WO1996021449A1

WO1996021449A1 - Pharmaceutical composition of indene derivatives for regulating apoptosis

Info

Publication number: WO1996021449A1
Application number: PCT/JP1995/002578
Authority: WO
Inventors: Tetsuro Kikuchi; Katsura Tottori; Yasufumi Uwahodo; Hideo Tanaka; Hiroyuki Ichikawa; Yukihisa Ono; Satoru Nakai
Original assignee: Otsuka Pharmaceutical Co., Ltd.
Priority date: 1995-01-11
Filing date: 1995-12-15
Publication date: 1996-07-18
Also published as: EP0749311A1; MX9603868A; CN1143322A; CA2185246A1; AU4188896A; AR001786A1

Abstract

The present invention provides a pharmaceutical composition for regulating apoptosis containing at least one active ingredient selected from the group consisting of 2,3-dihydro-1H-indene derivatives represented by general formula (I), (wherein R?1, R2, R3 and R4¿ are each the same or different and are lower alkyl groups; R5 is a lower alkoxy group; and A is a lower alkylene group) and salts thereof. The present invention also provides methods for treating and preventing various diseases by regulating apoptosis by use of said pharmaceutical composition.

Description

DESCRIPTION

PHARMACEUTICAL COMPOSITION OF INDENE DERIVATIVES FOR REGULATING APOPTOSIS

INDUSTRIAL FIELD OF THE INVENTION

The present invention relates to a pharma¬ ceutical composition for regulating apoptosis and methods for treating diseases in connection with the apoptosis by using said pharmaceutical composition .

RELATED ART STATEMENT

2 , 3-Dihydro-lH-indene derivatives represented by the general formula ( I ) :

(wherein R¹, R², R³ and R* are each the same or different, and are lower alkyl groups; R⁵ is a lower alkoxy group; and A is a lower alkylene group), and salts thereof are known compounds, they are disclosed, for example, in Japanese Patent Publication No. Hei-4- 77740 and US-A-4,895,847.

These 2,3-dihydro-lH-indene derivatives and salts thereof have activities for improving symptoms caused by deficiency of oxygen and syndrome accompanied therewith. Therefore, the 2,3-dihydro-lH-indene derivatives and salts thereof are known as agents for improving hypoxia, more particularly they are used effectively as agents for activating the brain, agents for curing amnesia, agents for curing senile dementia, agents for improving respiratory arrest accompanied with poisoning of potassium cyanide and hypoxia, as well as agents for preventing arrhythmia and heart failure caused by deficiency of oxygen.

Fatty emulsion preparation for other pharma¬ ceutical purpose containing said 2,3-dihydro-lH-indene derivatives is disclosed in EP-A-483,854, and other pharmaceutical preparation thereof is disclosed in Japanese Patent Kokai (Laid-open) No. Hei 3-112,928 and the intermediates for preparing said indene derivatives are disclosed in US-A-4,792,628 and J. Med. Chem. , 34, 2004-2013 (1991). Chemistry of synthesis of said indene derivatives is disclosed in J. Med. Che ., 34, 2014-2023 (1991).

Furthermore, pharmacological effects of said indene derivatives, other than the activity for regu¬ lating apoptosis, are disclosed in Drugs of the Future, ϋ, (8), 707-710 (1993), Neuropathol. , 11, 89-104 (1991), Journal of the Neurological Sciences, 109, 107-110 (1992) and Free Rad. Res. Comms. , 1__> (4), 223-230 (1991). BACKGROUND ART OF THE INVENTION

Hitherto, it is understood that cell deaths are caused by two types of mechanisms. One type of the cell death mechanisms is so called as "necrosis", which is a classic cell death. From the morphological stand¬ point, the necrosis is characterized by sequential changes in the cell, that is, there is arisen remark¬ able swelling of the mitochondria, swelling of the cytoplasm, degeneration of the nucleus, followed by collaps of the cell and autolysis of the whole cell. These sequential phenomina in the cell are took place passively and incidentally. Generally, it is recog¬ nized that such necrosis of cell (tissue) may be caused by physical injury (damage) and by chemical poison(s). Another type of the mechanisms of cell death is so called as "apoptosis (programmed cell death)". (Kerr, J. F. R. and Wyllie, A. H., Br. J. Cancer, 26, 239 (1972)). It is understood that the apoptosis is arisen under various physiological conditions. The cell to be taken apoptosis is morphologically charac¬ terized by, for example, defect in contacting with peripheral cells, tendency to condensation of the cyto¬ plasm, condensation of the chromatin and condensation of the nucleus, regarding activity of the endonuclease, and segmentation tendency of the nucleus. Furthermore, disappearance of microvillus on the cell surface, smoothing of the cell surface (formation of bulla (brister) on the cell surface, i.e., (membrance blebbing)), etc., are also observed. Yet, further, in accordance with activity of the endonuclease, it was observed a phenomenon that the nucleosome units in DNA were divided into fragments having 180 to 200 of the base pairs, thus there was dicussed the mechanism in that the final fragments of apoptic body of the cell were phagocytized by the adjoining cells. (Duvall, E. and Wyllie, A. H., Immunology Today, 2 (4), 115-119 (1986); Idem, Science, 245. 301-305 (1989)). Wyllie, one of the above-mentioned authors, also reported that apoptosis of the thymocyte which was induced by glucocorticoid involves activation of endonuclease in the cells. (Wyllie, A. H., Nature, 284. 555-556 (1980)). DNA in the cell to be taken apoptosis caused by the activity of endo-nuclease is divided into fragments of certain extent of oligonucleotide level, and the size of which can be confirmed easily by conducting an agarose gel electrophoresis.

The above-mentioned apoptosis were observed in the processes of genesis, differentiation and turnover of the tissues, thus it was considered as programmed cell death. (Wyllie, A. H., et al.. Int. Rev. Cytol., .68./ 251-306 (1980)).

When the concentration of calcium in the calcium ionophore was increased, or the concentration of c-AMP was increased in the thymocyte, then the cleavage of DNA, which is feature of the above- mentioned apoptosis, was promoted. (Wyllie, A. H., et al., J. Pathol., 142. 67-77 (1984)). Thus, it was guessed that both concentrations of calcium ion and c-AMP may be connected with the mechanism of apoptosis. Furthermore, apoptosis of HL-60 cell which was dif- ferentially induced by retinoic acid or calcium ionophore were reported as the examples of the above- mentioned guess. (Martin, S. J., et al., J. Immunol., 145. 1859-1867 (1990); Martin, S. J., et al., Clin. Exp. Immunol., T_, 448-453 (1990)). Some prior art literatures disclosed that, in view of the physiological cell death shown in the cells (e.g., liver, adrenal cortex and prostate gland, etc.) which is observed in the process of blastogenesis or in the process of vigorously version of normal cell, the above-mentioned apoptosis may be induced by treat¬ ment with glucocorticoid, cytotoxicity caused by the cytotoxic T-cell, atrophy of the hormone-dependent tissue, exposure to radiations, NK (natural killer) cell, killer cell, tumor necrosis factor (TNF), and cytokines such as lymphotoxin (LT), etc. (Wyllie, A. H., et al., Int. Rev. Cytol., 6jB, 251 (1980); Duvall, E. and Wyllie, A. H., Immunology Today, 1_, 115-119 (1986); Sellins, K. S., et al., J. Immunol., 139. 3199 (1987); Yamada, T. , et al.. Int. J. Radiat. Biol. , 53. 65 (1988); Wyllie, A. H., Nature, 284. 555 (1980);

Schmid, D. S., et al., Proc. Natl. Acad. Sci., U. S. A., B_, 1881-1885 (1986); John, C, et al., J. Immunol, 129. (4), 1782-1787 (1982); Howell, D. M. , et al. , J. Immunol., 140. 689-692 (1988); Gillian, B., et al., Eur. J. Immunol., 11_, 689-693 (1987)). In addition to the above, there were reported that even though some antibodies, for example, anti-CD3 antibody, anti-Apo-I antibody, anti-Fas antibody, etc., can be able to induce apoptosis. (Trauth, B. C, et al.. Science, 245. 301-305 (1989); Smith, C. A., et al.. Nature, 337. 181-184 (1989); and Tadakuma, T., et al., Eur. J. Immunol., 2_, 779 (1990)), and also even in the observation of autoregression of malignant tumor, apoptosis was confirmed. (Nakamura Yasuo, et al., RINSHO-HIFUKA (Clinical Dermatology), .35., (4), 289-295 (1981)).

While, actinomycin D which is an inhibitor of RNA-synthesis, cycloheximide which is an inhibitor of protein synthesis, as well as chelating agents for calcium ion (Ca^z+), etc. were reported as agents for inhibiting the above-mentioned apoptosis. Furthermore, there were reported that Cyclosporin A which is an immunosupressive agent, hematopoietic cytokines (IL-3

(interleukin-3) , GM-CSF (granulocyte-macrophage colony- stimulating factor), G-CSF (granulocyte colony- stimulating factor) and the like), IL-2 (interleukin- 2), bcl-2 gene product, etc. inhibit the apoptosis. (Cohen, J. J., J. Immunol., 132. 38 (1984); Wyllie, A. H., et al., J. Pathol., 142. 67 (1984); Shi, Y., et al.. Nature, 339. 625 (1989); Williams, G. T., et al.. Nature, 343. 76 (1990); Nielo, M. A., J. Immunol., 143. 4166 (1989); Vaux, D. L. , et al.. Nature, 335. 440 (1988)). Provided that, there were reported that the above-mentioned cycloheximide induces apoptosis of acute leukemia cells, actinomycin D induces apoptosis of intestinal crypt cells, and both cycloheximide and actinomycin D induce apoptosis of HL-60 cells, respec¬ tively. (Martin, S. J., et al., J. Immunol., 145. 1859-1867 (1990)). On the contrary, there were reported that cycloheximide inhibited the apoptosis of lymphoid tumor cells, existed before exposure of X-ray, and the number of which were increased after the exposure of X-ray. Also, there were reported that actinomycin D increased the above-mentioned apoptosis. Thus, there were suggested that inhibition or promotion of apoptosis may be arisen depend on type of the cells to be taken, various conditions and other mechanisms, etc. (Igarashi Tadahiko, et al., NIHON-KETSUEKI- GAKUKAISHI (Journal of Japanese Hematology Association), 5j,, (2), 144 (1988)). At all events, there was considered that differentiation, prolife¬ ration and maturation of the cells are closely related to the apoptosis. Thus any substance which may have biological activities in connection with the differ¬ entiation, proliferation, etc., to cells can be considered as related to the apoptosis.

Recently, treatment of cancer by using anti- Apo-I antibody is trying as a treating method in connection with the apoptosis. Among myelodysplastic syndrome (MDS), such as refractory anemia (RA) in which pancytopenia is the main cause, and sideroblastic anemia (RARS), the use of retinoic acid as to an agent for inducing differentiation of hemato-poietic cells, activated type-vitamin D₃ as well as the use in combi¬ nation with GM-CSF (granulocyte-macrophage colony- stimulating factor) and IL-3 (interleukin-3) as to regulating agents for inhibiting the excessive apoptosis of thrombocyte producing cells may be prefer- able. Furthermore, among myelodysplastic syndrome

(MDS), when RAEB in which proliferation of the blast is predominance and said RAEB is in transition period (RAEB-t), the above-mentioned retinoic acid and activated type-vitamin D₃ may be used as agents for inducing differentiation of the hematopoietic cells to formation of the blast. Also, Etoposide and aclarubicin which inhibit proliferation of blast may be act as agents for regulating the apoptosis, i.e., agents for promoting the apoptosis. (Shibuya, T., J. Clinical and Experimental Medicine, 160. (5), 319-323 (1992)).

Additionally, Murakami, et al. found the facts that about a half number of transgenic mice, which were expressing anti-erythrocyte autoantibody, onset autoimmune diseases due to loss of their self- tolerance, and they reported that such autoimmune diseases may be given by lack of elimination ability of autoantibody producing cells which was induced by apoptosis due to reaction of autoantigen with auto¬ antibody producing cells as in the case of normal mice. (Murakami, M. , et al.. Nature, 357. 77-80 (1992)). Watanabe-Fukunaga suggested that when Fas antigen relating to the apoptosis in MRL lpr/lpr mouse, involves abnormalities, and the negative selection (apoptosis) mechanism in autosensitized T-cell (autoimmune T-cell) in the thymus did not act properly, as the results autoimmune diseases become onset. (Watanabe-Fukunaga, R., et al. , Nature, 356, 314-317 (1992)).

Montagnier, et al. reported that there were observed apoptic band of DNA in the T-lymphocyte extract obtained from HIV-infected patient, and such phenomena were observed 90% of asympomatic HIV-infected patients, and 100% of AIDS and ARC (AIDS related complex) patients, such phenomena may be understood that induction of the apoptosis were promoted even though in HIV-infencted patients. (Montagnier, L. , et al., Sixieme Colloque des Cent Gardes, 9-17 (1991)).

There was reported that, in case of observing the cell death of chicken (avian) in the development phase, when NGF (nerve growth factor: a protein that promotes hypertrophy of the nerve cells at the ganglionic neurons of nerve cells and elongation of the nerve fiber) was administered in advance to the chicken embryo, then the nerve cell death in the development phase was completely inhibited. (Hamburger, V. et al.. J. Neurosci., _, 60 (1981)). On the contrary, there was reported that when the antibody against NGF was administered to the chicken embryo, then about 90% of juvenile sympathetic nerve cells were lost. (Levi- Montalchini, R. and Booker, B., Proc. Natl. Acad. Sci., U. S. A., _, 384 (1960) ) .

Clark proposed that the nerve cell death phenomena arisen in the nature may be classified into three types, and among these classified cell deaths, he concluded that morphological feature of type I of them was identical to apoptosis, further the cell death caused by deletion of growth factor resulted such cell death of type I, and segmentation of DNA was also occurred, to which he cosidered that such type I of cell death may be called as apoptosis. (Clark, P. G. H., Anat. Embryol., 181. 195 (1990); J. Neurosci., .1, 60 (1981); Proc. Natl. Acad. Sci., U. S. A., .46., 384 (1960); Rawson, C. L., et al., J. Cell. Biol. , 113. 671 (1991)). Edwards, et al. reported that programmed sympathetic nerve cell death can be inhibited by NGF (nerve growth factor), he concluded that the apoptosis may be inhibited with NGF. (Edwards, S. N., et al., J. Neuro-chemistry, 2 (6), 2140-2143 (1991)). According to the report by Fischer, et al., when NGF was administered to a rat having lerning disturbance by aging, said NGF affects to the cholinergic nerve cells at the forebrain basal field which is known as the area capable to receive disturbance of Alzheimer's disease, and observed that said lerning disturbance can be recovered. (Fischer, W., et al.. Nature, 329. 65 (1987); Barde Y-A, Neuron, 2, 1525 (1989); Hatanaka, H., Develop. Brain Res., 30. 47 (1986); Hatanaka, H. , et al.. Develop. Brain Res., 9., 85 (1988)). Hatanaka, et al. reported that the above-mentioned NGF was effective to differen¬ tiation, maturation, maintenance of survival and prevention of aging, thus Hatanaka suggested that NGF possesses capabilities of protective and recovery actions to disturbance of nerve cells and of preventive action to neuropathies involving aging of the brain, especially possesses capability for preventing the nerve cell death in Alzheimer's disease. (Hatanaka Hiroshi, TAISHA (Metabolism), 28, 891-899 (1991)).

There were suggested that, β-amyloid protein which is an important substance to consider the causal relationship to the onset mechanism of Alzheimer's disease, glutamic acid which is one of the neurotrans- mitters, death of neuron caused by l-methyl-4-phenyl- pyridinium ion (MPP⁺) which is known as a model of Parkinson's disease, as well as nervous disease caused by prion protein are related to apoptosis. (Enokido, Y. and Hatanaka, H. , GAN-TO-KAGAKU RYOHO (Cancer and Chemotherapy), 2_, (5), 615-620 (1994); Gianluigi Forloni, et al., Neuro. Report, 4., (5), 523-526 (1993)). Additionally, there were reported that multiple factors are related to the primary cause of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease and the like, while the secondary cause to these neurodegenerative diseases may be the apoptosis caused by decreasing of the functions of nerve growth factor. (Andrew Eisen and Charles Krieger, Can. J. Neurol. Sci., 20, (4), 286-296 (1993)).

There was reported that degeneration of black neuron appeared in Parkinson's disease may be related to apoptosis induced by dopamine. (Ilan Ziv, et al., Neuroscience Letters, 170. 136-140 (1994)). Further, in case of amyotrophic lateral sclerosis (ALS), the relationship between oxygen stress and disturbance of neuron was noticed, thus there was suggested that death of neuron being induced by oxygen was took place by a mechanism similar to the apoptosis through an inherent suicide mechanism in the cells. (Enokido, Y., and Hatanaka, H., GAN-TO-KAGAKU RYOHO (Cancer and Chemotherapy), 2_t (5), 615-620 (1994)).

There was thought that, in the hepatopathy of drug tolerance viral hepetitis, the acceleration of apoptosis which was took place directly by drug or viral infection, or through immunological mechanisms may be related to hepatopathy. (Bursh, W., et al., TiPS, i , 245-251 (1992)).

On the other hand, there was known that hepatocytes were proliferated with mitogen which bring about hyperplasticized state in the liver, such state may be recovered to normal state by decidual necrosis of hepatocytes, that is, by apoptosis. (Kerr, J. F., et al., Br. J. Cancer, 26, 239-257 (1972)). Said apoptosis phenomena are recognized as hyperplasticized liver, hyperplastic node and cancer of liver in the liver. (Columbano, A., et al.. Lab. Invest., 52, 670- 675 (1985); Columbano, A., et al.. Am. J. Pathol., 116, 441-446 (1984)). Kerr, et al. reported that apoptosis does not bring about inflammation or fibrosis. (Kerr, J. F., et al.. Lancet, 2_, 827-828 (1979)).

In view of these facts that if apoptosis can be regulated in connection with acute and chronic hepatitis, then it can be considered that these hepatitises can possibly be cured. In the process of changing over the chronic hepatitis to hepatic cirrhosis and to hepatic cancer, the above-mentioned apoptosis is in inhibitory state, and such state is thought to be allowing fibrosing of the hepatocyte followed by inflammation of the hepatocyte caused by cytotoxic T-cell, then development of the fibrosis to hepatic cirrhosis, thus it can be considered that if the apoptosis can be shifted into promotion state, then it can be considered that inhibition of hepatitis and development to hepatic cirrhosis can possibly be prevented. OBJECT AND SUMMARY OF THE INVENTION

The present inventors have made extensive research works on 2,3-dihydro-lH-indene derivatives represented by the above-mentioned general formula (I) and salts thereof, and as the result, we have found the facts that these 2,3-dihydro-lH-indene derivatives and salts thereof possess activities for regulating (i.e., activities for inhibiting and promoting) apoptosis which could not have been anticipated from the pharma¬ cological activities known to them previously. The present invention was completed successfully on the basis of said findings.

The present invention relates to a pharma¬ ceutical composition for regulating apoptosis contain¬ ing at least one active ingredient selected from 2,3- dihydro-lH-indene derivatives represented by the general formula (I) :

(wherein R¹, R², R³ and R* are each the same or different, and are lower alkyl groups; R⁵ is a lower alkoxy group; and A is a lower alkylene group) and salts thereof, as well as relates to methods for treating and preventing diseases in connection with apoptosis by use of said pharmaceutical composition.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided the pharmaceutical composition for regulating apoptosis containing at least one active ingredient selected from the group consisting of 2,3-dihydro-lH-indene derivatives represented by the general formula (I) and salts thereof.

The pharmaceutical composition of the present invention possesses the activities for regulating apoptosis, and on the basis of said activities, said composition is useful for treating and preventing various diseases as mentioned previously. More particularly, the pharmaceutical composition of the present invention can preferably be applied for treat¬ ing and preventing various diseases for example: cancer; HIV or HTLV-1 related complexes such as, AIDS, ARC (AIDS related complex), ATL (Adult T-cell leukemia), hairy cell leukemia, myelopathy (HAM/TSP), disturbance of respiratory organs (HAB/HABA), arthro- pathy (HAAP), uveitis (HAU), etc.; retro virus-related diseases, such as hepatitis C, etc.; autoimmune diseases such as collagen diseases of SLE (systemic lupus erythematosus) and chronic rheumatic arthritis (RA), etc., ulcerative collitis, Sjδgren syndrome, primary biliary cirrhosis, idiopathic thrombocytopenic purapura (ITP), autoimmune hemolytic anemia, myasthenia gravis, Hashimoto disease, insulin-dependent (I type) diabetes mellitus, etc.; various diseases accompanying with thrombocytopenia such as, myelodysplastic syndrome (MDS), periodic thrombocytopenia, hypoplastic anemia, idiopathic thrombocytopenia diffuse intravascular coagulation disease, etc.; hepatic diseases such as, vairal hepatitises C, A, B, F, etc., drug-induced hepatitis, hepatic cirrhosis, etc.; Alzheimer's disease and Alzheimer-type senile dementia; Parkinson's disease; Huntington's disease; prion disease; myocarditis; ARDS (adult acute respiratory disease); amyotrophic lateral sclerosis (ALS); infectious diseases; prostatic hypertrophy; uterine myoma; bronchial asthma; arteriosclerosis; various congenital malformations; renal disease; senile cataract; chronic fatigue syndrome (CFS) and myotonic dystrophy; disturbance of memories such as HIV-related disturbance of memory, etc. Thus the pharmaceutical composition for regulating apoptosis of the present invention is effective for various pharmaceutical fields.

Each one of the substituents respectively shown in the above-mentioned general formula (I) are described concretely as follows.

As to the lower alkyl group, a straight chain- or branched chain-alkyl group having 1 to 6 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, 1- methylpropyl, 2-methylpropyl, 1,1-dimethylpropyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2- dimethylpropyl, 2,3-dimethylpropyl, 1-methylpentyl, 1,1-dimethylbutyl and 1-ethylbutyl groups, etc.

As to the lower alkoxy group, a straight chain- or branched chain-alkoxy group having 1 to 6 carbon atoms, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy. pentyloxy and hexyloxy groups, etc.

As to the lower alkylene group, a straight chain- or branched chain-alkylene group having 1 to 6 carbon atoms, for example, methylene, ethylene, tri- methylene, 2-methyltrimethylene, 2,2-dimethyltri- methylene, 1-methyltrimethylene, methylmethylene, ethylmethylene, tetramethylene , pentamethylene and hexamethylene groups, etc.

Among the active ingredients to be contained in the pharmaceutical composition for regulating apoptosis of the present invention, l-[4-(3-methoxy- phenyl)-1-piperazinyl]acetylamino-2,3-dihydro-7- hydroxy-2,2,4,6-tetramethyl-lH-indene or salt thereof is particularly preferable. In addition to the ability for regulating apoptosis, the pharmaceutical composition for regulat¬ ing apoptosis of the present invention also possesses activity for inducing differentiation of cell, activity for inhibiting proliferation of cancer cell, carcino- static activity, anti-retro virus activity, activity for improving disturbance of memory, activity for inhibiting production of cytokines, etc., thus said composition can be preferably used as carcinostatic agent, agent for inhibiting metastasis of cancer, agent for improving disturbance of memory such as HIV-related disturbance of memory, agents for curing and preventing Alzheimer's disease, anti-retro virus agent, agent for inhibiting production of cytokines, etc.

In case of using the pharmaceutical composi¬ tion for regulating apoptosis of the present invention, for example as used for carcinostatic agent, by administration thereof, directly after the cancer cell was differentiated or without differentiated, induction of the apoptosis can be promoted or inhibited, then the carcinostatic activity of the pharmaceutical composi¬ tion comes out.

In this case, regardless of pharmaceutical preparation forms and administration routes, the pharmaceutical composition of the present invention can be used in combination with, for example, various other carcinostaic agents which are known as chemotherapeutic agents, or radiation therapy. Because of the active ingredient compounds of the present invention show excellent carcinostatic effects, they can further promote the effects of other carcinostatic agents which are used in combination therewith, so that synergestic effects can be expected. Therefore, in case of using other carcinostatic agent to be used in combination with the pharmaceutical composition of the present invention, even if the said carcinostatic agent is used in an amount considerably smaller than that of in a common usage, then sufficient therapeutic effects in treatment of cancer can be obtained, so that side-effects of the carcinostatic agent used in combination therewith can be reduced. As to the chemotherapeutic agents, 5-fluorouracyl

(5-FU, manufactured by Kyowa Hakko Kogyo Co., Ltd.), Mytomycin-C (manufactured by Kyowa Hakko Kogyo Co., Ltd.), Tegafur (Futraful, manufactured by Taiho Pharmaceutical Co., Ltd.), Cyclophosphamide (Endoxan, manufactured by Shionogi & Co., Ltd.) and Chromomycin A₃ (Toyomycin, manufactured by Takeda Chemical Industries, Ltd.) can be exemplified.

The pharmaceutical composition for regulating apotosis of the present invention is also useful as agent for curing and preventing Huntington's disease, Parkinson's disease and ALS. The effect in treating and preventing the above-mentioned diseases can be achieved by effects for inhibiting apoptosis of the pharmaceutical composition of the present invention. The regulating agent for apoptosis of the present invention is also useful as agent for treating and preventing Alzheimer's disease. In this case, for example, classical Alzheimer's disease and Alzheimer- type senile dementia, the pharmaceutical composition for regulating apoptosis of the present invention shows the effect similar to that of shown by NGF (nerve growth factor) according to the effects for inhibiting the apoptosis. The effect for treating and preventing the above-mentioned diseases can be achieved by the pharmaceutical composition of the present invention. In addition to the above, the pharmaceutical composi¬ tion for regulating apoptosis of the present invention can be used in combination with drugs for treating

Alzheimer's disease previously known in the art, such as, an agent for improving circulation of cerebral blood flow and agent for improving cerebral metabolism, etc., and said pharmaceutical composition assists the effects of these known drugs, and sometimes reduces the side-effects of these known agents.

The pharmaceutical composition for regulating apoptosis of the present invention possesses anti-retro virus activity, thus it can be used preferably as agents for treating and preventing retro virus-related diseases such as, the above-mentioned various HIV or HTLV-I related diseases and hepatitis C, etc.

The pharmaceutical composition for regulating apoptosis of the present invention can be applied to agents for treating and preventing disturbance of memories involving HIV-related disturbance of memory.

The pharmaceutical composition for regulating apoptosis of the present invention possesses activity for inhibiting production of cytokines, thus said pharmaceutical composition can be preferably applied as agent for inhibiting production of cytokines for curing various diseases accompanying with abnormal production of cytokines, particularly infectious diseases caused by various bacteria and parasites (IGAKU-NO-AYUMI (Course of Medicine), 159. (8), 467-470, 471-474 (1991)); RA (Rheumatic Arthritis) (Arthritic Rheum., 31. 1041 (1988); Ibid. .34., 1125 (1991); J. Immunol., ϋ , 4154 (1990); Ibid. 22, 1907 (1992); Bri. J.

Rheum., 31, 293 (1992); Eur. J. Immunol., .18., 1797 (1989)); ARDS (adult acute respiratory disease) (Nature, 324. 73 (1986)); fulmination of viral hepetitis (Lancet, ii, 1_ (1986)); CSF (colony stimulating factor) (NIHON-RINSHO (Japanese Clinical Medicine), 10., (11), 51-55 (1992); J. Infectious Diseases, 165. 994-1000 (1992)); hyper γ-globlinema, intraarterial myxoma (mucos tumor) accompanying with increasing of acute phase protein, and Castleman's syndrome (Blood, 7±, 1360 (1989); Eur. J. Immunol., 18. 1797 (1989)); mesangial proliferative glomerulone- phritis (PGN) (CHIRYO-GAKU (Curative Medicine), 2_, (1), 49 (1990)) and above-mentioned various autoimmune diseases and HIV or HTLV-related complex. The 2,3-dihydro-lH-indene derivatives repre¬ sented by the above-mentioned general formula (I) of the present invention include inevitably their optical isomers .

The 2,3-dihydro-lH-indene derivatives represented by the above-mentioned general formula (I) can be easily changed to their acid-addition salts by reacting with pharmaceutically acceptable acids. As to the pharmaceutically acceptable acids, inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid; organic acids such as acetic acid, oxalic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, malonic acid, methanesulfonic acid and benzoic acid can be exemplified. In the present invention, these acid- addition salts can be used as the active ingredient similar to the 2,3-dihydro-lH-indene derivatives represented by the general formula (I) in free form.

The 2,3-dihydro-lH-indene derivatives repre¬ sented by the above-mentioned general formula (I) or salts thereof can be used, generally in any forms of usual pharmaceutical preparations. The pharmaceutical preparations are prepared by using diluents or excipient such as fillers, diluents, binding agents, wetting agents, disintegrating agents, surface active agents, lubricants, etc. As to the pharmaceutical preparations, various forms of preparations can be selected depend on the objectives of medical treat¬ ments, and typical examples are tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, injection preparations (liquids, suspensions, etc.), ointments, etc..

In case of shaping the regulating agent in the form of tables, carriers which are widely known in this field can be used, for example excipients such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cel¬ lulose, silicic acid, etc.; binding agents such as water, ethanol, propanol, simple syrup, glucose solu¬ tions, starch solutions, gelatin solutions, carboxy- methylcellulose, shellac, methyl cellulose, potassium phosphate and polyvinylpyrrolidone, etc.; disintegrat¬ ing agents such as dried starch, sodium alginate, agar-agar powder, laminalia powder, sodium hydrogen carbonate, calcium carbonate, esters of polyoxyethylene sorbitan fatty acids, sodium laurylsulfate, mono- glyceride of stearic acid, starch, lactose, etc.; disintegration inhibitors such as white sugar, stearin, cacao butter, hydrogenated oils, etc.; absorption accelerators such as quaternary ammonium bases, sodium laurylsulfate, etc.; wetting agents such as glycerin, starch, etc.; adsorption agents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, etc.; and lubricants such as purified talc, stearic acid salts, boric acid powder, polyethylene glycols, etc. can be exemplified. If necessary, the tables can be further coated with usual coating materials to make them into coated tablets, for example tablets coated with sugar, tablets coated with gelatin film, tablets coated with enteric coating layers, tablets coated with films or double layer tablets as well as multiple layer tablets.

In case of shaping the regulating agent in the form of pills, any carrier which is known in this field can be widely used, for example excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oils, kaolin, talc, etc.; binding agents such as arabic gum powder, tragacanth gum powder, gelatin, ethanol, etc.; and disintegrating agents such as laminaran, agar-agar, etc., can be exemplified.

In case of shaping the regulating agent in the form of suppositories, carriers which are known in this field can be widely used, for example polyethylene glycols, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthesized glycerids, etc. can be exemplified.

Capsule preparations are prepared in accord¬ ance with usual method by mixing the active ingredient with the above-mentioned variuos carriers, and thus obtained mixture is filled into hard-gelatin capsules, soft-gelatin capsules, or the like.

In case of making injection preparations, solutions and suspensions prepared are further sterilized and are preferably isotonic to the blood.

In preparing the injection preparations in the form of solutions, emulsions and suspensions, the all of carriers which are known in this field can be used, for example water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. can be exemplified. In these instances, an adequate amount of sodium chloride, glucose or glycerin may be added to the desired injection preparation to make them isotonic. Furthermore, usual dissolving additives, buffer agent, analgesic agents may be added. Also if necessary, coloring agents, preservatives, perfumes, seasoning agents, sweetening agents and other medicines may be added in the desired pharmaceutical prepara¬ tions.

In case of making the preparations in the form of pastes, creams and gels, diluents such as, white petrolatum, paraffin, glycerin, cellulose derivatives, polyethylene glycols, silicones, bentonite, etc. can be added.

The amount of 2,3-dihydro-lH-indene deriva¬ tives represented by the general formula (I) or salts thereof to be contained in the pharmaceutical composi¬ tion for regulating apoptosis of the present invention is not specially restricted, and can be suitably selected from a wide range, and generally 1 to 70 % by weight thereof may be contained therein. Methods for administrating the above- mentioned pharmaceutical composition for regulating apoptosis are not specially restricted, they can be administered in various forms of pharmaceutical preparations depend on the age of the patient, the distinction of sex, the degree of symptoms and other conditions of the patient. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally. Injection prepara¬ tions are administered intravenously singly, or administered with usual injectable transfusions such as glucose solutions, amino acids solutions or the like; further if necessary, the injection preparations are administered singly intramuscularly, intracutaneously, subcutaneously or intraperitoneally. The suppositories are administered into the rectum. The ointment prepa¬ rations are coated on the skin.

The dosage of the above-mentioned pharma- ceutical composition for regulating apoptosis can be selected suitably according to the method of admin¬ istrations, the age of the patient, the distinction of sex and other conditions, as well as the degree of the symptoms. Generally, about 0.2 to 200 mg per kg of the body weight per day of 2,3-dihydro-lH-indene repre¬ sented by the general formula (I) or salt thereof, as the active ingredient, may be administered.

WORKING EXAMPLES OF THE INVENTION

Examples of pharmaceutical preparations and of pharmacological tests are shown as follows. Example of Pharmaceutical preparation - 1

l-[4-(3-Methoxyphenyl)-l-piperazinyl]- acetylamino-2,3-dihydro-7-hydroxy- 2,2,4,6-tetramethyl-lH-indene 200 mg Glucose 250 mg

Distilled water for injection σ.s.

Total amount 5 ml

1-[4-(3-Methoxyphenyl)-1-piperazinyl]- acetylamino-2,3-dihydro-7-hydroxy-2,2,4,6-tetramethyl- lH-indene (active ingredient) and glucose were dissolved in distilled water for injection, then this solution was filled in an ampule of 5 ml volume. After the air in the filled ampule was replaced with nitrogen gas, then the filled ampule was sterilized with hot steam under pressure at 121°C for 15 minute to obtain the injection preparation having the above-mentioned formulation.

Example of Pharmaceutical preparation - 2

1-[4-(3-Methoxypheny1)-1-piperaziny1]- acetylamino-2,3-dihydro-7-hydroxy-

2,2,4,6-tetramethyl-lH-indene 100 g

Avicel (a trademark for microcrystalline cellulose, manufactured by Asahi Chemical Industry Co., Ltd.) 40 g Corn starch 30 g Magnesium stearate 2 g

TC-5 (a trademark for hydroxypropyl methylcellulose, manufactured by Shin-Etsu Chemical Co., Ltd.) 10 g Polyethylene glycol-6000 3 g

Castor oil 40 g

Methanoi 40 q

1-14-(3-Methoxyphenyl)-1-piperazinyl]- acetylamino-2,3-dihydro-7-hydroxy-2,2,4,6-tetramethyl- lH-indene (active ingredient), Avicel, corn starch and magnesium stearate were admixed together and ground, then the mixture obtained was shaped into tablets by using a tablet machine (R=10 mm) . The tablets obtained were coated with a film coating consisting of TC-5, polyethylene glycol-6000, castor oil and methanoi to prepare the film coated tablets having the above- mentioned formulation.

Examples of pharmacological tests The following pharmacological tests were conducted by using l-[4-(3-Methoxyphenyl)-l- piperazinyl]acetylamino-2,3-dihydro-7-hydroxy-2,2,4,6- tetramethyl-lH-indene (hereinafter referred to as "test compound 1") as the test compound. Pharmacological test - 1

(Activity for inhibiting proliferation of human myelogenous leukemia cell strain HL-60)

Cells of strain HL-60 obtained by successive cultivation were centrifuged (centrifugal separator: Model 05PR-22, manufactured by Hitachi Ltd.) at 25°C, 1200 rp for 5 minutes, then the cells were suspended in RPMI-1640 culture medium (manufactured by Gibco Co.), containing 10% FBS (fetal bovine serum: Lot. 41K1295, manufactured by Gibco Co.). The living cells were dyed with 0.2% Trypan Blue (manufactured by Wako Pure Chemical Industries, Ltd.), and number of the dyed cells was counted by using an optical microscope (Model: BH-2, manufactured by Olympus Optical Co., Ltd. ) and the cell suspension was diluted so as to adjust number of the cells to 1.2 x 10⁵ cells/ml.

To each one of the wells on a 96-well microplate (Code No.: 25860, manufactured by Corning Glass Works) was added (dropped) a culture medium (RPMI-1640 culture medium containing 10% FBS) in an amount of 0.1 ml/well. Next, to each one of the wells in A-row on the microplate, 0.1 ml each of a diluted solution, containing the test compound in the concent¬ ration of 40 μ/ml, then the diluted solution was subjected to diploidic step dilution repeatedly to H-row (7 folds). To the whole wells on the 96-well microplate, keeping a series (from A-row to H-row) of the diluted solutions of the test compound, there were added (dropped) 0.1 ml each of the HL-60 cell suspen¬ sion (1.2 x 10⁵ cells/ml). Then said microplate was subjected to cultivation by keeping it in a C0₂- incubator (manufactured by Napco Co.) at 37°C, under the condition of 5% C0₂, for 3 days. After the cultivation, to each one of the wells on said micro¬ plate, there were added (dropped) a PBS(-) solution (manufacture by Nissui Pharmaceutical Co., Ltd.), containing 2.5 mg/ml of MTT (MTT (3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (manu¬ factured by Wako Pure Chemical Industries, Ltd.)), in an amount of 0.01 ml/well, then said microplate was further subjected to cultivation at 37°C, under the condition of 5% C0₂, for 3 hours. Then, to each one of the wells on said microplate, there was added (dropped) a dissolving solution (0.01 N hydrochloric acid, containing 10% of SDS (sodium dodecyl sulfate), manufactured by Wako Pure Chemical Industries, Ltd.) in an amount of 0.1 ml/well, and the optical density at 580 nm was measures by using an instrument (Model: Titertech-Multiscan MMC, manufac¬ tured by Flow Laboratories, Co. ) . Amount of the dye being taken in the living cells was determined from the optical density (optical absorbance), and the rate of inhibition for prolirefation of the cells (%) was calculated from the following formula: Rate of inhibition for proliferation of the cells (%) = (1-M/C) x 100

wherein M is an amount of dye being taken in the cells of the test group using test compound;

C is an amount of dye being taken in the cells of control group without using test compound.

The results are shown in Table 1 as follows.

Table 1

Pharmacological test - 2

(Activity for inhibiting proliferation of cell strain Hep 3B (Human))

Cells of the strain Hep 3B (Human) obtained by successive cultivation were washed twice with PBS (-) solution (manufactured by Nissui Pharmaceutical Co., Ltd.), after the cells were separated by using a 0.05% trypsin solution (manufactured by Flow Laboratories, Co.), the cells were suspended in Dulbecco MEM culture medium (manufactured by Flow Laboratories, Co.), 1% non-essential amino acids solution (manufactured by Flow Laboratories, Co.) and 2 mM of L-glutamine solution (manufactured by Flow Laboratories, Co.). The cells were washed twice by centrifugation (a centrifugal separator: Model 05PR-22, manufactured by Hitachi Ltd. ) at 25°C, 1200 rpm for 5 minutes, then again suspended in said culture medium. The living cells were dyed with 0.2% Trypan Blue (manufactured by Wako Pure Chemical Industries, Ltd.), and number of the dyed cells was counted by using an optical microscope (Model: BH-2, manufactured by Olympus Optical Co., Ltd.) and the cell suspension was diluted so as to adjust number of the cells to 3 x 10* cells/ml. 0.1 Milliliter each of the Hep 3B cell suspension (3 x 10⁴ cells/ml) was added (dropped) to each one of the wells on a 96-well microplate (Code: 25860 manufacture by Corning Glass Works), and the microplate was subjected to cultivation at 37°C under the condition of 5% C0₂, for 24 hours. Each one of test compound solutions, having the final concentration of 0 μg/ml, 0.1 μg/ml, 1 μg/ml and 10 μg/ml, respec¬ tively was added (dropped), in an amount of 0.1 ml/well, to each one of the wells on 96-well micro¬ plate. Then, this microplate was subjected to cultivation in an incubator (manufactured by Napco Co.) under the condition of 37°C, 5% C0₂, for 48 hours. After the cultivation, to each one of the wells on said microplate, there were added (dropped) a PBS (-) solu¬ tion (manufactured by Nissui Pharmaceutical Co., Ltd.), containing 2.5 mg/ml of MTT (MTT (3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (manufactured by Wako Pure Chemical Industries, Ltd.)), in an amount of 0.01 ml/well, then said microplate was further subjected to cultivation at 37°C, under the condition of 5% C0₂, for 3 hours.

Then, to each one of the wells on said micro¬ plate, there was added (dropped) a dissolving solution (0.01 N hydrochloric acid, containing 10% of SDS

(sodium dodecyl sulfate), manufactured by Wako Pure Chemical Industries, Ltd.) in an amount of 0.1 ml/well, and the optical density at 580 nm was measured by using an instrument (Model: Titertech Multiscan MMC, manufac- tured by Flow Laboratories, Co.). An amount of the dye being taken in the living cells was determined from the optical density (optical absorbance), and the rate of inhibition for proliferation of the cells (%) was calculated from the following formula. Statistical treatment of the results was conducted by Student t test by referring to the data obtained from the control group.

Rate of inhibition for proliferation of the cells (%) = (1-M/C) x 100 wherein M is an amount of the dye being taken in the cells of the test group using test compound: C is an amount of the dye being taken in the cells of control group without using test compound.

The results are shown in Table 2 as follows:

Table 2

As can be seen from the results obtained in above-mentioned Pharmacological test-1 and -2, that 2,3-dihydro-lH-indene derivative as the active ingredient of the regulating agent of the present invention show activity for inhibiting proliferation of various cell strains, therefore, it is understood that said regulating agent possesses activity for inducing apoptosis.

Pharmacological test-3 (Activity for inhibiting dopaminergic neurodegeneration induced by l-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine)

In conducting the test, ICR strain male mice (10-weeks age) were used as test animals, and 1-methyl- 4-phenyl-l,2,3,6-tetrahydropyridine hydrochloride

(MPTP, manufactured by Funakoshi Kabushiki Kaisha) was used as the agent for inducing neurodegeneration. MPTP was dissolved in physiological saline (as a solvent), and this solution was prepared in dose volume of 10 ml/kg, then was administered intraperitoneally. While, the test compound 1 was suspended in 5% gum arabic-physiological saline solution (as a vehicle), and the suspension was prepared in dose volume of 10 ml/kg, then was administered orally. The test animals were divided into 4 groups

(each one of the group is consisting of 10 mice), in that: (a ) Control group

(solvent, i.p. and vehicle, p.o.);

(b) MPTP singly administered group

(MPTP 20 mg/kg, i.p. and vehicle, p.o.); (c) Test compound 1 + MPTP administered group (test compound 30 mg/kg p.o. and MPTP 20 mg/kg i.p.); and (d) Test compound 1 singly administered group

(test compound 30 mg/kg p.o. and solvent, i.p.).

MPTP (20 mg/kg) was administered intra- peritoneally, once a day for 7 days continuously, and test compound 1 (30 mg/kg) was administered orally once a day, 30 minutes before the administration of MPTP. The next day after the final administration, the mouse was killed by irradiation of microwave. After cooled the killed mouse, the brain was enucleated from the head, then the corpus striatum was taken out by cutting under ice-cooling condition. Instantaneously, the weight of tissue of the corpus striatum was measured, 0.1 N aqueous solution of perchloric acid was added to the tissue, then was homogenized by using a super-sonic homogenizer under ice-cooling condition. Thus obtained homogenate was subjected to centrifugal separation at 4°C, 20000 g, for 15 minutes to obtain a supernatant. The contents of dopamine (DA), 3,4-dihydroxy- phenylacetic acid (DOPAC) and homovanillic acid (HVA) in the corpus striatum were measured respectively from said supernatant by means of a HPLC (high-performance liquid chromatography) equipped with an electrochemical detector. Significant tests of among the results obtained from 4 test groups were conducted by two- tailed t test.

The results are shown in Table 3 as follows.

Table 3

Test DA % DOPAC % HVA % group

(a) 15.16+0.81 100 1.16+0.07 100 1.6810.11 100

(b) 10.14+0.54^*** 67 0.8910.09^* 77 1.3710.05^* 82

(c) 15.6911.20" 103 1.1210.09 97 1.8110.12" 108

(d) 15.04±0.57^NS 99 1.0410.05^NS 90 1.6110.07^NS 96

[Note: The above-mentioned data are shown as mean 1 S.E., and the unit of concentrations are shown in nano mol/g by weight of tissue.

*: p < 0.05;

***: p < 0.001 vs (a) ;

##: p < 0.01 vs (b);

NS: not significant vs (a) As can be seen from the results shown in Table 3, it can be understood that 2,3-dihydro-lH- indene derivatives or their salts of the present inven¬ tion almost completely inhibit decreasing of the contents of DA, DOPAC and HVA in the corpus striatum induced by MPTP, and in case of single administration of a 2,3-dihydro-lH-indene derivative or their salt of the present invention, it does not give any significant effect to the contents of DA, DOPAC and HVA in the corpus striatum. These results suggest that 2,3- dihydro-lH-indene derivatives or their salts of the present invention inhibit the dopaminergic neurodegeneration induced by MPTP. Therefore, 2,3- dihydro-lH-indene derivatives or their salts of the present invention can be able to inhibit progress (aggravation) of Parkinson's disease.

Pharmacological test-4

(Activity for inhibiting production of γ-interferon) Effect to production of γ-interferon released when peripheral blood obtained from a normal and healthy person is stimulated with lipopolysaccharide (LPS) was investigated as follows.

1-[4-(3-Methoxyphenyl)-1-piperazinyl]- acetylamino-2,3-dihydro-7-hydroxy-2,2,4,6-tetramethyl- lH-indene as the test compound 1 was dissolved in dimethyl sulfoxide and further diluted with RPMI-1640 culture medium to the predetermined concentration. As to the reference, only dimethyl sulfoxide dulted simi¬ larly with the RPMI-1640 culture medium was used.

To RPMI-1640 culture medium (containing 100 unit/ml of penicillin and 0.1 μg/ml of streptomycin), there were added 10 % of peripheral blood of a normal and healthy person (heparin was added), the test compound and LPS (E. coli 055:B5, 1 μg/ml), then said mixture was subjected to cultivation in a 5% C0₂- incubator at 37°C for 18 to 24 hours. The supernatant of this cultured medium was recovered by means of centrifugal operation to obtain the supernatant sample of cultured medium. The concentration of γ-interferon in this supernatant sample was measured by method of enzyme-linked immunosorbent assay (ELISA), and the amount of γ-interferon in the sample was obtained from the standard curve of γ-interferon. Note, that limit of detection was 20 pg/ml.

Rate of inhibition for releasing γ-interferon (%) was calculated by the following formula.

Rate of inhibition for releasing γ-interferon (%) = (1-T/C) x 100 wherein T is amount of γ-interferon released in case of test compound was added; C is amount of γ-interferon released without added test compound. As the result, rate of inhibition for releasing γ-interferon was 69%, when the final concen¬ tration of the test compound (test compound 1) was 30 μg/ml.

Claims

CLAIMS 1. Method for treating and preventing diseases by regulating apoptosis by use of a pharmaceutical composition containing at least one active ingredient selected from the group consisting of 2,3-dihydro-lH- indene derivatives represented by the general formula

(I)*

(wherein R¹, R², R³ and R* are each the same or different, and are lower alkyl groups; R⁵ is a lower alkoxy group; and A is a lower alkylene group) and salts thereof.

2. Method for treating and preventing Huntington's disease by use of a pharmaceutical composition containing at least one active ingredient selected from the group consisting of 2,3-dihydro-lH- indene derivatives represented by the general formula

(wherein, R¹, R², R³, R⁴, R⁵ and A are the same as defined in Claim 1) and salts thereof. 3. Method for treating and preventing Parkinson's disease by use of a pharmaceutical composition containing at least one active ingredient selected from the group consisting of 2,

3-dihydro-lH- indene derivatives represented by the general formula

(I):

(wherein, R¹, R², R³, R⁴, R⁵ and A are the same as defined in Claim 1) and salts thereof.

4. Method for treating and preventing ALS (amyotrophic lateral sclerosis) by use of a pharma- ceutical composition containing at least one active ingredient selected from the group consisting of 2,3- dihydro-lH-indene derivatives represented by the general formula (I) :

5. Method for treating and preventing HIV- related disturbance of memory by use of a pharma¬ ceutical composition containing at least one active ingredient selected from the group consisting of 2,3- dihydro-lH-indene derivatives represented by the general formula (I) :

(I)

(wherein, R¹, R², R³, R*, R⁵ and A are the same as defined in Claim 1) and salts thereof.

6. Method for treating and preventing diseases by inhibiting the production of cytokines by use of a pharmaceutical composition containing at least one active ingredient selected from the group consisting of 2,3-dihydro-lH-indene derivatives represented by the general formula (I) :

7. The method for treating and preventing diseases by regulating apoptosis by use of the pharma¬ ceutical composition according to Claim 1, wherein the active ingredient is l-[4-(3-methoxyphenyl)-l- piperazinyl]acetylamino-2,3-dihydro-7-hydroxy-2,2,4,6- tetramethyl-lH-indene.

8. The method for treating and preventing Huntington's disease by use of the pharmaceutical composition according to Claim 2, wherein the active ingredient is l-[4-(3-methoxyphenyl)-l-piperazinyl]- acetylamino-2,3-dihydro-7-hydroxy-2,2,4,6-tetramethyl- lH-indene.

9. The method for treating and preventing Parkinson's disease by use of the pharmaceutical composition according to Claim 3, wherein the active ingredient is l-[4-(3-methoxyphenyl)-l-piperazinyl]- acetylamino-2,3-dihydro-7-hydroxy-2,2,4,6-tetramethyl- lH-indene.

10. The method for treating and preventing ALS (amyotrophic lateral sclerosis) by use of the pharma¬ ceutical composition according to Claim 4, wherein the active ingredient is l-[4-(3-methoxyphenyl)-l- piperazinyl]acetylamino-2,3-dihydro-7-hydroxy-2,2,4,6- tetramethyl-lH-indene.

11. The method for treating and preventing HIV-related disturbance of memory by use of the pharmaceutical composition according to Claim 5, wherein the active ingredient is l-[4-(3-methoxy- phenyl)-1-piperazinyl]acetylamino-2,3-dihydro- 7-hydroxy-2,2,4,6-tetramethyl-lH-indene.

12. The method for treating and preventing diseases by inhibiting the production of cytokines by use of the pharmaceutical composition according to Claim 6, wherein the active ingredient is l-[4-(3- methoxyphenyl)-1-piperazinyl]acetylamino-2,3-dihydro- 7-hydroxy-2,2,4,6-tetramethyl-lH-indene.

13. Use of 2,3-dihydro-lH-indene derivatives represented by the general formula (I) :

(wherein R¹, R², R³, R⁴, R⁵ and A are the same as defined in Claim 1) or salts thereof, for the production of a medicament for treating and preventing diseases by regulating apoptosis by use of a pharmaceutical com¬ position which comprises at least one active ingredient selected from the group consisting of 2,3-dihydro-lH- indene derivatives represented by the general formula (I) and salts thereof.