JP2005517014A - Cytoprotective benzofuran derivatives - Google Patents

Abstract

Cytoprotective compounds, many of which are benzofuran derivatives, are certain ischemic or inflammatory conditions including but not limited to stroke, myocardial infarction, congestive heart failure, and skin disorders characterized by inflammation or oxidative disorders Useful for the treatment of They are also useful in the manufacture of pharmaceutical and cosmetic formulations for the treatment of such conditions. The invention also relates to a method of treating inflammation by reducing C-reactive protein (CRP). The invention also relates to cosmetic formulations for the treatment of skin inflammation and other skin disorders.

Description

(Field of Invention)
The present invention relates to specific compositions having cytoprotective activity, and in particular to a series of benzofuran derivatives. The present invention also relates to formulations and methods for treating stroke, myocardial infarction and chronic heart failure and other oxidative stress related conditions typically responsive to cellular enzyme regulation. The invention also relates to a method of treating inflammation by reducing C-reactive protein (CRP). The invention also relates to cosmetic formulations for the treatment of skin inflammation and other skin disorders.

(Background information)
The present invention relates to cytoprotective compounds, which are benzofuran derivatives, which include stereoisomers, mixtures of stereoisomers and pharmaceutically acceptable salts thereof.

  The compositions of the present invention have, for example, efficacy in certain ischemic or inflammatory conditions, including but not limited to stroke, myocardial infarction, congestive heart failure, and skin disorders characterized by inflammation or oxidative damage. It is active in the specific experimental model to be predicted. The present invention therefore relates to the use of cytoprotective derivatives in such situations.

  2,3-dihydro-5-oxy-4,6,7-trimethyl-2-optionally substituted alkylbenzofuran is described in US Pat. No. 5,114,966 as an antioxidant with anti-ischemic properties. It is disclosed as a sex pharmaceutical product. Hydroxamine derivatives of 2,3-dihydrobenzofuran carboxylic acid are disclosed in US Pat. No. 5,480,645. 2,3-dihydrofuran derivatives useful in preventing and treating neovascularization are disclosed in US Pat. Nos. 5,719,167 and 5,798,356. 5-hydroxybenzofuran is disclosed in US Pat. No. 5,674,876 for the treatment of pathological cell proliferative disorders. A method for inhibiting mammalian leukotriene biosynthesis with 6-hydroxybenzofuran is disclosed in US Pat. No. 4,714,711.

  A variety of drugs have been provided for such conditions, but provide new treatments for conditions characterized by oxidative stress, especially cerebral ischemia, UV exposure or inflammation Agents that are effective even when initially administered after a significant period of time (eg, about 5 hours or more), particularly after ischemic or oxidative damage, are desired. .

(Summary of the Invention)
The present invention relates to certain novel and related cytoprotective compounds that are particularly active in restoring or preserving metabolic integrity in oxidative cells that have been subjected to hypoxia. Such compounds are primarily benzofuran derivatives to treat a number of conditions characterized by oxidative stress, even when administered after a significant time interval following ischemic or oxidative injury. Are useful in the manufacture of pharmaceutical compositions, and are particularly useful in providing protection in the event of cerebral ischemia or UV exposure, or inflammation. In particular, the compositions of the invention are useful in the treatment of stroke, as demonstrated by providing neuroprotection in a standard experimental model of focal cerebral ischemia. They also include myocardial ischemia (myocardial infarction) and other indications characterized by oxidative stress and / or inflammation (diabetes, kidney disease, premenstrual syndrome, asthma, cardiopulmonary inflammatory disorders, chronic heart failure, rheumatoid arthritis Useful for the treatment of allograft tissue for transplantation, including, but not limited to, muscle fatigue, intermittent claudication). In particular, with respect to dermatological conditions, the compounds, formulations and methods of the present invention are useful in regulating skin conditions, in regulating signs of skin aging, and in treating a number of conditions (aging). Useful for, but not limited to, prevention or protection of skin tissue against damage or damage resulting from, for example, harmful (UV) radiation, stress and fatigue. These compounds, formulations and methods of the present invention also include, for example, contact dermatitis, wounds, irritation (including retina-induced irritation), hirsutism, hair growth regulation, disorders in pigmentation, or psoriasis. It is useful in treatment and can be used as a bactericidal, antifungal and antibacterial agent. The compounds of the present invention also show activity for reduced elevated CRP levels associated with a number of diseases and disorders, including but not limited to cardiovascular disease, diabetes and infectious diseases.

  The present invention provides compounds of formula I:

によって表される化合物（１つの立体異性体、立体異性体の混合物、ならびにその薬学的に受容可能な塩を含む）に関し、
ここで：
Ｒ^１は、水素、必要に応じて置換されたアルキル、必要に応じて置換されたアルケニル、必要に応じて置換されたアリール、（必要に応じて置換されたアルコキシ）カルボニルまたはハロゲンであり；
Ｒ^２およびＲ^３は、必要に応じて置換されたアルキル、必要に応じて置換されたアルケニル、必要に応じて置換されたシクロアルキルから独立して選択され；
Ｒ^４は、水素、必要に応じて置換されたアリール、（必要に応じて置換されたアルキル）カルボニル、（必要に応じて置換されたアリール）カルボニル、（必要に応じて置換されたヘテロシクリル）カルボニル、（必要に応じて置換されたヘテロシクリルアルキル）カルボニル、（必要に応じて置換されたアルコキシ）カルボニル、（必要に応じて置換されたアルケニルオキシ）カルボニル、（必要に応じて置換されたアミノ）カルボニル、カルボキシ、ホルミル、またはヒドロキシ（必要に応じて置換された）アルキルであり；
Ｒ^５は、水素、アルキル、アルケニル、（必要に応じて置換されたアルコキシ）カルボニル、カルボキシ、（必要に応じて置換されたアミノ）カルボニル、または必要に応じて置換されたアリールであり；
ただし、Ｒ^４またはＲ^５のうちの１つは、水素であり、そしてＲ^４が水素である場合、Ｒ^５は、水素ではなく、かつ、Ｒ^５が水素である場合、Ｒ^４は、水素ではない；
Ｒは、水素、アルキル、アシル、ホスホリル、またはポリアルコキシであり；もしくは
ＲおよびＲ^１は、それらが結合する原子とともに、必要に応じて置換された環を形成する。

With respect to compounds represented by (including one stereoisomer, a mixture of stereoisomers, and pharmaceutically acceptable salts thereof),
here:
R ¹ is hydrogen, alkyl optionally substituted, alkenyl which is optionally substituted, optionally substituted aryl, (alkoxy optionally substituted) carbonyl or halogen;
R ² and R ³ are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl;
R ⁴ is hydrogen, optionally substituted aryl, (optionally substituted alkyl) carbonyl, (optionally substituted aryl) carbonyl, (optionally substituted heterocyclyl) carbonyl , (Optionally substituted heterocyclylalkyl) carbonyl, (optionally substituted alkoxy) carbonyl, (optionally substituted alkenyloxy) carbonyl, (optionally substituted amino) carbonyl , Carboxy, formyl, or hydroxy (optionally substituted) alkyl;
R ⁵ is hydrogen, alkyl, alkenyl, (optionally substituted alkoxy) carbonyl, carboxy, (optionally substituted amino) carbonyl, or optionally substituted aryl;
Provided that when one of R ⁴ or R ⁵ is hydrogen and R ⁴ is hydrogen, R ⁵ is not hydrogen and when R ⁵ is hydrogen, R ⁴ is hydrogen is not;
R is hydrogen, alkyl, acyl, phosphoryl, or polyalkoxy; or R and R ¹ together with the atoms to which they are attached form an optionally substituted ring.

A preferred embodiment of the present invention relates to compounds of formula I wherein R ² and R ³ are (C ₁ -C ₆ ) alkyl, preferably methyl, and a subset of compounds of formula I wherein R is hydrogen included.

In another embodiment, the invention relates to a compound of formula I, wherein R ² and R ³ are (C ₁ -C ₆ ) alkyl, preferably methyl, R is hydrogen, and R ⁵ Is hydrogen and R ⁴ is optionally substituted aryl, (optionally substituted alkyl) carbonyl, (optionally substituted aryl) carbonyl, (optionally substituted Heterocyclyl) carbonyl, (optionally substituted heterocyclylalkyl) carbonyl, (optionally substituted alkoxy) carbonyl, (optionally substituted alkenyloxy) carbonyl, (optionally substituted) Amino) carbonyl, carboxy, formyl, or hydroxy (optionally substituted) alkyl, especially where the aryl , Phenyl which is unsubstituted or substituted with alkyl, alkoxy, hydroxy, phenyl substituted by one or more substituents selected (optionally substituted alkoxy) carbonyl, nitro, halo, and cyano.

In another embodiment, the present invention is concerned with compounds of formula I wherein R ² and R ³ are (C ₁ -C ₆ ) alkyl, preferably methyl, R is hydrogen and R ⁴ is Is hydrogen and R ⁵ is optionally substituted aryl, preferably unsubstituted phenyl or alkyl, alkoxy, hydroxy, (optionally substituted alkoxy) carbonyl, nitro, halo And phenyl substituted by one or more substituents selected from cyano.

In another embodiment, the invention relates to compounds of formula I, wherein R and R ¹ form a ring, preferably R and R ¹ are unsubstituted phenyl or alkyl, alkenyl, Forming a furan ring substituted with phenyl substituted by one or more substituents selected from hydroxy, alkoxy, nitro, cyano, carboxy, carboxy ester, haloalkyl and halo.

  Certain embodiments of the present invention provide novel and preferred combinations of substituents depending from different inventive formulas.

  In another aspect, the present invention provides a pharmaceutical and / or cosmetic composition comprising a therapeutically effective amount of a compound of formula I mixed with at least one pharmaceutically acceptable excipient, or a pharmacy thereof Relating to chemically acceptable salts. Particularly preferred are pharmaceutical or cosmetic compositions wherein the compound of formula I is selected from the preferred compounds.

  Another aspect of the present invention is the treatment of a mammal suffering from a condition characterized by oxidative stress with a therapeutically effective amount of a compound represented by Formula I (single stereoisomers, mixtures of stereoisomers and pharmaceuticals thereof) For the treatment by administering a pharmaceutically acceptable salt).

  In one embodiment, the present invention relates to a method for the treatment of cardiovascular conditions, cerebrovascular conditions, neurological conditions, inflammatory conditions, autoimmune conditions and / or dermatological conditions. In another embodiment, the invention is a condition selected from stroke, cerebral ischemia, myocardial infarction, chronic heart failure, retinal ischemia, postoperative cognitive dysfunction, peripheral neuropathy, spinal cord injury, head injury and surgical trauma Relates to a method for treating In another embodiment, the condition includes an inflammatory component or an autoimmune component.

  In another embodiment, the present invention relates to a method for treating a skin condition characterized by the following oxidative stress: regulation of skin condition, regulation of signs of skin aging, contact dermatitis, wound wound, skin pigmentation , Hair growth regulation, stimulation (including retina-induced stimulation), psoriasis, age-related damage and damage resulting from harmful (UV) radiation, damage resulting from stress or fatigue, but is not limited thereto. In another embodiment, the compound of formula I or composition thereof is administered topically. In another embodiment, the condition is dermatological and promotes production by instructing the user to apply to the skin a pharmaceutical or cosmetic composition incorporating the above compound of formula I The method further includes.

  In yet another embodiment, the present invention relates to methods for treating stroke and other oxidative stress related conditions, which conditions are regulated by cellular enzyme modulation (eg, cerebral ischemia, myocardial infarction, chronic in mammals). Responsive to heart failure and exposure to UV radiation. This treatment is by administering to a mammal in need of such treatment a therapeutically effective amount of any compound of Formula I, or a pharmaceutically acceptable salt thereof.

  In yet another aspect, the present invention relates to a method for reducing the level of C-reactive protein (CRP) associated with inflammation, which includes cardiovascular inflammatory conditions, respiratory inflammatory conditions, sepsis, diabetes, muscle fatigue, Systemic lupus erythematosus (SLE), end-stage renal disease (ERSD), periodontal disease, and inflammatory skin conditions include, but are not limited to.

  Particular preference is given to the method of treatment and its use in the manufacture of pharmaceutical and / or cosmetic compositions, wherein the compound of formula I is selected from the preferred compounds.

(Detailed description of the invention)
(Definition)
As used herein, the following words and phrases are generally intended to have the meanings indicated below, except to the extent that the context in which they are used otherwise indicates.

  The term “optional” or “as appropriate” means that the event or situation subsequently described may or may not occur, and that this description may be used if and when the event or situation described above occurs. It means to include cases that do not occur. For example, “optionally substituted alkyl” means either “alkyl” or “substituted alkyl” as defined below. For any group that contains one or more substituents, such groups may not be sterically practical and / or not intended to introduce any substitution or substitution pattern that is not synthetically feasible. Will be understood by those skilled in the art.

Abbreviations for specific compounds, reactants, or reaction parameters are defined as follows:
“DCM” refers to dichloromethane or methylene chloride.
-"T-Bu" means t-butyl-"DCC" means 1,3-dicyclohexylcarbodiimide-"DIC" means N, N-diisopropylcarbodiimide-"DIPEA" means diisopropyl “DMAP” refers to 4-N, N-dimethylaminopyridine, “DMF” refers to N, N-dimethylformamide, “Eq.” Refers to equivalent, “EDCl” "Refers to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride." MeOH "refers to methanol." THF "refers to tetrahydrofuran." EtOAc "refers to ethyl acetate. Say.

  The term “acyl” refers to the group —C (O) —H, —C (O) — (optionally substituted alkyl), —C (O) — (optionally substituted cycloalkyl). ) Group, -C (O)-(optionally substituted alkenyl) group, -C (O)-(optionally substituted cycloalkenyl) group, -C (O)-(optional) Substituted aryl) group, -C (O)-(optionally substituted heteroaryl) group and -C (O)-(optionally substituted heterocyclyl) group.

  The term “alkenyl” refers to a monoradical, branched or unbranched, unsaturated or polyunsaturated having about 2 to 20 carbon atoms (more preferably about 2 to 10 carbon atoms). The hydrocarbon chain. The terms include ethenyl, but-2-enyl, 3-methyl-but-2-enyl (also referred to as “prenyl”), octa-2,6-dienyl, 3,7-dimethyl-octa-2,6-dienyl. (Also referred to as “geranyl”) and the like.

  The term “substituted alkenyl” refers to an alkenyl group in which one or more (up to about 5, preferably up to about 3) hydrogen atoms are replaced by, for example, the following substituents: hydroxy, alkoxy, Carboxy, cyano, halogen or nitro.

  The term “alkoxy” refers to an —O-alkyl group, —O-alkenyl group, —O-cycloalkyl group, —O-cycloalkenyl group, and —O-alkynyl group. Preferred alkoxy groups are -O-alkyl and -O-alkenyl and are, for example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n- Hexoxy, 1,2-dimethylbutoxy, 3,7-dimethyl-octa-2,6-dienyloxy and the like can be mentioned.

The term “substituted alkoxy” refers to —O— (substituted alkyl) group, —O— (substituted alkenyl) group, —O— (substituted cycloalkyl) group, —O— ( Substituted cycloalkenyl) group, -O- (substituted alkynyl) group and -O- (optionally substituted alkylene) -alkoxy group. One preferred substituted alkoxy group is “polyalkoxy” or —O— (substituted alkylene) -alkoxy, and includes, for example, —OCH ₂ OCH ₃ groups, —OCH ₂ CH ₂ OCH ₃ groups, and ( Or PEG) groups (eg, —O (CH ₂ CH ₂ O) _x CH ₃ and —O (CH ₂ CH ₂ O) _x H, where x is about 2-20, preferably about 2 10 and more preferably an integer of about 2-5.

  The term “alkyl” refers to a monoradical branched or unbranched saturated hydrocarbon chain (preferably about 1 to 20 carbon atoms, more preferably about 1 to 10 carbon atoms, and even more preferably Has about 1 to 6 carbon atoms). This term is exemplified by, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl, and the like.

  The term “substituted alkyl” means that one or more (up to about 5, preferably up to about 3) hydrogen atoms are replaced by the following groups: ═O, ═S, acyl, acyloxy, optionally. Alkoxy, optionally substituted amino, azide, carboxyl, (optionally substituted alkoxy) carbonyl, (optionally substituted amino) carbonyl, cyano, optionally substituted cyclo Substituted by a substituent independently selected from alkyl, optionally substituted cycloalkenyl, heterocyclyl, carboxy, halogen, hydroxyl, nitro, cyano, sulfanyl, sulfinyl, and sulfonyl, or two substitutions An alkyl group in which the group together with the carbon to which they are attached can form a ring. One of the preferred optional substituents for alkyl is a hydroxyalkyl group (eg, 2-hydroxyethyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl, etc.); a dihydroxyalkyl group (glycol) (eg, 2,3-dihydroxypropyl, 3,4-dihydroxybutyl, 2,4-dihydroxybutyl, etc.); and hydroxy exemplified by polyethylene glycol, polypropylene glycol and polybutylene glycol. A preferred “substituted alkyl” wherein the substituent groups form a ring is 6-hydroxy-3-methyl- [1,3] oxazinan-6-yl.

The term “amino” refers to the group —NH ₂ .

  The term “substituted amino” refers to an —NHR or —NRR group. Wherein each R is as follows: optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted cycloalkenyl, optionally From optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, acyl, optionally substituted alkoxy, carboxy and alkoxycarbonyl An independently selected group, wherein RR together with the nitrogen to which they are attached form a cyclic amine, which is optionally selected from O, N and S Incorporate one or more additional heteroatoms.

  The term “aryl” refers to an aromatic cyclic hydrocarbon group of 6 to 20 carbon atoms having a single ring (eg, phenyl) or a condensed (fused) ring (eg, naphthyl or anthryl). Say. Preferred aryl includes phenyl, naphthyl and the like.

  The term “substituted aryl” means 1 to 5 substituents (preferably 1 to 3 substituents) independently selected from the group consisting of, unless constrained by the definition regarding aryl substituents: ) -Substituted aryl group as defined above: = O, = S, acyl, acyloxy, optionally substituted alkenyl, optionally substituted alkoxy, optionally substituted Alkyl (eg tri-halomethyl), optionally substituted alkynyl, optionally substituted amino, optionally substituted aryl, optionally substituted aryloxy, azide, Carboxyl, (optionally substituted alkoxy) carbonyl, (optionally substituted amino) carbonyl, cyano, optionally substituted cycl Alkyl, optionally substituted cycloalkenyl, halogen, optionally substituted heteroaryl, optionally substituted heteroaryloxy, optionally substituted heterocyclyl, optionally substituted Heterocyclooxy, hydroxyl, nitro, sulfanyl, sulfinyl, and sulfonyl. Preferred aryl substituents include: optionally substituted alkenyl, alkyl, alkoxy, substituted amino, halo, hydroxyl, alkoxycarbonyl, carboxy, cyano, nitro, phosphoryl.

  The term “carbonyl” refers to the diradical “—C (═O) —”, also illustrated as “—C (O) —”.

  The term “(optionally substituted alkoxy) carbonyl” refers to the following groups: —C (O) O— (optionally substituted alkyl), —C (O) O— (necessary). Optionally substituted cycloalkyl), —C (O) O— (optionally substituted alkenyl), —C (O) O— (optionally substituted alkynyl), —C (O ) O— (optionally substituted aryl), —C (O) O— (optionally substituted heteroaryl), and —C (O) O— (optionally substituted heterocyclyl). ). These moieties are also called esters.

  The term “(optionally substituted amino) carbonyl” refers to the group —C (O) — (optionally substituted amino). This moiety is also referred to as a primary carboxamide, a secondary carboxamide, or a tertiary carboxamide, and the “substituted amino” can be a cyclic amine.

  The term “carboxy” or “carboxyl” refers to a “—C (O) OH” moiety, also illustrated as “—COOH”.

  The term “compound of formula I” is intended to include the derivatives of the invention as disclosed and / or pharmaceutically acceptable salts of such compounds. Furthermore, the compounds used in the present invention include the individual stereochemical isomers (resulting from the choice of substituents) and mixtures of isomers.

  The term “cosmetics” includes makeup products, foundation products, and skin care products. The term “make-up” refers to products that leave a color on the face and include foundation, black and brown, ie mascara, concealer, eyeliner, brow color, eye shadow, blusher, lip color, and the like. The term “foundation” refers to products such as liquids, creams, mousses, pancakes, compacts, concealers, etc. that make the overall coloration of the skin uniform. Foundations are typically manufactured to perform well on moisturized and / or oiled skin. The term “skin care product” refers to a product used to treat or care to moisturize, improve or clean the skin. Products contemplated by the phrase “skin care products” include, but are not limited to: adhesives, bandages, toothpastes, anhydrous occlusive humectants, antiperspirants, deodorants, powder cleaners, for soft finishes Towels, occlusive drug delivery patches, nail polish, powder, tissue, wipes, solid emulsion compacts, anhydrous hair conditioners, medicated shampoos, scalp treatments and more.

  The term “CRP” or “C-reactive protein” refers to a biochemical marker of inflammation. The presence of elevated levels of CRP has been shown to be associated with various inflammatory conditions, for example: cardiovascular disease or cardiovascular disorders (eg, atrial fibrillation, unstable angina) , Coronary artery disease, peripheral arterial disease, cardiovascular vasculopathy (CAVD)); mastitis; pre-clamplucia; inflammatory bowel condition; stroke; tissue infarction; estrogen / progestin hormone replacement therapy (HRT); infection (bacteria, viruses and protozoa); bacterial meningitis; trauma; surgery; transplantation of medical materials; smoking; diabetes; neurodegenerative diseases (eg, Alzheimer) Infectious diseases (eg, myocarditis, cardiomyopathy, acute endocarditis, pericardium Atherosclerosis; systemic inflammatory response syndrome (SIRS) / sepsis; adult respiratory distress syndrome (ARDS); asthma; rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus; airway responsiveness (AHR) Bronchial hyperresponsiveness; chronic obstructive pulmonary disease (COPD); congestive heart failure (CHF); inflammatory complications of type I and type II diabetes; metabolic syndrome; end-stage renal disease (ESRD), premenstrual Syndrome (PMS) or muscle fatigue or inflammation; multiple organ dysfunction syndrome (MODS); airway hyper-responses (AHR); bronchial hyperresponsiveness; Acute allergic reaction; gingivitis and skin condition. CRP is reported by Spanheimer (2001, Postgrad. Med. 109 (4) 26) and Ridkler et al. (2000, NEJM 342 (12) 836-43) as a marker for systemic inflammation. ing.

  The term “cycloalkyl” refers to a monovalent saturated group consisting of one or more rings, which unless otherwise indicated, are optionally substituted with: hydroxy, cyano, Lower alkyl, lower alkoxy, thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, dialkylamino, aminocarbonyl, carbonylamino, aminosulfonyl, sulfonylamino or sulfonyl. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl and the like.

  The term “dermatologically acceptable”, as used herein, indicates that the composition or its components are described without undue toxicity, incompatibility, instability, allergic response, etc. Means suitable for use in contact with human skin.

  The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

  The term “heteroaryl” refers to about 1 to 40 (preferably about 3 to 15) carbon atoms and about 1 to 10 heteroatoms (preferably nitrogen, sulfur, phosphorus, in at least one ring). And / or an aromatic cyclic hydrocarbon group having about 1 to 4 heteroatoms selected from oxygen. Such heteroaryl groups have a single ring (eg, pyridyl or furyl) or multiple condensed rings (eg, indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, [2,2 '] bipyridinyl, pyrrolyl and furyl.

  The terms “heterocycle”, “heterocyclic” and “heterocyclyl” mean about 1 to 40 (preferably about 3 to 15) carbon atoms and about 1 to 10 heteroatoms (preferably about 3 to 15) in the ring. Monoradical, saturated or unsaturated, non-aromatic cyclic hydrocarbon group having about 1 to 4 heteroatoms selected from nitrogen, sulfur, phosphorus, and / or oxygen. Such heterocyclic groups can have a single ring or multiple condensed rings. Preferred heterocyclic groups include morpholino, piperidinyl, 1,3-oxazinane and the like.

  The terms “substituted (substituted) heterocycle”, “substituted (substituted) heterocyclic” and “substituted (substituted) heterocyclyl”, unless otherwise restricted by the definition relating to heterocyclic, from the group consisting of Refers to a heterocyclyl group as defined above substituted with 1 to 5 independently selected substituents (preferably 1 to 3 substituents): ═O, ═S, acyl, Acyloxy, optionally substituted alkenyl, optionally substituted alkoxy, optionally substituted alkyl (eg, tri-halomethyl), optionally substituted alkynyl, optionally substituted Amino, optionally substituted aryl, optionally substituted aryloxy, azide, carboxyl, (optionally substituted alkoxy) carbonyl, (required) Optionally substituted amino) carbonyl, cyano, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, halogen, optionally substituted heteroaryl, optionally substituted Heteroaryloxy, optionally substituted heterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro, sulfanyl, sulfinyl, and sulfonyl.

  The term “heterocycloalkyl” refers to “-alkylene-heterocycle” moieties, each having a meaning as defined herein.

  The term “substituted heterocycloalkyl” means “-(optionally substituted alkylene) — (optionally), each having the meaning as defined herein. Optionally substituted heterocyclic) "moiety.

  The term “inflammation”, “inflammatory condition”, or “inflammatory condition” includes, but is not limited to: muscle fatigue, osteoarthritis, rheumatoid arthritis, inflammatory bowel syndrome or disorder, dermatitis (Eg, atopic dermatitis, contact dermatitis, allergic dermatitis), dryness, eczema, rosacea, seborrhea, psoriasis, atherosclerosis, burns and radiation injury, pressure ulcers, oily skin, Caused by wrinkles, excess cellulite, excess pore size, intrinsic skin aging, photoaging, photo damage, harmful UV damage, keratinization abnormalities, stimuli including retinoid-induced stimuli, hirsutism, alopecia, hyperpigmentation, wounds Inflammation, scar or skin atrophy streaks, loss of elasticity, skin atrophy and gingivitis.

  The term “ischemia” refers to a blood deficiency to an organ or tissue resulting from functional contraction or actual occlusion of a blood vessel. Cerebral ischemia, also known as stroke, usually results from blockage or reduction of blood and oxygen to the blood vessels of the brain; more rarely, this can be the result of bleeding. Signs of seizures include paralysis, obscure language, general confusion, gait disturbance, loss of cortical sensation across toes, feet and legs, and urinary incontinence, to name just a few. Many types of heart disease, including cardiac arrhythmias or diseases resulting from cardiac structural abnormalities, can result in cerebral embolism. Atrial fibrillation from any cause, including rheumatic valvular disease, can result in created emboli that can migrate into the arteries of the brain. Embolization and embolization can occur as a result of atherosclerotic cardiovascular disease and myocardial infarction. Embolization is also a clear risk for intracardiac surgery and prosthetic valve replacement. Cardiac bypass surgery and angioplasty can result in the formation of microemboli that can travel into the arteries of the brain, causing a series of blockages in many arteries, resulting in mental disorders. Cerebral embolism is also a major complication in artificial heart transplantation. Furthermore, the overall risk of seizures after any type of systemic surgery is between 0.2% and 1%. A pathological growth of acute and subacute bacterial endocarditis can produce emboli that can occlude the main intracranial arteries. The population at risk of ischemia includes patients scheduled for coronary artery bypass graft surgery (CABG), patients at risk of postoperative complications, patients with subarachnoid hemorrhage (SAH), first or second Patients with acute ischemic stroke, Patients with acute ischemic stroke, Patients undergoing cardiopulmonary resuscitation (CPR), Patients undergoing temporary lobectomy, Patients with dominant hemispherectomy, Prophylactic brain irradiation Patients receiving, patients with closed head trauma with neurological deficits, patients with microvascular multiple cerebral infarction dementia, homozygous and heterozygous MELAS (mitochondrial myopathy, brain damage, lactoacidosis, stroke) Patients with atherosclerosis or progressive supranuclear palsy, patients with symptomatic and asymptomatic Huntington's disease, neonatal asphyxia Patients with meningitis or encephalitis, patients with postherpetic neuropathy, patients with intermittent claudication, patients with spinal cord injury, Huntington's disease, amyotrophic lateral sclerosis (ALS) or Fleetrich Patients with ataxia, patients with diabetic neuropathy, or systemic disease, carcinoma, vasoconstriction (including reversible cerebral vasoconstriction (eg, migraine, trauma, idiopathic disease)) or venous condition (dehydration, pulmonary artery) Including, but not limited to, patients with diseases associated with hypercoagulable conditions after embolism, pericranial infection, postpartum and postoperative conditions, and systemic cancer).

  The term “personal care product” refers to health and cosmetic beauty aid products that are generally recognized to be formulated to beautify and tailor the skin and hair. For example, personal care products include sunscreen products (eg, lotions, skin creams, etc.), cosmetics, toiletries, and public health products intended for topical use.

  As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial agents and Antifungal agents, isotonic agents, absorption delaying agents and the like can be mentioned. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

  The term “pharmaceutically acceptable salt” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and which are not biologically or otherwise undesirable. In many cases, the compounds of the present invention may form acidic and / or basic salts due to the presence of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Examples of the salt derived from an inorganic base include sodium salt, potassium salt, lithium salt, ammonium salt, calcium salt, and magnesium salt by way of example only. Salts derived from organic bases include, but are not limited to: primary amines, secondary amines, and tertiary amines (eg, alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di ( Substituted alkyl) amine, tri (substituted alkyl) amine, alkenylamine, dialkenylamine, trialkenylamine, substituted alkenylamine, di (substituted alkenyl) amine, tri (substituted alkenyl) amine, cycloalkylamine, di (cycloalkyl) Amine, tri (cycloalkyl) amine, substituted cycloalkylamine, disubstituted cycloalkylamine, trisubstituted cycloalkylamine, cycloalkenylamine, di (cycloalkenyl) amine, tri (cycloalkenyl) amine, substituted cycloalkenylamine, two Place Cycloalkenylamine, trisubstituted cycloalkenylamine, arylamine, diarylamine, triarylamine, heteroarylamine, diheteroarylamine, triheteroarylamine, heterocyclic amine, diheterocyclic amine, triheterocyclic amine , Mixed diamines and mixed triamines, wherein at least two substituents on the amine are different and are alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, Selected from the group consisting of aryl, heteroaryl, heterocyclic, etc. In addition, amines in which two or three substituents taken together with the nitrogen of the amino group form a heterocyclic group or a heteroaryl group included.

Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Examples of salts derived from inorganic acids include salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Salts derived from organic acids include: acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid Acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-
Salts such as toluenesulfonic acid and salicylic acid.

The term “phosphoryl” refers to the group —P (O) (OR ″) ₂ , where R ″ is independently selected from hydrogen or alkyl and aryl, this group is sometimes also referred to as “phosphono” Or “phosphate” or “phosphonic acid”.

  “Regulating skin condition” includes prophylactically and / or therapeutically adjusting a skin condition that includes visible and / or tactile discontinuities in the skin. This includes, for example, but is not limited to: adjusting visible discontinuities in skin tissue and / or adjusting palpable discontinuities, reducing hyperpigmentation after inflammation Regulating skin non-melanin discoloration, regulating skin moisture retention and barrier function, regulating skin epithelial differentiation, regulating skin peeling, thickening the skin Reducing atrophy, regulating skin elasticity, reducing oily skin, regulating cellulite in the skin, regulating itching in the skin, and To promote wound healing. As used herein, prophylactically regulating skin conditions includes delaying, minimizing and / or preventing visible and / or palpable discontinuities in the skin. To do. As used herein, therapeutically adjusting the skin condition includes improving, eg, reducing, minimizing and / or eliminating discontinuities in the skin. Regulating the skin condition relates to improving the appearance and / or feel of the skin. Regulating skin conditions includes regulating body / cranium hair growth (including slowing and / or preventing body and / or hair growth). Regulating skin conditions includes regulating inflammation (including retinoid-induced inflammation). As used herein, regulating the skin includes the use of the compounds of the invention as fungicides, fungicides and antibiotics.

  “Regulating skin aging signs” includes prophylactically adjusting and / or therapeutically adjusting one or more of the following signs: Similarly, pre-determining skin aging (E.g., line, wrinkle or gap) includes adjusting this symptom prophylactically and / or therapeutically). As used herein, prophylactically modulating such symptoms includes delaying, minimizing and / or preventing skin aging signs. As used herein, therapeutically modulating such symptoms includes improving, eg, reducing, minimizing and / or eliminating skin aging symptoms.

  “Skin aging signs” include visually and tactilely perceivable manifestations on all surfaces, and any other macro or micro effects, due to skin aging, It is not limited to these. Can such signs be induced by intrinsic or extrinsic factors (eg, aging over time and / or environmental damage (eg, sunlight, UV, smoking, ozone, pollutants, stress, etc.)) Or can be caused. These symptoms may arise from processes such as, but are not limited to: development of tissue discontinuities such as wrinkles, including both wrinkles on fine surfaces and rough and deep wrinkles Wrinkles, facial eyebrow wrinkles, facial expression wrinkles, rhytides, cutaneous sun dermatosis, photodamage, premature skin aging, tears, aneurysms, dents, large gaps (eg sweat gland ducts, sebaceous glands or hairs) Associated with the structure of appendages such as sachets), “citrus skin” skin appearance, dryness, scaliness, flakiness, and / or other forms of skin unevenness or roughness; , Acne), papules, amputation; excessive skin lipid problems (eg, excessive production of sebum, fat, facial balance, collapse of support structure); abnormal flaking (or De), or abnormal epithelial differentiation (eg, abnormal skin turnover) such as scaliness, frankiness, keratosis, hyperkeratosis; skin barrier damage, as caused by dry environment Improper skin moisturization (or hydration); elastic fibrosis, loss of skin elasticity (including swelling in the eye area and jaw) (loss of functional skin elasticity and / or inability), skin Loss of firmness, loss of skin toughness, loss of skin recoil from deformation; bear under eyes, wrinkles (eg due to bruising, eg uneven reddishness), poor bloodiness ( Pale color), discoloration of non-melanin skin, such as discoloration caused by telangiectasia or spider vassels; age spots (liver spots, brown spots) And melanin-related hyperpigmented (or pigmented spotty) skin areas such as freckles; after inflammatory events (eg acne lesions, growing hair, insect / spider bite or sting, scratches Post-inflammation hyperpigmentation, such as, but not limited to, aging or steroid use; substrate (eg, hyaluronic acid) Other histochemical or microscopic changes in skin components such as glycosaminoglycans, collagen ruptures and structural changes or structural abnormalities (eg stratum corneum, dermis, epidermal epithelium, cutaneous vasculature) (Eg, changes in telangiectasia or spider vessels); tissue response to invasion (eg, itc ) Or itching (pruritus); and basic tissues (e.g., subcutaneous fat, cellulite, muscles, trabeculae, septal (Septae), etc.), in particular changes to underlying tissue proximate to the skin.

  The term “therapeutically effective amount” refers to any compound of formula I that, when administered to a mammal in need of treatment as defined below, is sufficient to effect such treatment. Say quantity. This therapeutically effective amount depends on the subject and the disease state being treated, the weight and age of the subject, the severity of the disease state, the particular compound selected, the dosage regimen to be followed, the timing of administration, the method of administration, etc. All of which can be easily determined by one skilled in the art.

The term “treatment” or “treating” means any treatment of a disease or disorder in a mammal and includes the following:
Preventing or protecting against the disease or disorder, ie not developing its clinical condition;
Inhibiting the disease or disorder, i.e., preventing or suppressing the onset of clinical symptoms; and / or alleviating the disease or disorder, i.e., causing regression of the clinical condition.
Distinguish between “prevention” and “suppression” in human medicine because the event ultimately induced may be unknown and latent, or the patient is not confirmed until well after the onset of the event It will be appreciated by those skilled in the art that this is not always possible. Thus, as used herein, the term “prevention” is a component of “treatment” to encompass both “prevention” and “suppression” as defined herein. Intended as. The term “protection” as used herein is meant to include “prevention”.

  As used herein, the term “topical administration” means to apply or spread the composition of the invention onto the surface of the skin.

(Nomenclature)
In general, the nomenclature used in this application is based on the AUTONOM ^™ v2.1, Beilstein Institute computer system for the creation of IUPAC systematic nomenclature.

  The compounds of the invention are named and numbered as described below.

式Ｉａは、式Ｉに従う化合物を表し、ここで、Ｒ、Ｒ^１、Ｒ^５は、水素であり、Ｒ^２およびＲ^３は、メチルであり、そしてＲ^４は、ｐ−ニトロ−フェニルであり、これは、６，７−ジメチル−２−（４−ニトロ−フェニル）−ベンゾフラン−５−オールと命名され得る。

Formula Ia represents a compound according to Formula I, wherein R, R ¹ , R ⁵ are hydrogen, R ² and R ³ are methyl, and R ⁴ is p-nitro-phenyl This can be termed 6,7-dimethyl-2- (4-nitro-phenyl) -benzofuran-5-ol.

式Ｉｂは、式Ｉに従う化合物を表し、ここで、Ｒ、Ｒ^１、Ｒ^４は、水素であり、Ｒ^２およびＲ^３は、メチルであり、そしてＲ^５は、フェニルであり、これは、６，７−ジメチル−３−フェニル−ベンゾフラン−５−オールと命名され得る。

Formula Ib represents a compound according to Formula I, wherein R, R ¹ , R ⁴ are hydrogen, R ² and R ³ are methyl, and R ⁵ is phenyl, which is It can be named 6,7-dimethyl-3-phenyl-benzofuran-5-ol.

(Synthesis of the compound of the present invention)
(Synthetic reaction parameters)
The term “solvent”, “inert organic solvent” or “inert solvent” means a solvent which is inert under the reaction conditions described in connection therewith. Examples of the solvent used in the synthesis of the compound of the present invention include the following: methanol, acetone, water, acetonitrile, 1,4-dioxane, dimethylformamide ("DMF"), benzene, toluene, tetrahydrofuran ("THF "), Chloroform, methylene chloride (ie dichloromethane (" DCM ")), diethyl ether, pyridine and the like, as well as mixtures thereof. Unless specified to the contrary, the solvents used in the reactions of the present invention are inert organic solvents.

  The term “qs.” Means adding an amount sufficient to achieve the stated function (eg, an amount sufficient to bring the solution to the desired volume (ie, 100%)).

  Unless stated to the contrary, the reactions described herein are -10 ° C to 110 ° C (preferably 0 ° C to 40 ° C; most preferably "room temperature" or "ambient temperature" (eg 20 ° C). ) At atmospheric pressure within the temperature range. Further, unless otherwise specified, reaction times and reaction conditions are approximate (eg, from about −10 ° C. to about 110 ° C. (preferably about −10 ° C. to about 40 ° C .; most preferably about “room temperature” or “ Within the temperature range of “ambient temperature” (eg, about 20 ° C.) over a period of about 1 hour to about 10 hours (preferably about 5 hours). The parameters given in the examples are intended to be specific and not approximate.

  Isolation and purification of the compounds and intermediates described herein may be performed using any suitable separation or purification procedure (eg, filtration, extraction, crystallization, column chromatography, thin layer chromatography, if desired). Or by thick-layer chromatography, or a combination of these procedures). Specific illustrations of suitable separation and isolation procedures can be made with reference to the following examples herein. However, other equivalent separation or isolation procedures can of course also be used.

(Starting material)
Starting materials (eg, 2,3-dimethylhydroquinone) are commercially available from, for example, Aldrich Chemical Company, Milwaukee, Wis., Or can be readily prepared by one skilled in the art using commonly used methodologies.

反応スキーム１を参照して、式１０１の２，３−置換ヒドロキノンを、溶媒（例えば、アセトン、ＴＨＦ、またはＤＭＦ）中、塩基（例えば、炭酸ナトリウム、炭酸カリウムまたは炭酸セシウム）の存在下で、１当量の２−ハロ−１−置換−エタノン、好ましくは、２−ブロモ−１−置換−エタノン、最も好ましくは、２−ブロモ−１−置換フェニルエタノンとともに処理して、式１０２の２−（４−ヒドロキシ−２，３−置換−フェノキシ）−１−置換−エタノンを得る。式１０２の化合物は、続いて、不活性な溶媒（トルエンまたはキシレン）中で酸（ポリリン酸、塩酸、または硫酸）により処理されて、式１０３のベンゾフラン化合物へと環化し得る。

Referring to Reaction Scheme 1, a 2,3-substituted hydroquinone of formula 101 is reacted in a solvent (eg, acetone, THF, or DMF) in the presence of a base (eg, sodium carbonate, potassium carbonate, or cesium carbonate). Treatment with 1 equivalent of 2-halo-1-substituted-ethanone, preferably 2-bromo-1-substituted-ethanone, most preferably 2-bromo-1-substituted phenylethanone, yields 2- (4-Hydroxy-2,3-substituted-phenoxy) -1-substituted-ethanone is obtained. The compound of formula 102 can be subsequently treated with an acid (polyphosphoric acid, hydrochloric acid, or sulfuric acid) in an inert solvent (toluene or xylene) to cyclize to the benzofuran compound of formula 103.

  Similarly, the 2,3-substituted hydroquinone of formula 102 is treated with 2 equivalents or more of 2-halo-1-substituted ethanone in the presence of a base and then cyclized in the presence of an acid to give formula 105 Can be obtained.

  (Reaction Scheme 2)

スキーム２を参照して、式２０１の化合物（ここで、「Ｐｒｏｔ」は、ヒドロキシ保護基、好ましくはテトラヒドロピラニル基であり、溶媒（例えば、アセトン、ＴＨＦまたはＤＭＦ）中、塩基（例えば、炭酸ナトリウム、炭酸カリウムまたは炭酸セシウム）の存在下で、２−ハロ−１−置換エタノン、好ましくは２−ブロモ−１−置換エタノンと反応させ、次いで、反応スキーム１に記載される通りの酸の存在下で環化すると、式２０２の化合物を得ることができる。

Referring to Scheme 2, a compound of formula 201 (where “Prot” is a hydroxy protecting group, preferably a tetrahydropyranyl group, and a base (eg, carbonic acid) in a solvent (eg, acetone, THF or DMF). Sodium, potassium carbonate or cesium carbonate) in the presence of 2-halo-1-substituted ethanone, preferably 2-bromo-1-substituted ethanone, and then the presence of acid as described in Reaction Scheme 1. Cyclization below can give compounds of formula 202.

(Preferred compound)
The following combinations and permutations of substituents (subgrouped in increasing order of preference, respectively) are items and compound compositions for use in methods and pharmaceutical and cosmetic compositions according to the present invention: Preferred compounds are defined as products.

Any compound of formula I, wherein R ² and R ³ are optionally substituted alkyl, in particular R ² and R ³ are methyl.

Preferably R ¹ is hydrogen or halogen.

      In particular R is hydrogen.

Any compound of Formula I wherein R ¹ is hydrogen or halo.

Preferably R ¹ is hydrogen.

Any compound of formula I, wherein R is hydrogen, alkyl, acyl, phosphoryl or polyalkoxy, preferably hydrogen, and R ¹ is hydrogen.

1. In particular R ² and R ³ are methyl.

In particular, in the compounds of formula I, R ⁵ is optionally substituted aryl, where the substituents are alkyl, alkoxy, hydroxyl, (optionally substituted alkoxy) carbonyl, nitro, Selected from halo and cyano.

In particular R ⁵ is optionally substituted phenyl.

In particular R ⁵ is optionally substituted phenyl and R ⁴ is hydrogen.

Particularly in compounds of formula I, R ⁴ is an optionally substituted aryl.

In particular, R ⁴ is optionally substituted phenyl.

Preferably R ⁴ is para-substituted phenyl, wherein the substituent is selected from alkyl, alkoxy, hydroxy, (optionally substituted alkoxy) carbonyl, nitro, halo and cyano. .

Preferably, R ⁴ is 4-nitrophenyl or 4-cyanophenyl, and R ⁵ is hydrogen.

In particular, in the compound of formula I, R ⁴ is formyl, (optionally substituted alkyl) carbonyl, (optionally substituted aryl) carbonyl, (optionally substituted heterocyclyl). Carbonyl, (optionally substituted heterocyclylalkyl) carbonyl.

In particular, R ⁴ is (optionally substituted aryl) carbonyl.

In particular, R ⁴ is (optionally substituted alkyl) carbonyl.

In particular, R ⁴ is substituted with alkylcarbonyl, hydroxy or heterocyclyl, especially morpholin-1-yl, optionally substituted with halogen.

In particular, R ⁴ is formyl.

Preferably R ⁴ is formyl, phenylcarbonyl, bromoacetyl, morpholin-1-yl-acetyl and acetyl; and R ⁵ is hydrogen.

In particular, R ⁴ is (optionally substituted alkoxy) carbonyl, (optionally substituted alkenyloxy) carbonyl, (optionally substituted amino) carbonyl, carboxy or hydroxy (optionally Substituted).

In particular, R ⁴ represents (optionally substituted alkoxy) carbonyl (preferably alkoxy is polyalkoxy) or optionally substituted alkenyloxy) carbonyl (preferably alkenyloxy is geranyloxy. Yes).

In particular, R ⁴ is (optionally substituted amino) carbonyl (preferably amino is substituted with optionally substituted alkyl (especially hydroxyalkyl) or amino is a cyclic amine. Yes).

Preferably, R ⁴ is the following: morpholin-1-yl-carbonyl; bis- (2-hydroxy-ethyl) -amide, 2-hydroxy-ethyl-amide, carboxylic acid; carboxylic acid methyl ester; carboxylic acid 3,7 -Dimethyl-octa-2,6-dienyl ester; [2- (2-methoxy-ethoxy) -ethoxy] ethyl ester; and R ⁵ is hydrogen.

In particular, R ⁴ is hydroxy (optionally substituted) alkyl.

In particular, R ⁴ is hydroxymethyl, 1-hydroxy-2-morpholin-4-ylethyl or 6-hydroxy-3-methyl- [1,3] oxazinan-6-yl.

Any compound of formula I wherein R and R ¹ and the atom to which they are attached form an optionally substituted ring.

In particular, R and R ¹ and the atom to which they are attached form a furan ring substituted with an unsubstituted phenyl ring or a substituted phenyl ring, wherein one or more substituents are alkyl, alkenyl, haloalkyl Independently selected from hydroxy, alkoxy, carboxy, ester, haloalkyl and halo.

Preferred compounds for use in the present invention include the following and their stereoisomers, salts and mixtures (where appropriate):
(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl) -phenyl-methanone;
(5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -morpholin-4-yl-methanone;
1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone;
Acetic acid 2- (2-bromo-acetyl) -6,7-dimethyl-benzofuran-5-yl-ester;
2- (1-hydroxy-2-morpholin-4-yl-ethyl) -6,7-dimethyl-benzofuran-5-ol;
Acetic acid 6,7-dimethyl-2- (2-morpholin-1-yl-acetyl) -benzofuran-5-yl ester;
Acetic acid 2- (6-hydroxy-3-methyl- [1,3] oxazinan-6-yl) -6,7-dimethyl-benzofuran-5-yl ester;
1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -2-morpholin-4-yl-ethanone;
1- (4-bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone;
2-hydroxymethyl-6,7-dimethyl-benzofuran-5-ol;
6,7-dimethyl-3-phenyl-benzofuran-5-ol;
6,7-dimethyl-2- (4-nitrophenyl) -benzofuran-5-ol;
5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde;
4- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -benzonitrile;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 3,7-dimethyl-octa-2,6-dienyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid bis- (2-hydroxy-ethyl) -amide;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 2- [2- (2-methoxy-ethoxy) -ethoxy] -ethyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid (2-hydroxy-ethyl) -amide;
3- (4-methoxy-phenyl) -6,7-dimethyl-benzofuran-5-ol;
3- (4-chloro-phenyl) -6,7-dimethyl-benzofuran-5-ol;
3- (4-fluoro-phenyl) -6,7-dimethyl-benzofuran-5-ol;
4,5-dimethyl-1,8-diphenyl-benzo [1,2-b; 4,3-b ′] difuran;
1,8-bis- (4-fluoro-phenyl) -4,5-dimethyl-benzo [1,2-b; 4,3-b ′] difuran; and 1,8-bis- (4-methoxy-phenyl) ) -4,5-dimethyl-benzo [1,2-b; 4,3-b ′] difuran.

(Usability, testing and administration)
(General usefulness)
The compounds, compositions / formulations and methods of the present invention are useful in treating a number of disorders, particularly those characterized by oxidative stress and / or inflammation. In particular, the compounds of the present invention have cerebral ischemia (“stroke”), myocardial ischemia (myocardial infarction and other forms of heart disease), diabetes, kidney disease, premenstrual syndrome, asthma, cardiopulmonary inflammatory disorders, chronic heart failure, chronic It can be used in treating rheumatoid arthritis, muscle fatigue, intermittent claudication, and for preservation of allograft tissue for transplantation.

  The compounds of the present invention also show use in reducing C-reactive protein (CRP) associated with inflammation and / or inflammatory conditions, including cardiovascular diseases and disorders, including, for example: Atrial fibrillation, unstable angina pectoris, coronary arteritis disease, peripheral arterial disease, cardio allograft vasopathy (CAVD), mastitis, pre-clampsia, inflammatory bowel Disease, stroke, tissue infarction, lumbosciatica, estrogen / progestin hormone replacement therapy (HRT), infection (bacteria, viruses and protozoa), bacterial meningitis, trauma, surgery, biomaterial transplantation, Smoking, obesity, neurodegenerative diseases (eg Alzheimer's disease), inflammatory Diseases (eg, myocarditis, cardiomyopathy, acute endocarditis or epicarditis, arteriosclerosis, systemic inflammatory response (SIRS) / sepsis, adult respiratory distress syndrome (ARDS), asthma, chronic Rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, airway hyperresponsiveness (AHR), bronchial hyper-reactivity, chronic obstructive pulmonary disease (COPD), congestive heart failure (CHF) Intrinsic inflammatory complications, metabolic syndrome, late renal disease (ESRD), premenstrual syndrome (PMS) or muscle fatigue, or inflammation, multiple organ dysfunction (MODS), increased airway responsiveness (AHR), high bronchial reactivity, aging, acute allergic reaction, teeth Peripheral diseases such as gingivitis and skin conditions (including inflammatory skin conditions).

  The compounds, formulations and methods of the present invention are useful in treating a number of dermatological conditions, including but not limited to: age-related injury or disorder (eg, detrimental) Prevention and skin protection against damage resulting from ultraviolet (UV) radiation, stress and fatigue. Such compounds, formulations and methods are likewise useful for hair care and scalp treatment, for example by incorporation into medicinal shampoos, anhydrous hair conditioners and the like. Such compounds, formulations and methods are likewise useful in reducing hair growth.

  For example, exposure to sunlight can create a number of dangers to the skin. The major short-term risk of prolonged exposure to sunlight is erythema (ie, sunburn), which results primarily from UVB radiation having a wavelength of about 290 nm to about 320 nm. However, over long periods of time, such long exposures can often cause malignant changes that occur on the skin surface. Epidemiological studies demonstrate a strong link between sun exposure and human skin cancer. Another long-term danger of ultraviolet radiation is premature aging of the skin, which is mainly caused by UVA radiation having a wavelength of about 320 nm to about 400 nm. This condition is characterized by skin wrinkles and pigment changes, along with other physical changes such as cracks, telangiectasia, sun dermatosis, ecchymosis, and loss of elasticity. Individuals, especially those with light skin that are easily burned and less tanned by tanning, and those who have been exposed to the sun significantly in childhood, may have It can show significant changes in the skin: wrinkles, leatheriness, yellowing, relaxation, rough skin, dryness, spot formation (hyperpigmentation) and various premalignant growths (often latent). These accumulating effects of sunlight are often referred to as “photoaging”. Although anatomical degeneration of the skin is most advanced in old age, the destructive effects of excessive sun exposure are already apparent by the 20th year. Serious microscopic changes in the epidermis and dermis occur decades before they become clinically visible. Wrinkles, yellowing, hardening and loss of elasticity are very slow changes.

  The composition of the present invention is useful for regulating the growth of body hair and / or hair, and particularly useful for reducing hair growth. Typically, the composition can be applied to the face, particularly to the facial folds (ie, cheeks, neck, upper lip, and jaw). The composition can also be applied to the feet, arms, torso and armpits. This composition is particularly suitable for the treatment of hirsutism. In order to achieve a perceived reduction in hair growth, in humans the composition should be applied once or twice daily or more frequently for at least 3 months.

  Other skin conditions that may benefit from the methods of the present invention include, but are not limited to, diaper rash (the normal form of contact dermatitis and irritation that occurs in infants and adults wearing diapers). US Pat. No. 6,211,186 (incorporated herein by reference) describes possible etiology and methods for treating this condition. It is generally considered that one or more fecal and lipolytic enzymes, as well as ammonia, bacteria, urine pH, excess water and Candida albicans can be involved in the development of skin irritation and inflammation associated with diaper rash It has been. It is also that the physiological response of the skin to irritants (eg, the production of cytokines by keratinocytes) contributes to ensuring the appearance of erythema, papules, scaling and ulceration characteristic of this condition, It seems likely. Furthermore, the compositions and methods of the present invention may be useful in treating wounds (skin conditions characterized by severe inflammatory components) and stimuli (including retinoid-induced stimuli).

  The compositions of the present invention are also useful for regulating skin conditions, including visible and / or tactile skin discontinuities, particularly skin surfaces; such discontinuities are generally undesirable. . Such discontinuities can be induced or caused by intrinsic and / or extrinsic factors and include the signs of skin aging described herein. Clear discontinuities include pigmentation disorders.

  The compositions of the present invention are useful for modulating signs of skin aging, particularly visible and / or tactile discontinuities in aging-related skin tissue. The present invention is not limited to the regulation of “indications of skin aging” resulting from the above-described mechanisms for skin aging, but encompasses the regulation of such signs regardless of the origin of the mechanism of such signs. It should be understood that is intended.

(test)
This section describes how compositions incorporating the compositions of the invention are selected using, for example, in vitro and / or in vivo animal models, and three exemplary indications (ie, stroke, chronic heart failure and myocardial Describe how it is used as a therapeutic intervention in (infarction).

  Induces cell death by injury to the brain (as ischemia interrupts the blood supply of the brain or interrupts the oxygen supply of the brain as hypoxia (low oxygen) or anoxia (no oxygen)) Neuron imbalance is rapidly produced (Flynn, CJ, et al., 1989, G. Siegel et al., (Ed.), Basic Neurochemistry, Raven Press, NY). Studies of cellular and molecular mechanisms that lead to neuronal damage and inflammation for various types of cerebral ischemia are performed using in vitro model systems (eg, primary cell cultures) that retain the metabolic characteristics of neurons in vivo. obtain. The use of such cell-based models has led to advances in identifying biochemical mechanisms that lead to neuronal death in conditions such as anoxia, hypoglycemia, excitotoxicity, and exposure to reactive oxygen species. Neuronal cell lines such as the pheochromocytoma cell line, PC12, are also useful models for studying the effects of oxidative stress on the structure and function of neuron-specific proteins expressed in these cell lines. Since many neuronal cell lines do not express all the characteristics of true neurons, primary neuronal cultures are now widely used as an in vitro model to identify processes occurring in the intact brain.

  In vitro models of ischemia mimic in vivo conditions, for example, by placing neuronal cultures in large anoxic or hypoxic chambers and replacing the culture medium with a medium of deoxygenated defined ionic composition Approximates oxygen and glucose deficiency. Toxic overstimulation of neuronal glutamate receptors (especially N-methyl-D-aspartate (NMDA) receptors) causes hypoxic ischemic neuronal damage (Choi, DM, 1988, Neuron 1: 623-634), response. Induction of ischemia of reactive oxygen species (ROS) (Watson, BD, et al., 1988, Ann NY Acad Sci., 59: 269-281), excessive calcium influx (Grotta, J.C., 1988, Stroke 19) : 447-454), increased arachidonic acid (Siesjo, BK, 1981, J. Cereb. Blood Flow Metab. 1: 155-186) and DNA damage (MacManus, JP, et al., 1993, Neurosci. Lett., 164: 89-92) (each Contributes to cause) the through-modified cascade.

  Primary embryonic hippocampal neuronal cells are widely recognized as useful in models of neuronal function. The hippocampus is generally a relatively uniform population of source neurons with well-characterized characteristics typical of central nervous system (CNS) neurons. Pyramidal neurons (major cell type in the hippocampus) are estimated to account for 85% to 90% of the total neuronal population (Banker and Goslin, 1998, Cultured Nerve Cells, 2nd edition, The MIT Press, Cambridge, Massachusetts). The hippocampus also has a remarkable ability for activity-dependent changes in synaptic function, such as long-term potentiation (Hawkins RD, Kandel ER, Siegelbaum SA. (1993) Learning to modulated transmitter release: themes and variations in the theory of it. ], Ann.Rev Neurosci.16: 625-665.).

  In experiments carried out in support of the present invention according to the methods described in detail in the Examples, anoxia / ischemia is induced in primary cultures of hippocampal neuronal cells and the compounds are used to prevent cell death. Were tested for their ability. Compounds found to have activity in such in vitro assays are then used in one or more animal models of cerebral ischemia (“seizures”), such as the middle cerebral artery occlusion (MCAO model in rats). Further testing was performed.

  Briefly, primary cultures of hippocampal neurons are used to test compounds for activity in neuronal protection. Hippocampal cultures are typically prepared from fetal rats on days 18-19. At this age, the development of pyramidal neurons (starting with rats in E15) is essentially complete. The brain tissue at this stage dissociates relatively easily, the meninges are easily removed, and the number of glial cells is still relatively moderate (Park LC, Calingasan NY, Uchida K, Zhang H, Gibson GE (2000) Metabolic impulse eliminations brain cell type-selective changes in cell stress in culture. (J) Neurochem 1: 24)

  To assess the activity of the compounds of the present invention, the test compound is capable of protecting cells against one or more standard stressors (including hypoxia), as described in detail in the Examples. To evaluate. In general, the desired therapeutic compound candidate is effective in this model at a concentration of less than about 1 mM, and even more preferably at a concentration of less than about 100 μM. Effective (effective) such compounds protect at least 20%, preferably 30%, more preferably 40%, even more preferably 50% or more of the cells being tested from stressor-induced death. Means that. By way of example, compounds that are effective in providing protection over concentrations in the range of about 1-1000 μM are expected to provide neuroprotection in vivo. Since the exact value may vary depending on the particular conditions under which the neuroprotective cell assay is performed, the subsequent test rather than providing an absolute concentration at which the compounds of the invention are deemed effective. It is the intent of the present disclosure to provide the above criteria as a guideline in the form of criteria for comparing compounds. Typically, compounds that are found to be neuroprotective in such in vitro cell lines are then in vivo animal models of neuroprotection (eg, the rat middle cerebral artery occlusion model described below, or models well known in the art). In other suitable models).

  Cerebral ischemic injury is modeled in animals by occluding a blood vessel to or into the skull (Molinari, GF, 1986, HJM Barnett, et al., (Ed)). Stroke: Pathology, Diagnosis and Management, Vol. 1, Churchill Livingstone, NY). The rat middle cerebral artery occlusion (MCAO) model is the most widely used technique for inducing transient focal cerebral ischemia that approximates cerebral ischemic injury in humans (eg, humans who experience seizures). One of them. The middle cerebral artery used as an ischemic trigger in this model is the most affected blood vessel in human stroke. This model also involves a period of reperfusion, which typically occurs in human seizures. MCAO, including a 2 hour occlusion, was found to produce the largest size cortical infarction that could be obtained without an increase in mortality at the 24 hour time point.

  Briefly, nylon filaments are implanted into the rat right carotid artery. To effect occlusion, the rats are anesthetized and the filament is advanced 18-20 mm from the bifurcation of the internal and external arteries to the internal carotid artery, and a suture is tightly bound around the filament for 2 hours. After 2 hours of occlusion, the animals are anesthetized again and the filaments are removed and reperfused for the remainder of the experiment. The test drug can be administered during this process, before, during, or after occlusion, and can include one or more of various means (including but not limited to intracerebroventricular (ICV) infusion, intravenous (IV) Infusion, intraperitoneal (IP) administration, and enteral administration (eg, gavage)). Animals are maintained at normal temperature during the experiment as described in the Examples. At predetermined times after occlusion and reperfusion, animals are sacrificed and their brains removed and processed for assessment of damage as measured by infarct volume. In general, the compounds in this model are less than about 10 mg / kg, preferably less than 1 mg / kg, more preferably less than 100 μg / kg, and even more preferred when they are administered ICV or IV. Is considered active if it provides a significant reduction in total infarct volume at a dose that is less than about 1 μg / kg. A significant reduction in total infarct volume means a reduction of at least 20%, preferably at least 30%, more preferably at least 40%, and even more preferably about 50% compared to a control value. .

  Further confirmation of efficacy in neuroprotection can be assessed in functional tests (eg grip strength test or rotrod test). Animals treated with compounds that exhibit neuroprotection show a significant decrease in infarct size and their MCAO grip strength values after MCAO compared to untreated animals that exhibit loss of sensorimotor function. maintain. Similarly, animals treated with compounds that exhibit neuroprotection are also post-MCAO compared to untreated animals that showed a significant reduction in rotrod score, indicating loss of sensorimotor function at higher brain levels. Maintained their pre-MCAO rotorod activity score.

  Similarly, primary cultures of muscle cells can be used to test compounds in vitro for the ability to provide protection against heart damage resulting from, for example, myocardial ischemia or congestive heart failure. The preparation of cardiomyocytes from neonatal rats is described in the examples. Such cells are typically used to study molecular models of myocardial ischemia (Webster, KA, Discher, DJ & Bishopric, NH. 1995. J. Mol. Cell Cardiol. 27: 453-458; Camilleri , L, Moins, N, Papon, J, Maubrant, J, Baily, P, de Riverols, C & Veyre, A. 1997. Cell Biol. & Toxicol. 13: 435-444; , Melkonyan, HS, Piot, C, Wolfe, CL, Tomei, LD, Hannun, YA & Umansky, SR. 1997. Am. J. Pathol. 151: 1257-126. ), Therefore, it is accepted as an indicator of myocardial protective activity. An exemplary stressor assay for this purpose is provided in the examples. For example, cardiomyocytes in culture show contractile (“beating”) activity; the contraction of each cardiomyocyte is associated with an increase in intracellular calcium called “calcium transient”. These calcium transients can be measured using Fluo-4, a fluorescent dye that exhibits a large increase in fluorescence intensity when bound to calcium. This assay tests the ability of potential cytoprotective molecules to be cell based and protect against ischemic damage and allow cells to maintain their contractile function.

  Further validation of the compounds can be performed in whole organ assays, such as the isolated Langendorff model of cardiac function. Similarly, compounds can be further identified as is well known in the art in additional animal models of disease (eg, diabetes, renal failure, rash, muscle fatigue, inflammation).

  This section describes methods for selecting compositions incorporating the compositions of the present invention using in vitro and in vivo animal models and for use as therapeutic treatments in dermatological signs. Many cell screening assays for mediators of inflammatory responses are well known in the art. Such mediators include, but are not limited to: inflammatory cytokines, interleukin-1β, and tumor necrosis factor α (TNF.α). Other molecules have been reported for use as markers of inflammation, including C-reactive protein (CRP), certain adhesion molecules, and proteins such as leukotrienes, thromboxanes and isoprostanes.

  In vitro assessment of anti-inflammatory activity can be determined by well-characterized assays (eg, E-selectin (ELAM) production assay or CRP assay exemplified in Example 9), and in vivo assessment is carrageenan induced It can be determined by sex paw edema assay. The ELAM assay measures the activity of a test compound in reduced ELAM expression in activated endothelial cells. Briefly, endothelial cells are activated by adding known activators (eg, lipopolysaccharide, TNF, or IL-1β) alone or in some combination. Activated cells produce ELAM, which can be measured using, for example, an E-selectin monoclonal antibody based ELISA assay. In studies carried out in support of the present invention, ELAM production was reduced. In vivo assessment of anti-inflammatory activity, as described in Example 10, can be determined by a well-characterized assay (Gabor, M., Mouse Ear Inflammation Models and therological Applications, 2000). Carrageenan-induced paw edema is a model of inflammation, which causes the formation of time-dependent edema following administration of carrageenan to the inner plantar surface of the rat foot. Application of arachidonic acid (AA) to the mouse ear results in immediate vasodilation and erythema, followed by a rapid development of edema (this is greatest at 40-60 minutes). The development of edema coincides with extravasation of proteins and leukocytes. After 1 hour, edema rapidly decreases and the inflammatory cells leave the tissue, so that at 6 hours the ear recovers almost normal. These assays measure the ability of test compounds to treat these inflammatory processes via the systemic and local routes of administration, respectively.

  Cytoprotective activity on the skin can be assessed in cell culture using an Epiderm Skin Model (EPI-100) from Mattek Corporation of Ashland, Mass, as described in Example 7. Neonatal foreskin cell culture was cultured according to manufacturer's instructions and taken up by the cells 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) Assay for percent cell viability by measuring the amount of dye.

  Activities related to hair growth inhibition are described in Example 8.

(Administration)
The compound of formula I is administered at a therapeutically effective dosage (eg, a dosage sufficient to provide treatment for the disease states described above). Administration of a compound of the present invention or a pharmaceutically acceptable salt thereof can be obtained by any of the accepted modes of administration for agents that provide similar utilities.

  In general, daily dosages are about 0.01-2.0 mg / kg body weight, preferably about 0.1-kg / kg body weight, although human dosage levels must be optimized for the compounds of the invention. 1.5 mg, and most preferably about 0.3-1.0 mg / kg body weight. Thus, for administration to a 70 kg human, the dosage range is about 0.7-140 mg per day, preferably about 7.0-105 mg per day, and most preferably about 21-70 mg per day. The amount of active compound administered will, of course, be dependent on the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician.

  The compositions of the present invention are suitable to provide protection (preferably in a care product) against the harmful effects of ultraviolet radiation. More preferably, the compositions of the present invention are sunscreens to provide protection for human skin from the harmful effects of UV radiation, including but not limited to sunburn and premature skin aging. Suitable for use as an agent. Thus, the present invention further relates to a method for protecting human skin from the harmful effects of UV radiation. Such methods generally involve attenuating or reducing the amount of UV radiation that reaches the skin surface. In the present case, the method of treatment for the harmful effects of ultraviolet radiation also includes administration of the composition of the present invention after exposure to UV radiation has already taken place. In order to protect the skin from UV radiation, a safe and effective (photoprotective) amount of the composition is applied topically to the skin. “Topical application” refers to application of the composition of the present invention by application, spraying, etc. on the surface of the skin. The exact amount applied can vary depending on the level of UV protection desired. A composition of about 0.5 mg per square centimeter of skin to about 25 mg of composition per square centimeter of skin is typically applied.

  The compounds and methods of the invention can be used in any skin care application where a reduced inflammatory response is desired. For example, the compounds and compositions of the present invention can be used in leave-on and rinse-off pressure ulcer preparations, facial milk and conditioners, shower gels, foaming and non-foaming facial cleansers, cosmetics, hand lotions. And can be incorporated into body lotions, leave-on humectants, cosmetic and cleansing wipes, poison ivy, chickenpox or ointments for pruritus and the like. In general, for dermal application, topical application is preferred; however, systemic administration is also possible, as described elsewhere herein.

  The compositions of the present invention can also be used in cosmetic compositions. The cosmetic composition of the present invention is ideal for applying permanent or semi-permanent colors to the lips, ideally having a glossy or glossy finish, especially in the form of lipsticks or lip balms, and Ideally suited for use in treating lips. Cosmetic compositions also provide protection against harmful weather (including wind and rain), dry and / or hot environments, exposure to environmental pollution (eg ozone, smoke, etc.) or exposure to excessive doses of sunlight. Can be used in treating skin and / or lips with a skin care agent. These compositions also provide protection from sunlight, hair and skin humidification and / or conditioning, improved skin feel, controlled skin texture, fine lines and wrinkle reduction, hair or Useful for reducing skin oily shine, lightening skin and reducing skin or hair odor.

  Thus, the cosmetic composition is applied to the skin and / or lips in a conventional manner with or without a conventional holder or applicator to provide a decorative and / or protective film to the skin and / or lips. obtain.

  In using the compounds of the present invention for the treatment of the above conditions, any of the pharmaceutically acceptable modes of administration can be used. The compounds of formula I can be administered either alone or with a pharmaceutically acceptable excipient, including solid, semi-solid, liquid or aerosol dosage forms (eg, tablets, capsules, powders) Liquids, suspensions, suppositories, aerosols, etc.). The compounds of formula I are also preferably in unit dosage forms suitable for single administration of precise dosages, in sustained or controlled release dosage forms (accumulation injection) for long term administration of the compounds at a given rate. Osmotic pumps, pills, transdermal (including electrotransport) patches, etc.). The composition typically comprises a conventional pharmaceutical carrier or excipient and a compound of formula I or a pharmaceutically acceptable salt thereof. In addition, these compositions include other medical agents, pharmaceutical agents, carriers, adjuvants, etc. (including but not limited to anticoagulants, clot lysing agents, permeation enhancers and sustained release formulations). Can be included.

  Generally, depending on the intended mode of administration, the pharmaceutically acceptable composition is about 0.1% to 90% by weight, preferably about 0.5% to 50% by weight. Including a compound or salt of formula I, the remainder being suitable pharmaceutical excipients, carriers, and the like.

  One preferred method of administration for the conditions described in detail above is oral, using a conventional daily dosing regimen that can be adjusted according to the degree of pain. For such oral administration, the pharmaceutically acceptable non-toxic composition can be combined with any commonly used excipient (eg, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose , Croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, etc.). Such compositions take the form of solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations and the like.

  Preferably, the composition is in the form of a pill or tablet, so that the composition, together with the active ingredient, is a diluent such as lactose, sucrose, dicalcium phosphate, etc .; a lubricant such as magnesium stearate, etc. Agents; and binders such as starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.

  Liquid pharmaceutically administrable compositions can contain, for example, an active compound as defined above and any pharmaceutical adjuvant in a carrier (eg, water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc.). Can be prepared by dissolving, dispersing, etc., thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain small amounts of nontoxic auxiliary substances such as wetting, emulsifying, or solubilizing agents, pH buffering agents, and the like (eg, sodium acetate, sodium citrate, etc. , Cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc.)). Actual methods of preparing such dosage forms are known or apparent to those skilled in the art; see, eg, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 15th Edition, 1975. about. The composition or formulation to be administered will, in any event, contain an amount of the active compound in an amount effective to alleviate the symptoms of the subject being treated.

  Dosage forms or compositions containing the active ingredient in the range of 0.005% to 95% with the remainder made up of non-toxic carrier can be prepared.

  For oral administration, pharmaceutically acceptable non-toxic compositions are prepared using commonly used excipients (eg, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talc, cellulose derivatives, croscarmellose Sodium, glucose, sucrose, magnesium carbonate, sodium saccharin, talc, etc.). Such compositions take the form of solutions, suspensions, tablets, capsules, powders, sustained-release formulations and the like. Such compositions may contain from 0.01% to 95% active ingredient, preferably from 0.1 to 50% active ingredient.

  For solid dosage forms, solutions or suspensions (eg, in propylene carbonate, vegetable oils, or triglycerides) are preferably encapsulated in gelatin capsules. Such diester solutions, and their preparation and encapsulation are disclosed in US Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For liquid dosage forms, the solution (eg, in polyethylene glycol) can be diluted with an amount of a pharmaceutically acceptable liquid carrier (eg, water) sufficient to be readily measured for administration.

  Alternatively, liquid or semi-solid oral formulations are prepared by dissolving or dispersing the active compound or salt in a vegetable oil, glycol, triglyceride, propylene glycol ester (eg, propylene carbonate) and the like and dissolving these solutions or suspensions. It can be prepared by encapsulating in a hard gelatin capsule shell or soft gelatin capsule shell.

  Other useful formulations include those set forth in US Reissue Pat. No. 28,819 and US Pat. No. 4,358,603.

  The formulation can be administered in a single unit dosage form for continuous treatment, or optionally in a single unit dosage form, especially if relief of symptoms is required. For example, the formulation can be administered as a bolus or as a continuous intravenous infusion after the onset of symptoms of stroke, myocardial infarction, or chronic heart failure.

  Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional manner, either as liquids, solutions or suspensions, solid forms suitable for solutions or suspensions in liquids prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like. In addition, if desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, solubility enhancers and the like (eg, sodium acetate, sorbitan Monolaurate, triethanolamine oleate, cyclodextrin and the like)).

  More recently developed approaches for parenteral administration perform implantation of sustained or sustained systems so that a constant level of dosage is maintained. See, eg, US Pat. No. 3,710,795. The percentage of active compounds contained in such parenteral compositions is highly dependent on their particular nature, as well as the activity of the compound and the needs of the subject. However, a percentage of active ingredient of 0.01% to 10% in solution is higher if the composition is a solid that can be used and is later diluted to the above percentage. Preferably, the composition comprises 0.2-2% active agent in solution.

  Nasal solutions with the active compound alone or in combination with other pharmaceutically acceptable excipients may also be administered.

  Formulations of the active compounds or salts can also be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a fine powder for inhalation, alone or with an inert carrier such as lactose. In such cases, the particles of the formulation have a diameter of less than 50 microns, preferably less than 10 microns.

  The dermatological formulations of the present invention typically comprise a cytoprotective derivative of formula I, and optionally a polar solvent. Suitable solvents for use in the formulations of the present invention include any polar solvent that can dissolve the cytoprotective derivative. Suitable polar solvents include: water; alcohols (eg, ethanol, propyl alcohol, isopropyl alcohol, hexanol, and benzyl alcohol); polyols (eg, propylene glycol, polypropylene glycol, butylene glycol, hexylene glycol, Maltitol, sorbitol and glycerin); and panthenol dissolved in glycerin; flavor oils and mixtures thereof. Mixtures of these solvents can also be used. Exemplary polar solvents are polyhydroxy alcohols and water. Examples of preferred solvents include glycerin, panthenol in glycerin, glycols (eg, propylene glycol and butylene glycol, polyethylene glycol), water, and mixtures thereof. Further preferred polar solvents for use are alcohols, glycerin, panthenol, propylene glycol, butylene glycol, hexylene glycol and mixtures thereof.

  Typically, the formulations of the present invention are about 0.1% to about 80%, preferably about 0.5% to about 60%, more preferably about 1% to about 30%, and most preferably Contains about 3% to about 18% polar solvent.

  Emollients can also be added to the cosmetic / dermatological compositions of the present invention. Emollient ingredients include fats, oils, fatty alcohols, fatty acids, and esters, which aid in application and adhesion, produce a gloss, and most importantly provide occlusive moisturization To do. Suitable emollients for use are isostearic acid derivatives, isopropyl palmitate, lanolin oil, diisopropyl dimerate, maleated soybean oil, octyl palmitate, isopropyl isostearate, cetyl lactate, cetyl ricinoleate, tocopherol acetate , Acetylated lanolin alcohol, cetyl acetate, phenyl trimethicone, glyceryl oleate, tocopheryl linoleate, wheat malt glyceride, arachidyl propionate, myristyl lactate, decyl oleate, propylene glycol ricinoleate, isopropyl lanolate (Lanolate), pentaerythrityl tetrastearate, neopentyl glycol dicaprylate (dica rylate / dicaplate, hydrogenated coco-glyceride, isononyl isononanoate, isotridecyl isononanoate, myristal myristate, triisocetyl citrate, cetyl alcohol, octyldodecanol, Oleyl alcohol, panthenol, lanolin alcohol, linoleic acid, linolenic acid, sucrose esters of fatty acids, octyl hydroxystearate and mixtures thereof. Examples of other suitable emollients can be found in Cosmetic Bench Reference, pages 1.19-1.22 (1996), incorporated herein by reference. Suitable emollients include polar emollient emulsifiers (eg, linear or branched polyglycerol esters) and nonpolar emollients. The emollient ingredient is typically about 1% to about 90%, preferably about 10% to about 80%, more preferably about 20% to about 70%, and most preferably about the cosmetic composition. About 40% to about 60%.

  “Polar emollient” as used herein has at least one polar moiety and the solubility (at 30 ° C.) of the cytoprotective derivative compound in the polar emollient is about Mean any emollient emulsifier greater than 1.5%, preferably greater than about 2%, more preferably greater than about 3%. Suitable polar emollients include, but are not limited to, polyol esters and polyol ethers (eg, linear or branched polyglycerol esters and polyglycerol ethers). Non-limiting examples of such emollients include PG3 diisostearate, polyglyceryl-2-sesquiisostearate, polyglyceryl-5-distearate, polyglyceryl-10-distearate, polyglyceryl-10-diisostearate Acetylated monoglyceride, glycerol ester, glycerol tricaprylate / caprate, glyceryl ricinoleate, glyceryl isostearate, glyceryl myristate, glyceryl linoleate, polyalkylene glycol (eg PEG 600), monoglyceride, 2-monolaurin, sorbitan Examples include esters and mixtures thereof.

  “Nonpolar emollient” as used herein means any emollient emulsifier that does not have a permanent electrical moment. Suitable non-polar emollients include, but are not limited to, esters and straight or branched chain hydrocarbons. Non-limiting examples of such emollients include isononyl isononanoate, isopropyl isostearate, octyl hydroxystearate, diisopropyl dimerate, lanolin oil, octyl palmitate, isopropyl palmitate, paraffin, isopararifin. ), Acetylated lanolin, sucrose fatty acid ester, isopropyl myristate, isopropyl stearate, mineral oil, silicone oil, dimethicone, allantoin, isohexadecane, isododecane, petrolatum, and mixtures thereof. The solubility of the compound in polar emollients or non-polar emollients is determined according to methods known in the art.

  Suitable oils include esters, triglycerides, hydrocarbons and silicones. These can be a single substance or a mixture of one or more substances. These usually constitute 0% to about 100% of the emollient ingredient, preferably about 5% to about 90%, and most preferably about 70% to about 90%.

  Oils that act as emollients also impart viscosity, tackiness, and drug properties to cosmetic compositions such as lipsticks. Examples of suitable oils include: caprylic acid triglyceride; capric acid triglyceride; isostearic acid triglyceride; adipic acid triglyceride; propylene glycol myristyl acetate; lanolin; lanolin oil; polybutene; isopropyl palmitate; isopropyl myristate; Isopropyl acid; Diethyl sebacate; Diisopropyl adipate; Tocopheryl acetate; Tocopheryl linoleate; Hexadecyl stearate; Ethyl lactate; Cetyl oleate; Cetyl ricinoleate; Oleyl alcohol; Hexadecyl alcohol; Octyl hydroxy stearate; Malt oil; hydrogenated vegetable oil; castor oil; petrolatum; modified lanolin; branched chain hydrocarbons; Corn oil; cottonseed oil; olive oil; palm seed oil; rapeseed oil; jojoba oil; jojoba oil; avocado oil; mineral oil, shea butter, octyl palmitate, maleated modified soybean oil, grocerol Trioctanoate, diisopropyl dimerate, and volatile and non-volatile silicone oils (including phenyl trimethicone).

  Suitable oils for use herein include acetylglycerides, octanoic acid esters and decanoic acid esters of alcohols and polyhydric alcohols (eg, octanoic acid and decanoic acid esters of glycols and glycerol), alcohols and ricinol of polyhydric alcohols. Acid esters (eg cetyl ricinoleate), PG-3 diisostearate, polyglycerol ether, polyglycerol esters, caprylic acid triglyceride, capric acid triglyceride, isostearic acid triglyceride, adibic acid triglyceride, phenyl trimethicone, lanolin oil, Polybutene, isopropyl palmitate, isopropyl isostearate, cetyl ricinoleate, octyldodecanol, oleyl alcohol, hardened plant , Castor oil, modified lanolin, octyl palmitate, lanolin oil, maleic acid modified soybean oil, cetyl ricinoleate, glyceryl trioctanoate, diisopropyl dimer, synthetic lanolin derivatives and branched chain alcohols, sucrose esters of fatty acids, hydroxystearic acid Octyl as well as mixtures thereof.

  Preferably, the oil used is the majority (at least about 75%, preferably at least about 80% and most preferably at least about 99%) of the type of oil used is greater than about 1 to about 0.1. It is selected to have a solubility parameter that does not differ, preferably does not differ by more than about 0.8 to about 0.1.

  Surfactants can also be added to the compositions of the present invention to impart beneficial cosmetic or application properties. Suitable surfactants for use are surfactants that can form emulsions and / or associated structures. Surfactant emulsifiers can be 0% to about 20%, preferably 0% to about 15% and most preferably about 1% to about 10% of the formulation. Examples of suitable emulsifiers can be found in Dunphy et al. US Pat. No. 5,085,856 and El-Nokaly et al. US Pat. No. 5,688,831. Examples of other suitable emulsifiers are available from Cosmetic Bench Reference, pp. 1.22, 1.24-1.26 (1996), all of which are incorporated herein by reference.

  Also useful herein are surfactants that form when an associated structure (preferably a layered or hexagonal liquid crystal) is mixed with a polar solvent at ambient temperature. Ambient temperature / room temperature as used herein typically means about 20 ° C. In general, the ambient temperature can range from about 18 ° C. to about 27 ° C., preferably from about 20 ° C. to about 27 ° C., depending on variables such as geographical location (ie, subtropical) relative to the temperature region. It can be in the range of about 25 ° C. One skilled in the art can readily determine whether an association structure forms at ambient temperature. Surfactants suitable for use generally have a Kraft point of about ambient temperature (about 20 ° C) or lower, or generally about 18 ° C to about 27 ° C or lower, preferably about 20 ° C to about 25 ° C or lower. .

  The definition of craft point is well known in the art and one skilled in the art can readily determine the craft point of a surfactant. In general terms, the Kraft point is the melting point of the hydrocarbon chain of the surfactant. This can also be expressed as a temperature at which the solubility of the associated colloid in water suddenly increases due to the formation of micelles above the critical micelle concentration.

  In preparing a sample mixture of surfactant and polar solvent to demonstrate the ability to form an association structure, the surfactant is sufficient in the polar solvent so that the association structure can form at ambient temperature. Must be soluble in One skilled in the art can determine a compatible interaction.

  Any surfactant that forms an association structure at ambient temperature and is suitable for use in cosmetics is suitable for use herein. Surfactants suitable for use in cosmetics do not exhibit dermatological or toxicological problems. Anionic surfactants, nonionic surfactants, cationic surfactants, amphoteric surfactants and mixtures thereof are suitable for use. Preferably, anionic and nonionic surfactants, cationic surfactants, amphoteric surfactants, and mixtures thereof having a Kraft point below about ambient temperature are used. More preferably, nonionic surfactants, cationic surfactants, amphoteric surfactants, and mixtures thereof having a Kraft point below about ambient temperature are used.

  The surfactant is at a level of about 4% to about 97%, preferably about 5% to about 95%, more preferably about 20% to about 90%, and most preferably about 30% to about 70% of the associative structure. Can be used in

  The cosmetic composition of the present invention is referred to herein as a “coagulant”, either singly or collectively, and is one type effective to coagulate a particular liquid base material used in the cosmetic composition. These materials can be included. (As used herein, the term “coagulates” refers to the formation of a solid or semi-solid at ambient conditions (ie, having a stable physical structure and on the skin under normal use conditions). Refers to the physical and / or chemical changes of the liquid base material so that it forms a final composition that can be deposited on the surface.) As specified by those skilled in the art, specific for use in cosmetic compositions The choice of coagulant depends on the particular type of composition desired (ie, gel or wax-based), the desired rheology, the liquid base material used and the other materials used in the composition. The coagulant is preferably present at a concentration of about 0 to about 90%, more preferably about 1 to about 50%, even more preferably about 5% to about 40%, and most preferably about 3% to about 20%. To do.

  Embodiments of the wax cosmetic stick of the present invention preferably comprise from about 5% to about 50% (by weight) of a waxy coagulant. The term “waxy coagulant” as used herein means a coagulant having a waxy character. Such waxy materials can also act as emollients. Among the waxy materials useful herein are high melting waxes (ie, having a melting point of about 65 ° C. to about 125 ° C.), such as beeswax, spermaceti, carnauba wax, baysberry, candelilla Wax, montan wax, ground wax, ceresin, paraffin, synthetic wax (eg, Fisher-Tropsch wax), microcrystalline wax, and mixtures thereof. Ceresin, ground wax, white beeswax, synthetic wax, and mixtures thereof are useful waxes herein and are published in US Pat. No. 4,049,792, Elsnau, September 20, 1977 (herein). Which is incorporated by reference in its entirety. Low melting waxes having a melting point of about 37 ° C. to about 75 ° C. are preferred for use in the wax stick embodiments of the present invention. Wax stick embodiments of the present invention comprise a volatile silicone oil as the liquid base material, and preferably have a low melting point of about 10% to about 35%, more preferably about 10% to about 20% (by weight). Contains wax. Such materials include fatty acids, fatty alcohols, fatty acid esters and fatty acid amides, and mixtures thereof having a fatty chain of about 8 to about 30 carbon atoms. Preferred waxy materials include cetyl alcohol, palmitic acid, stearyl alcohol, behenamide, tallow fatty acid, mono- and difatty acid esters of polyethylene glycol, and mixtures thereof. Stearyl alcohol, cetyl alcohol, and mixtures thereof are particularly preferred. Additional fatty acids, fatty alcohols, and other waxy materials useful in the present invention are also well known in the art.

  The following preparations and examples are provided to enable those skilled in the art to more clearly understand and to practice the present invention. These examples should not be construed as limiting the scope of the invention, but merely as exemplifications and representative examples thereof.

(General characterization method)
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DTX300 spectrometer, most often using tetramethylsilane (TMS) as an internal reference. Mass spectra were obtained on an Agilent 1100 LC / MSD instrument using either electrospray ionization (positive or negative mode) (ESI) or atmospheric pressure chemical ionization (positive or negative mode) (APCI).

(Example 1)
(Determination of activity using neuronal cell stress assay)
(A. Isolation and culture of primary hippocampal neuronal cells)
(material)
Neurobasal / R27i: 1 × B27 supplement (Life Technologies), Neurobasal medium (Life Technologies, MD) containing 0.5 μM L-glutamine, 25 μM L-glutamate, and 1 × penicillin / streptomycin.
Hanks basal salt solution (without HBSS, Ca / Mg) supplemented with HEPES (10 mM, pH 7.3), sodium bicarbonate (0.35%), 1 × penicillin / streptomycin, and 1 mM pyruvate Prepared by preparing 1 × Hanks CMF (Gibco).
Poly-D-lysine (Sigma, St. Louis, MO), 50 μg / ml solution filtered through a 0.2 μm filter tube.
Sigmacote (Sigma, St. Louis, MO).
Poly -D- lysine (Sigma, St.Louis, MO) plastic culture flasks (T75 cm ²⁾ treated with or 12-well cell culture plate.

(Preparation of primary hippocampal neuronal cells)
Female pregnant mice (E18-E19) were euthanized with CO ₂ followed by removal of the uterus. The uterus was then placed in a sterile plastic petri dish. Embryos were removed from the sac, embryonic brains were removed and soaked in cold (4 ^° C.) buffered saline (HBSS; Ca / Mg free; Life Technologies) in small petri dishes. The hippocampus was then removed from the brain under a dissecting microscope and placed on a paraffin-covered dish. The meninges were peeled and the dissected hippocampus was collected in HBSS in a small petri dish. The hippocampus was transferred to a 15 ml centrifuge tube filled with HBSS (usually 10-12 brains). The test tube containing the brain was centrifuged at 1000 rpm for 2 minutes in a tabletop centrifuge. The supernatant was removed, 2 ml of HBSS was added to the hippocampus in the centrifuge tube, and the resulting suspensions were each treated with a siliconized glass pipette with a long tip and a gradually reduced opening. Use (starting with a pipette with a standard size opening (about 1.0 mm diameter), then a pipette with about a half size opening (about 0.5 mm diameter), then (Use a pipette with an opening about ½ of its size (0.25 mm diameter)). The suspension was then centrifuged again at 1000 rpm for 2 minutes in a tabletop centrifuge, the supernatant discarded, and 2 ml Neurobasal / B27i (containing antibiotics) added to the centrifuge tube. The above trituration procedure was then repeated for this suspension.

Cell density was determined for small aliquots of cells using standard counting procedures, corrected for cell viability with trypan blue stain exclusion. Using this procedure, the estimated yield is 3 × 10 ^{5 to} 6 × 10 ⁵ cells / brain. The cells were then coated with PDL containing Neurobasal / B27I at a density of about 1.5 × 10 ⁶ cells (T75 flask) or about 70,000 cells / well (24 well plate), 24 well plate, flask or Added to MetTek dish. Plated cells were incubated at 37 ° C. in an atmosphere of 5% CO ₂ /95% O ₂ . The medium was renewed after 3-4 days by replacing half of the medium with fresh Neurobasal / B27m medium (containing 5 μM cytosine arabinoside (AraC)). Seven to eight days after the initial culture, the medium was renewed by collecting half or all of it and replacing it with an equal volume of fresh Neurobasal / B27m medium (no Ara-C).

(B. Hippocampal oxygen deficiency-reoxygenated cell death assay)
Using this assay, ischemia was induced in cultured hippocampal neuronal cells by hypoxia-reoxygenation. Test compounds were added to assess efficacy and efficacy against ischemia-induced neuronal cell injury and cell death.

(material)
Neurobasal medium, NoG neurobasal medium, B27 supplement and B27 supplement (without AO) were obtained from Invitrogen Life Technologies.
Neurobasal / B27 medium was prepared with 2 × B27 (no AO) supplement, 0.5 mM L-glutamine and 0.25 × penicillin / streptomycin.
Cell Tracker Green was obtained from Molecular Probes and a new 5 μM solution was prepared from a 10 mM stock just prior to use.
LoG-Neurobasal contains NoG neurobasal medium and 1 mM glucose, 0.5 mM L-glutamine 0.25 × penicillin / streptomycin, and 10 mM Hepes (pH 7.4).
Primary hippocampal neuronal cells were prepared according to the method described above and cultured for 10-11 days in a 24-well plate coated with poly-D-lysine before use.

Deoxygenated LoG-Neurobasal medium (100 ml) was prepared by pre-equilibrating the medium overnight in a T150 cm ² flask in a low oxygen chamber. After preincubation under hypoxic conditions, the LoG-Neurobasal medium was lightly bubbled with 100% N ₂ for 30 minutes to completely deoxygenate the medium. An additional 20 ml of LoG-Neurobasal was pre-equilibrated in a T75 cm ² flask and incubated overnight in a normal incubator (5% CO ₂ ). Reoxygenated medium was prepared by placing Neurobasal / B27 medium overnight in a culture incubator (5% CO ₂ /95% O ₂ ).

Ten to 11 days after hippocampal nerve plating, the existing culture medium (Neurobasal / B27m) is removed from the cells by aspiration. The cells are washed once with 600 μl / well (24 well culture plate) of BSS without glucose. Neurons are supplemented with deoxygenated LoG-Neurobasal (400 μl / well for each well of a 24-well plate). Test compounds were added directly to each well (usually 3 concentrations of compound and a positive control (3 replicates each)). Most test compounds were dissolved in 100% DMSO; however, the concentration was adjusted so that the final DMSO concentration in the cell medium never exceeded 0.5%. Plates containing cells with test compounds were placed in a hypoxic chamber for 4-5 hours with the plate lid half open. For normoxia control, pre-equilibrated normoxia LoG-Neurobasal medium was added to the cells in each well and the plates were repositioned in a normal culture incubator for 4-5 hours. After 4-5 hours of hypoxia, the existing medium was carefully aspirated and 400 μL of fresh reoxygenated (pre-equilibrated) Neurobasal / B27 was added to each well. The same test compound (same concentration) was added back into the corresponding well. Plates were placed in a cell culture incubator _{(5% CO 2/95%} O 2), and reoxygenation 20-24 hours. After 20-24 hours of reoxygenation, surviving neurons were quantified using the cell tracker green fluorescence method described below.

  To test for cell viability, the existing culture medium was aspirated from each well of a 24-well plate and the neurons were washed once with 1 ml HBSS (pH 7.4, pre-warmed to 30-37 ° C.). . To each well, 500 μL of 5 μM Cell Tracker Green fluorescent dye dissolved in HBSS was added. Plates were protected from light and left at room temperature for 15 minutes, then washed with 1 ml HBSS. 500 μL of HBSS was then added to each well and fluorescent cells were counted using a fluorescence microscope. Significantly increased cell viability compared to control cells is an indication of a protective compound.

  When tested as described above, the compounds of the present invention provided protection against stressor-induced cell death in at least 20% of the cells tested at concentrations ranging from about 1-1000 μM.

(Example 2)
(Myocyte Calcium Contractility Assay)
(A. Isolation and culture of primary neonatal myocytes)
(material)
• 10 × Heart Dissection Solution (HDS) contains the following components (g / l) in cell culture grade water: NaCl, 68; HEPES, 47.6; NaH ₂ PO ₄ , 2; Glucose , 10; KCl, 4; MgSO ₄ , 1, pH was adjusted to 7.4. Prior to filter sterilization of the diluted (1 × HDS) solution, 10 mg phenol red was added to each 500 ml medium.
Transferrin and bovine insulin were obtained from Life Technologies and resuspended in tissue culture grade water at a concentration of 4 mg / ml.
DMEM-F12 (DMEM-F12, powder containing glutamine and pyridoquine hydrochloride 1: 1) was purchased from Life Technologies. To a 1 liter equivalent of powder, 2.43 g sodium bicarbonate and 10 ml 100 × penicillin / streptomycin in 950 ml tissue culture grade water was added with stirring. The pH was adjusted to 7.2 with 1M HCl and the volume was adjusted to 1 liter. After this solution was sterilized by filtration, 2.5 ml of 4 mg / ml transferrin, 250 μl of 4 mg / ml insulin and 30.7 mg bromodeoxyuridine were added.
DMEM-F12 containing 4% fetal bovine serum (FBS) was also prepared for pre-coating tissue culture plates and for initial suspension of cardiomyocyte pellets.
Collagenase solution (49 mg collagenase was resuspended in 120 ml 1 × HDS).

(Preparation of primary neonatal myocardial culture)
Tissue culture ware was pre-coated with DMEM-F12-4% FBS by incubating 50 μl per well of a 96 well plate and 0.25 ml per well of a 12 well plate at 37 ° C.

  Two-day-old rat pups are separated from their mothers and placed in sterile containers. The pups were quickly soaked in 70% alcohol, then decapitated, and the body was placed in an empty sterile tissue culture dish. The incision was started from the neck and progressed towards the abdomen and cut through the sternum. The heart was removed and placed in a tissue culture dish containing 1 × HDS. The atria were excised and the remaining ventricles (these were cut into 3-4 pieces each) were placed in separate tissue culture dishes containing 1 × HDS. The ventricles were then transferred to a sterile 250 ml glass flask and 1 × HDS was removed. 20 ml of prewarmed collagenase solution was added to the ventricle followed by incubation at 37 ° C. with shaking. After 30 minutes, the collagenase solution was removed and replaced with 20 ml of fresh pre-warmed collagenase. Incubation is continued for another 30 minutes. At the end of the incubation, any tissue mass was processed, after which collagenase (including isolated cardiomyocytes) was removed from the disrupted tissue pieces. Isolated myocytes were added to a 50 ml Falcon tube containing 2 ml fetal bovine serum (FBS). The remaining tissue pieces are subjected to secondary digestion by the addition of 20 ml of fresh pre-warmed collagenase and incubated as above for 30 minutes. The secondary digest was then centrifuged at 1000 rpm for 10 minutes (desktop centrifuge). The resulting supernatant was discarded and the cell pellet was suspended in 4 ml FBS. The resulting cell suspension was placed in an incubator at 37 ° C. This process was repeated several more times to collect additional material.

  A Percoll gradient was prepared by adding 2.5 ml of 10 × HDS to 22.5 ml of Percoll (Life Technologies) with mixing (Percoll stock). A top gradient solution (11 ml Percoll stock and 14 ml 1 × HDS) and a bottom gradient solution (13 ml Percoll stock and 7 ml 1 × HDS) were prepared. 4 ml of the top gradient solution was transferred to a 6 × 15 ml sterile Falcon tube. 3 ml of bottom gradient solution was placed in each tube by inserting a serological pipette at the bottom of the tube and slowly adding liquid.

  All digests (5) were pooled into one 50 ml Falcon tube and centrifuged at 1000 rpm for 10 minutes in a tabletop centrifuge. The supernatant was discarded and the cell pellet was resuspended in 12 ml 1 × HDS. 2 ml of cell suspension was added to the top of each gradient. The gradient tube was then centrifuged at 3000 rpm for 30 minutes without breaking in a Beckman Allegra 6 centrifuge (GH 3.8A rotor). Following centrifugation, the cells were separated into two sharp bands at the two interfaces. The lower of the two bands was hatched for cardiomyocytes; there was also a cardiomyocyte pellet at the bottom of the tube. The upper band was hatched for fibroblasts and other non-cardiomyocytes. The top of the gradient was aspirated just above the cardiomyocyte phase. The cardiomyocyte layer was then carefully removed with the pellet, and the two fractions were pooled into a 50 ml Falcon tube that was sterilized with the corresponding fraction from a further gradient tube; To the total volume of. The tube was centrifuged at 1000 rpm for 7 minutes. The supernatant was discarded and resuspended in 25 ml 1 × HDS. An additional 25 ml of 1 × HDS was added and the centrifugation step was repeated. The cell pellet was carefully and thoroughly resuspended in 40-50 ml of DMEMF 12-4% FBS.

Small aliquots of cell suspension were counted with a hemocytometer. DMEM / F12-FBS coated medium was aspirated from the tissue culture dish. Cardiomyocytes were added to the dishes at a plating density of 7.5 × 10 ⁴ / well per 96 wells in 200 μL and a plating density of 1.5 × 10 ⁵ / well per 12 wells in 3 ml. Cultures were incubated overnight at 37 ° C. with 5% CO ₂ . The original medium was removed and fresh DMEM / F12-5% FBS was added to each culture and incubated for an additional 48 hours at 37 ° C. under 5% CO ₂ before use.

(B. Contractility assay)
(material)
Complete DMEM-F12: DMEM / F12 (powder, 1: 1, containing glutamine and pyridoxine hydrochloride) was purchased from Life Technologies (Invitrogen Life Technologies, Carlsbad, Calif.). Enough powder and 2.43 g of sodium bicarbonate to prepare 1 l of buffer were mixed in 950 ml of tissue culture grade water. The pH was adjusted to 7.2 with 1M HCl and the remaining water was added to 1 liter. After sterile filtration, 10 ml 100 × penicillin / streptomycin, 2.5 ml 4 mg / ml transferrin, 250 μl 4 mg / ml insulin and 30.7 mg bromodeoxyuridine are added and the mixture is allowed to Incubated at 0 ° C.

  • 1 mM glucose in DMEM was made from DMEM (purchased from Life Technologies) that does not contain L-glutamine, glucose, or sodium pyruvate.

  • 20 μM Fluo-4: Cell persistent AM ester of Fluo-4 was obtained from Molecular Probes (Eugene, Oreg.) As a dry powder stored at −20 ° C. This fluorescent dye is photosensitive and should be replaced with 1 mM in DMSO before use to prevent photolysis.

• 10 mM CaCl ₂ solution was replaced daily in 1 × HBSS and incubated at 37 ° C. before use. Neonatal cardiomyocytes were isolated as described above. The cardiomyocytes were plated in a 96-well format at a density of 7.5 × 10 ⁴ / well (black clear bottom plate) and in the presence of 5% FBS prior to use in the assay Grown for 2 days.

Physiological ischemia is simulated by placing cardiomyocytes in an anaerobic chamber (0% O ₂ , 85% N ₂ , 5% CO ₂ and 10% H ₂ ) in DMEM containing 1 mM glucose. I did. Positive control cells are treated with DMEM-F12 containing 25 mM glucose, which protects against hypoxia.

  Test compounds were made in DMEM (1 mM) glucose in 96 deep well mother plates and diluted appropriately for use in the assay. This medium was removed from the cells and replaced with either 200 μl DMEM-F12 or 1 mM DMEM with or without test compound. The plate was then placed inside a 37 ° C. incubator in an anaerobic chamber and incubated for 16 hours. The plate was then removed and reoxygenated by the addition of DMEM-F12. DMEM with or without test compound was carefully removed from the cells and replaced with pre-warmed DMEM-F12 containing 5% FBS. Since hypoxia treatment can damage the cells and / or kill the cells, it is necessary for this step to remove the cells from the bottom of the well by gentle aspiration of the medium. The cells were then placed in a normal incubator at 37 ° C. and incubated for 2 hours to reoxygenate the cells.

A working solution of 20 μM Fluo-4 was added to pre-warmed 1 × HBSS. The cells were first loaded with Fluo-4 by removing the medium from the cells and replacing with 100 μl of 20 μM Fluo-4. Unloaded control cells were treated with 1 × HBSS alone in parallel. All cells were then incubated for 30 minutes at 37 ° C. Prior to performing fluorescence measurements, cells were washed in medium without indicator (HBSS) to remove any non-specifically associated dye on the cell surface. The cells were then incubated for an additional 20 minutes at room temperature. Basal Fluo-4 fluorescence is measured with a microplate fluorometer (Fluorskan ^™ , Thermo Labsystems Oy, Helsinki, Finland) using a pair of 485 nm excitation filter and 538 nm emission filter. Each well was read for 160 milliseconds to obtain a baseline reading and then stimulated to contact by the addition of 10 mM CaCl ₂ . After incubating at 37 ° C. for 30 minutes, a stimulated fluorescence reading was taken after 90 minutes.

When tested as described above, compounds of the present invention as shown below show an amount of at least 20% protection and the ability to protect against ischemic injury and allow cells to maintain their contractile function. Indicates the presence of calcium transients in:
(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl) -phenyl-methanone;
Acetic acid 6,7-dimethyl-2- (2-morpholin-1-yl-acetyl) -benzofuran-5-yl ester;
2- (1-hydroxy-2-morpholin-4-yl-ethyl) -6,7-dimethyl-benzofuran-5-ol;
Acetic acid 2- (2-bromo-acetyl) -6,7-dimethyl-benzofuran-5-yl ester;
1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -2-morpholin-4-yl-ethanone;
1- (4-bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone;
1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone;
4- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -benzonitrile;
5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde;
2-hydroxymethyl-6,7-dimethyl-benzofuran-5-ol;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid bis- (2-hydroxy-ethyl) -amide;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 2- [2- (2-methoxy-ethoxy) -ethoxy] -ethyl ester; and 5-hydroxy-6,7-dimethyl-benzofuran-2- Carboxylic acid bis- (2-hydroxy-ethyl) -amide.

Example 3
(Model of cerebral ischemic rat middle cerebral artery occlusion (MCAO))
(A. Preparation of animals)
Male Wistar rats (Harlan, IN) weighing 300-350 g are commonly used for these experiments. Animals are given free access to water and commercially available rodent food under standard laboratory conditions. Room temperature is maintained at 20-23 ° C and room lighting is a 12/12 hour light / dark cycle. Animals are acclimated to the laboratory environment for 5-7 days prior to the study and fasted overnight prior to surgery (water is free to drink)
(B. Middle cerebral artery occlusion (MCAO))
Anesthesia is maintained by inhalation of 3.0% isoflurane in 0.8% oxygen (Aerlane, Front Dodge, IA). Prior to surgery, the animal's neck was shaved and sterilized. Body temperature was controlled and maintained at 37.5 ° C. + / − 1 ° C. via an external heating and cooling device. To lower the body temperature, animals are placed in a cooling chamber that uses ice to cool the circulating air. Throughout the study, body temperature is recorded between rat scapulae using a temperature transponder (BMDS Inc., Seaford, DL) implanted subcutaneously at the time of MCAO. This temperature transponder allows the user to read body temperature via a pocket scanner (BMDS Inc., Seaford, DL). Body temperature can also be measured by inserting a temperature probe into the rectum of the animal. Body temperature is recorded every hour for 6 hours after obstruction, but is frequently measured so that body temperature can be maintained at the normal temperature of the animal.

  The animals were subjected to 2 hours of MCAO using the modified intraluminal filament technique as follows. A midline incision is made on the ventral side of the neck to expose the external and internal carotid arteries. The right external carotid artery and common carotid artery were ligated with sutures (silk 5/0, Carlisle Laboratories, Farmers Branch, TX), and the right internal artery was microvascular clip (Fine Science Tool Inc., Foster City, CA). ) To temporarily ligate. A small incision was made in the common carotid artery. Nylon filaments, whose tips are rounded by heating, are prepared from fishing lines (Stren Fishing Lines, Wilmington, DE) and inserted through the right common carotid artery. The filament is advanced into the internal carotid artery 18-20 mm from the bifurcation of the internal and external carotid arteries, and the suture is tightly ligated around the filament. Two hours after occlusion, the animals are re-anesthetized and re-perfusion is allowed for the remainder of the experiment by removal of the filament.

(C. Drug administration)
The test compound can be administered by any of a number of routes (eg, as described below). The compound may be administered before, during or after occlusion, as appropriate to the protocol.

(A) Intraventricular (ICV) injection)
Anesthetized animals are placed on a stereotaxic device (Harvar Apparatus, S. Natick, MA). During all procedures, anesthesia is maintained by inhalation of 3.0% isoflurane (Aerrane, Front Dodge, IA) in 0.8% oxygen. Prior to surgery, the scalp is shaved and sterilized. A mid-sagittal incision approximately 3 cm long is made slightly behind the eye to expose the skull. Rub the skull with a rounded spatula to remove the periosteal connective tissue. A hole in the bar is placed 1.5 mm outside and 1 mm behind the left side of the bregma to mark the left ventricle. A brain infusion cannula (ALZET Co., Palo Alto, Calif.) Is inserted into this hole at a depth of 4 mm. The desired depth is adjusted by attaching a spacer to the cannula. A cannula attached to a 4 cm silastic catheter (Helix Medical Inc., Carpinteria, Calif.) Is secured in place using dental cement (Ketac-cement, Norrtown, PA). The catheter is attached to either a prepared osmotic pump placed subcutaneously between the scapula for continuous infusion or a syringe for short infusion.

(B) Intravenous (IV) osmotic pump implantation into the jugular vein)
During all procedures, anesthesia is maintained by inhalation of 3.0% isoflurane (Aerrane, Front Dodge, IA) in 0.8% oxygen. Prior to surgery, the animal's neck is shaved and sterilized. A midline incision is made on the ventral side of the neck and the jugular vein is exposed. The veins were isolated and the rostral side to the incision point ligated with sutures (Silk 5/0, Carrisle Laboratories, Farmers Branch, TX) and microvascular clips (Fine Science Tool Inc., Foster City, CA). Is placed near the heart. A small incision is made between the two ligatures. A 2 cm silastic catheter (Helix Medical Inc.) attached to a PE-60 tube (Becton. Dickinson and Co. Sparks, MD) connected to an ALZET (ALZET Co., Palo Alto, CA) pump was introduced toward the heart. Advance 2 mm into the jugular vein. The microvascular clip is removed and the catheter is secured in place with sutures (silk 5/0, Carlisle Laboratories, Farmers Branch, TX). Place the pump in a subcutaneously created pocket between the scapulas and allow the catheter to reach the jugular vein across the neck with sufficient looseness to allow free movement of the neck and head .

(C) IV injection through the femoral vein)
During all procedures, anesthesia is maintained by inhalation of 3.0% isoflurane (Aerrane, Front Dodge, IA) in 0.8% oxygen. Prior to surgery, the external site of the right femoral vein is shaved and sterilized. A 3 cm incision is made in the right groin area and the femoral vein is isolated. A small incision is made on the femoral vein, temporarily ligated with a microvascular clip, a polyethylene (PE-50) catheter (Becton Dickinson and Co. Sparks, MD) is introduced and advanced. The catheter is secured in place with sutures (silk 5/0, Carlisle Laboratories, Farmers Branch, TX). The other end of the catheter is attached to a syringe filled with heparinized saline for bolus injection. A pocket is made subcutaneously on the animal's back using a hemostatic material to allow the PE catheter to be lifted to the point of extraction at the neck of the neck for either bolus injection or continuous injection with an osmotic pump. .

(D) Intraperitoneal (IP) injection)
Awake rats are maintained in a standard hand hold position and a 23 3 / 4G needle is injected slightly away from the midline and past the peritoneum into the lower right quadrant of the abdomen. . Pull back the syringe plunger slightly to avoid organ injection. If the fluid is not withdrawn, the syringe contents are delivered to the abdominal cavity.

(D. Behavioral evaluation)
One hour after MCAO, the animals were held gently on the tail and observed for forelimb flexion. Animals are then placed on the floor and observed for gait patterns; only animals with a score of 3 in the Bederson rating system (Table 1) are included in this study.

  (Table 1 Bederson rating system for neurological evaluation)

（Ｅ．虚血損傷の評価）
いくつかの実験においてＭＣＡＯの２４時間後、またはより長い時間後、動物をＣＯ_２窒息（ドライアイス）によって屠殺する。標準の手段を使用して、脳を頭蓋骨からすばやく取りだし、冷却した生理食塩水溶液でリンスし、そしてラット脳組織スライサー（ＡＳＩ ｉｎｓｔｒｕｍｅｎｔ，ＭＩ）上に配置した。２ｍｍの厚さの７つの冠状切片を、剃刀の刃を使用して各脳から切り出す。これらの切片を、１．０％の塩化２，３，５−トリフェニルテトラゾリウム（ｔｒｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｕｍｅ）（ＴＴＣ）（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を含有する０．９％生理食塩水中に浸し、そして３７℃の水浴中で、３０分間インキュベートした。

(E. Evaluation of ischemic damage)
In some experiments, animals are sacrificed by CO ₂ asphyxiation (dry ice) 24 hours or longer after MCAO. Using standard means, the brain was quickly removed from the skull, rinsed with chilled saline solution, and placed on a rat brain tissue slicer (ASI instrument, MI). Seven coronal sections 2 mm thick are cut from each brain using a razor blade. These sections were soaked in 0.9% saline containing 1.0% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma Chemical Co., St. Louis, MO). And incubated in a 37 ° C. water bath for 30 minutes.

  After staining, each 2 mm section is taken using a TMC-7 camera (JH Technologies, Ca), which is connected directly to a desktop PC and images of each brain section are captured and stored. This image is used for measurement of the area of interest using a computer-based image processing system (Metamorph).

For each area measurement, the area of interest is selected using a freehand selection tool and the area is automatically calculated by selecting a measurement command. Measurements for the primary area of interest are right hemisphere, left hemisphere, total infarction, subcortical infarction, total penumbra and subcortical penumbra. After all areas of interest have been measured for all seven sections of the brain, Excel ^™ macro statistical final is used to classify by section number and corresponding area of interest. The macro also calculates cortical penumbra, cortical infarction and total ischemic damage for each section; the corresponding area of each rat's brain) is combined to produce a single measurement for each area. Since the ipsilateral hemisphere will swell after MCAO, the edema volume is calculated and reported as the volumetric difference between the right and left hemisphere of each brain section. The whole volume is calibrated for edema using the% hemisphere that swells. The damage volume is measured using the following calculation for each rat brain.

（Ｆ．統計的分析）
サンプルの大きさを、有意な結果の９０％の可能性を達成するように選択する。各ラットの脳の７つの切片中で目的の同じ領域を示す測定値を一緒にし、各動物における総梗塞、皮質下梗塞、皮質梗塞、総半影、皮質下半影、皮質半影、総虚血損傷および水腫についての単一の測定値を得る。グループデータを、平均＋／−ＳＥＭとして示す。ｐ＜０．０５のレベルの差異を、統計的に有意であるとみなす。グループの間で、目的の各領域の比較を、片側スチューデントｔ検定（２つのグループの間）または一方向ＡＮＯＶＡにより、次いでＢｏｎｆｅｒｒｏｎｉの複数比較あるいは非母数Ｄｕｎｎｅｔｔ試験（コントロールグループと薬物処理グループの間）によって行う。

(F. Statistical analysis)
The sample size is chosen to achieve 90% probability of significant results. Measurements representing the same area of interest in seven sections of the brain of each rat are combined and total infarct, subcortical infarction, cortical infarction, total penumbra, subcortical penumbra, cortical penumbra, total imaginary in each animal A single measurement for blood damage and edema is obtained. Group data are shown as mean +/- SEM. Differences at a level of p <0.05 are considered statistically significant. Between groups, each region of interest was compared by one-sided student t-test (between two groups) or one-way ANOVA, followed by Bonferroni's multiple comparisons or non-parametric Dunnett test (between control and drug treatment groups). ).

  The compounds of the invention can be tested as described above.

(Example 4)
(Myocardial infection model: left coronary artery ligation (rat))
Male Sprague-Dawley weighing 250-320 g is allowed free access to water and a commercial rodent diet under standard laboratory conditions. Room temperature is maintained at 20-23 ° C. and room lighting is set to a 12/12 hour light / dark cycle. Animals are acclimated to the laboratory environment 5-7 days prior to the study and fasted overnight prior to surgery.

(Surgical procedure for acute experiments :)
Rats are anesthetized with urethane (1.2-1.5 gm / kg). Core body temperature is maintained at 37 ° C. by using a heating blanket. The surgical area is shaved and a ventral midline incision is made to expose the trachea and jugular vein area. A catheter (PE50) is placed in the jugular vein for compound administration and maintenance of anesthesia. The trachea is opened and a 14-16 gauge modified intravenous catheter is inserted and tied in place as an endotracheal tube. The animal is placed in the right recumbent position and initially fitted with a Harvard ventilator with a tidal volume of 5-10 ml / kg. 100% O ₂ is delivered to the animal by the ventilator. An ECG electrode is placed and a standard lead II ECG is recorded. The surgical site is cleaned with an alcohol swab and a skin incision is made at the ribs over the fourth to fifth intercostal spaces. Carefully dissect the underlying muscle to avoid the lateral thoracic vein to expose the intercostal muscle. The thoracic cavity is extended through the fourth to fifth intercostal spaces and the incision is widened so that the heart is visible. Open the pericardium to expose the heart. A 6-0 silk suture threaded through a tapered needle is threaded around the left coronary artery near its origin, which is about 1 mm from the left atrial appendage insertion and is in contact with the left edge of the pulmonary artery cone. To do. A piece of tube is placed over the suture to form an occluder. The coronary artery is occluded over 30 minutes by sliding the tube until resistance is felt and holding the tube in place with a vascular clamp. ECG is monitored for ST changes indicative of ischemia. After 30 minutes, the occluder is removed and the suture is left in place. ECG is monitored for the first 10 minutes of reperfusion. The rat is transferred to a pressure controlled ventilator for the rest of the protocol. The rats in 10 to 15 cm H ₂ up to the suction pressure and 60-110 breaths / min respiration rate of O, artificial respiration by a small animal ventilator. The heart is reperfused for 90 minutes.

(Surgical procedure for 24-hour study :)
Rats are anesthetized by intraperitoneal injection of ketamine / xylazine (95 and 5 mg / kg) and a 14-16 gauge modified intravenous catheter is intubated. The anesthesia level is checked every 15 minutes by tow pinch. Core body temperature is maintained at 37 ° C. by using a heating blanket. The surgical area is shaved and scrubbed. A ventral midline incision is made to expose the jugular vein. A catheter (PE50) is placed in the jugular vein for compound administration and maintenance of anesthesia. The animal is placed in the right recumbent position and initially a ventilator with a tidal volume of 5-10 ml / kg H ₂ O or a maximum inhalation pressure of 8-15 cm H ₂ O and 60-110 breaths / min Attach a pressure-controlled ventilator with a respiration rate of 100% O ₂ is delivered to the animal by the ventilator. An ECG electrode is placed and a standard lead II ECG is recorded. The surgical site is cleaned with a surgical swab and alcohol. A skin incision is made at the ribs over the fourth to fifth intercostal spaces. Carefully dissect the underlying muscle to avoid the lateral thoracic vein to expose the intercostal muscle. The thoracic cavity is extended through the fourth to fifth intercostal spaces and the incision is widened so that the heart is visible. Open the pericardium to expose the heart. A 6-0 silk suture threaded through a tapered needle is threaded around the left coronary artery near its origin, which is about 1 mm from the left atrial appendage insertion and is in contact with the left edge of the pulmonary artery cone. To do. A piece of tube is placed over the suture to form an occluder. The coronary artery is occluded over 30 minutes by sliding the tube until resistance is felt and holding the tube in place with a vascular clamp. ECG is monitored for ST changes indicative of ischemia. After 30 minutes, the occluder is removed and the suture is left in place. ECG is monitored for the first 10 minutes of reperfusion. The three-layer incision is closed. The IV catheter is removed or passed under the skin and out between the scapulae to allow blood drainage or further drug treatment. Ventilate until the rat is able to breathe on its own. Tubes are removed from these rats and allowed to recover on a heating pad. Once awake, these rats are returned to their cages. Animals are administered buprenorphine (0.01-0.05 mg / kg SQ) for post-operative analgesia. After a predetermined reperfusion time (24 hours), these animals are anesthetized and deeply anesthetized and the heart is removed.

(Treatment protocol)
(Food :)
Animals are fed a conventional diet before or after coronary artery ligation. The length of treatment varies from study to study. For 300 gm rats, doses are calculated based on the average consumption of 15 gm diet per day. Rat weight is monitored during the study. Weigh the food that has not been consumed and estimate the consumption rate.

(IV treatment :)
A ventral incision is made to expose the jugular vein area. For compound administration, a catheter (PE50) is placed in the jugular vein. Animals are administered by bolus injection and / or continuous infusion. The duration and duration of treatment varies from protocol to protocol.

(Organization processing)
After reperfusion, each animal is administered 200 units of heparin (IV) intravenously with general anesthesia and the heart is removed and placed in cold saline. After removal, the coronary artery is ligated with sutures already in place. The heart is placed on a perfusion device and Evans Blue dye is injected to depict the area at risk. The heart is then cut into five 2 mm thick transverse slices from the top to the base. These slices are incubated for 20 minutes at 37 ° C. in 1% triphenyltetrazolium chloride (TTC) in 0.9% saline. Tetrazonium reacts with NADH in the presence of the dehydrogenase enzyme and stains viable tissue in a deep red color, which is easily distinguished from infarcted lightly stained necrotic tissue. For the staining procedure, these slices are placed top-down in a small petri dish lid. The bottom of the dish is placed on these slices and keeps them flat. These slices are photographed from bottom to top and from edge to bottom. The area of infarcted tissue, the area at risk and the area of the entire left ventricle are determined using a computer image analysis system. Combine the total area of each region to get the total for the whole heart. Infarct size is expressed as both the percentage of total ventricles and the percentage of area at risk.

(Statistical analysis)
Group data are expressed as mean +/- SEM. Comparison between treatment groups is performed using ANOVA, where p <0.05 is considered significant. Subsequent comparisons can be made using either the Dunnett test or the Tukey test.

  The compounds of the present invention show activity when tested by this method.

(Example 5)
(Evaluation of sensorimotor behavior)
(A. Grip strength of forelimbs and hind limbs in rats)
Animals with cerebral infarction induced by transient or permanent unilateral occlusion of the central cerebral artery (MCA) and sham-operated rats are standard models of grip strength (neuromuscular function and sensorimotor formation) ) Using a rat computer grip strength meter (Dual Stand Model, Columbia Instruments, Columbia, OH).

  Animals are transferred to the testing room for 30 minutes prior to testing. Prior to testing, each gauge is calibrated with a known set of weights and the device is adjusted to the size of the animal according to the manufacturer's instructions. Measurement of the forelimbs is performed with this meter in the fully extended state, and the reading when the test animal leaves the grip bar is fixed. Measurements of the hind limbs are performed with this meter in a maximally compressed manner and the reading is fixed when the hind limb of the subject animal is pulled from the bar toward the meter. Each animal is held by the researcher's hand while pulling over the grip bar using consistent techniques, allowing the forelimbs and hind limbs to freely grip the gripbar.

  The study is performed in a blinded random manner on the second day after the operation and is repeated twice a week at predetermined intervals. Typically, three consecutive readings are taken for each animal with a test interval long enough to record the data and zero both meters for the next test.

(B. Rota-Rod test in rats)
(apparatus)
Rota-Rod Trademill for rats (made by 7750 Accelerating Model, UGO BASILE, COMERIO-ITARY).

(procedure)
Animals with cerebral infarction induced by transient or permanent unilateral occlusion of the central cerebral artery (MCA), and sham-operated rats were treated with a Rota-Rod Treadmill for rats (7750 Accelerating Model, UGO Basile, Comerio). , Italy) in this study. These animals are transferred to the test room 30 minutes before the test. Prior to testing, all rats will receive 2-3 minutes training runs 2-3 times at 2-3 hour intervals.

  The cylinder on the device is actuated before placing the rat in place. The motor is set to a constant selected speed at 7700 in RESET mode and rats are placed one by one in that section.

  The study is performed in a blinded random manner on the second day after the operation and is repeated twice a week at predetermined intervals. Typically, three consecutive readings are taken for each animal with a test interval long enough to record the data and zero both meters for the next test.

(Example 6)
(Model of congestive heart failure)
(Preparation for experiment)
225-275 g male Sprague-Dawley rats are anesthetized with ketamine / xylazine (95 mg / kg and 5 mg / kg) and a 14-16 gauge modified intravenous catheter is intubated. Core body temperature is maintained at 37 ° C. by using a heating blanket. Pick the operating area and rub it. The animals in the right lateral recumbency and initially placed on the ventilator having a 10 to 15 cm H ₂ peak inspiratory pressure and 60-110 breaths / min breathing of O. 100% O ₂ is delivered to the animal by the ventilator. An ECG electrode is placed and a standard lead II ECG is recorded. An incision is made at the ribs over the fourth to fifth intercostal spaces. Carefully dissect the underlying muscle to avoid the lateral thoracic vein to expose the intercostal muscle. The chest cavity is entered through the fourth to fifth intercostal spaces and the incision is widened so that the heart is visible. Open the pericardium to expose the heart.

  A 6-0 silk suture threaded through a tapered needle is threaded around the left coronary artery near its origin. This starting point is about 1 mm from the insertion part of the left atrial appendage. The coronary artery is occluded by tying a suture around the artery. ECG is monitored for ST changes indicative of ischemia. If the animal develops ventricular fibrillation, a gentle heart massage is used to turn the animal into a normal beat. The sham-operated control is subjected to the same procedure, but the suture is not tied. The incision is closed in three layers. Infected or moribund animals are excluded from this study.

  After 4 weeks of operation, the animals are anesthetized and a catheter is placed in the right carotid artery and advanced to the left ventricle for hemodynamic measurements. Pressure traces are recorded and analyzed for heart rate, left ventricular systolic and diastolic blood pressure, left ventricular deployment blood pressure, and dP / dt maximum and dP / dt minimum. After taking the measurement, 2 ml of blood is taken and placed in a serum tube and a plasma tube. The heart is removed and the Langendorff device is placed as follows.

(Langendorff procedure)
Buffer preparation: Krebs-Henseleit (KH) buffer solution (NaCl 118 mmol / L, KCl 4.7 mmol / L, MgSO ₄ 1.2 mmol / L, KHPO ₄ 1.2 mmol / L, glucose 11 mmol / L, NaHCO ₃ 25 mmol / L and CaCl ₂ 2.5 mmol / L (Sigma)) are made fresh daily using Nanopure pyrogen-free water.

Animals are given 200 units of heparin, the chest is opened, the heart is quickly excised and placed in ice-cold KH buffer solution. After the cardiac contractile activity ceases, the heart is cut and the ascending aorta is removed from the connective tissue. The heart is quickly weighed and then the aorta cannulated and the heart is attached to a non-recirculating Langendorff perfusion device (Radnoti Glass Technology, Inc., Monrovia, Calif.). The heart is perfused in a retrograde fashion through the aorta using KH buffer solution oxygenated with 95% O ₂ and 5% CO ₂ to maintain pH 7.4 at 37 ° C. To assess contractile function, a latex balloon is inserted through the mitral valve opening into the left ventricle and connected to the pressure transducer by rigid polyethylene tubing. The balloon is inflated with water to a left ventricular final diastolic pressure (LVEDP) of 1-10 mmHg. The flow rate is started at 12 ml / min and adjusted during the first 15 minutes of baseline to obtain a perfusion pressure between 65 mmHg and 75 mmHg. Target parameters for baseline are as follows: perfusion pressure 65-75 mmHg; LVEDP 10 mmHg.

  Allow the heart to stabilize for 15 minutes. After this time, functional measurements are made, after which a pressure volume curve is generated by adjusting the balloon volume in 0.05 ml increments and recording the ventricular pressure. Left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVEDP), left ventricular deployed (developed) pressure (LVDP), first derivative of rise and fall of left ventricular pressure (dp / dt max, dp / dt Minimal), perfusion pressure and heart rate are automatically recorded using a computerized data acquisition system.

(Other measurements)
The heart, lungs and liver were weighed after removal. The lungs and liver are weighed and dried overnight to determine the wet to dry ratio.

  After completion of the Langendorff procedure, the heart is placed in cold saline to stop pulsation and then cut into five 2 mm thick transverse slices from the apex to the base of the heart. Slice # 3 is incubated for 20 minutes at 37 ° C. in 1% triphenyltetrazolium chloride (TTC) in 0.9% saline. Tetrazonium reacts with NADH in the presence of a dehydrogenase enzyme and stains viable tissue in a deep red color. This is easily distinguished from infarcted light unstained necrotic tissue. For the staining procedure, this slice is placed top-down in a small petri dish lid. Place the bottom of the dish on the slices and keep the slices flat. The slice is then photographed and the area of the infarcted tissue, left ventricle and right ventricle is determined using a computer image analysis system. Infarct size is expressed as a percentage of the total ventricle. Measure the total area of the left and right ventricles. The remaining portion is divided into right and left ventricles and frozen for TBARS and glutathione assays.

(Processing protocol)
No treatment is given to the sham operation group or the control group.

(Measurement for CHF research)
In vivo measurements include heart rate (HR), left ventricular systolic blood pressure (LVSP), left ventricular last diastolic blood pressure (LVEDP), dP / dt minimum and maximum values, right ventricular systolic pressure (RVSP), right Ventricular diastolic pressure (RVDP), right ventricular end diastolic pressure (RVEDP), and total weight. Ex vivo measurements consist of HR, LVSP, LVEDP, dP / dt minimum and maximum values, and pressure volume curves. Heart weight, infarct size, glutathione peroxidase (GPX), catalase, thiobarbital reactive substance (TBARS), glutathione ratio (GSH / GSSG), lung and liver wet to dry weight ratio, serum isoprostane and interleukin- 6 (IL-6) is also measured ex vivo.

  Certain compounds of the present invention can be tested by this method.

(Example 7)
(Skin protection assay)
Skin cytoprotective activity was determined by Mattek Corporation, Ashland, Mass. Can be evaluated in cell culture using the epithelial model obtained from (EPI-100). Neonatal foreskin cell cultures are cultured according to the manufacturer's instructions. It is then assayed for percent cell survival by measuring the amount of 3- (4,5-dimethyltazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) dye taken up by the cells. . Living cells take up this dye and convert it to insoluble formazine crystals. The crystals are present in the mitochondria of the cell until extracted with alcohol. The amount of MTT converted to extractable formazine crystals is directly proportional to the viability of the cell culture. MTT is measured spectrophotometrically.

Cells are obtained at a rate of 1.5 erythemal dose (MED) per hour per square centimeter for a total dose UV of about 31.5 mJ / cm ² (to obtain a wavelength in the region of 290-400 nm). The effect of the test compound protecting the cell culture from the generation of free radicals resulting from the UV light when exposed to UV light from a solar simulator) filtered in the presence of a cell protective compound or mixture thereof. Measure.

  The control for this study is a cell culture with no test compound added (positive control). All cell cultures are also exposed to UV light and cell viability is determined compared to cultures that do not contain cytoprotective agents or cell protective mixtures (negative control). This latter measurement is assumed to be equal to 100% viability.

  Cell cultures treated with the cytoprotective compounds of the present invention show more survival than positive control cell cultures when tested as described above.

(Example 8)
(Inhibition of hair growth)
The decrease in hair growth is due to a decrease in hair removal frequency, or when the subject perceives a lack of hair on the treatment site, or quantitatively, the weight of hair removed by shaving (i.e., the amount of hair). ) Is demonstrated when it decreases. Golden Syrian hamsters present one oval abdominal organ (each outer diameter about 8 mm) on each side, which produces dark black and rough hair similar to a human heel. It is considered to be an accepted model for human cocoon growth. These organs produce hair in hamsters in response to androgens.

  To assess the effectiveness of certain inhibitors in reducing hair growth, hair removal is performed on each flank organ of a group of hamsters by applying a thioglycolate-based chemical hair loss drug (Surgex). . 10 μL of vehicle alone is applied once a day to one organ of each animal, while an equal volume of vehicle containing the inhibitor is applied to the other organ of each animal. After 13 applications (one application per day for 5 days per week), the flank organs are shaved and the amount of body hair (hair volume) collected from each is weighed. The percent reduction in body hair growth is calculated by subtracting the test compound treated body hair mass (mg) value from the vehicle treated body hair mass. Thereafter, the obtained Δ value is divided by the body hair value on the vehicle treatment side, and the obtained number is multiplied by 100.

  The compounds of the invention can be tested in this assay.

Example 9
(In vitro cell inflammation assay)
A. Human Hep3B cell-CRP assay The Hep3B cell line is obtained from the American Type Culture Collection (ATCC catalog number HB-8064). The Hep3B cell line was derived from 8-year-old black male liver tissue. The cells are epithelial in shape and produce tumors in nude mice. The cells produce α-fetoprotein, hepatitis B surface antigen, albumin, α-2-macroglobulin, α-1-antitrypsin, transferrin, plasminogen, complement C3 and β-lipoprotein (Knowles BB). Et al., Science, 1980, 209: 497-499). This cell line has been used extensively to study hepatocyte cytokines and acute phase protein release (eg, Damtew B et al., 1993, J Immunol 150: 4001-4007).

  HEP3B cells supplemented with 10% fetal bovine serum (FBS; Hyclone), 1 × penicillin / streptomycin (GIBCO, catalog number 15140-122) and 0.1 mM non-essential amino acids (GIBCO, catalog number 11140-050), minimal Growing in essential medium (MEM; GIBCO). Cells are thawed and transferred to warm medium according to standard methods known in the art.

Cells are incubated in flasks at 37 ° C. with 5% CO ₂ in an atmospheric incubator. The HEP3B growth medium is changed every 2 days until the cells reach 70-80% confluence (approximately 3-4 days). For the assay, the cells are transferred to a 96-well plate, seeded at 5000 cells per well in culture medium, and grown for 7 days in a 37 ° C. incubator (air supplemented with 5% CO ₂ ). The medium is changed daily until the assay.

  Test compounds in “stimulation buffer” (MEM medium containing 0.1 mM non-essential amino acids, 1 × penicillin / streptomycin, 10% FBS and 10 ng / ml IL-1β, 20 ng / ml IL-6 and 1 μM dexamethasone) Dilute to The medium is returned from the cells and replaced with 200 μl of test diluent. Cells are returned to the incubator for 3 days at 37 ° C. A CRP ELISA is then performed on the cell supernatant as described below.

  Costar EIA / RIA plates are coated for 45 minutes at 37 ° C. with rabbit anti-human CRP (DAKO) diluted 1: 4000 in carbonate buffer (100 μl / well). The plates are then washed 5 times with CRP wash buffer (50 mM Tris-HCl, 0.3 mM NaCl, 0.5 ml Tween-20, pH 8.0) using an automated plate washer. Plates can be dried, covered and refrigerated until use. Supernatant (100 μl) is removed from each well of the test plate and added to the corresponding well of the pre-coated ELISA plate.

100 μl of HRP-conjugated rabbit anti-human CRP (DAKO) diluted 1: 500 (in CRP wash buffer) is added to each well followed by incubation at 37 ° C. for 30 minutes. The plate is washed 5 times with CRP wash buffer using an automatic plate washer. 200 μl of 3,3 ′, 5,5′-tetramethylbenzidine (TMB) liquid substrate system (Sigma, St. Louis, Mo.) is added to each well and then incubated in the dark for 15 minutes at room temperature. To do. Finally, 50 μl of 1 MH ₂ SO ₄ is added to each well and the absorbance at 450 nm is immediately measured in a microtiter spectrophotometer.

  CRP measured as above is normalized to the number of cells per well using a cell viability assay (eg, Cell Tracker Green assay). To do this, the rest of the medium is obtained from the cell test plate and the cells are washed with 200 μl of pre-warmed 1 × Hanks basic salt solution (HBSS; GIBCO) and 100 μl of 5 μM Cell Tracker Green ( (Molecular Probes, Eugene, OR) is added to each well. The plate is then incubated for 30 minutes at 37 ° C. Then, it is washed twice with preheated 1 × HBSS. The plate is read immediately using a Fluoroskan® fluorometer equipped with a 485 excitation filter / 538 emission filter pair.

In CRP assays such as those described herein, compounds such as the following are effective in reducing CRP levels with an EC ₅₀ between about 10 μM and about 40 μM:
6,7-dimethyl-3-phenyl-benzofuran-5-ol;
1- (4-bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone;
1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -2-morpholin-4-yl-ethanone;
Acetic acid 2- (6-hydroxy-3-methyl- [1,3] oxazinan-6-yl) -6,7-dimethyl-benzofuran-5-yl ester;
1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone;
3- (4-methoxy-phenyl) -6,7-dimethyl-benzofuran-5-ol;
4,5-dimethyl-1,8-diphenyl-benzo [1,2-b; 4,3-b ′] difuran; and 1,8-bis- (4-fluoro-phenyl) -4,5-dimethyl- Benzo [1,2-b; 4,3-b ′] difuran.

(B. Cell-ELAM)
Endothelial leukocyte adhesion molecule (ELAM) (also known as E-selectin) is expressed on the surface of endothelial cells. In this assay, lipopolysaccharide (LPS) and IL-1β are used to stimulate ELAM expression; according to studies showing that decreased leukocyte adhesion to the endothelial cell surface is associated with decreased cytotoxicity. Test reagents are tested for the ability to reduce expression (eg, Takada, M. et al., Transplantation 64: 1520-25, 1997; Steinberg, JB. Et al., J. Heart Lung Trans. 13: 306-313, 1994).

  Endothelial cells can be selected from any of a number of sources and can be cultured according to methods known in the art; these cells include coronary artery endothelial cells, human brain microvascular endothelial cells (HBMEC; Hess, D. et al. C. et al., Neurosci. Lett. 213 (1): 37-40, 1996), or lung endothelial cells. Cells are cultured conveniently in 96 well plates. In the presence of the test reagent, a solution containing 10 μg / ml LPS and 100 pg / ml IL-1β is added to each well for 6 hours to stimulate the cells (the specific concentration and time depends on the cell type). Can be adjusted). Remove processing buffer and replace with pre-warmed Fixing Solution (100 μL / well) for 25 minutes at room temperature. Cells are then washed 3 times and then incubated with blocking buffer (PBS + 2% FBS) for 25 minutes at room temperature. Blocking buffer containing monoclonal E-selectin antibody (1: 750, Sigma Catalog # S-9555) is added to each well. Seal the plate and store at 4 ° C. overnight. Wash the plate 4 times with 160 μL blocking buffer per well. The secondary antibody-HRP diluted 1: 5000 in blocking buffer is then added (100 μL / well) and the plate is incubated for 2 hours at room temperature (protected from light). The plates are then washed 4 times with blocking buffer at room temperature before adding 100 μL of ABTS substrate solution (Zymed, Catalog # 00-2024). The wells are allowed to develop for 35 minutes and then measured at 402 nm in a Fluoroskan® reader with a 10 second shaking program. A positive result is recorded as a decrease in the ELAM concentration in the test well compared to the control well.

In ELAM assays such as those described herein, certain compounds of the invention with EC ₅₀ in the range of 10-1000 μM, particularly in the range of 200-600 μM, could reduce ELAM expression.

(C. Interleukin-1β microglial cell assay)
(Materials and equipment)
A. Materials for cell preparation and experiment-Mouse microglia cell line-DMEM high glucose medium (Gibco Catalog # 11965-092)
-FBS (Hyclone Catalog # SH30070.03)
−100 × penicillin / streptomycin (Gibco Catalog # 15140-122)
-LPS (Sigma Catalog # L2537)
Interferon-γ (Sigma Catalog # 14777)
-Cell Tracker Green (Molecular Probes Catalog # C2925)
-HBSS buffer (950 ml pyrogen-free water, 2.44 g / L MgCl2.6H2O, 3.73 g / L KCl, 59.58 g / L Hepes, 58.44 g / L NaCl, 1.36 g / L KH2PO4, 1.91 g / L CaCl2.2H2O and HCl to pH 4.5)
-Sterile 96-well plate pre-coated with poly-D-lysine (Corning Catalog # 3665)
-96-well deep well mother plate, DyNA Block 1000 (VWR Catalog # 40002-008).

B. Material for Il-1β Elisa-Mouse IL-1β Duo Set (R & D Systems Catalog # DY401)
-Substrate solution (R & D Systems Catalog #DY 999)
Bovine serum albumin fraction V (BSA V) (Sigma Catalog # A4503)
-96-well Costar EIA high binding plate (VWR Catalog # 29442-302)
Plate seal (VWR Catalog # 29442-310)
-PBS (Irvine Scientific Catalog # 9240)
-Cell culture grade water (Irvine Scientific Catalog # 9312)
-Tween 20 (Sigma Catalog #P 1379)
-Sucrose (Sigma Catalog # S7903)
-Sodium azide (Sigma Catalog #S 8032)
_{-H 2 SO 4 5N (VWR Catalog} # JT 5691-2).

(Experimental preparation and procedure)
(Mouse IL-1β Elisa)
solution:
Wash buffer: PBS 1 L + 500 μl Tween 20 (final 0.05%) pH 7.2-7.4.
Blocking buffer: 500 ml PBS + 5 g BSA V (1%) + 25 g sucrose (5%) + 0.25 g sodium azide (0.05%).
Reagent dilution: 500 ml PBS + 5 g BSA V (1%) pH 7.2-7.4, and filter is sterilized through 0.2 μm.
Stop solution: Make 2N sulfuric acid by adding 10 ml of 5NH2SO4 to 15 ml of ddH2O.

Duo Set preparation:
1. The IL-1β capture antibody was reconstituted with 1 ml PBS to give a final concentration of 720 μg / ml and the working concentration was 4 μg / ml. To coat one 96 well plate (at 100 μl / well), 56 μl of 720 μg / ml stock was diluted in 10 ml PBS.
2. The IL-1β standard was reconstituted with 0.5 ml reagent diluent (70 ng / ml). For high standards of 1 ng / ml (100 μl each plus 2 wells sufficient for serine dilution), 7.1 μl of 70 ng / ml standard was diluted in 0.5 ml reagent dilution.
3. The IL-1β detection antibody is reconstituted with 1 ml reagent diluent to give a final concentration of 18 μg / ml and the working concentration is 100 ng / ml. For one 96-well plate (at 100 μl / well), 56 μl of 18 μg / ml stock was diluted into 10 ml of reagent diluent.

(IL-1β ELISA procedure)
Plate preparation:
1. Costar EIA Hi binding plates were coated with capture antibody at 4 μg / ml. 56 μl of 720 μg / ml stock was taken in one plate and added to 10 ml PBS. Each well was coated with 100 μl and the plate was sealed and incubated overnight at room temperature.
2. Each well was aspirated and washed 3 times with wash buffer. Each well was filled to the top, dispensed, and any remaining buffer was removed by inverting the plate and removed by gently blotting on a clean paper towel.
3. Non-specific binding sites were blocked by adding 300 μl of blocking buffer to each well and incubated at room temperature for at least 1 hour after sealing.
4). After washing, the plate was now prepared for the sample.

Assay procedure:
5. Either 100 μl of standard or sample was added to each well of the capture coated and preblocked plate. Plates were sealed and incubated for 2 hours at room temperature followed by washing as in step 2.
6. 100 μl of detection antibody (100 ng / ml) was added to each well. For one 96-well plate, 56 μl of 18 μg / ml stock was diluted in 10 ml reagent diluent.
7). Plates were sealed and incubated for 2 hours at room temperature followed by washing as in step 2.
8. 100 μl streptavidin-HRP working dilution was added and the plate was sealed and incubated at room temperature in the dark for 20 minutes followed by washing as in step 2.
9. A new substrate solution was prepared by mixing color reagent A (H ₂ O ₂ ) and color reagent B (tetramethylbenzidine) in a 1: 1 ratio. 100 μl of this substrate solution mixture was added to each well and the plate was incubated for 20 minutes in the dark at room temperature.
10.50 μl of stop solution was added to each well and mixing was ensured by tapping gently.
11. Each plate was read once at 450 nm with Spectramax. If wavelength correction is possible, set to 540 nm or 570 nm.

  The compounds of the invention can be tested for the ability to reduce inflammation in this model.

Example 10 In Vivo Cell Inflammation Assay
These assays measure the ability of test compounds to prevent or reduce inflammation following oxazolone and arachidonic acid.

A. Arachidonic acid. Albino male CD-1 mice (7-9 weeks old) were used for this study. Prepare a 20% (w / v) arachidonic acid solution in acetone. 20 μl of arachidonic acid solution is applied to the back left ear of the mouse. Immediately thereafter, the test compound (20 μl in 70% ethanol / 30% propylene glycol) is applied to the left ear. The untreated right ear is the control. One hour after treatment, mice are sacrificed with CO ₂ inhalation. The left and right ears are removed and a 7 mm punch biopsy is taken from each. Weigh the punch biopsy and calculate the difference.

  B. Oxazolone. CD-1 mice are induced by applying 3% oxazolone (Sigma) (corn oil: prepared to 30 mg / ml in acetone) to the shaved abdomen. After 5 days, mice are challenged with 2% oxazolone in acetone (20 mg / ml) in the left ear (the right ear was an untreated control). One hour after challenge, test compounds in 70% ethanol / 30% propylene glycol are applied to the left ear. After 24 hours, animals are sacrificed and 7 mm ear punches are removed. An ear punch is placed on the balance scale and the difference between the untreated ear and the treated ear is determined. The% inhibition relative to the vehicle group is calculated by comparing the means of each group. (In this test, hydrocortisone is used as a positive control).

  The compounds of the invention can be tested for the ability to reduce inflammation in this model.

  Example 11: 6,7-Dimethyl-3-phenyl-benzofuran-5-ol

（工程１：）
アセトン（３０ｍＬ）中、２，３−ジメチル−１，４−ジヒドロキノン（１．０８ｇ、７．８２６ｍｍｏｌ）、２−ブロモアセトフェノン（１．４５ｇ、７．２９ｍｍｏｌ）および炭酸カリウム（１．７８ｇ、１２．９０ｍｍｏｌ）の混合物を、室温で３時間攪拌した。次いで、この混合物を水に注ぎ、沈殿の形成を生じた。この沈殿を水およびヘキサンで洗浄し、乾燥して、モノ生成物およびビス生成物を得た。シリカゲルカラム（ヘキサン中３０％ ＥｔＯＡｃで溶離する）で精製し、２−（４−ヒドロキシ−２，３−ジメチル−フェノキシ）−１−フェニル−エタノン（０．６５ｇ）および２−［２，３−ジメチル−４−（２−オキソ−２−フェニル−エトキシ）−フェノキシ］−１−フェニル−エタノン（０．５ｇ）を得た。

(Step 1 :)
2,3-Dimethyl-1,4-dihydroquinone (1.08 g, 7.826 mmol), 2-bromoacetophenone (1.45 g, 7.29 mmol) and potassium carbonate (1.78 g, 12) in acetone (30 mL). .90 mmol) was stirred at room temperature for 3 hours. The mixture was then poured into water resulting in the formation of a precipitate. The precipitate was washed with water and hexane and dried to give mono and bis products. Purify on a silica gel column (eluting with 30% EtOAc in hexanes) to give 2- (4-hydroxy-2,3-dimethyl-phenoxy) -1-phenyl-ethanone (0.65 g) and 2- [2,3- Dimethyl-4- (2-oxo-2-phenyl-ethoxy) -phenoxy] -1-phenyl-ethanone (0.5 g) was obtained.

(Process 2 :)
A mixture of 2- (4-hydroxy-2,3-dimethyl-phenoxy) -1-phenyl-ethanone (1.2 g, 4.70 mmol) and polyphosphoric acid (about 100 mg) in xylene (10 ml) Stir for hours. The mixture was poured into water, extracted with ethyl acetate, washed and dried to give 6,7-dimethyl-3-phenyl-benzofuran-5-ol (560 mg) as a light brown solid.

ブロモアセトフェノンを適切な置換ブロモアセトフェノンと置き換える以外は、上記の手順に従って、同様に、以下の化合物を得た：
・３−（４−フルオロ−フェニル）−６，７−ジメチル−ベンゾフラン−５−オール；

According to the above procedure, except that bromoacetophenone was replaced with the appropriate substituted bromoacetophenone, the following compound was obtained as well:
3- (4-Fluoro-phenyl) -6,7-dimethyl-benzofuran-5-ol;

・３−（４−メトキシ−フェニル）−６，７−ジメチル−ベンゾフラン−５−オール

3- (4-Methoxy-phenyl) -6,7-dimethyl-benzofuran-5-ol

・３−（４−クロロ−フェニル）−６，７−ジメチル−ベンゾフラン−５−オール；）

3- (4-Chloro-phenyl) -6,7-dimethyl-benzofuran-5-ol;)

（実施例１２）
（４，５−ジメチル−１，８−ジフェニル−ベンゾ［１，２−ｂ；４，３−ｂ’］ジフラン）

(Example 12)
(4,5-Dimethyl-1,8-diphenyl-benzo [1,2-b; 4,3-b ′] difuran)

キシレン（１０ｍＬ）中の、実施例１１に記載されるようにして合成した２−［２，３−ジメチル−４−（２−オキソ−２−フェニル−エトキシ）−フェノキシ］−１−フェニル−エタノン（４８０ｍｇ、１．２８ｍｍｏｌ）、過剰のブロモアセトフェノン、およびポリリン酸（ＰＰＡ、１００ｍｇ）の混合物を、１５０℃で１０時間撹拌した。次いで、この混合物を、水に注ぎ入れ、ＥｔＯＡｃで抽出し、有機層を水およびブラインで洗浄し、ＭｇＳＯ_４で乾燥し、そして濃縮した。残渣を、ヘキサン中１０〜２０％のＥｔＯＡｃで溶出するシリカゲルカラムにより精製して、１９０ｍｇの４，５−ジメチル−１，８−ジフェニルベンゾ［１，２−ｂ；４，３−ｂ’］ジフラン（２５０ｍｇ）を得た。

2- [2,3-Dimethyl-4- (2-oxo-2-phenyl-ethoxy) -phenoxy] -1-phenyl-ethanone synthesized as described in Example 11 in xylene (10 mL) A mixture of (480 mg, 1.28 mmol), excess bromoacetophenone, and polyphosphoric acid (PPA, 100 mg) was stirred at 150 ° C. for 10 hours. The mixture was then poured into water and extracted with EtOAc, the organic layer was washed with water and brine, dried over MgSO ₄ and concentrated. The residue was purified by silica gel column eluting with 10-20% EtOAc in hexane to give 190 mg of 4,5-dimethyl-1,8-diphenylbenzo [1,2-b; 4,3-b ′] difuran. (250 mg) was obtained.

同様に、実施例１２の手順に従うことによって、以下の化合物を得た：
・１，８−ビス−（４−フルオロ−フェニル）−４，５−ジメチル−ベンゾ［１，２−ｂ；４，３−ｂ’］ジフラン；

Similarly, following the procedure of Example 12 yielded the following compound:
1,8-bis- (4-fluoro-phenyl) -4,5-dimethyl-benzo [1,2-b; 4,3-b ′] difuran;

・１，８−ビス−（４−メトキシ−フェニル）−４，５−ジメチル−ベンゾ［１，２−ｂ；４，３−ｂ’］ジフラン：

1,8-bis- (4-methoxy-phenyl) -4,5-dimethyl-benzo [1,2-b; 4,3-b ′] difuran:

（実施例１３）
（（５−ヒドロキシ−３，６，７−トリメチル−ベンゾフラン−２−イル）フェニル−メタノン）

(Example 13)
((5-Hydroxy-3,6,7-trimethyl-benzofuran-2-yl) phenyl-methanone)

（工程１）
ジクロロメタン（１００ｍＬ）中の２，３−ジメチル−１，４−ジヒドロキノン（１１．５８ｇ）およびトリエチルアミン（２５ｍＬ）の懸濁液に、無水酢酸（１６ｍＬ）をゆっくりと添加した。次いで、この混合物を、固体が溶解するまで、室温で２時間撹拌した。この混合物を水で洗浄し、ＭｇＳＯ_４で乾燥し、そして乾固するまで濃縮して、固体を得た。この固体を、ヘキサンおよびエーテルで洗浄して、出発物質の１，４−ジアセテート誘導体（１５．４０ｇ）を得た。

(Process 1)
To a suspension of 2,3-dimethyl-1,4-dihydroquinone (11.58 g) and triethylamine (25 mL) in dichloromethane (100 mL) was slowly added acetic anhydride (16 mL). The mixture was then stirred at room temperature for 2 hours until the solid dissolved. The mixture was washed with water, dried over MgSO ₄ and concentrated to dryness to give a solid. This solid was washed with hexane and ether to give the starting 1,4-diacetate derivative (15.40 g).

(Process 2)
A suspension of Step 1 diacetate (5.08 g) in boron trifluoride acetic acid complex (15 mL) was stirred at 110 ° C. for 1 hour. After cooling, the mixture was poured onto ice and the mixture was extracted with dichloromethane. The organic layer was washed with water and dried. After evaporation, the residue was recrystallized from EtOAc-hexanes to give 4.37 g of 1- (2-hydroxy-5-acetoxy-3,4-dimethyl-phenyl) -ethanone.

(Process 3)
Step 2 phenylethanone (500 mg) was dissolved in MeOH (20 mL) and potassium carbonate (1 eq) was added followed by water (1 mL). The mixture was stirred at room temperature for 1 hour and then poured into water. The solution was acidified with HCl and a precipitate formed. The precipitate was collected and air dried to give about 350 mg of 1- (2,5-dihydroxy-3,4-dimethyl-phenyl) -ethanone.

(Process 4)
Step 3 1- (2,5-dihydroxy-3,4-dimethyl-phenyl) -ethanone (275 mg) was dissolved in dichloromethane (20 mL) and 3,4-dihydro-2H-pyran (0.2 mL). Followed by pyridinium p-toluenesulfonate (PPTS) (30 mg). The mixture was stirred at room temperature for 4 hours. The mixture was dried over MgSO ₄ and purified on a silica gel column eluted with 30% EtOAc in hexane to give 397 mg of 1- [2-hydroxy-3,4-dimethyl-5- (tetrahydro-pyran-2- (Iloxy) -phenyl] -ethanone was obtained as a yellow solid.

(Process 5)
A mixture of Step 4 tetrahydrofuran (250 mg), 2-bromo-1-phenyl-ethanone (250 mg), and potassium carbonate (300 mg) in DMF was stirred at room temperature for 3 hours. The mixture was poured into water and extracted with ethyl acetate, the organic layer was dried and evaporated. Purification on a silica gel column eluting with 30% EtOAc in hexanes afforded 260 mg of a colorless solid which was then dissolved in DMF followed by the addition of cesium carbonate (2 eq). The mixture was stirred overnight at room temperature, then poured into water and extracted with ethyl acetate, the organic layer was dried and evaporated. The crude product was dissolved in MeOH containing dilute HCl and stirred for 1 hour. It was then poured into water and extracted with ethyl acetate, the organic layer was washed, dried and evaporated. Purification on a silica gel column eluting with 30% EtOAc in hexanes afforded (5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl) -phenyl-methanone (187 mg).

（実施例１４）
（１−（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノン）

(Example 14)
(1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone)

ＭｅＯＨ（２０ｍＬ）中の、１−［６，７−ジメチル−５−（テトラヒドロ−ピラン−２−イルオキシ）−ベンゾフラン−２−イル］−エタノン（１．１ｇ）（実施例１３の工程３、４および５の手順に従って、２，５−ジヒドロキシ−３，４−ジメチル−ベンズアルデヒドから調製した）、および濃ＨＣｌ（１０滴）の混合物を、室温で２時間撹拌した。この混合物に水を注ぎ入れ、そして冷蔵庫中で一晩沈殿させた。この沈殿物を回収し、ヘキサンで洗浄し、そして乾燥して、１−（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノンを薄茶色の個体（４９０ｍｇ）として得た。

1- [6,7-Dimethyl-5- (tetrahydro-pyran-2-yloxy) -benzofuran-2-yl] -ethanone (1.1 g) (1.1 g) in MeOH (20 mL) (Steps 3 and 4 of Example 13). A mixture of 2,5-dihydroxy-3,4-dimethyl-benzaldehyde) and concentrated HCl (10 drops) was stirred at room temperature for 2 hours. Water was poured into the mixture and allowed to settle overnight in the refrigerator. The precipitate was collected, washed with hexane and dried to give 1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone as a light brown solid (490 mg).

（実施例１５）
（１−（３−ブロモ−５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノン）

(Example 15)
(1- (3-Bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone)

クロロホルム（２０ｍＬ）中の１−［６，７−ジメチル−５−（テトラヒドロ−ピラン−２−イルオキシ）−ベンゾフラン−２−イル］−エタノン（２００ｍｇ）の溶液に、臭素（１２０ｍｇ）を添加した。１時間後、この溶液を水で洗浄し、ＭｇＳＯ_４で乾燥し、そして濃縮した。残渣を、ＤＣＭ中１０％のヘキサンで溶出するシリカゲルカラムにより精製して、１−（３−ブロモ−５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノンを黄色の固体として得た。

To a solution of 1- [6,7-dimethyl-5- (tetrahydro-pyran-2-yloxy) -benzofuran-2-yl] -ethanone (200 mg) in chloroform (20 mL) was added bromine (120 mg). After 1 hour, the solution was washed with water, dried over MgSO ₄ and concentrated. The residue is purified by silica gel column eluting with 10% hexane in DCM to give 1- (3-bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone as a yellow solid. It was.

（実施例１６）
（２−ブロモ−１−（５−アセトキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノン）

(Example 16)
(2-Bromo-1- (5-acetoxy-6,7-dimethyl-benzofuran-2-yl) -ethanone)

１−（５−アセトキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノン（８３ｍｇ）（アセトアルデヒドおよびピリジンを用いて、実施例１４の１−（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノンから調製した）を、クロロホルム（１０ｍＬ）に溶解した。臭素（５０ｍｇ）を添加し、そしてこの溶液を７０℃で１０分間撹拌した。臭素の色が消えると、この混合物を乾固するまでエバポレートした。残渣を、ヘキサン中３０％のＥｔＯＡｃで溶出するシリカゲルカラムにより精製して、８５ｍｇの２−ブロモ−１−（５−アセトキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノンを白色固体として得た。

1- (5-acetoxy-6,7-dimethyl-benzofuran-2-yl) -ethanone (83 mg) (1- (5-hydroxy-6,7-dimethyl-benzofuran of Example 14 using acetaldehyde and pyridine) -2-yl) -ethanone) was dissolved in chloroform (10 mL). Bromine (50 mg) was added and the solution was stirred at 70 ° C. for 10 minutes. When the bromine color disappeared, the mixture was evaporated to dryness. The residue was purified by silica gel column eluting with 30% EtOAc in hexane to give 85 mg of 2-bromo-1- (5-acetoxy-6,7-dimethyl-benzofuran-2-yl) -ethanone as a white solid. Obtained.

（実施例１７）
（酢酸６，７−ジメチル−２−（２−モルホリン−４−イル−アセチル）−ベンゾフラン−５−イルエステル）

(Example 17)
(Acetic acid 6,7-dimethyl-2- (2-morpholin-4-yl-acetyl) -benzofuran-5-yl ester)

アセトン（１０ｍＬ）中の、実施例１６の２−ブロモ−１−（５−アセトキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノン（４７ｍｇ）、モルホリン（１５ｍｇ）、および炭酸カリウム２５ｍｇ）の溶液を、室温で３０分間撹拌した。次いで、これを乾固するまでエバポレートし、そして残渣を、ＤＣＭ中５％のＭｅＯＨで溶出するシリカゲルカラムにより精製して、４３ｍｇの酢酸６，７−ジメチル−２−（２−モルホリン−４−イル−アセチル）−ベンゾフラン−５−イルエステルを油状物として得た。塩酸塩に変換して、黄色の固体を得た。

2-bromo-1- (5-acetoxy-6,7-dimethyl-benzofuran-2-yl) -ethanone of Example 16 (47 mg), morpholine (15 mg, and potassium carbonate 25 mg) in acetone (10 mL) Was stirred at room temperature for 30 minutes. It was then evaporated to dryness and the residue was purified on a silica gel column eluting with 5% MeOH in DCM to yield 43 mg 6,7-dimethyl-2- (2-morpholin-4-yl acetate). -Acetyl) -benzofuran-5-yl ester was obtained as an oil. Conversion to the hydrochloride gave a yellow solid.

（実施例１８）
（１−（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−２−モルホリン−４−イル−エタノン）

(Example 18)
(1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -2-morpholin-4-yl-ethanone)

ＭｅＯＨ（１０ｍＬ）中の、酢酸６，７−ジメチル−２−（２−モルホリン−４−イル−アセチル）−ベンゾフラン−５−イルエステル（２３ｍｇ）（実施例１７のようにして調製した）の溶液を、炭酸水素ナトリウム）１０ｍｇ）を添加しながら撹拌した。次いで、この混合物を室温で一晩撹拌し、次いで乾固するまでエバポレートした。残渣を、ジクロロメタン中５％のＭｅＯＨで溶出するシリカゲルカラムにより精製して、１１ｍｇの１−（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−２−モルホリン−４−イル−エタノンを油状物として得た。ＨＣｌ塩に変換して、黄色の固体を得た。

A solution of acetic acid 6,7-dimethyl-2- (2-morpholin-4-yl-acetyl) -benzofuran-5-yl ester (23 mg) (prepared as in Example 17) in MeOH (10 mL). Was stirred with addition of 10 mg). The mixture was then stirred at room temperature overnight and then evaporated to dryness. The residue was purified by silica gel column eluting with 5% MeOH in dichloromethane to give 11 mg of 1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -2-morpholin-4-yl-ethanone. Was obtained as an oil. Conversion to the HCl salt gave a yellow solid.

（実施例１９）
（２−（１−ヒドロキシ−２−モルホリン−４−イル−エチル）−６，７−ジメチル−ベンゾフラン−５−オール）

(Example 19)
(2- (1-hydroxy-2-morpholin-4-yl-ethyl) -6,7-dimethyl-benzofuran-5-ol)

酢酸６，７−ジメチル−２−（２−モルホリン−４−イル−アセチル）−ベンゾフラン−５−イルエステル（３５ｍｇ）（実施例１７において調製した）の、ＭｅＯＨ（１０ｍＬ）中の溶液を、ＮａＢＨ４（４０ｍｇ）を添加しながら撹拌し、そしてこの混合物を、室温でさらに４時間攪拌した。この混合物を水に注ぎ、そして酢酸エチルで抽出した。有機層をＭｇＳＯ_４で乾燥し、そしてエバポレートした。その残渣を、ＤＣＭ中５％のＭｅＯＨで溶出するシリカゲルカラムによって精製し、２１ｍｇの２−（１−ヒドロキシ−２−モルホリン−４−イル−エチル）−６，７−ジメチル−ベンゾフラン−５−オールを油状物として得た。ＨＣｌ塩への変換により、黄色固体を得た。

A solution of acetic acid 6,7-dimethyl-2- (2-morpholin-4-yl-acetyl) -benzofuran-5-yl ester (35 mg) (prepared in Example 17) in MeOH (10 mL) was added NaBH4. (40 mg) was added with stirring, and the mixture was stirred at room temperature for an additional 4 hours. The mixture was poured into water and extracted with ethyl acetate. The organic layer was dried over MgSO ₄ and evaporated. The residue was purified by silica gel column eluting with 5% MeOH in DCM to yield 21 mg 2- (1-hydroxy-2-morpholin-4-yl-ethyl) -6,7-dimethyl-benzofuran-5-ol. Was obtained as an oil. Conversion to the HCl salt gave a yellow solid.

（実施例２０）
（２−（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−４−メチル−モルホリン−２−オール）

(Example 20)
(2- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -4-methyl-morpholin-2-ol)

２−ブロモ−１−（５−アセトキシ−６，７−ジメチル−ベンゾフラン−２−イル）−エタノン（３０ｍｇ）、メチルアミノエタノール（７ｍｇ）、および炭酸カリウム（２０ｍｇ）の、アセトン（１０ｍＬ）中の混合物を、室温で１時間攪拌した。この混合物を水に注ぎ、そして酢酸エチルで抽出した。有機層を乾燥し、そしてエバポレートし、次いで、ＤＣＭ中５％のＭｅＯＨで溶出するシリカゲルカラムクロマトグラフィーによって精製して、２０ｍｇの２−（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−４−メチル−モルホリン−２−オールを油状物として得、そしてＨＣｌ塩に変換した。ＮＭＲは、ヘミケタール形態とケトン形態との平衡を示した。

2-Bromo-1- (5-acetoxy-6,7-dimethyl-benzofuran-2-yl) -ethanone (30 mg), methylaminoethanol (7 mg), and potassium carbonate (20 mg) in acetone (10 mL). The mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic layer was dried and evaporated, then purified by silica gel column chromatography eluting with 5% MeOH in DCM to give 20 mg of 2- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl. ) -4-Methyl-morpholin-2-ol was obtained as an oil and converted to the HCl salt. NMR showed an equilibrium between the hemiketal form and the ketone form.

（実施例２１）
（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−カルボン酸メチルエステル）

(Example 21)
(5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester)

（工程１：）
２，５−ジヒドロキシ−３，４−ジメチル−ベンズアルデヒド（２．０ｇ）を、ジクロロメタン（５０ｍＬ）に溶解し、そして３，４−ジヒドロ−２Ｈ−ピラン（１．５ｇ）を添加し、次いで、ｐ−トルエンスルホン酸一水和物（２００ｇ）を添加した。この溶液を室温で１時間撹拌し、そして重炭酸ナトリウム溶液（１ｍＬ）を添加することによって、クエンチした。次いで、ジクロロメタン溶液をＭｇＳＯ_４で乾燥し、そして濃縮した。その残渣を、ヘキサンおよび酢酸エチル（８：２）で溶出するシリカゲルカラムによって精製して、１．５ｇの２−ヒドロキシ−３，４―ジメチル−５−（テトラヒドロ−ピラン−２−イルオキシ）ベンズアルデヒド生成物を得た。

(Step 1 :)
2,5-dihydroxy-3,4-dimethyl-benzaldehyde (2.0 g) is dissolved in dichloromethane (50 mL) and 3,4-dihydro-2H-pyran (1.5 g) is added, then p. -Toluenesulfonic acid monohydrate (200 g) was added. The solution was stirred at room temperature for 1 hour and quenched by adding sodium bicarbonate solution (1 mL). The dichloromethane solution was then dried over MgSO ₄ and concentrated. The residue was purified by silica gel column eluting with hexane and ethyl acetate (8: 2) to produce 1.5 g of 2-hydroxy-3,4-dimethyl-5- (tetrahydro-pyran-2-yloxy) benzaldehyde. I got a thing.

(Process 2)
2-hydroxy-3,4-dimethyl-5- (tetrahydro-pyran-2-yloxy) benzaldehyde from step 1 (150 mg), methyl bromoacetate (180 mg) and potassium carbonate (200 mg) in DMF (10 mL). The mixture was stirred at room temperature for 2 hours. The solution was then poured into water and extracted with ethyl acetate. The ethyl acetate was washed with water and brine, dried over MgSO ₄ and concentrated. The residue was purified by silica gel column eluting with hexane and ethyl acetate (9: 1) to give 100 mg [6-formyl-2,3-dimethyl-4- (tetrahydro-pyran-2-yloxy) -phenoxy]. Acetic acid methyl ester was obtained, which was then dissolved in DMF (15 mL). To this solution was added cesium carbonate (200 mg) and the mixture was stirred at room temperature overnight. The solution was then poured into water and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over MgSO ₄ and concentrated to give 6,7-dimethyl-5- (tetrahydro-pyran-2-yloxy) -benzofuran-2-carboxylic acid methyl ester. . The tetrahydropyranyl ether was dissolved in methanol, p-toluenesulfonic acid monohydrate (20 mg) was added, and the solution was stirred at room temperature for an additional 30 minutes. After the addition of a small amount of NaHCO ₃ , methanol was evaporated. The residue was purified by silica gel column chromatography eluting with hexane and ethyl acetate (7: 3) to give 30 mg of 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester. ¹ H NMR (CDCl ₃ , 300 MHz) δ: 7.39 (s, 1H), 6.87 (s, 1H), 4.75 (s, 1H), 3.95 (s, 3H), 2.50 2.26 (2s, 6H) ppm.

(Example 22)
(5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 2- [2- (2-methoxy-ethoxy) ethoxy] -ethyl ester)

酸（５０ｍｇ）、２−［２−（２−メトキシ−エトキシ）エトキシ］−エタノール（１００μＬ）、ＤＣＣ（１２０ｍｇ）、ＤＭＡＰ（５０ｍｇ）の、ＤＣＭ（２０ｍＬ）中の混合物を、室温で一晩撹拌した。この混合物を水に注ぎ、そして酢酸エチルで抽出した。有機層を水およびブラインで洗浄し、ＭｇＳＯ_４で乾燥し、そして濃縮した。その残渣を、ヘキサン中４０％のＥｔＯＡｃで溶出するシリカゲルカラムクロマトグラフィーによって精製して、５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−カルボン酸２−［２−（２−メトキシ−エトキシ）エトキシ］−エチルエステルのテトラヒドロピラニルエーテルを得た（４０ｍｇ）。この中間体をＭｅＯＨに溶解し、希ＨＣｌを添加し、そしてこの混合物を３０分間撹拌した。この混合物を水に注ぎ、酢酸エチルで抽出した。その有機層を水およびブラインで洗浄し、ＭｇＳＯ４で乾燥し、そして濃縮した。ＤＣＭ中２％のＭｅＯＨで溶出するシリカゲルカラムによって精製して、３０ｍｇの５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−カルボン酸２−［２−（２−メトキシ−エトキシ）エトキシ］−エチルエステルを得た。

A mixture of acid (50 mg), 2- [2- (2-methoxy-ethoxy) ethoxy] -ethanol (100 μL), DCC (120 mg), DMAP (50 mg) in DCM (20 mL) was stirred at room temperature overnight. did. The mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over MgSO ₄ and concentrated. The residue was purified by silica gel column chromatography eluting with 40% EtOAc in hexanes to give 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 2- [2- (2-methoxy-ethoxy). The tetrahydropyranyl ether of ethoxy] -ethyl ester was obtained (40 mg). This intermediate was dissolved in MeOH, dilute HCl was added, and the mixture was stirred for 30 minutes. The mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over MgSO 4 and concentrated. Purify by silica gel column eluting with 2% MeOH in DCM to give 30 mg of 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 2- [2- (2-methoxy-ethoxy) ethoxy] -ethyl. An ester was obtained.

同様に、上記手順に従って、２−［２−（２−メトキシ−エトキシ）エトキシ］−エタノールを適切なアルコールに置き換えて、以下の化合物を調製した：
ゲラニオールは、５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−カルボン酸３，７−ジメチル−オクタ−２，６−ジエニルエステルを与えた；

Similarly, following the procedure above, substituting 2- [2- (2-methoxy-ethoxy) ethoxy] -ethanol with the appropriate alcohol, the following compound was prepared:
Geraniol gave 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 3,7-dimethyl-octa-2,6-dienyl ester;

２−アミノエタノールは、５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−カルボン酸（２−ヒドロキシ−エチル）アミドを与えた；

2-aminoethanol gave 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid (2-hydroxy-ethyl) amide;

２−（２−ヒドロキシ−エチルアミノ）−エタノールは、５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−カルボン酸ビス−（２−ヒドロキシ−エチル）−アミドを与えた；

2- (2-hydroxy-ethylamino) -ethanol gave 5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid bis- (2-hydroxy-ethyl) -amide;

モルホリンは、（５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−イル）−モルホリン−４−イル−メタノンを与えた；^１Ｈ ＮＭＲ（ＤＭＳＯ−ｄ_６−Ｄ_２Ｏ、３００ＭＨｚ）δ：７．２２（ｓ、１Ｈ）、５．８８（１Ｈ）、３．６５（ｍ、８Ｈ）、２．３４（ｓ、３Ｈ）、２．１５（ｓ、３Ｈ）ｐｐｍ。ＭＳ ２７６（Ｍ＋Ｈ^＋）。

Morpholine gave (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -morpholin-4-yl-methanone; ¹ H NMR (DMSO-d ₆ -D ₂ O, 300 MHz) δ: 7 .22 (s, 1H), 5.88 (1H), 3.65 (m, 8H), 2.34 (s, 3H), 2.15 (s, 3H) ppm. MS 276 (M + H ^< + ^> ).

(Example 23)
(2-hydroxymethyl-6,7-dimethyl-benzofuran-5-ol)

実施例２に記載されるように調製された５−ヒドロキシ−６，７-ジメチル−ベンゾフラン−２−カルボン酸メチルエステル（２３１ｍｇ）を、ＴＨＦに溶解し、水素化リチウムアルミニウム（９３ｍｇ）を添加した。この混合物を、室温で４時間攪拌した。次いで、溶液を、水に注ぎ、酢酸エチルで抽出した。酢酸エチルを水およびブラインで洗浄し、ＭｇＳＯ_４で乾燥し、そして濃縮した。ヘキサン中の５０％ ＥｔＯＡｃで溶出するシリカゲルカラムによって精製し、８９ｍｇの２−ヒドロキシメチル−６，７−ジメチル−ベンゾフラン−５−オールを得た。

5-Hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester (231 mg) prepared as described in Example 2 was dissolved in THF and lithium aluminum hydride (93 mg) was added. . The mixture was stirred at room temperature for 4 hours. The solution was then poured into water and extracted with ethyl acetate. The ethyl acetate was washed with water and brine, dried over MgSO ₄ and concentrated. Purification by silica gel column eluting with 50% EtOAc in hexanes afforded 89 mg of 2-hydroxymethyl-6,7-dimethyl-benzofuran-5-ol.

（実施例２４）
（６，７-ジメチル−２−（４−ニトロ−フェニル）−ベンゾフラン−５−オール）

(Example 24)
(6,7-dimethyl-2- (4-nitro-phenyl) -benzofuran-5-ol)

ＤＭＦ（１０ｍＬ）中の、実施例２１のように調製された２−ヒドロキシ−３，４−ジメチル−５−（テトラヒドロ−ピラン−２−イルオキシ）−ベンゾアルデヒド（２００ｍｇ）、ｐ−ニトロベンジルブロミド（２００ｍｇ）、および炭酸カリウム（２２０ｍｇ）の混合物を、室温で一晩攪拌した。次いで、この溶液を、水に注ぎ、酢酸エチルで抽出した。酢酸エチルを水およびブラインで洗浄し、ＭｇＳＯ４で乾燥し、そして濃縮した。残渣を、ヘキサン：酢酸エチル（８：２）で溶出するシリカゲルカラムによって精製し、６，７-ジメチル−２−（４−ニトロ−フェニル）−ベンゾフラン−５−オールのテトラヒドロピラニルエーテル２４２ｍｇを得た。ＤＭＦ（１０ｍＬ）中のエーテル（２４０ｍｇ）とＮａＯＭｅ（１７ｍｇ）との混合物を、７０℃で２時間攪拌した。次いで、溶液を、水に注ぎ、酢酸エチルで抽出した。有機層を、水およびブラインで洗浄し、ＭｇＳＯ_４で乾燥し、そして濃縮した。残渣を、ＭｅＯＨ中に溶解し、ｐ−トルエンスルホン酸一水和物（２０ｍｇ）を添加し、溶液を、室温で１．５時間攪拌した。少量のＮａＨＣＯ_３の添加後、メタノールをエバポレートし、残渣を、ヘキサンおよび酢酸エチル（７：３）で溶出するシリカゲルカラムクロマトグラフィーによって精製し、６，７-ジメチル−２−（４−ニトロ−フェニル）−ベンゾフラン−５−オール１７０ｍｇを得た。

2-hydroxy-3,4-dimethyl-5- (tetrahydro-pyran-2-yloxy) -benzaldehyde (200 mg), p-nitrobenzyl bromide (200 mg) prepared as in Example 21 in DMF (10 mL). 200 mg), and a mixture of potassium carbonate (220 mg) was stirred overnight at room temperature. The solution was then poured into water and extracted with ethyl acetate. The ethyl acetate was washed with water and brine, dried over MgSO4 and concentrated. The residue was purified by silica gel column eluting with hexane: ethyl acetate (8: 2) to give 242 mg of 6,7-dimethyl-2- (4-nitro-phenyl) -benzofuran-5-ol tetrahydropyranyl ether. . A mixture of ether (240 mg) and NaOMe (17 mg) in DMF (10 mL) was stirred at 70 ° C. for 2 hours. The solution was then poured into water and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over MgSO ₄ and concentrated. The residue was dissolved in MeOH, p-toluenesulfonic acid monohydrate (20 mg) was added and the solution was stirred at room temperature for 1.5 hours. After addition of a small amount of NaHCO ₃ , the methanol was evaporated and the residue was purified by silica gel column chromatography eluting with hexane and ethyl acetate (7: 3) to give 6,7-dimethyl-2- (4-nitro-phenyl). ) -Benzofuran-5-ol 170 mg was obtained.

上記手順に同様に従って、以下の化合物を、調製した：４−（５−ヒドロキシ−６，７-ジメチル−ベンゾフラン−２−イル）−ベンゾニトリル；

Following the above procedure, the following compound was prepared: 4- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -benzonitrile;

（実施例２５）
（５−ヒドロキシ−６，７-ジメチル−ベンゾフラン−２−カルボアルデヒド）

(Example 25)
(5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde)

ＤＭＦ（１０ｍＬ）中の２−ヒドロキシ−３，４−ジメチル−５−（テトラヒドロ−ピラン−２−イルオキシ）−ベンゾアルデヒド（３００ｍｇ）、ブロモアセトアルデヒドジメチルアセタール（３００ｍｇ）および炭酸カリウム（３３０ｍｇ）の混合物を、１４０℃で２時間攪拌した。次いで、この溶液を、水に注ぎ、酢酸エチルで抽出した。有機層を、水およびブラインで洗浄し、ＭｇＳＯ４で乾燥し、そして濃縮した。残渣を、ヘキサン：酢酸エチル（８：２）で溶出するシリカゲルカラムクロマトグラフィーによって精製し、５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−カルボアルデヒドのテトラヒドロピラニルエーテル１０２ｍｇを得た。酢酸（１５ｍＬ）中のこの中間体（１００ｍｇ）の溶液を、２時間還流した。酢酸をエバポレートし、残渣を、ヘキサン中の３０％酢酸エチルで溶出するシリカゲルカラムクロマトグラフィーで精製し、７０ｍｇの５−ヒドロキシ−６，７−ジメチル−ベンゾフラン−２−カルボアルデヒドを得た。ジクロロメタンおよび酢酸エチル（９：１）で溶出するシリカゲルによるさらなる精製によって、純粋生成物（３６ｍｇ）を得た。

A mixture of 2-hydroxy-3,4-dimethyl-5- (tetrahydro-pyran-2-yloxy) -benzaldehyde (300 mg), bromoacetaldehyde dimethyl acetal (300 mg) and potassium carbonate (330 mg) in DMF (10 mL). , And stirred at 140 ° C. for 2 hours. The solution was then poured into water and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over MgSO4 and concentrated. The residue was purified by silica gel column chromatography eluting with hexane: ethyl acetate (8: 2) to give 102 mg of tetrahydropyranyl ether of 5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde. A solution of this intermediate (100 mg) in acetic acid (15 mL) was refluxed for 2 hours. Acetic acid was evaporated and the residue was purified by silica gel column chromatography eluting with 30% ethyl acetate in hexanes to give 70 mg of 5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde. Further purification on silica gel eluting with dichloromethane and ethyl acetate (9: 1) gave the pure product (36 mg).

本発明は、その特定の実施形態を参照して記載されてきたが、本発明の真の精神および範囲から逸脱することなく、種々の変更がなされ、等価物が置換され得ることが、当業者に理解されるべきである。さらに、多くの改変が、特定の状況、材料、物質の組成、プロセスまたは処理工程を、本発明の目的、精神および範囲に対して適合され得る。このような改変の全ては、添付の特許請求の範囲内であることが意図される。上記の特許および刊行物の全ては、本明細書中で参考として援用される。

Although the present invention has been described with reference to specific embodiments thereof, those skilled in the art will recognize that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Should be understood. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process or process step, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims. All of the above patents and publications are incorporated herein by reference.

Claims (26)

Compound represented by Formula I

Where R 1 is: hydrogen, optionally substituted (C 1 -C 6 ) -alkyl, optionally substituted (C 2 -C 10 ) -alkenyl, optionally Substituted aryl, (optionally substituted (C 1 -C 6 ) -alkoxy) carbonyl, or halogen;
R 2 and R 3 are optionally substituted (C 1 -C 6 ) -alkyl, optionally substituted (C 2 -C 10 ) -alkenyl, or optionally substituted ( C 3 ~C 8) - are independently selected from cycloalkyl;
R 4 is: hydrogen, optionally substituted aryl, (optionally substituted (C 1 -C 6 ) -alkyl) carbonyl, (optionally substituted aryl) carbonyl, (optionally Optionally substituted heterocyclyl) carbonyl, (optionally substituted heterocyclylalkyl) carbonyl, (optionally substituted (C 1 -C 6 ) -alkoxy) carbonyl, (optionally substituted) (C 2 -C 10 ) -alkenyloxy) carbonyl, (optionally substituted) carbonyl, carboxy, formyl, or hydroxy (optionally substituted) (C 1 -C 6 ) -alkyl ;
R 5 is: hydrogen, (C 1 -C 6 ) -alkyl, (C 2 -C 10 ) -alkenyl, (optionally substituted alkoxy) carbonyl, carboxy, (optionally substituted amino) Carbonyl, or optionally substituted aryl;
Provided that one of R 4 or R 5 is hydrogen, when R 4 is hydrogen, R 5 is not hydrogen, and when R 5 is hydrogen, R 4 is not hydrogen;
R is hydrogen, (C 1 -C 6 ) -alkyl, (C 1 -C 6 ) -alkylcarbonyl, phosphoryl, or polyalkoxy; or R and R 1 together with the atoms to which they are attached, Compounds that form an optionally substituted ring; including single stereoisomers, mixtures of stereoisomers, and pharmaceutically acceptable salts of the compounds of Formula I. 2. A compound according to claim 1 wherein R < 1 > is hydrogen or halogen. A compound according to claim 1, R 2 and R 3, (C 1 ~C 6) - alkyl compound. A compound according to claim 3, R 1 is hydrogen, compound. 5. A compound according to claim 4, wherein R is hydrogen. A compound according to claim 5, R 5 is phenyl optionally substituted with one or more substituents, (C 1 ~C 6) - alkyl, (C 1 -C 6 ) - alkoxy, hydroxy, carboxy, (C l -C 6) - alkoxycarbonyl, nitro, halo, and are independently selected from cyano compound. A compound according to claim 6, R 4 is hydrogen, compound. A compound according to claim 5, R 4 is phenyl optionally substituted with one or more substituents, (C 1 ~C 6) - alkyl, (C 1 -C 6) - alkoxy, hydroxy, carboxy, (C 1 ~C 6) - alkoxycarbonyl, nitro, halo, and are independently selected from cyano compound. A compound according to claim 8, R 4 is 4-nitrophenyl or 4-cyanophenyl, R 5 is hydrogen, compound. A compound according to claim 5, R 4 is formyl, phenylcarbonyl which is optionally substituted, optionally substituted (C 1 ~C 6) - alkylcarbonyl or hydroxy (needs, Optionally substituted (C 1 -C 6 ) -alkyl), wherein the one or more substituents are independently selected from hydroxy, hydroxyalkyl, halogen, cyano, and heterocyclyl. A compound according to claim 10, R 4 is formyl, phenylcarbonyl, acetyl, bromoacetyl, morpholin-1-yl - acetyl, hydroxymethyl, 1-hydroxy-2-morpholinyl-4-ylethyl or 6, A compound, which is hydroxy-3-methyl- [1,3] oxazinan-6-yl; R 5 is hydrogen. A compound according to claim 5, R 4 is, (C 1 ~C 6) - alkenyloxy carbonyl or aminocarbonyl optionally substituted, - alkoxycarbonyl, (C 2 ~C 10) Wherein the one or more substituents are independently selected from (C 1 -C 6 ) -alkyl and hydroxy- (C 1 -C 6 ) -alkyl. 13. A compound according to claim 12, wherein R 4 is bis- (2-hydroxy-ethyl) -amide, 2-hydroxy-ethyl-amide, carboxylic acid; carboxylic acid methyl ester; carboxylic acid 3,7 dimethyl- A compound wherein octa-2,6-dienyl ester; [2- (2-methoxy-ethoxy) -ethoxy] ethyl ester or morpholin-1-ylcarbonyl; R 5 is hydrogen. A compound according to claim 1, R and R 1, together with the atoms to which they are attached, form a ring optionally substituted, compound. A compound according to claim 14, R and R 1, together with the atoms to which they are attached form a furan ring substituted with phenyl, said phenyl, alkyl, alkenyl, hydroxy A compound optionally substituted with one or more substituents independently selected from: alkoxy, nitro, cyano, carboxy, ester, haloalkyl, and halo. 2. The compound of claim 1 selected from the following group:
(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl) -phenyl-methanone;
(5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -morpholin-4-yl-methanone;
1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone;
Acetic acid 2- (2-bromo-acetyl) -6,7-dimethyl-benzofuran-5-yl ester;
2- (1-hydroxy-2-morpholin-4-yl-ethyl) -6,7-dimethyl-benzofuran-5-ol;
Acetic acid 6,7-dimethyl-2- (2-morpholin-1-yl-acetyl) -benzofuran-5-yl ester;
Acetic acid 2- (6-hydroxy-3-methyl- [1,3] oxazinan-6-yl) -6,7-dimethyl-benzofuran-5-yl ester;
1- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -2-morpholin-4-yl-ethanone;
1- (4-Bromo-5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -ethanone;
-2-hydroxymethyl-6,7-dimethyl-benzofuran-5-ol;
-6,7-dimethyl-3-phenyl-benzofuran-5-ol;
6,7-dimethyl-2- (4-nitro-phenyl) -benzofuran-5-ol;
-5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde;
4- (5-hydroxy-6,7-dimethyl-benzofuran-2-yl) -benzonitrile;
-5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 3,7-dimethyl-octa-2,6-dienyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid bis- (2-hydroxy-ethyl) -amide;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 2- [2- (2-methoxy-ethoxy) -ethoxy] -ethyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid (2-hydroxy-ethyl) -amide;
(5-hydroxy-3,6,7-trimethyl-benzofuran-2-yl) -phenyl-methanone;
Acetic acid 2- (2-bromo-acetyl) -6,7-dimethyl-benzofuran-5-yl ester;
-2-hydroxymethyl-6,7-dimethyl-benzofuran-5-ol;
-6,7-dimethyl-3-phenyl-benzofuran-5-ol;
6,7-dimethyl-2- (4-nitro-phenyl) -benzofuran-5-ol;
-5-hydroxy-6,7-dimethyl-benzofuran-2-carbaldehyde;
-5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid methyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid bis- (2-hydroxy-ethyl) -amide;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid 2- [2- (2-methoxy-ethoxy) -ethoxy] -ethyl ester;
5-hydroxy-6,7-dimethyl-benzofuran-2-carboxylic acid (2-hydroxy-ethyl) -amide;
3- (4-methoxy-phenyl) -6,7-dimethyl-benzofuran-5-ol;
3- (4-Chloro-phenyl) -6,7-dimethyl-benzofuran-5-ol;
3- (4-Fluoro-phenyl) -6,7-dimethyl-benzofuran-5-ol;
-4,5-dimethyl-1,8-diphenyl-benzo [1,2-b; 4,3-b '] difuran;
1,8-bis- (4-fluoro-phenyl) -4,5-dimethyl-benzo [1,2-b; 4,3-b ′] difuran; and 1,8-bis- (4-methoxy) -Phenyl) -4,5-dimethyl-benzo [1,2-b; 4,3-b '] difuran. A pharmaceutical or cosmetic formulation comprising a compound according to any of claims 1-16 mixed with at least one acceptable excipient. 17. A compound according to any one of claims 1 to 16 for use as a medicament. Use of one or more compounds according to any one of claims 1 to 16 for the manufacture of a medicament for the treatment or prevention of diseases characterized by oxidative stress. 20. The use according to claim 19, wherein the condition is seizure, cerebral ischemia, renal ischemia, myocardial infarction, chronic heart failure, postoperative cognitive dysfunction, peripheral neuropathy, spinal cord injury, head injury, and surgical trauma. Including, use. 20. Use according to claim 19, wherein the condition is associated with an inflammatory component or an autoimmune component. 20. Use according to claim 19, wherein the condition is a skin condition. 23. Use according to claim 22, wherein the conditions are skin condition regulation, skin aging indication regulation, hair growth regulation regulation, as well as contact dermatitis, skin pigmentation, retinoid-induced irritation, wrinkles Use, including treatment of stimuli including acne, psoriasis, age-related damage and harmful (UV) radiation, damage resulting from stress or fatigue. Use of one or more compounds according to any one of claims 1 to 16 for the manufacture of a medicament for the treatment or prevention of inflammatory conditions characterized by elevated C-reactive protein levels. 25. Use according to claim 24, wherein the inflammatory condition is a cardiovascular inflammatory condition, a respiratory inflammatory condition, sepsis, diabetes, muscle fatigue, systemic lupus erythematosus (SLE), end stage renal disease (ERSD), periodontal disease. Use, including inflammatory skin conditions. The invention described herein.