 1 groups were not statistically different from that in the positive control group.
  • the cerebral infarct volume of the positive control group was statistically significantly smaller than that of the vehicle group. From the above results, it is suggested that Compound 1 and Synthesized Compound 1 have the cerebral infarct volume reducing effect.
  • Test Example 3 Evaluation of dose-response in a rat model of transient focal cerebral ischemia Test substance Three different concentrations, 0.005, 0.05, and 0.5 mg/mL, of Compound 1 were tested. Physiological saline (pH 6.0) and Edaravone (Mitsubishi Tanabe Pharma Corporation, commercial name: RADICUT (registered trademark) injection, 30 mg) were used as a vehicle and a positive control, respectively. Preparation of transient focal cerebral ischemia model in rats The transient focal cerebral ischemia model was prepared in rats in the same manner as Test Example 2.
  • test substance The test substance or vehicle was administered 3 minutes before and 2 hours after the MCA reperfusion on the day of the surgery.
  • physiological saline pH 6.0
  • Compound 1 was administered intravenously via the caudal vein at doses of 0.01, 0.1 and 1 mg/2 mL/kg, respectively.
  • Compound 1 was administered intravenously via the caudal vein at a dose of 1 mg/kg (i.e., high dose) in each injection at the same schedule, then Edaravone was administered intravenously via the caudal vein at a dose of 3 mg/kg in each injection immediately before and 30 minutes after the MCA reperfusion.
  • Edaravone was administered intravenously via the caudal vein at a dose of 3 mg/kg in each injection immediately before and 30 minutes after the MCA reperfusion.
  • cerebral infarct volume The cerebral infarct volumes were calculated in the same manner as Test Example 2. Statistical analysis As for the results of the cerebral infarct volume, Dunnett's multiple comparison test was performed to compare the low, middle or high dose group with the vehicle group. Student's t-test was performed to compare the vehicle group or the combination group with the positive control group. Statistical analysis was performed using SAS 9.1.3 [EXSUS Version 7.7.1, SAS Institute Japan Ltd. (CAC EXICARE Corporation)]. Significance level was set to 0.05 (5%, both sides); and the results were shown with the attained significance level of 1% or 5%, if any.
  • Results Study results are shown in Fig. 2 and Table 4.
  • the cerebral infarct volumes in the vehicle, low, middle and high dose, positive control, and combination groups were 358.0 ⁇ 40.7 mm 3 , 322.1 ⁇ 19.6 mm 3 , 293.6 ⁇ 29.7 mm 3 , 259.7 ⁇ 80.0 mm 3 , 263.0 ⁇ 50.7 mm 3 , and 262.0 ⁇ 52.2 mm 3 , respectively, in which each value represents the mean ⁇ S.D.
  • Compound 1 reduced the cerebral infarct volume with an increase in dose.
  • the cerebral infarct volumes of the middle and high dose groups were statistically significantly smaller than that of the vehicle group. No statistical significance was attained between the high dose group and the combination group; and between the combination group and the positive control group.
  • Test Example 4 Behavioral pharmacological study in a rat model of transient focal cerebral ischemia Test substance We studied Compound 1 (0.5 mg/mL) as test substance. The vehicle and positive control were the same as those in Test Example 3. Preparation of transient focal cerebral ischemia model in rats The transient focal cerebral ischemia model was prepared in rats in the same manner as Test Example 2.
  • test substance group Compound 1 was administered intravenously via the caudal vein at a dose of 1 mg/2 mL/kg at immediately before and 2 and 8 hours after the MCA reperfusion on the day of the surgery (Day 0). Additionally, from Day 1 to Day 6, Compound 1 was administered intravenously via the caudal vein twice daily at 9:00 and 21:00. In the positive control group, Edaravone was administered intravenously via the caudal vein at a dose of 3 mg/2 mL/kg at immediately before and 30 minutes after the MCA reperfusion on the day of the surgery (Day 0). Further, from Day 1 to Day 6, the same scheme as that in the test substance group applied to the positive control group.
  • Rota-rod test Rota-rod test was performed one or two days before the surgery and on Days 3, 7, and 14. On Days 3, 7, and 14, the rota-rod test was performed after the observation of the neurological symptoms. The time on rod (sec) was measured using the rota-rod at a rotation speed of 8 rpm. The cut-off time was 120 seconds. For two successive days during three days before the surgery, rats were trained so that they stay on the rota-rod. The rats that were able to stay on the rota-rod for 120 seconds were given surgery to prepare the model.
  • Test Example 5 Pharmacological study in a rat model of cerebral ischemia with autologous thrombus Test substance Compound 1 (0.5 mg/mL) was tested. Physiological saline (pH 6.0) was used as a vehicle. As a thrombolytic agent rt-PA (alteplase, Kyowa Hakko Kirin Co., Ltd., Commercial name: ACTIVACIN (registered trademark) for Injection 6,000,000) was used.
  • the mixture was transferred into a polyethylene tube (PE8040, outer diameter: 0.80 mm, inner diameter: 0.40 mm, Natsume Seisyakusyo Co., Ltd.) and placed in an incubator maintained at 37°C for 24 hours.
  • a polyethylene tube PE8040, outer diameter: 0.80 mm, inner diameter: 0.40 mm, Natsume Seisyakusyo Co., Ltd.
  • the shape of the thrombus was observed with a stereoscopic microscope, and the thrombus was cut into a piece of 25 mm.
  • the rats were held supine position under anesthesia by inhalation of 1.5 to 2.0% isoflurane using an inhalation anesthesia instrument for small experimental animals [NARCOBIT-E (Type II), Natsume Seisyakusyo Co., Ltd.].
  • NARCOBIT-E Type II
  • the right common carotid artery, external carotid artery, and internal carotid artery were exposed by median section at the cervical region, and then external carotid artery was ligated with a suture.
  • Teflon tube (SLW-AWG32, inner diameter: 0.203 mm, outer diameter: 0.355 mm, Hagitec Inc.) stuffed with the thrombus was inserted to the origin of middle cerebral arteries through the right external carotid artery and right internal carotid artery, and then the thrombus was injected. After the injection, the tube was removed and the dissected part was sutured, and then rats were released from anesthesia.
  • test substance The rats were held supine position under anesthesia by inhalation of 1.5 to 2.0% isoflurane. Sutures at the neck were released and a Teflon-tube was inserted into the internal carotid artery. Two hours after the injection of the thrombus rt-PA (dose: 200,000 IU/0.5 mL/rat) or physiological saline (volume: 0.5 mL/rat) was administered through the tube using infusion pump (Telfusion Syringe Pump Type 35 TE-351, Terumo Corp.) at the injection speed of 2 mL/h for 15 minutes. In addition, the rats were maintained at 37°C with heating pad (BWT-100, Bioresearch Center Co. Ltd.) to avoid hypothermia during infusion.
  • heating pad BWT-100, Bioresearch Center Co. Ltd.
  • Compound 1 (dose: 1 mg/2 mL/kg) was administered intravenously via the caudal vein four times (just before the start of the rt-PA or physiological saline infusion or 2, 8 and 14 hours after the end of those).
  • Results Study results are shown in Fig. 5 and Table 7. Dead rats were excluded from the results.
  • the neurological symptoms scores of the vehicle, rt-PA, Compound 1, and combination groups were 10.0 ⁇ 2.7, 7.5 ⁇ 3.1, 7.6 ⁇ 3.3, and 6.4 ⁇ 3.8,respectively, wherein each value represents the mean ⁇ S.D.
  • the neurological symptoms scores of the Compound 1 and combination groups were significantly lower than that of the vehicle group (Fig. 5).

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Abstract

The object of the present invention is to provide a pharmaceutical composition for treating cerebral infarction. The present invention provides a pharmaceutical composition for treating cerebral infarction comprising a compound of formula (I) or a pharmaceutically acceptable salt. In formula (I), m is an integer of 1 or 2, n is an integer of 2 or 3, and A is dialkylamino or a certain ring structure containing at least one nitrogen atom.

Description

PHARMACEUTICAL COMPOSITION FOR TREATING CEREBRAL INFARCTION
The present invention relates to a pharmaceutical composition for treating cerebral infarction comprising a VAP-1 inhibitor, which is a benzene derivative having a certain structure.
Background
Cerebral stroke has been one of the major therapeutic targets. In Japan, the number of cerebral stroke patients has increased and is estimated to reach three million in 2020. Further, such patients are highly likely to suffer from aftereffects of the strokes. This also gives major challenges from the perspective of welfare and medical economics.
Cerebral stroke broadly classified into two subtypes. One subtype is ischemic stroke such as cerebral infarction, which is caused by blockage in the blood vessels in the brain; and the other subtype is hemorrhage stoke such as cerebral hemorrhage or subarachnoid hemorrhage, which is caused by bleeding due to rupture of an artery in the brain. In general, cerebral infarction occurs when blood supply to the brain is interrupted due to e.g., occlusion or stenosis of cerebral arteries. Such cerebral ischemia leads to insufficient oxygen or nutrients supply, causing brain tissues to necrotize completely or partially. Cerebral infarction includes, for example, lacunar infarction, which is caused by lesions in intracerebral small artery; atherothrombotic cerebral infarction, which is caused by atherosclerosis of relatively large artery in cervical region or skull; and cardiogenic embolism, which is caused by a cardiac disease.
For the acute stage of cerebral infarction, thrombolytic treatment using tissue plasminogen activator (tPA) is the first-line choice. Plasminogen is an enzyme that strongly dissolves thrombi formed in blood vessels. After cerebral infarction is induced by thrombi in the brain artery, tPA is administered to dissolve the thrombi and make the blood flow reperfuse. The recovery rate, representing the rate of the patients who has recovered from the disease to the feasible level of living an independent life, with tPA treatment has been reported 40 to 50%. Administration of tPA is limited for the patients within 4.5 hours from the onset of acute stroke, considering the risk of cerebral hemorrhage when administered after a lapse of prolonged time from the onset. An intravascular therapy, which removes thrombi from the blood vessels with mechanical method, has gradually used as a therapeutic option which can be applied within six hours from the onset of the acute stroke. Nevertheless further enhancement of the recovery rate and expansion of the time window for the treatment are strongly desired in clinical practice. Developing a novel therapy for cerebral infarction that decreases the risk of brain hemorrhage and enlarges the patient population to be treated will be highly appreciated.
Vascular adhesion protein-1 (VAP-1) is a protein which is synonymously referred to as semicarbazide-sensitive amine oxidase (SSAO). Membrane-bound VAP-1 is present at the surfaces of vascular endothelium and circulating SSAO is present in serum. The former is mainly involved in inflammation and works as an adhesive molecule to leukocytes such as granulocytes related to inflammation, and lymphocytes or monocytes related to inflammation and immunity. The latter is responsible for detoxicating amines through the amine oxidase activity. VAP-1/SSAO activity increases in plasma and various tissues of patients of diseases such as diabetes, atopic dermatitis, psoriasis, obesity, arteriosclerosis, and cardiac diseases. Therefore, VAP-1/SSAO inhibitors are expected to treat the diseases through suppressing and normalizing the increased VAP-1/SSAO activity.
Regarding cerebral infarction, stroke patients who have high VAP-1 activity in plasma were reported to have poor functional prognosis (Non Patent Literature 1). VAP-1 inhibitors were reported to produce a neuroprotective effect in rat models of cerebral infarction in which cerebral ischemia was caused by autologous thrombi or a plug (Non Patent Literatures 1 and 2).
Benzene, thiophene, and thiazole derivatives having VAP-1 inhibitory effect have been known and their use in prevention or treatment of VAP-1 associated diseases has been suggested (Patent Literatures 1 and 2).
WO2009/096609 WO2009/145360
Stroke. 2010;41:1528-1535 Journal of Neurochemistry (2012) 123 (Suppl. 2), 116-124
Summary
An object of the present invention is to provide a pharmaceutical composition for treating cerebral infarction. Further object of the present invention is to provide a novel benzene derivative.
In an aspect, the present invention provides a pharmaceutical composition for treating cerebral infarction comprising a compound of formula (I):
Figure JPOXMLDOC01-appb-C000001
 wherein
m is an integer of 1 or 2,
n is an integer of 2 or 3, and
A is selected from the group consisting of
Figure JPOXMLDOC01-appb-C000002
 wherein R and R' are independently selected from C1-C6 alkyl,
or a pharmaceutically acceptable salt thereof.
In another aspect, the present invention provides a compound of formula (I'):
Figure JPOXMLDOC01-appb-C000003
 wherein
m is an integer of 1 or 2,
n is an integer of 2 or 3, and
A' is selected from the group consisting of
Figure JPOXMLDOC01-appb-C000004
 or a pharmaceutically acceptable salt thereof. All compounds represented by formula (I') are also covered by formula (I).
Fig. 1 shows effects of Compound 1 and Synthesized Compound 1 on the cerebral infarct volume in a rat model of transient focal cerebral ischemia. Each value represents the mean ± standard deviation (S.D.) of 10 rats. *P < 0.05 and **P < 0.01 represent significant differences between the vehicle group and the Compound 1, Compound 2, or Synthesized Compound 1 group (Dunnett's multiple comparison test). ##P < 0.01 represents significant difference between the vehicle group and the positive control group (Student's t-test). No significant difference between the vehicle group and the Compound 2 group (Dunnett's multiple comparison test).Further, no significant difference between the positive control group and the Compound 1, Compound 2 or Synthesized Compound 1 group was observed (Dunnett's multiple comparison test). Fig. 2 shows effect of Compound 1 on the cerebral infarct volume in a rat model of transient focal cerebral ischemia. Each value represents the mean ± S.D. of 10 rats. **P < 0.01 represents significant difference between the vehicle group and the Compound 1 group (Dunnett's multiple comparison test). ##P < 0.01 represents significant difference between the vehicle group and the positive control group (Student's t-test). No significant difference between the vehicle group and the low dose group (Dunnett's multiple comparison test). No significant difference between the positive control group and the combination of Compound 1 (high dose) and positive control agent group was observed (Student's t-test). Fig. 3 shows effect of Compound 1 on the neurological symptoms score in a rat model of transient focal cerebral ischemia. Each value represents the mean ± S.D. of 10 or 11 rats. *P < 0.05 represents significant difference between the vehicle group and the Compound 1 group (Steel-Dwass test). #P < 0.05 represents significant difference between the vehicle group and the positive control group (Steel-Dwass test). Further, no significant difference between the vehicle group and the Compound 1 group or positive control group on Day 3 and 7 (Dunnett's multiple comparison test). No significant difference between the Compound 1 group and the positive control group was observed (Steel-Dwass test). Fig. 4 shows effect of Compound 1 in the rota-rod test in a rat model of transient focal cerebral ischemia. Each value represents the mean ± S.D. of 10 or 11 rats. *P < 0.05 represents significant difference between the vehicle group and the Compound 1 group (Tukey-Kramer test). #P < 0.05 represents significant difference between the vehicle group and the positive control group (Tukey-Kramer test). Further, no significant difference between the vehicle group and the Compound 1 or positive control group on Day 3 and 7 (Dunnett's multiple comparison test). No significant difference between the Compound 1 group and the positive control group was observed (Tukey-Kramer test). Fig. 5 shows effect of Compound 1 and effect of the combination of rt-PA and Compound 1 on the neurological symptoms score in a rat model of cerebral ischemia caused by autologous thrombus. *P < 0.05 represents significant difference between the vehicle group and the Compound 1 group or the combination group (Wilcoxon test).
The terms used in the specification and claims are explained in detail below. Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise.
In formulae (I) and (I’), substituents A and A' may be attached to any suitable position on the pyridine ring.
As used herein the term "alkyl" means a straight chain or branched chain alkyl having 1 to 6 carbon atoms, for example 1 to 4 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, tert-pentyl, and hexyl.
In one embodiment, the pharmaceutical composition may comprise a compound represented by the following formula
Figure JPOXMLDOC01-appb-C000005
 or
Figure JPOXMLDOC01-appb-C000006
 or a pharmaceutically acceptable salt thereof.
The term "pharmaceutically acceptable salt" as used herein means any nontoxic pharmaceutically acceptable salt, including salts with an inorganic or organic base and acid addition salts. Examples of the salt with an inorganic or organic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, an ammonium salt, and amine salts such as triethylamine salt and N-benzyl-N-methylamine salt. Examples of the acid addition salt include salts derived from mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and salts derived from organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, maleic acid, benzoic acid, glycol acid, gluconic acid, succinic acid, and arylsulfonic acids such as p-toluenesulfonic acid.
The compounds of formula (I) and the pharmaceutically acceptable salts thereof can be prepared with reference to known literatures such as WO2009/096609 or WO2009/145360, or in accordance with synthesis examples shown in the specification.
The term "cerebral infarction" as used herein means damages in the brain caused by cerebral ischemia and related symptoms, including, but not limited to, lacunar infarction, atherothrombotic cerebral infarction, and cardiogenic embolism, and also including sequelae of cerebral infarction such as neurologic symptoms, higher brain dysfunction, and affective disorder. The cause of cerebral ischemia includes, but not limited to, cerebral thrombosis, cerebral embolism, vasospasm, hypotension, and hypoxemia.
The term "pharmaceutical composition for treating cerebral infarction" as used herein means a pharmaceutical composition that is administered to a subject for the purpose of treating cerebral infarction in the subject. The term "treating cerebral infarction" means alleviating, relieving, and/or healing cerebral infarction, or delaying or suppressing the progression of cerebral infarction.
The subject to be treated by the pharmaceutical composition is a mammal such as a human, mouse, rat, pig, dog, cat, horse, and cow, especially a human.
The pharmaceutical composition may be administered via any route. For example, it may be administered systemically or locally via an administration route such as oral, subcutaneous, percutaneous, transmucosal, intravenous, intra-arterial, intramuscular, intraperitoneal, intranasal, subdural, rectal, or gastrointestinal route, or by inhalation. Spinal injection, epidural injection, or administration into cerebral ventricle is also possible. A physician who treats the disease may properly determine the administration route of the pharmaceutical composition. The pharmaceutical composition may be administered via two or more routes, wherein one route may provide more rapid and effective activity than the other. The administration route is not limited to the routes exemplified above.
The pharmaceutical composition may be administered for treating cerebral infarction in the acute phase. The term "acute phase" means a period of 14 days from the onset of cerebral infarction. The pharmaceutical composition may be administered shortly after the onset of cerebral infarction in a mammalian subject, especially a human, for example, within 3, 4.5, 6, 12, or 24 hours or longer during the acute phase after the onset of cerebral infarction.
When administered to a mammalian subject, especially a human, the pharmaceutical composition should provide the compound of formula (I) or a pharmaceutical acceptable salt thereof at a dose that is sufficient to effect a desired response in the subject for a reasonable period of time. Such dose is also called as a therapeutically effective amount. Those of ordinary skill in the art understand that the dose is appropriately determined according to various factors including the activity of the used compound, the species, age, sex, and body weight of the subject, and the symptom and severity of cerebral infarction. The dose also depends on the route, timing and frequency of the administration, the nature and degree of the possible adverse effect caused by the used compound itself or the desired physiological activity of the compound. Those of ordinary skill in the art understand that plural times of administration may be necessary in accordance with the symptom or severity of cerebral infarction. Suitable dose and administration schedule may be determined within the common technical knowledge of those of ordinary skill in the art.
The pharmaceutical composition may be administered to provide the compound of formula (I) or a pharmaceutical salt thereof, for example, at a dose of about 0.001 to about 100 mg/kg body weight/day, about 0.005 to about 50 mg/kg body weight/day, about 0.01 to about 25 mg/kg body weight/day, or about 0.1 to about 10 mg/kg body weight/day. The pharmaceutical composition may be administered one, two, three, four or more times per day, or in a sustained manner.
The term "about" as used herein means that the recited value may vary in the range of ± 30%, preferably ± 20%, more preferably ± 10%.
The pharmaceutical composition may comprise the compound of formula (I) or a pharmaceutically acceptable salt thereof as the active ingredient in an amount sufficient for treating cerebral infarction together with a pharmaceutically acceptable carrier. The carrier is not particularly limited and may be any carrier which is generally used in pharmaceutical compositions, unless it has unsuitable physicochemical property, for example unfavorable solubility or reactivity with the compound, or is not suitable for the administration route.
The amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical composition may vary in accordance with the formulation of the composition, for example, being 0.00001 to 10.0% by weight, 0.001 to 5% by weight, or 0.001 to 1% by weight.
The pharmaceutical composition may be formulated in various forms without particular limitation to achieve the treatment of cerebral infarction. The composition may be formulated in a form suitable for oral administration or parenteral administration and may comprise the compound of formula (I) or a pharmaceutically acceptable salt thereof alone or two or more thereof in combination. Characteristics and properties of the composition may be determined in view of chemical properties of the active ingredient including solubility and the selected administration route according to standard pharmaceutical practice. Examples of the composition to be used for oral administration include solid dosage forms such as a capsule, tablet, or powder, or liquid forms such as a solution or suspension. Examples of the composition to be used for parenteral administration include an injection and infusion in the form of an aseptic solution or suspension. The solid oral composition may contain an excipient generally known in the art. The liquid oral composition may contain various additives such as flavoring agent, colorant, preservative, stabilizer, solubilizer, and suspending agent. The composition may contain various isotonicity agents.
The pharmaceutical composition may also contain another active ingredient unless the effect of the invention is inhibited. In one embodiment, the present invention provides a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutical salt thereof and another active ingredient. In another embodiment, the present invention provides a kit or package comprising the pharmaceutical composition comprising the compound of formula (I) or a pharmaceutical salt thereof and a composition comprising another active ingredient. The kit or package may be associated with a written instruction for the administration.
The pharmaceutical composition may be administered to a subject in combination with another active ingredient concomitantly, sequentially, or at different times unless the effect of the invention is inhibited. The compound of formula (I) and another active ingredient may be provided in a single composition or in separate compositions. The administration route of the separate compositions may be same or different.
Examples of the another active ingredient or active compound include, but not limited to, thrombolytic agents such as tPA, rt-PA (alteplase), desmoteplase, urokinase, ozagrel sodium, and aspirin, selective thrombin inhibitors such as argatroban, heparin, low molecular weight heparin, heparinoid, cerebroprotective agents such as edaravone, hypertonic glycerol, and hypertonic mannitol. Additionally, administration of the pharmaceutical composition may be combined with a surgical therapy such as mechanical thrombectomy, decompressive craniectomy, emergency carotid endarterectomy, acute recanalization therapy, and acute carotid revascularization (angioplasty/stent implantation).
In a further embodiment, the present invention provides use of the compound of formula (I) or a pharmaceutically acceptable salt thereof for manufacturing a pharmaceutical composition for treating cerebral infarction. The compound of formula (I) or a pharmaceutically acceptable salt thereof, which is a VAP-1 inhibitor, is useful for manufacturing a pharmaceutical composition for preventing or treating a disease associated with VAP-1, especially cerebral infarction.
In a further embodiment, the present invention provides a method for treating cerebral infarction comprising administering a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof.
In a further embodiment, the present invention provides the compound of formula (I) or a pharmaceutically acceptable salt thereof for use in treating cerebral infarction.
Particularly, the present invention provides the following embodiments among others.
[1] A pharmaceutical composition for treating cerebral infarction comprising a compound of formula (I):
Figure JPOXMLDOC01-appb-C000007
 wherein
m is an integer of 1 or 2,
n is an integer of 2 or 3, and
A is selected from the group consisting of
Figure JPOXMLDOC01-appb-C000008
 in which R and R' are independently selected from C1-C6 alkyl,
or a pharmaceutically acceptable salt thereof.
[2] The pharmaceutical composition according to item [1] wherein A is
Figure JPOXMLDOC01-appb-C000009
 [3] The pharmaceutical composition according to item [1] or [2] wherein A is
Figure JPOXMLDOC01-appb-C000010
 [4] The pharmaceutical composition according to item [3] wherein R is methyl.
[5] The pharmaceutical composition according to any one of items [1] to [4] wherein the cerebral infarction is acute cerebral infarction.
[6] The pharmaceutical composition according to any one of items [1] to [5] wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is to be administered concomitantly, sequentially, or at different times, with one or more further active ingredient.
[7] The pharmaceutical composition according to item [6] wherein the further active ingredient is another active ingredient for treating cerebral infarction.
[8] The pharmaceutical composition according to item [6] or [7] wherein the further active ingredient is a thrombolytic agent.
[9] The pharmaceutical composition according to item [8] wherein the thrombolytic agent is tPA.
[10] A compound of formula (I')
Figure JPOXMLDOC01-appb-C000011
 wherein
m is an integer of 1 or 2,
n is an integer of 2 or 3, and
A' is selected from the group consisting of
Figure JPOXMLDOC01-appb-C000012
 or a pharmaceutically acceptable salt thereof.
[11] The compound according to item [10] wherein the compound is selected from the group consisting of
Figure JPOXMLDOC01-appb-C000013
 [12] A compound of formula (I) for use in treating cerebral infarction:
Figure JPOXMLDOC01-appb-C000014
 wherein
m is an integer of 1 or 2,
n is an integer of 2 or 3, and
A is selected from the group consisting of
Figure JPOXMLDOC01-appb-C000015
 in which R and R' are independently selected from C1-C6 alkyl,
or a pharmaceutically acceptable salt thereof.
[13] A method for treating cerebral infarction comprising administering a therapeutically acceptable amount of a compound of formula (I):
Figure JPOXMLDOC01-appb-C000016
 wherein
m is an integer of 1 or 2,
n is an integer of 2 or 3, and
A is selected from the group consisting of
Figure JPOXMLDOC01-appb-C000017
 in which R and R' are independently selected from C1-C6 alkyl,
or a pharmaceutically acceptable salt thereof.
[14] Use of the compound of formula (I):
Figure JPOXMLDOC01-appb-C000018
 wherein
m is an integer of 1 or 2,
n is an integer of 2 or 3, and
A is selected from the group consisting of
Figure JPOXMLDOC01-appb-C000019
 in which R and R' are independently selected from C1-C6 alkyl,
or a pharmaceutically acceptable salt thereof for manufacturing a pharmaceutical composition for treating cerebral infarction.
The present invention provides pharmaceutical composition comprising the compound of formula (I), which is a benzene derivative, or a pharmaceutically acceptable salt thereof for use in treating cerebral infarction. The compound or salt is a VAP-1 inhibitor and thus can treat cerebral infarction through a mechanism different from that of the known thrombolytic agent, which may cause brain hemorrhage, and can be used without limitation of the timing at which it is applied.
The following examples, including synthesis and test examples, illustrate the present invention in detail; and they do not restrict or limit the scope of the invention.
Synthesis Example 1:
Synthesis of 2-{4-[2-(3,4'-bipyridine-6-yl)ethyl]phenyl}acetohydrazide
Step 1
To a solution of 5-bromopyridine-2-carbaldehyde (4.97 g, 26.9 mmol) in toluene (50 mL), ethylene glycol (8.34 g, 134 mmol) and p-toluenesulfonic acid monohydrate (51.2 mg, 0.27 mmol) were added. The mixture was heated to reflux for 13 hours while the generated water was separated by a water-measuring tube. The reaction mixture was cooled to room temperature. A saturated aqueous solution of sodium hydrogen carbonate was added, and the mixture was stirred and stood still. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (50 mL) twice. The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (FUJI SILYSIA CHEMICAL LTD. BW-300SP 120 g, ethyl acetate : hexane = 2 : 3) to give 5-bromo-2-(1,3-dioxolane-2-yl)pyridine (5.24 g, 22.8 mmol, yield 85%) as a yellow solid.
Step 2
A mixture of bromo-2-(1,3-dioxolane-2-yl)pyridine (2.08 g, 9.00 mmol), pyridine-4-yl boronic acid (1.28 g, 10.4 mmol), palladium acetate (101 mg, 1.3 μmol), triphenylphosphine (708 mg, 2.70 mmol), sodium carbonate (1.91 g, 18.0 mmol), dioxane (50 mL) and water (50 mL) was heated to reflux for 25 hours. The mixture was cooled to room temperature and water (50 mL) was added. The mixture was extracted with ethyl acetate (150 mL) three times. The organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (FUJI SILYSIA CHEMICAL LTD. BW-300SP 120 g, dichloromethane : methanol = 30 : 1 to 10 : 1) to give 6-(1,3-dioxolane-2-yl)-3,4'-bipyridine(1.97 g, 8.60 mmol, yield 96%) as a pale yellow solid.
Step 3
6-(1,3-dioxolane-2-yl)-3,4'-bipyridine (961 mg, 4.21 mmol), p-toluenesulfonic acid monohydrate (640 mg, 3.37 mmol), acetonitrile (25 mL) and water (5 mL) were mixed and stirred for 8 hours at 80°C. The mixture was cooled to room temperature. A saturated aqueous solution of sodium hydrogen carbonate (25 mL) and water (25 mL) was added. The mixture was extracted with dichloromethane three times. The organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (FUJI SILYSIA CHEMICAL LTD. BW-300SP 100 g, acetone) to give 3,4'-bipyridine-6-carbaldehyde (525 mg, 2.85 mmol, yield 68%) as a yellow solid.
Step 4
To a suspension of [4-(2-methoxy-2-oxoethyl)benzyl](triphenyl)phosphonium bromide (906 mg, 1.79 mmol) in tetrahydrofuran (7 mL), t-butoxy potassium (216 mg, 1.79 mmol) was added at 0°C and the mixture was stirred for 15 minutes. 3,4'-bipyridine-6-carbaldehyde (300 mg, 1.63 mmol) was added. The mixture was stirred for 30 minutes and water (10 mL) was added. The mixture was extracted with ethyl acetate three times. The organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give a mixture of methyl{4-[2-(3,4'-bipyridine-6-yl)ethenyl]phenyl}acetate and triphenylphosphine oxide (580 mg).
Step 5
The mixture of methyl {4-[2-(3,4'-bipyridine-6-yl)ethenyl]phenyl}acetate and triphenylphosphine oxide obtained in Step 4 was dissolved in a solvent consisting of ethyl acetate and ethanol (1:1, 30 mL). 10% palladium on carbon (wetted with 50% water, 400 mg) was added and the solution was hydrogenated at room temperature under atmospheric pressure. The reaction mixture was filtered through Celite (registered trademark) and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (FUJI SILYSIA CHEMICAL LTD. BW-300SP 60 g, ethyl acetate) to give methyl{4-[2-(3,4'-bipyridine-6-yl)ethyl]phenyl}acetate (218 mg, 0.66 mmol, yield through Steps 4 and 5: 40%).
Step 6
To a solution of methyl{4-[2-(3,4'-bipyridine-6-yl)ethyl]phenyl}acetate (218 mg, 0.66 mmol) in methanol (20 mL), hydrazine monohydrate (334 mg, 6.67 mmol) was added and the mixture was stirred for 19 hours at 50°C. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (FUJI SILYSIA CHEMICAL LTD. BW-300SP 50 g, dichloromethane : methanol = 30 : 1). The fraction containing the desired product was concentrated to give crystals, which were washed in suspension with a mixture of ethyl acetate and diisopropylether (1 : 1, 5 mL). The crystal was filtered and dried under reduced pressure to give the title compound (150 mg, 0.45 mmol, yield 68%) as a white solid.
1H-NMR(CDCl3): 8.83 (d, J = 2.4 Hz, 1H), 8.69 (dd, J = 4.4 Hz, 1.8 Hz, 2H), 7.82 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 7.50 (dd, J = 4.4 Hz, 1.8 Hz, 2H), 7.29 - 7.13 (m, 5H), 6.58 (brs, 1H), 3.83 (brs, 2H), 3.53 (s, 2H), 3.19 - 3.05 (m, 4H)
High Resolution MS (ESI): 355.1515 [M+Na]+ (measured), 355.1529 [M+Na]+ (calculated)
Synthesis Example 2:
Synthesis of 2-{4-[2-(3,3'-bipyridine-6-yl)ethyl]phenyl}acetohydrazide
Step 1
In a similar manner as in Step 2 of Synthesis Example 1, from 5-bromo-2-(1,3-dioxolane-2-yl)pyridine (2.03 mg, 8.82 mmol) and pyridine-3-yl boronic acid (1.25 g, 10.2 mmol), 6-(1,3-dioxolane-2-yl)-3,3'-bipyridine (1.78 g, 7.81 mmol, yield 89%) was obtained as a pale yellow solid.
Step 2
In a similar manner as in Step 3 of Synthesis Example 1, from 6-(1,3-dioxolane-2-yl)-3,3'-bipyridine (1.77 g, 7.75 mmol), 3,4'-bipyridine-6-carbaldehyde (797 mg, 4.32 mmol, yield 56%) was obtained as a yellow solid.
Step 3 to Step 5
In a similar manner as in Steps 4 to 6 of Synthesis Example 1, from [4-(2-methoxy-2-oxoethyl)benzyl](triphenyl)phosphonium bromide (778 mg, 1.54 mmol) and 3,4'-bipyridine-6-carbaldehyde (258 mg, 1.54 mmol), the title compound (335 mg, 0.94 mmol, yield 61%) was obtained as a white solid.
1H-NMR(CDCl3): 8.85 (d, J = 2.0 Hz, 1H), 8.79 (d, J = 2.0 Hz, 1H), 8.64 (dd, J = 4.7 Hz, 1.7 Hz, 1H), 7.88 (ddd, J = 7.7 Hz, 2.0 Hz, 1.7 Hz, 1H), 7.79 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.41(dd, J = 7.7 Hz, 4.7 Hz, 1H), 7.25 (d, J = 12.2 Hz, 2H), 7.19 (d, J = 12.2 Hz, 2H), 6.60 (brs, 1H), 3.84 (brs, 2H), 3.55 (s, 2H), 3.20 - 3.06 (m, 4H)
High Resolution MS (ESI): 355.1522 [M+Na]+ (measured), 355.1529 [M+Na]+ (calculated)
Synthesis Example 3:
Synthesis of 2-{4-[2-(5-pyrimidine-5-ylpyridine-2-yl)ethyl]phenyl}acetohydrazide
Step 1
In a similar manner as in Step 2 of Synthesis Example 1, from 5-bromo-2-(1,3-dioxolane-2-yl)pyridine (2.06 g, 8.95 mmol) and pyrimidine-5-yl boronic acid (1.28 g, 10.3 mmol), 5-[6-(1,3-dioxolane-2-yl)pyridine-3-yl]pyrimidine (1.24 g, 5.41 mmol, yield 60%) was obtained as a pale yellow solid.
Step 2
In a similar manner as in Step 3 of Synthesis Example 1, from 5-[6-(1,3-dioxolane-2-yl)pyridine-3-yl]pyrimidine (1.24 g, 5.41 mmol), 5-pyrimidine-5-ylpyridine-2-carbaldehyde (453 mg, 2.45 mmol, yield 45%) was obtained as a yellow solid.
Step 3 to Step 5
In a similar manner as in Steps 4 to 6 of Synthesis Example 1, from [4-(2-methoxy-2-oxoethyl)benzyl](triphenyl)phosphonium bromide (900 mg, 1.78 mmol) and 5-pyrimidine-5-ylpyridine-2-carbaldehyde (300 mg, 1.62 mmol), the title compound (162 mg, 0.49 mmol, yield 52%) was obtained as a white solid.
1H-NMR(CDCl3): 9.25 (s, 1H), 8.95 (s, 2H), 8.79 (d, J = 2.2 Hz, 1H), 7.78 (dd, J = 8.0 Hz, 2.2 Hz, 1H), 7.34 - 7.10 (m, 5H), 6.58 (brs, 1H), 3.83 (brs, 2H), 3.53 (s, 2H),3.20 - 3.06 (m, 4H)
High Resolution MS (ESI): 356.1461 [M+Na]+ (measured), 356.1482 [M+Na]+ (calculated)
Test Example 1: Analysis of SSAO and MAO-A/B inhibitory activities
Using the method described in WO2009/145360 (Patent Literature 2), we measured SSAO (VAP-1) and human monoamineoxydase (MAO)-A/B inhibitory activities of the compounds synthesized in Synthesis Examples 1 to 3 (hereinafter referred to as Synthesized Compounds 1 to 3, respectively) and Compounds 1 and 2 as shown in Table 1 below. Results are shown in the table below.
Figure JPOXMLDOC01-appb-T000001
Test Example 2: Evaluation of pharmacological activity in rat model of transient focal cerebral ischemia
Test substance
We studied as test substance, Compound 1, Compound 2, and Synthesized Compound 1 in solution (the concentration of each compound : 0.2 mg/mL). Physiological saline (pH 4.0) and Edaravone (Mitsubishi Tanabe Pharma Corporation, commercial name: RADICUT (registered trademark) injection, 30 mg) was used as a vehicle and a positive control, respectively.
Figure JPOXMLDOC01-appb-T000002
Preparation of transient focal cerebral ischemia model in rats
Eight weeks old male rats [strain: Crl:CD(SD), Charles River Laboratories Japan, Inc.] having 260 to 350 g of body weight were used for preparing a model of transient focal cerebral ischemia. The rats were anesthetized by inhalation of 2% isoflurane (Mylan Seiyaku Ltd.) and were restrained in the supine position. The right common carotid artery, external carotid artery, and internal carotid artery were exposed. The right common carotid artery and the external carotid artery were ligated to occlude the middle cerebral artery (MCA). A 19 mm-long 4-0 nylon suture (plug) precoated with silicone (Heraeus Kulzer Japan Co., Ltd.) was inserted into the bifurcation of the external and internal carotid arteries to occlude the MCA. The plug was removed 2 hours after the MCA occlusion to restore the blood flow to the MCA. After the MCA occlusion surgery, benzylpenicillin potassium (Meiji Seika Pharma Co., Ltd.) at a dose of 20,000 units/kg was intramuscularly injected. Flexion of the forelimb opposite to the MCA occlusive site was confirmed 30 minutes after the surgery.
Administration of the test substances
The test substances or vehicle was administered 3 minutes before and 2 hours after the MCA reperfusion on the day of the surgery. In the vehicle group, physiological saline (pH 4.0) was administered intravenously via the caudal vein at a volume of 5 mL/kg (n = 10). In the test substance groups, i.e. Compound 1, Compound 2, and Synthesized Compound 1 groups, Compound 1, Compound 2, and Synthesized Compound 1 were respectively administered intravenously via the caudal vein at a dose of 1 mg/5 mL/kg in each injection (n = 10 in each group). In the positive control group, Edaravone at a dose of 3 mg/2 mL/kg in each injection was administered intravenously via the caudal vein, 3 minutes before and 30 minutes after the MCA reperfusion on the day of the surgery.
Measurement of cerebral infarct volume
The rats were decapitated 24 hours after the MCA occlusion, and their brains were cut into 2-mm thick brain coronal sections. The brain coronal sections were stained in 1% (W/V) 2,3,5-Triphenyltetrazolium chloride (Nacalai Tesque, Inc.) and photographed. The cerebral infarct areas in the photographs were measured by graphical analysis. Based on the measured areas, the cerebral infarct volume was calculated.
Statistical analysis
As for the results of the cerebral infarct volume, Dunnett's multiple comparison test was performed to compare the vehicle or the positive control group with each test substance group. Student's t-test was performed to compare the vehicle group with the positive control group. Statistical analyses were obtained using SAS 9.1.3 [EXSUS Version 7.7.1, SAS Institute Japan Ltd. (CAC EXICARE Corporation)]. Significance level was set to 0.05 (5%, both sides); and the results were shown with the attained significance level of 1% or 5%, if any.
Results
Study results are shown in Fig. 1 and Table 3. The cerebral infarct volumes in the vehicle, Compound 1, Compound 2, Synthesized Compound 1, and positive control groups were 344.0 ± 36.3 mm3, 287.7 ± 42.9 mm3, 305.2 ± 41.2 mm3, 273.2 ± 57.9 mm3, and 273.7 ± 56.6 mm3, respectively; in which each value represents the mean ± S.D. The cerebral infarct volumes in the Compound 1 and Synthesized Compound 1 groups were statistically reduced as compared with that in the vehicle group. The cerebral infarct volumes in Compound 1, Compound 2, and Synthesized Compound 1 groups were not statistically different from that in the positive control group. The cerebral infarct volume of the positive control group was statistically significantly smaller than that of the vehicle group. From the above results, it is suggested that Compound 1 and Synthesized Compound 1 have the cerebral infarct volume reducing effect.
Figure JPOXMLDOC01-appb-T000003
Test Example 3: Evaluation of dose-response in a rat model of transient focal cerebral ischemia
Test substance
Three different concentrations, 0.005, 0.05, and 0.5 mg/mL, of Compound 1 were tested. Physiological saline (pH 6.0) and Edaravone (Mitsubishi Tanabe Pharma Corporation, commercial name: RADICUT (registered trademark) injection, 30 mg) were used as a vehicle and a positive control, respectively.
Preparation of transient focal cerebral ischemia model in rats
The transient focal cerebral ischemia model was prepared in rats in the same manner as Test Example 2.
Administration of test substance
The test substance or vehicle was administered 3 minutes before and 2 hours after the MCA reperfusion on the day of the surgery. In the vehicle group physiological saline (pH 6.0) was administered intravenously via the caudal vein at a volume of 2 mL/kg (n = 10). In the low, middle and high dose groups (n = 10 in each group), Compound 1 was administered intravenously via the caudal vein at doses of 0.01, 0.1 and 1 mg/2 mL/kg, respectively. Furthermore, in the combination group of Compound 1 and Edarabone, Compound 1 was administered intravenously via the caudal vein at a dose of 1 mg/kg (i.e., high dose) in each injection at the same schedule, then Edaravone was administered intravenously via the caudal vein at a dose of 3 mg/kg in each injection immediately before and 30 minutes after the MCA reperfusion. In the positive control group, Edaravone was administered intravenously via the caudal vein at a dose of 3 mg/kg in each injection immediately before and 30 minutes after the MCA reperfusion.
Measurement of cerebral infarct volume
The cerebral infarct volumes were calculated in the same manner as Test Example 2.
Statistical analysis
As for the results of the cerebral infarct volume, Dunnett's multiple comparison test was performed to compare the low, middle or high dose group with the vehicle group. Student's t-test was performed to compare the vehicle group or the combination group with the positive control group. Statistical analysis was performed using SAS 9.1.3 [EXSUS Version 7.7.1, SAS Institute Japan Ltd. (CAC EXICARE Corporation)]. Significance level was set to 0.05 (5%, both sides); and the results were shown with the attained significance level of 1% or 5%, if any.
Results
Study results are shown in Fig. 2 and Table 4. The cerebral infarct volumes in the vehicle, low, middle and high dose, positive control, and combination groups were 358.0 ± 40.7 mm3, 322.1 ± 19.6 mm3, 293.6 ± 29.7 mm3, 259.7 ± 80.0 mm3, 263.0 ± 50.7 mm3, and 262.0 ± 52.2 mm3, respectively, in which each value represents the mean ± S.D. Compound 1 reduced the cerebral infarct volume with an increase in dose. The cerebral infarct volumes of the middle and high dose groups were statistically significantly smaller than that of the vehicle group. No statistical significance was attained between the high dose group and the combination group; and between the combination group and the positive control group.
Figure JPOXMLDOC01-appb-T000004
Test Example 4: Behavioral pharmacological study in a rat model of transient focal cerebral ischemia
Test substance
We studied Compound 1 (0.5 mg/mL) as test substance. The vehicle and positive control were the same as those in Test Example 3.
Preparation of transient focal cerebral ischemia model in rats
The transient focal cerebral ischemia model was prepared in rats in the same manner as Test Example 2.
Administration of test substance
In the test substance group, Compound 1 was administered intravenously via the caudal vein at a dose of 1 mg/2 mL/kg at immediately before and 2 and 8 hours after the MCA reperfusion on the day of the surgery (Day 0). Additionally, from Day 1 to Day 6, Compound 1 was administered intravenously via the caudal vein twice daily at 9:00 and 21:00. In the positive control group, Edaravone was administered intravenously via the caudal vein at a dose of 3 mg/2 mL/kg at immediately before and 30 minutes after the MCA reperfusion on the day of the surgery (Day 0). Further, from Day 1 to Day 6, the same scheme as that in the test substance group applied to the positive control group.
Evaluation of neurological symptoms score
The neurological symptoms were observed on the morning of Days 3, 7 and 14 according to the method of Bederson et al. (See, for details, Bederson J.B., Pitts L.H., Tsuji M. Nishimura M.C., Davis R.L., Bartkowski H. Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination. Stroke. 1986; 17: 472-476).
Rota-rod test
Rota-rod test was performed one or two days before the surgery and on Days 3, 7, and 14. On Days 3, 7, and 14, the rota-rod test was performed after the observation of the neurological symptoms. The time on rod (sec) was measured using the rota-rod at a rotation speed of 8 rpm. The cut-off time was 120 seconds. For two successive days during three days before the surgery, rats were trained so that they stay on the rota-rod. The rats that were able to stay on the rota-rod for 120 seconds were given surgery to prepare the model.
Statistical analysis
The neurological symptoms score of each day were compared among each group using Steel-Dwass test. The time on rod of each day were compared among each group using Tukey-Kramer test. Statistical analysis was performed using SAS 9.1.3 [EXSUS Version 7.7.1, SAS Institute Japan Ltd. (CAC EXICARE Corporation)].
Results
Study results are shown in Figs. 3 and 4 and Tables 5 and 6 below.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
On Day 14, the neurological symptoms score of the Compound 1 group was as low as that of the positive control group and significantly lower than that of the vehicle group (Fig. 3). The time on rod was also extended in the Compound 1 group in the similar manner (Fig. 4).
Test Example 5: Pharmacological study in a rat model of cerebral ischemia with autologous thrombus
Test substance
Compound 1 (0.5 mg/mL) was tested. Physiological saline (pH 6.0) was used as a vehicle. As a thrombolytic agent rt-PA (alteplase, Kyowa Hakko Kirin Co., Ltd., Commercial name: ACTIVACIN (registered trademark) for Injection 6,000,000) was used.
Preparation of the rat cerebral ischemia model with autologous thrombus
Eight-weeks old male rats (strain: Slc:Wistar, Japan SLC, Inc.) having 180 to 260 g of body weight were used for preparing a model of cerebral ischemia with autologous thrombus.
Under anesthesia by inhalation of 1.5 to 2.0% isoflurane, approximate 2 mL of blood was collected from subclavian vein and mixed with Thrombin JP 150 Unit and Human fibrinogen powder 40 mg. Immediately after the mixing, the mixture was transferred into a polyethylene tube (PE8040, outer diameter: 0.80 mm, inner diameter: 0.40 mm, Natsume Seisyakusyo Co., Ltd.) and placed in an incubator maintained at 37°C for 24 hours. Just before use, the shape of the thrombus was observed with a stereoscopic microscope, and the thrombus was cut into a piece of 25 mm.
The rats were held supine position under anesthesia by inhalation of 1.5 to 2.0% isoflurane using an inhalation anesthesia instrument for small experimental animals [NARCOBIT-E (Type II), Natsume Seisyakusyo Co., Ltd.]. The right common carotid artery, external carotid artery, and internal carotid artery were exposed by median section at the cervical region, and then external carotid artery was ligated with a suture. Teflon tube (SLW-AWG32, inner diameter: 0.203 mm, outer diameter: 0.355 mm, Hagitec Inc.) stuffed with the thrombus was inserted to the origin of middle cerebral arteries through the right external carotid artery and right internal carotid artery, and then the thrombus was injected. After the injection, the tube was removed and the dissected part was sutured, and then rats were released from anesthesia.
Administration of test substance
The rats were held supine position under anesthesia by inhalation of 1.5 to 2.0% isoflurane. Sutures at the neck were released and a Teflon-tube was inserted into the internal carotid artery. Two hours after the injection of the thrombus rt-PA (dose: 200,000 IU/0.5 mL/rat) or physiological saline (volume: 0.5 mL/rat) was administered through the tube using infusion pump (Telfusion Syringe Pump Type 35 TE-351, Terumo Corp.) at the injection speed of 2 mL/h for 15 minutes. In addition, the rats were maintained at 37°C with heating pad (BWT-100, Bioresearch Center Co. Ltd.) to avoid hypothermia during infusion.
Under the administration or non-administration of rt-PA, Compound 1 (dose: 1 mg/2 mL/kg) was administered intravenously via the caudal vein four times (just before the start of the rt-PA or physiological saline infusion or 2, 8 and 14 hours after the end of those).
Assessment of neurological symptoms
Neurological symptoms were evaluated 24 hours after the injection of the thrombus according to the method of W. E. Hoffman et al. (William E. Hoffman, Eberhard Kochs, Christian Werner, Chinamma Thomas, and Ronald F. Albrecht, Dexmedetomidine improves neurologic outcome from incomplete ischemia in the rat. Anesthesiology 75: 328-332, 1991).
Statistical analysis
For the data on neurological symptoms score, Wilcoxon test was performed to compare the vehicle group with the rt-PA group, Compound 1 group, or group administered Compound 1 and rt-PA (hereinafter refer to the group as the combination group). Steel test was performed to compare the combination group with the rt-PA group or Compound 1 group. Statistical analysis was performed using SAS 9.3 (SAS Institute Inc.). All analyses were conducted as one-side test. Significance level was set to 0.05 (5%, both sides); and the results were shown with the attained significance level of 1% or 5%, if any.
Results
Study results are shown in Fig. 5 and Table 7. Dead rats were excluded from the results. The neurological symptoms scores of the vehicle, rt-PA, Compound 1, and combination groups were 10.0 ± 2.7, 7.5 ± 3.1, 7.6 ± 3.3, and 6.4 ± 3.8,respectively, wherein each value represents the mean ± S.D. The neurological symptoms scores of the Compound 1 and combination groups were significantly lower than that of the vehicle group (Fig. 5).
Figure JPOXMLDOC01-appb-T000007

Claims (14)

  1. A pharmaceutical composition for treating cerebral infarction comprising a compound of formula (I):
    Figure JPOXMLDOC01-appb-C000020
     wherein
    m is an integer of 1 or 2,
    n is an integer of 2 or 3, and
    A is selected from the group consisting of
    Figure JPOXMLDOC01-appb-C000021
     in which R and R' are independently selected from C1-C6 alkyl,
    or a pharmaceutically acceptable salt thereof.
  2. The pharmaceutical composition according to claim 1, wherein A is
    Figure JPOXMLDOC01-appb-C000022
  3. The pharmaceutical composition according to claim 1 or 2, wherein A is
    Figure JPOXMLDOC01-appb-C000023
  4. The pharmaceutical composition according to claim 3, wherein R is methyl.
  5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cerebral infarction is acute cerebral infarction.
  6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is to be administered concomitantly, sequentially, or at different times, with one or more further active ingredient.
  7. The pharmaceutical composition according to claim 6, wherein the further active ingredient is another active ingredient for treating cerebral infarction.
  8. The pharmaceutical composition according to claim 6 or 7, wherein the further active ingredient is a thrombolytic agent.
  9. The pharmaceutical composition according to claim 8, wherein the thrombolytic agent is tPA.
  10. A compound of formula (I'):
    Figure JPOXMLDOC01-appb-C000024
     m is an integer of 1 or 2,
    n is an integer of 2 or 3, and
    A' is selected from the group consisting of
    Figure JPOXMLDOC01-appb-C000025
     or a pharmaceutically acceptable salt thereof.
  11. The compound according to claim 10, wherein the compound is selected from the group consisting of
    Figure JPOXMLDOC01-appb-C000026
  12. A compound of formula (I) for use in treating cerebral infarction:
    Figure JPOXMLDOC01-appb-C000027
     wherein
    m is an integer of 1 or 2,
    n is an integer of 2 or 3, and
    A is selected from the group consisting of
    Figure JPOXMLDOC01-appb-C000028
     in which R and R' are independently selected from C1-C6 alkyl,
    or a pharmaceutically acceptable salt thereof.
  13. A method for treating cerebral infarction comprising administering a therapeutically acceptable amount of a compound of formula (I):
    Figure JPOXMLDOC01-appb-C000029
     wherein
    m is an integer of 1 or 2,
    n is an integer of 2 or 3, and
    A is selected from the group consisting of
    Figure JPOXMLDOC01-appb-C000030
     in which R and R' are independently selected from C1-C6 alkyl,
    or a pharmaceutically acceptable salt thereof.
  14. Use of the compound of formula (I):
    Figure JPOXMLDOC01-appb-C000031
     wherein
    m is an integer of 1 or 2,
    n is an integer of 2 or 3, and
    A is selected from the group consisting of
    Figure JPOXMLDOC01-appb-C000032
     in which R and R' are independently selected from C1-C6 alkyl,
    or a pharmaceutically acceptable salt thereof for manufacturing a pharmaceutical composition for treating cerebral infarction.
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