JPH11510144A - Use of non-estrogenic polycyclic phenolic compounds for the manufacture of a medicament for imparting neuroprotection to cells - Google Patents

Abstract

(57) The present invention relates to a method for the manufacture of a medicament for imparting neuroprotection to cells in a patient, the method comprising a terminal phenolic group having at least a second ring structure in the structure and a molecular weight of less than 1,000 daltons. (E.g., naphthols, phenanthrenes or steroids).

Description

DETAILED DESCRIPTION OF THE INVENTION Non-estrogenic polycyclic ferrites for the manufacture of a medicament for imparting neuroprotection to cells Use of knol compoundsTechnical fields   The present invention is directed to protecting cells in the central nervous system of a patient from cell death and Set for stimulating neuronal survival in patients with neurodegenerative degenerative conditions Related to compositions and methods.background   Pathological conditions resulting from accelerated or ongoing neuronal death are In many societies, with chronic conditions such as Alzheimer's disease and Parkinson's disease Disease, acute illness such as stroke, brain cell loss following myocardial infarction and spinal cord injury and And acute illnesses such as acute neuronal injury associated with head and head trauma. Chronic And acute neurodegenerative degenerative diseases and acute neuronal injury and associated deaths And pathological conditions are often incurable. Of the disability of the patient resulting from those conditions The result is a high cost to society of patient care as well as a significant reduction in quality of life. is there. Effective in preventing or reducing neuronal death or damage associated with the above conditions Treatment attempts are necessary. Currently, the condition in the brain that results from neuronal loss The most important attempts at developing therapeutics for treatment include relatively non-toxic, female Suitable for use in both sexes and men, with easy access to the brain through the blood-brain barrier Getting effective medicines that work well.   Estrogen compounds protect neurons from cell death and reduce Alzheimer's disease Found useful in slowing the progression of neurodegenerative degenerative diseases such as (WO 95/12402 by Simpkins et al., Biochem by Behl et al. . Biophys. Res. Commun. (1995), 216: 473-482, Molecula by Bishop et al. r and Cellular Neuroscience (1994), 5: 303-308, Neuro by Simpkins et al. obiology of Aging (1994), 15: s195-s197). In addition, WO by Simpkins et al. 95/12402 previously stated that it was considered biologically inert. The α-isomer of estrogen compounds has been shown to delay neurodegenerative degeneration. And is shown to be effective. This proof is, for the first time, He gave men the opportunity to administer estrogen therapeutically without any side effects.   Estrogen compounds have several different physiological and biological roles in neurons Has been shown to have chemical effects, but the compounds have neuroprotective effects There is no known mechanism. For example, estrogens cause the production of neurophilic substances It has been shown to stimulate, thereby stimulating neuronal growth. (REF) Estrogen compounds are also found in NMDA-induced cells in primary cell cultures. It has also been found to prevent death (Bahl et al., Biochem. Biophys Res. Com. mun. (1995), 216: 973, by Goodman et al. Neurochem (1996), 66: P. 1836), furthermore it can remove oxygen free radicals and prevent lipid peroxidation. Has been found (Droescher et al., SO95 / 13076). But, The potential effects of free radicals on the neuron itself have not been proven. Droeschl have shown that some estrogens, like vitamin E, have activity. A cell-free "in vitro" assay system using lipid peroxidation as a defined endpoint ing. Estradiol has also been reported to reduce membrane lipid peroxidation. (Niki's Chem. Phys. Lipids (1987), 44: 227, Bio by Nakano et al.) chem. Biophys. Res. Comm. (1987), 142: 919, Hall et al. Cer. Bloo d Flow Metab. (1991), 11: 292). Specific 21-amino steroids and glue Cococorticosteroids have been found to act as antioxidants and Their use in spinal cord injury as well as trauma, ischemia and stroke has been tested ( J. Wilson et al. Trauma (1995), 39: 473, J. Levitt et al. Cardiov asc. Pharmacol (1994), 23: 136, Stroke by Akhter et al. (1994), 25:41. 8 pages).   As noted above, several factors are involved in neuroprotection. Single incarnation Therapeutic agents selected on the basis of a biological mechanism cure a disease or trauma in a patient. It may limit the generalized utility in treating. For example, estrogen To achieve the in vitro antioxidant effect used, Dröscher et al. An amount of estrogen was used. Such doses are Even if effective, due to toxicity-related problems with long-term use of high doses In addition, it limits its usefulness in treating chronic neurological symptoms.   Non-gender, degenerative neurodegenerative diseases, acute symptoms caused by stroke and aging Cell death can also be used in non-toxic doses for chronic treatment Compounds that have been shown to have biological efficacy in protecting neurons from It is helpful to identify. About compositions capable of inducing neuroprotection Understanding structural requirements has increased neuroprotection, simultaneously reducing side effects To provide a basis for designing new medicines.Overview   The present invention provides neurons from degeneration and cell death resulting from disease, stroke or aging. It is effective in protecting and provides male and female patients with minimal side effects. The above for certain types of compounds that can be used to achieve a similar effect Meet the need. In a preferred embodiment of the present invention, at least having a molecular weight of less than 1,000 Daltons Also a non-estrogenic compound having a terminal phenol group in the structure having the second ring. Providing the compound and stimulating the compound in a population of cells. A method comprising administering a dose effective to confer protection comprises the steps of: Conferring cell protection in a population of   In another embodiment of the present invention, there is provided a terminal phenol having a molecular weight of less than 1,000 daltons. A ring and at least one additional ring structure covalently linked to the phenol ring. Selecting an effective dose of a compound having the compound and administering the compound to a patient There is provided a method of treating a neurodegenerative degenerative condition in a patient, comprising:   In another embodiment of the present invention, the compound used in the method of the present invention has a tetracyclic structure Has a tricyclic or bicyclic structure, the tetracyclic structure of which achieves a plasma concentration of less than 500 nM. Can be administered at an effective dose. The molecular weight of the compound is greater than 170D Can get.   In another aspect of the invention, the tricyclic structure comprises tetrahydrophenanthrene and octa Hydrophenanthrene, more particularly phenanthrene methanol or phenan Phenanthrenation which can be further selected from the group consisting of trencarboxaldehydes It is a compound.   In another aspect of the invention, the bicyclic structures can be fused to form naphthol and naphthalene. Or a non-fused bicyclic structure having a linking group.   In another aspect of the invention, the terminal phenolic ring comprises a non-steroidal compound. Departure The clear embodiment uses a compound having a phenol A ring.   In yet another aspect of the invention, the dose of the neuroprotective compound is less than 500 nM, more Specifically, in the range of 0.02 nM to 500 nM, and more specifically, in the range of 0.1 nM to 1 nM Plasma concentration.Drawing   These and other features of the present invention are described in the following description, claims and accompanying figures. It is better understood by referring to the plane.   In that figure, Figure 1 shows the number of viable SK-N-SH cells in the absence of serum for 48 hours. 3,17β-estradiol and 3,17β-estradiol-3- at% change in The effect of O-methyl ether is shown. Analysis of variance and Scheffe's F test  F test), the original data is compared to each serum-free control group, Were normalized to the serum-free group (= 100%).^*= P <serum-free control 0.05. The data are expressed as mean ± SEM for 6 wells per group.   FIG. 2 shows that all SK-N-SH viable cell counts at 48 hours serum loss were all 2 3,17β-estradiol, estra-2,3,17β-triol (2-OH- Stradiol), 1,3,5 (10) -estraditrien-3-ol (estraditrien- 3 shows the effects of 3-ol) and 2,3-O-methylestradiol.^*= Serum-free control In contrast, p <0.05.^**= Serum-free controls and their respective 2,3-O-methyl estradio P <0.05 vs. The data were expressed as mean ± SEM for 5 wells per group. To represent.   FIG. 3 shows that all SK-N-SH cell counts in the absence of serum for 48 hours were all 2 3,17β-estradiol, estriol, 17α-ethynyl-3,1 at a concentration of nM 7β-estradiol (ethynylestradiol) and their 3-O-methyl Ether: estriol-3-O-methyl ether (estriol-3-O- ME) and ethinyl estradiol-3-O methyl ether (ethinyl estra) Diol-3-O-ME).^*= Serum-free controls and their respective 2,3-O-methyl P <0.05 vs steroid.^**= P <0.05 for all other groups. That de Data are expressed as mean ± SEM for 5 wells per group.   FIG. 4 shows that the number of viable SK-N-SH cells in the absence of serum for 48 hours was all 2 3,17β-estradiol at a concentration of nM, diethylstilbestrol (DE S), DES mono O-methyl ether (DES mono-O-ME) and DES di-O- The effect of methyl ether (DES di-O-ME) is shown.^*= Serum-free control and DES P <0.05 relative to the di-O-methyl ether group. The data into 6 wells per group And are expressed as mean ± SEM.   FIG. 5 shows 2 nM of SK-N-SH dead cell count in serum-free for 48 hours. 3,17β-estradiol, estrone, [2S- (2a, 4aα, 10aβ) −1 at concentrations , 2,3,4,4a, 9,10,10a-Octahydro-7-hydroxy-2-methyl-2-phenanthrene Tanol (PAM) and [2S- (2a, 4aα, 10aβ) -1,2,3,4,4a, 9,10,10a-octa Hydro-7-hydroxy-2-methyl-2-phenanthrenecarboxaldehyde (PA CA).^*= P <0.05 relative to serum-free control (SF). The data Are expressed as mean ± SEM for 6 wells per group.   FIG. 6 shows 2 nM in live SK-N-SH cell counts in the absence of serum for 48 hours. 3,17β-estradiol, estrone, [2S- (2a, 4aα, 10aβ) -1,2,3,4,4 a, 9,10,10a-Octahydro-7-hydroxy-2-methyl-2-phenanthrene methanol (PAM) and [2S- (2a, 4aα, 10aβ) -1,2,3,4,4a, 9,10,10a-octahydro-7- Effect of hydroxy-2-methyl-2-phenanthrene carboxaldehyde (PACA) The result is shown.^*= P <0.05 relative to serum-free control (SF). The data Expressed as mean ± SEM for 6 wells.   FIG. 6a shows the percentage increase in viable cell number relative to serum-free controls in serum deprivation for 48 hours. [2S- (2a, 4aα, 10aβ) -1,2,3,4,4a, 9,10,10a-octahi at 2 nM Effects of Dro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM) Show.^*= P <0.05 relative to serum-free control (SF). Statistical analysis is based on the raw data It was made according to. The data were expressed as mean ± SEM for 8 wells per group Express.   FIG. 6b shows the percentage increase in viable cell number relative to the serum-free control in the absence of serum for 48 hours. [2S- (2a, 4aα, 10aβ) -1,2,3,4,4a, 9,10,10a-octahydro-7-hydr Effects of Roxy-2-methyl-2-phenanthrenecarboxaldehyde (PACA) Show.^*= P <0.05 relative to serum-free control (SF). Statistical analysis is based on the raw data It was made according to. The data were expressed as mean ± SEM for 8 wells per group Express.   FIG. 7 shows that β-amyloid protein (Aβ25-35) (20 μm ), 3,17β-estradio in toxicity induced by neurotoxic fragments (0.2 or nM) and 3,17α-estradiol (2 nM) You. SK-N-SH neurons are 3,17β-estradiol, 3,17α-estradiol All and Aβ25-35 were exposed alone or in combination. Raw after 4 days of exposure Cell numbers were determined.^*= P <0.05 relative to serum-free control (SF). That data , Expressed as mean ± SEM for 6 wells per group. Another group without Aβ Exposed to 3,17β-estradiol and 3,17α-estradiol. Source of injury In the absence of a factor, the addition of the steroid has no effect on cell number. won.   FIG. 8 shows the results at 100 μg / kg body weight two hours before the middle cerebral artery occlusion. Ovariectomy by injection (oil) or by subcutaneous injection with 3,17β-estradiol 2 shows the effect of treatment on aged rats. Maximum lesion size in the transcerebral part of the dissected animal Are recorded and reported as% of brain cross-sectional area. A, B, C, D and E Corresponds to the brain part 7, 9, 11, 13 and 15 mm after the olfactory bulb, respectively.   FIG. 9 shows 2 nM in live SK-N-SH cell counts in the absence of serum for 48 hours. Shows the effect of progesterone. The data were averaged over 6 wells per group Expressed as ± SEM.   FIG. 10 shows the viability of SK-N-SH in the absence of serum for 48 hours at 2 nM. Shows the effects of corticosterone. The data were averaged for 6 wells per group. Expressed as mean ± SEM.   FIG. 11 shows a tricyclic compound: [2S- (2a, 4aα, 10aβ) -1,2,3,4,4a, 9,10,10a-octa Hydro-7-hydroxy-2-methyl-2-phenanthrenecarboxaldehyde (PA CA) shows the structure.   FIG. 12 shows a generalized core ring structure with numbered carbons, (a) tetracyclic structure , (B) a tricyclic structure, (c) a bicyclic structure (fused) and a bicyclic structure (unfused).Detailed description   Neuroprotection, as used herein and in the claims, promotes neurons that lead to cell death. Deterioration of behavioral degradation.   Non-estrogenic compounds are referred to herein as Steraloids Inc. (Wilton, New Hampshire) It is defined as a compound other than an estrogen compound. A phenol ring, in the present description and claims, is a component consisting of more than one carbon ring. It is described as "terminal" if it is the last carbocycle in the nucleophile.   Steroids are referred to herein and in the claims as a number arranged in a tetracyclic structure. Are defined as compounds having labeled carbon atoms [J. American Chemical Soc , 82: 5525-5581 (1960) and Pure and Applied Chemistry 31: 285-32. 2 (1972)].   “Neurodegenerative degenerative disorder”, as used herein and in the claims, refers to the progression of neurons. Gender impairment is defined as a disorder that occurs in the peripheral or central nervous system. Neurodegenerative changes Sexual diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, sugar Chronic neurodegeneration such as uremic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis Sexual degenerative diseases; aging; and stroke, traumatic brain injury, schizophrenia, peripheral nerve injury, hypotension Acute neurodegenerative degenerative diseases including glycemia, spinal cord injury, epilepsy and anoxia and hypoxia Is included.   These examples are not meant to be inclusive, but rather, "neurodegenerative degeneration It is merely provided as an illustration of the term "disease".   The present invention is a method of neuroprotection using a novel class of neuroprotective compounds, The class of compounds is the set here first identified as a determinant of neuroprotection. Specified by the characteristics of This method also provides for neurodegenerative changes in human patients. Suitable for the treatment of sexually transmitted diseases, trauma and aging.   Protection of neurons from severe degenerative degeneration is a matter of acute or chronic neurodegenerative degeneration. An important aspect of the treatment of patients with sexually transmitted diseases, a chronic example is Alzheimer's disease. You. For Altiheimer patients, the method of the invention is an important method of therapeutic use. You. Other diseases for which such methods are effective include Parkinson's disease, Huntington Disease, AIDS dementia, Wernicke-Korsakoff's related dementia) (alcohol-induced dementia), age-related dementia, age-related memory impairment Head injury, stroke, hypoglycemia, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, blood Brain cell loss due to tumor and epilepsy; spinal cord due to any of the conditions listed in Brain cell loss Vesicle impairment; as well as peripheral neuropathy. Due to the observed cytoprotective properties, One pathway for action with cyclic phenolic compounds is suggested to be apoptosis inhibition Is done.   A unique set of features that identify types of neuroprotective compounds include (a) 1,000 daltons The presence of more than one ring in the compound having a size range of less than It includes a knol ring and (c) a dose effective to produce a neuroprotective effect in vivo.   The classes of neuroprotective compounds of the invention described herein include (a) two carbon atoms Compounds characterized by a ring (Table II); (b) characterized by three carbocycles Compounds (Figs. 11 and 12); and steroids characterized by four carbocycles (Fig. 12) is included. Neuroprotective compounds must exceed a total molecular weight of 1,000 daltons Further includes five or more carbocycles.   According to the present invention, SK under stress to determine neuroprotection by test compounds Assays as described in Example 1 using -N-SH neuron cells were used. Can be 3,17β-estradiol has shown its neuroprotective effects (Simpkin (WO 95/12402). Neuroprotective activity of control compound The properties are shown in FIG. FIG. 1 shows that in the absence of serum for 48 hours, 2 nM 3,17β-E In the presence of stradiol, the percentage of live SK-N-SH cells increases to 100%.   The present invention relates to a method for administering polycyclic phenolic compounds at an effective dose that provides low plasma concentrations. Security is achieved. More specifically, the neuroprotective effect is a plasma concentration of less than 500 nM. In particular, a plasma concentration of between 0.1 nM and 1 nM can be achieved. The present invention Relatively effective use of neuroprotective compounds that can provide neuroprotective effects Quantities are in direct contrast to other views where estrogen was tested in in vitro assays It is a target. For example, by Droescher et al., Free radical acids in lipid peroxidation assays. IC of 12.8 μM estrogen to prevent oxidation₅₀Is described. The indirect lipid peroxidation assay for cytoprotection used by Droescher et al. A biochemical assay used in the present invention to directly measure cellular response Is not comparable to the neuron cell assay used. As a result, the two Are not directly comparable, and the results of one assay are It cannot be the basis for inference. Advantages of the neuroprotection assay used herein Is that live and dead cells are used directly to determine neuroprotection. You.   The amount of neuroprotective drug used to treat the patient, especially if the compound Within a range that is relatively non-toxic to patients when used for desirable. According to the present invention, significant neuroprotection was achieved at a concentration of 2 nM Obtained in cell culture (FIGS. 1-6), and furthermore, significant neurons in rats Protection is achieved at 100 μg / kg body weight. Conversion at 100μg / kg body weight The dose of the compound gives a plasma concentration of about 0.4 nM to 2 nM of the compound (FIG. 8).   According to the neuronal cell assay described in Example 1, neuroprotection provided phenol. It was shown that a hydroxyl group in the A ring was required. To the terminal phenol group Substitution of a hydroxy group, for example, with a methyl group, in the A significant loss of gender was observed (FIGS. 1-6, Tables I and II). Usually progestero Lack of hydroxyl groups on terminal carbocycles, such as carbohydrate and corticosterone Shows little or no neuroprotection (FIGS. 9 and 10). The present inventors furthermore In order to maintain neuroprotection, the hydroxyl group at the terminal phenol group It was determined that it could be located on any available carbon.   The neuroprotective compound for use in the method of the invention has a terminal phenolic ring. It was decided that it was necessary. These compounds are charcoal containing hydroxyl groups. R group substitution at any suitable carbon in the terminal phenolic ring other than Optionally, those R groups can be in the α or β isomeric configuration. Furthermore, itself Is not neuroprotective and linear substitution of phenolic compounds Not transprotective (Table III). However, a terminal phenol ring and at least one Compounds with other carbocycles are neuroprotective. For example, a non-fused bicyclic structure, Diethylstilbestrol showed neuroprotective properties according to the invention (Table II, (Fig. 4). This compound has a terminal phenol linked to the second phenol ring by a linking group. Having a ring structure. Removal of the hydroxyl group at the terminal phenolic ring This results in a loss of protective activity.   In addition, it is non-steroidal and has a terminal phenolic ring and at least two additional Compounds having a carbocyclic structure include [2S- (2a, 4aα, 10aβ) -1,2,3,4,4a, 9,10,10 a-octahydro-7-hydroxy-2-methyl-2-phenanthrenemethanol (PAM ) And [2S- (2a, 4aα, 10aβ) -1,2,3,4,4a, 9,10,10a-octahydro-7-hydroxy Illustrated by ci-2-methyl-2-phenanthrenecarboxaldehyde (PACA) Compounds containing tricyclic compounds (Figures 11 and 12) have neuroprotective effects (See FIGS. 5 and 6, 6a and 6b). The structure of those compounds is This is shown in FIG. At 2 nM concentration of PAM or PACA, approximately 15% of neurons It has been found to increase cell survival. Terminal phenol ring and low Compounds having at least three additional carbocycles are provided by 3,17β-estradiol It has been found to have a neuroprotective effect as illustrated (FIG. 1). Maximum size of compounds of the invention that are neuroprotective and have a terminal phenolic ring Does not depend much on the number of carbocycles in the structure, but rather the compound It depends on whether it is small enough to pass through the gate.   Recommended routes of administration of the neuroprotective compound include oral, intramuscular, transdermal, buccal, Intravenous and subcutaneous are included. The method of administering the compounds of the present invention may be by dose or controlled. Depends on controlled release vehicle.   In a preferred embodiment of the present invention, a terminal phenol ring and at least a second carbon ring Compounds. In addition to their required structures, the compounds A phenol ring or any other, provided that the phenolic structure is maintained May have several R groups attached to available sites. Those R groups are inorganic Alternatively, it may be selected from organic atoms or molecules. Later, examples of the number of different types of R groups Although described, the invention is not limited to those examples. (a) R groups include halogen, amide, sulfate, nitrate, fluoro, Inorganic R groups containing a bromo or bromo group are included. In addition, sodium and potassium And / or an R group selected from ammonium salts is bonded to the α or β position, Replaces hydrogen at any available carbon in the system. The R group is organic Obtained or may comprise a mixture of organic molecules and ions. Organic R groups include linear or Contains alkanes, alkenes or alkynes having up to 6 carbons in a branched arrangement Can be For example, additional R group substituents include methyl, ethyl, propyl, butyl, Pentyl, hexyl, heptyl, dimethyl, isobutyl, isopentyl, tert- Butyl, sec-butyl, isobutyl, methylpentyl, neopentyl, isohexyl , Hexenyl, hexadiene, 1,3-hexadiene-5-yne, vinyl, allyl, Isopropenyl, ethynyl, etyridine, vinylidine, isopropylidene; Len, sulfate, mercapto, methylthio, ethylthio, propylthio, Tylsulfinyl, methylsulfonyl, thiohexanyl, thiobeny l), thiopenol, thiocyanato, sulfoe Tylamide, thionitrosyl, thiophosphoryl, p-toluenesulfonate, No, imino, cyano, carbamoyl, acetamido, hydroxyamino, nitro Seo, nitro, cyanate (cyanato), celecyanato (celecyanato), Arco Arcosine, pyridinium, hydrazide, semicarbazone, carboxy Methylamide, oxime, hydrazone, sulfrutrimethylammonium, o- Carboxymethyl oxime, aldehyde hemiacetate, methyl ether, ethyl Ether, propyl ether, butyl ether, benzyl ether, methyl ether Rubonate, carboxylate, acetate, chloroacetate, trimethylacetate Acetate, cyclopentylpropionate, propionate, phenylpropionate Carboxylic acid methyl ether, formate, benzoate, butyrate, Caprylate, cinnamate, decylate, hepti Rate, enanthate, glucosiduronate, succine , Hemisuccinate, palmitate, nonanoate, stearate, tosire , Valerate, valproate, decanoate, hexahydrobenzoate , Laurate, myristate, phthalate, hydroxyl, ethylene ketal, Diethylene ketal, formate, chloroformate, formyl, dichloroacetate Tate, keto, difluoroacetate, ethoxycarbonyl, trichloroforme , Hydroxymethylene, epoxy, peroxy, dimethyl ketal, aceto Nide, cyclohexyl, benzyl, phenyl, diphenyl, benzylidene and A chloropropyl group may be included. The R group can be attached to any of the constituent rings, , Pyriazine, pyrimidine or v-triazine. Other R group substitution The group includes either the 6-membered ring or the 5-membered ring mentioned in b below. (b) Aroma having any of the substitutions described in (a) above, in addition to the phenol A ring Or a non-aromatic phenol ring, for example, having the following structure: phenanthrene , Naphthalene, naphthol, diphenyl, benzene, cyclohexane, 1,2-pyra 1,4-pyran, 1,2-pyrone, 1,4-pyrone, 1,2-dioxin, 1,3-dioxin ( Dihydro form), pyridine, pyridazine, pyrimidine, pyrazine, piperazine, s- Triazine, as-triazine, v-triazine, 1,2,4-oxazine, 1,3,2-oxa Gin, 1,3,6-oxazine (pentoxazole), 1,2,6-oxazine, 1,4-oxa Gin, o-isoxazine, p-isoxazine, 1,2,5-oxathiazine, 1,2,6-oxo Sadiazine, 1,4,2-oxadiazine, 1,3,5,2-oxadiazine, morpholine (te Having a heterocyclic carbocycle which can be selected from one or more of (trahydro-p-isoxazine) Any of the above-mentioned six-membered ring structures It can be a terminal group. In addition, any of the above carbocyclic structures may be directly or , Furan, thiophene, (thiofuran); pyrrole (azole); isopillo (Isoazole); 3-isopyrrole (isoazole); pyrazole (1,2-die Azole); 2-isoimidazole (1,3-isodiazole); 1,2,3-triazole 1,2,4-triazole; 1,2-dithiol; 1,2,3-oxathiol; isoxazo (Furo (a) monozole); oxazole; (furo (b) monazole); thiazole Isothiazole; 1,2,3-oxadiazole; 1,2,4-oxadiazole; 1,2, 5-o Oxadiazole; 1,3,5-oxadiazole; 1,2,3,4-oxatriazole; 1,2, 3,5-oxatriazole; 1,2,3-dioxazole; 1,2,4-dioxazole; 1,3, 2-dioxazole; 1,3,4-dioxazole; 1,2,5-oxathiazole; 1,3-oxo Sathiol; any other heteroaromatic or non-aromatic, including cyclopentane May be attached to a group carbocycle. Those compounds then have one of the available sites Bond with an R group selected from the preceding (a) part or (b) part substituted on the carbocycle I got it. (c) containing the groups listed above to form a cyclopentanophena (a) nthrene ring compound Obtain, for example, 1,3,5 (10), 6,8-estradipentaene, 1,3,5 (10), 6,8,11-est Lapentaene, 1,3,5 (10), 6,8,15-estradipentaene, 1,3,5 (10), 6-estradi Tetraene, 1,3,5 (10), 7-estratetraene, 1,3,5 (10), 8-estrateratee 1,3,5 (10), 16-estraditetraene, 1,3,5 (10), 15-estraditetraene, 1, Selected from the group consisting of 3,5 (10) -estraditriene and 1,3,5 (10) 15-estraditriene Any compound that can be used. (d) Complete with hydroxyl groups present at carbons 1, 2, 3, and 4 of the terminal phenol ring Raloxifen, tamoxifen (t amoxifen), a precursor or derivative selected from androgen compounds and salts thereof Any of the compounds comprising (e) any compound in the form of a prodrug, which is metabolized and has neuroprotective activity An active polycyclic phenol compound having the formula:   Administration of any of the compounds of the present invention may involve a single compound or a mixture of neuroprotective compounds. Includes the use of compounds. Example 1: Assay for identifying neuroprotective compounds   Cell line, SK-N-SH, well characterized for testing potential neuroprotective drugs The attached human neuroblastoma cell line was used. This cell line is Pharmaceutical neuroprotective agents against human diseases and injuries It is widely considered to be a suitable assay system for the evaluation of   SK-N-SH cells are from the American Type Tissue Collection (Amer ican Type Tissue Collection (Rockville, MD). Cell cultures were prepared with 10% fetal bovine serum (FBS), 100 U / ml penicillin G and 100 5% at 37 ° C. in RPMI-1640 medium supplemented with mg / ml streptomycin. CO_TwoAnd grown in 95% air to confluency. Medium for one week SK-N-SH cells were changed every three days, and returned every 5 to 7 days. ) And maintained the cell line and the cells used in the experiments below were passaged between 7 and 12 passages. Number (in passages). First decant the growth medium and bring the cells to 0.0 Rinse with 2% EDTA, then discard EDTA. Next, another 0.02% EDTA Aliquots were added and after 30 minutes incubation at 37 ° C, Neubauer Count cells with a hemocytometer (Fisher Scientific Inc., Orlando, FL) And resuspended in a suitable medium. 1.0 x 10 in a suitable treatment medium⁶Cell / ml back culture The experiment was started by back-culture of SK-N-SH at the nutrient concentration. The cell density is 3,17β- Has no effect on response to estradiol or 3,17α-estradiol .   Neuroprotection assays were performed at 48 hours to serum deprivation and 2 nM concentration of each test compound. During continuous exposure of SK-N-SH cells during This concentration was 3,17-β and 3,17α-d It is at least 10 times higher than the concentration required for the neuroprotective effect of stradiol. 3,17β -Estradiol was used as a control. This compound is available in the absence of serum Results in dose-dependent protection of SK-N-SH cells with an ED of 0.13 nM₅₀And 0.2n It had significant neuroprotection at M concentrations (FIG. 1). This effect can be seen in eight separate trials And was potent at 2 nM concentration of 3,17β-estradiol showing neuroprotection (FIG. 1). To FIG. 4). Live cells in treated wells versus cell numbers in serum-free wells Neuroprotection was determined by the magnitude of the difference in numbers. Original data for each experiment Statistical analysis was performed on the data. The significance of differences between groups was determined by one-sided analysis of variance. Specified. Designed comparison between groups using Scheffe's F-test I went there. In all tests, p <0.05 was considered significant. Analytical Later, the response of the 3,17β-estradiol group to each study was calculated using the following calculation: Data were normalized to% Answer.   Cell viability was assessed by treatment with trypan blue exclusion for 48 hours. At an appropriate time, the medium was decanted and each well was filled with 0.02% EDTA. Rinse with 0.2 ml and incubate the cells with 0.2 ml of 0.02% EDTA for 30 minutes at 37 ° C. To prepare a cell suspension. Repeat EDTA on the cells Cells were suspended by pet addition. 100 ul aliquot from each cell suspension 100 ul of 0.4% trypan blue dye [Sigma Chemical  Co.) for 5 minutes at room temperature. After adding trypan blue Within 15 minutes, all suspensions were counted on a Neubauer hemocytometer. Each Aliko Two separate measurements of live cells were performed on the cells. Example 2: Comparison of neuroprotection provided by different tetracyclic and bicyclic formulations   3,17α-estradiol, 1,1,3,5 (10) -estraditrien-3-ol and 1,3, 5 (10) Estra-2,3,17β-triol (2,3-catechol estradiol) , 17β-estradiol found to be equivalent in their degree of neuroprotection (FIG. 2, Table 1). Estrone, estriol and 17α-ethynyl 3,17β Estradiol was significantly neuroprotective, but not 3,17β estradiol. Less activity (Figure 3 and Table II). Bicyclic non-fused diethylstil Vesterol (DES) was active as a neuroprotectant, but one, but Not both phenolic hydroxyl groups are replaced by O-methyl ether groups If so, almost complete neuroprotection was maintained (Figure 4 and Table 2). Similarly, 3- When the hydroxyl group is replaced with an O-methyl ether group, all steroids Becomes inactive (FIGS. 1-4 and Tables I and II), eliminating the acid and hydrophilicity of the A ring. A replacement has been made. The 3-O methyl ether of 3,17β-estradiol is as high as 20 nM Was inactive even at high concentrations (Figure 1). These data indicate that the C-3 hydro Xylated estratrienes have been shown to be neuroprotective. Difeno Similarly located phenolic hydroxyl groups perform the same function in .   Two 19-carbon steroids at 2 nM for neuroprotection in the assay evaluated. Viable cells / ml for 5-6 cultures / group (mean ± SEM X 10^Three / Ml) gave the following results: Study 1: Serum-free control = 94 ± 7, Tests Teron = 87 ± 6, dihydrotestosterone = 90 ± 7, cholesterol = 65 + 4. Study 2: Serum-free control = 177 ± 18, 3,17β-estradiol = 329 ± 33 (serum-free versus Prednisolone = 187 ± 16, 6α-methylprednisolone = 173 ± 13, aldosterone = 132 ± 18. Neuroprotective effects of non-phenolic steroids There were no fruits.   Two androgens with a C3 ketone, ie, a moiety without the phenol A ring Dihydrogen of partially unsaturated testosterone and saturated compounds without phenol A ring Both rotestosterone were inactive. Similarly, the tested C-3 ketone All five 21-carbon pregnane derivatives having a group and △^Four-Steroids, Logesterone and aldosterone and △^1.4-Steroids, prednisolone and And 6-methylprednisolone were inactive. Finally, cholesterol is completely Cholesterol because it has a hydroxyl group in the highly saturated A ring Tested and was inactive. Flat phenol ring conformation and / or cyclohexa The increased activity of phenols against knol provides the observed neuroprotective activity It is important in that.   In all studies, cells were cultured in RPMI-1640 medium (serum free, SF group), 10% RPMI-1640 medium supplemented with FBS (FBS group) or 2 nM (as indicated elsewhere) Steroids at the concentration of (if not present): 3,17β-estradiol (1,3,5 ( 10) -estraditriene-3,17β-diol); 3,17α-estradiol (1,3,5 (10 ) -Estratriene-3β, 17α-diol); 3,17β-estradiol 3-O-methyl Ether (1,3,5 (10) -estraditriene-3,17β-diol 3-O-methyl ether 3,17α-estradiol 3 acetate (1,3,5 (10) -estraditrien 3β , 17α-diol 3 acetate); estrone (1,3,5 (10) -estraditriene 3-O -17-one); estrone 3-O-methyl ether (1,3,5 (10) -estraditrie 3-ol-17-one 3-O-methyl ether); estriol (1,3,5 (10) -est) Latriene-3β, 16α, 17β-triol); estriol 3-O-methyl ether (1,3,5 (10) -estraditriene-3β, 16α, 17β-triol 3-O-methyl ether); 17α-ethynyl estradiol (1,3,5 (10) -estradi Triene-17α-ethynyl 3β, 17α-diol); ethinyl estradiol 3-O- Methyl ether (1,3,5 (10) -estraditriene-17α-ethynyl 3β, 17α-dio 3-O-methyl ether); 2-hydroxyestradiol (1,3,5 (10) -est Latriene-2,3,17β triol); 2,3-methoxyestradiol (1,3,5 (10)- Estratriene-2,3,17β-triol-2,3-O-methyl ether) or estra Trien-3-ol (1,3,5 (10) -estradien-3-ol); cortisone, Logesterone, prednisolone (1,4-pregnadien-11β, 17,21-triol-3 , 20-dione); and aldosterone (4-pregnene-11β, 21-diol-3,18,20- Trion); methylprednisolone (6α-methyl-1,4-pregnadien-11β, 17, (21-triol-3,20-dione) supplemented with RPMI-1640 medium. All steroids are available from Steraloids, Inc. (New Hamp) (Wilton, Shah), first dissolved in 1 mg / 1 ml absolute alcohol, Diluted to a final concentration of 2 nM with RPMI-1640 medium. In the treated well Serum-free medium (SF group) and FBS medium to control possible ethanol effects (FBS group) was supplemented with absolute ethanol at a concentration of 544 pl / ml. For all research In this, at least 4 and usually 6 replicate wells were treated with each medium. Example 3: Tricyclic structure having neuroprotective properties   [2S- (2a, 4aα10αβ)]-1,2,3,4,4a, 9,10a-octaidro-7-hydroxy-2-methyl Ru-2-phenanthrene methanol (PAM) and [2S- (2a, 4aα, 10αβ)]-1,2,3,4 , 4a, 9,10a-Octidotro-7-hydroxy-2-methyl-2-phenanthrene carboxy Aldehyde (PACA) contains SK-N-SH cells simultaneously with serum deprivation Medium. After 24 or 48 hours, transfer cell viability to dye-colored material Determined by a dye-forming method that requires mitochondrial activity for the reduction of Goodwin et al. (1995); Immunol. Methods, 179: 95]. P Both ACA and PAM show neuroprotective activity with peak response at 2 nM concentration (FIGS. 5a and 6a). Neuroprotective activity was positive control, 3,17β-estradiated Similar to diol (FIGS. 5 and 6). Example 4: "in vivo" dose study   In rats injected with 2 hours prior to occlusion of the middle cerebral artery with some compounds, 10 Tested at a dose of 0 ug / kg body weight. The injection should be between 100 and 200 pg / ml (0.4 Plasma estradiol concentrations of up to 0.8 nM). Ovariectomized rats And a maximum lesion of 25% of the cross-sectional area of the brain was observed. With 3,17β-estradiol When treated, the maximum lesion area was reduced by about 50%. These data are Only low nM concentration of 3,17β-estradiol to protect from internal ischemic lesions Only that it is necessary.   * P <0.05 vs serum-free control group as shown in FIGS.   † Here are the common names.   The ‡ 3-O-conjugate was not available for evaluation.   * P <0.05 relative to serum-free control group

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Green, Patti S             United States, Florida 32604, Ge             Innsville, Essex Thurty             --Forth Street 2220, apartment             Statement 162 (72) Inventor Gordon, Catherine             Massachusetts, United States             01890, Winchester, Parker Lo             Mode 5

Claims (1)

[Claims] 1. (I) having a terminal phenol group having at least a second ring in the structure, 1,0 Providing a non-estrogenic compound having a molecular weight of less than 00 daltons;   (ii) administering the compound to a population of cells at a dose effective to confer neuroprotection. thing   A method of providing neuroprotection in a population of cells in a patient, comprising: 2. 2. The method of claim 1, wherein said compound comprises a tetracyclic structure. 3. 3. The method of claim 2, wherein the effective dose reaches a plasma concentration of less than 500 nM. 4. 2. The method of claim 1, wherein said compound comprises a tricyclic structure. 5. 5. The method of claim 4, wherein the tricyclic is non-steroidal. 6. 2. The method of claim 1, wherein said compound comprises a bicyclic structure. 7. 7. The method of claim 6, wherein the bicyclic compound is non-steroidal. 8. The method according to claim 5, wherein the tricyclic compound is a phenanthrene compound. 9. When the phenanthrene compound is tetrahydrophenanthrene and octahydrofuran 9. The method of claim 8, wherein the method is selected from the group consisting of enanthrene. Ten. When the phenanthrene compound is phenanthrene methanol or phenanthrene 9. The method of claim 8, wherein the method is selected from the group consisting of ruboxaldehyde. 11． 7. The bicyclic structure is selected from the group consisting of naphthol and naphthalene. The method described in. 12． Terminal phenolic groups have hydroxyl groups on any of carbons 1-4 The method of claim 1, wherein 13. At least the second ring structure is linked by a terminal phenol group and a linking group The method of claim 1. 14. At least the second ring structure is covalently linked to the terminal phenol group without a linking group The method of claim 1, wherein 15． 2. The method of claim 1, wherein the effective dose reaches a plasma concentration of less than 500 nM. Law. 16． Wherein the effective dose reaches a plasma concentration ranging from 0.02 nM to 500 nM. Item 16. The method according to Item 15. 17． 2. The method of claim 1, wherein the effective dose reaches a plasma concentration ranging from 0.1 nM to 1 nM. 6. The method according to 6. 18． The method of claim 1, wherein the compound has a molecular weight greater than 170 daltons. Law. 19. 2. The method of claim 1, wherein the terminal phenol group is a phenol A ring. 20. (a) having a terminal phenol and at least a second ring structure, less than 1,000 daltons Preparing an effective dose of a formulation containing a compound having a molecular weight of   (b) administering the dose to the patient   A method of treating neurodegenerative degenerative disease in a patient, comprising: twenty one. 18. The compound of claim 17, wherein the compound has a molecular weight greater than 170 Daltons. Method. twenty two. 21. The method of claim 20, wherein the terminal phenol is a phenol A ring.