HK1197650B

HK1197650B - Telomerase activating compounds and methods of use thereof

Info

Publication number: HK1197650B
Application number: HK14111336.4A
Authority: HK
Inventors: 阿维夫．加齐特; 希蒙．斯拉温; 艾瑟．普里尔; 萨拉．伊兹扎克
Original assignee: 本古里安大学内盖夫研究发展局; 希蒙．斯拉温; 阿维夫．加齐特
Priority date: 2007-06-04
Filing date: 2014-11-07
Publication date: 2018-08-03

Description

Telomerase activating compounds and methods of use thereof

The application is a divisional application of a Chinese patent application No. 200880024705.X with the same name, the original international application number is PCT/IL2008/000756, and the international application date is 2008/6 and 4.

Technical Field

The present invention relates to the use of a series of compounds and compositions comprising them to promote telomerase expression and/or activation, and to treat diseases, disorders, and/or conditions associated therewith.

Background

Telomerase is a ribonucleoprotein that catalyzes the addition of telomeric repeats to the telomeric end. Telomeres are long stretches of repetitive sequences that terminate at the ends of chromosomes. In humans, telomeres are typically 7-10kb in length and contain multiple repeat units. Telomerase is not expressed in most adult cells, and telomere length is shortened with successive cycles of replication.

Telomerase acts as a reverse transcriptase in the elongation of telomeres, which prevents telomere deletion due to end replication problems. Without telomerase, telomeres are shortened in each cell division leading to senescence, apoptosis and cell death caused by chromosome instability. Telomerase is inactive in somatic cells, whereas in 90% of cancer cells, telomerase is reactivated. Although telomerase activation can be dangerous because it can mimic the process of carcinogenesis, telomerase enhancers can theoretically be used clinically in certain medical conditions as anti-aging agents. In contrast, telomerase inhibitors may be useful against cancer. Cancer and aging are closely related: cancer prevention measures can lead to premature aging, and cellular immortalization is required for the formation of malignant cancer cells. Although there is a theoretical risk of activating carcinogenesis, activating telomerase can lead to slowing of aging.

Assessment of telomere length is important in understanding the biological and clinical significance of telomeres. The length of telomeres is useful as an indicator in studying chromosome stability, telomerase activity and/or expression, cell proliferative capacity and the aging process. The clinical value of telomerase can be demonstrated in its importance in the following cases: cancer, premature aging syndrome or segmental premature aging; genetic abnormalities, diseases caused by chromosome instability, such as bloom syndrome (a rare genetic disorder characterized by high frequent interruptions and rearrangements of chromosomes in affected persons) and diseases associated with aging, such as Warner's syndrome (a rare disease that manifests rapid aging in young people). The kinetics of telomere length have different expression patterns in the development of a particular disease. Therefore, it has great value in predicting disease.

Telomere length can be determined by southern blotting, hybridization protection assays, fluorescence in situ hybridization, flow cytometry, in situ priming, quantitative-polymerase chain reaction, and single telomere length analysis.

Lack of telomerase activity and/or expression and short telomeres can lead to congenital dyskeratosis, aplastic anemia, increased death from cardiovascular disease, stroke or infection, hypertension or chronic stress.

It was demonstrated that transduction of telomerase in telomerase inactivated mice prevented damage in the liver.

In addition to the role of telomerase in telomere length maintenance, the accumulated data suggest that the telomerase reverse transcriptase (TERT) protein has an additional physiological function, i.e., protection of cells and protection of mice from various insults by mechanisms not involving telomere extension (not yet understood).

Telomerase-activating compounds are thus useful in a variety of clinically relevant protocols

Disclosure of Invention

The present invention provides, in one embodiment, a method of stimulating or increasing telomerase activity and/or expression in a cell or tissue, comprising contacting the cell or tissue with a compound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphorus, arsenic, silicon or germanium;

R₁to R₉Identical or different, are H, D, OH, halogen, nitro, CN, nitriloylamino, amidosulfide, amino, aldehyde, substituted ketone, -COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino(ii) alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, alkylheteroaryl; or R₃、R₄Or R₇Form a fused cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with a predominantly aromatic ring; and is

R₁₀Is absent, H, D, OH, halogen, oxo, nitro, CN, nitriloamido, amidosulfide, amino, aldehyde, substituted ketone, -COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, alkylheteroaryl;

or an isomer, a pharmaceutically acceptable salt, a pharmaceutical product, a hydrate, an N-oxide, a crystal, or any combination thereof.

[ O11] the present invention provides in one embodiment a method of treating a disease capable of being affected by telomerase activation and/or expression in a subject, comprising administering to the subject a compound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphorus, arsenic, silicon or germanium;

R₁to R₉Identical or different, are H, D, OH, halogen, nitro, CN, nitriloylamino, amidosulfide, amino, aldehyde, substituted ketone, -COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenylAlkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, alkylheteroaryl; or R₃、R₄Or R₇Form a fused cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with a predominantly aromatic ring; and is

or an isomer, a pharmaceutically acceptable salt, a pharmaceutical product, a hydrate, an N-oxide, a crystal, or any combination thereof;

wherein the compound stimulates or promotes telomerase activity and/or expression.

In another embodiment, the disease is an HIV infection or a degenerative disease. In another embodiment, the degenerative disease is a neurodegenerative disease, a bone or joint degenerative disease, macular degeneration, atherosclerosis, or anemia. In another embodiment, the disease is an inflammatory disease. In another embodiment, the disease is congenital dyskeratosis. In another embodiment, the disease is aplastic anemia. In another embodiment, the disease is cancer.

The present invention provides, in one embodiment, a method of treating acute or chronic skin disorders comprising contacting the skin with a compound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphorus, arsenic, silicon or germanium;

R₁to R₉The same or different is H, D, OH, halogen, nitro, CN, nitriloamido, amidosulfide, amino, aldehyde, substituted ketone, -COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, alkylheteroaryl; or R₃、R₄Or R₇Form a fused cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with a predominantly aromatic ring; and is

In another embodiment, the acute or chronic disease is a wound, burn, abrasion, incision, graft site, lesion caused by an infectious agent, chronic venous ulcer, diabetic ulcer, pressure ulcer, mucosal ulcer, melanoma or keloid formation.

The present invention provides, in one embodiment, a method of inhibiting, abrogating, or delaying cellular senescence in a subject comprising administering to the subject a compound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphorus, arsenic, silicon or germanium;

R₁₀Is absent, H, D, OH, halogen, oxo, nitro, CN, nitrilo-amido, amidothioA hydride, amino, aldehyde, substituted ketone, -COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, alkylheteroaryl;

In another embodiment, the method of inhibiting, abrogating, or delaying cellular senescence in said subject extends the lifespan of said subject.

The present invention provides, in one embodiment, a method of reducing or eliminating the effects of aging in a subject, comprising administering to the subject a compound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphorus, arsenic, silicon or germanium;

R₁to R₉The same or different is H, D, OH, halogen, nitro, CN, nitriloylamino, amidosulfide, amino, aldehyde, substituted ketone, -COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heteroalkylsulfide, amide, aldehyde, substituted ketone, amideCycloalkylalkyl, heteroaryl, heteroarylalkyl, alkylheteroaryl; or R₃、R₄Or R₇Form a fused cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with a predominantly aromatic ring; and is

In another embodiment, the aging effect comprises an effect on the skin, eye, muscle tissue, or bone of the subject.

In another embodiment, the structure of formula I is represented by the structure of formula IV:

wherein R is₁，R₃、R₄，R₆，R₇，R₉And R₁₀As described above.

In another embodiment, the structure of formula I is represented by the structure of formula VI:

wherein R is₁'，R₃'，R₄'，R₆'，R₇' and R₉' same or different, including halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, monoalkylamino, dialkylamino, or arylamino; and is

R₁₀As described above.

In another embodiment, the structure of formula I is represented by the structure of formula VII:

in another embodiment, the structure of formula I is represented by the structure of formula VIII.

In another embodiment, the structure of formula I is represented by the structure of formula IX:

in another embodiment, the structure of formula I is represented by the structure of formula X:

in another embodiment, the structure of formula I is represented by the structure of formula XI:

in another embodiment, the structure of formula I is represented by the structure of formula XII:

in another embodiment, the structure of formula I is represented by the structure of formula XIII:

xm [028] in another embodiment, the structure of formula I is represented by the structure of formula XIV:

in another embodiment, the structure of formula I is represented by the structure of formula XV:

in another embodiment, the structure of formula I is represented by the structure of formula XVI:

in another embodiment, the invention utilizes a pharmaceutical composition comprising a compound as described herein for any of the methods as described herein.

Drawings

The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

FIG. 1: u-251 cells were treated with the compounds of the invention and telomerase activity was determined by the TRAP assay. A. Telomerase activity in cells treated with compound 68. B. Quantification results and% telomerase activity.

FIG. 2: the compounds of the invention result in a time-dependent increase in telomerase protein levels. A. Western blot analysis using anti-human telomerase antibody. B. Quantification of telomerase protein levels.

FIG. 3: compound 68 increased telomerase RNA expression. A. Northern blot analysis using htERT-specific cDNA probes. AD is actinomycin D. Quantification of rna levels.

FIG. 4: effect of treatment with the compounds of the present invention on survival and proliferation of hmscs.

FIG. 5: activation of telomerase expression in hmscs by compounds of the invention. A. Hmscs were treated with 250nM of compounds 79, 77 and 68 for 6 hours. Immunofluorescence assay (red) was performed with anti-hTERT antibody and nuclei were stained with DAPI (blue). B. Hmscs 6 and 24h were treated with compound 79. Telomerase activity was determined by real-time PCR using a quantitative telomerase detection kit (allied biotech inc., USA).

FIG. 6: the effect of the compounds of the invention on human keratinocytes aged in vitro.

FIG. 7: A. the effect of the compounds of the invention on human retinal pigment epithelial cells under oxidative stress. B. Effect of compounds on telomerase activity in RPE cells (determined by quantitative telomerase detection kit-real-time PCR).

FIG. 8: telomerase activating compounds 68 and 77 extended the life span of nematode c.

FIG. 9: activation of telomerase expression in rat endometrial cells by the compounds of the invention. Histological analysis. Immunohistochemical analysis using specific anti-hTERT antibodies. G. Telomerase activity in nuclear extracts derived from rat endometrium injected with a compound of the invention. Telomerase activity in nuclear extracts from rat endometrium injected with a compound of the invention was determined by real-time PCR using a quantitative telomerase detection kit (allied biotech inc., USA).

FIG. 10: the compounds of the invention activate telomerase of rat brain cortical cells.

FIG. 11: the compounds of the invention activate telomerase in the mouse CNS.

FIG. 12: telomerase protein was increased in rats treated with the compounds of the invention.

FIG. 13: compound 79 was used to prevent glutamate-induced apoptosis in mice.

FIG. 14: telomerase expression in mouse hearts was activated with compound 79.

FIG. 15: compound 68 prevents the effects of drugs on the damage of rat embryonic development.

Detailed Description

In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

The present invention, in certain embodiments, relates to the use of a novel class of triphenyl compounds and compositions comprising them in the treatment of diseases or disorders that can be affected, inter alia, by enhanced telomerase expression and/or telomerase activation.

The present invention utilizes such compounds that stimulate and/or increase telomerase expression and/or activity in cells and tissues of a subject, wherein the activity is decreased, absent, altered, or normal. Such diseases include in particular: a) alzheimer's disease; b) parkinson's disease; c) huntington's disease; d) nerve injury, motor neuron disease, Multiple Sclerosis (MS), peripheral and central nervous system injury, including spinal cord injury and cerebrovascular accidents; e) stroke; f) diseases or disorders associated with aging, e.g. skin aging, such as dermal atrophy and thinning, elastolysis and skin wrinkling, sebaceous gland hyperplasia or hypoplasia, age-related lentigines, hyperpigmentation, graying and thinning of hair or thinning (alopecia, baldness) or chronic skin ulcers; g) degenerative joint disease; h) osteoporosis, osteoarthritis and other degenerative diseases of the skeletal system i) sarcopenia and other degenerative diseases of muscle tissue; j) age and stress related diseases of the vascular system including atherosclerosis, calcification, thrombosis and aneurysm; k) age-related macular degeneration; 1) AIDS; m) age and stress related immune system damage including impaired tissue turnover following natural aging, cancer therapy, acute or chronic infection, degenerative inflammatory diseases or following genetic diseases leading to accelerated cell turnover, and associated anemia and other degenerative diseases; n) healing of wounds, burns, abrasions or other acute or chronic disorders of the epidermis; o) congenital dyskeratosis; p) luteal phase defect; q) premature ovarian failure (major ovarian insufficiency or hypergonadotropic hypogonadism); and/or r) increasing telomerase expression and/or activity in memory T cells, thereby potentiating an immune memory response and a response to a vaccine; s) increasing telomerase expression and/or activity in healthy tissue, thereby extending the life of the subject while maintaining good health of the subject.

The compounds of the invention:

in one embodiment, the method of the present invention comprises the use of a triphenyl compound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphorus, arsenic, silicon or germanium;

or isomers, pharmaceutically acceptable salts, pharmaceutical products, hydrates, N-oxides, crystals, or any combination thereof and compositions containing the same.

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula II:

wherein

Z is carbon, nitrogen, phosphorus, arsenic, silicon or germanium;

R₁，R₃、R₄，R₆，R₇and R₉The same or different is H, D, OH, halogen, nitro, CN, nitriloamido, amidosulfide, amino, aldehyde, substituted ketone, -COOH, ester, trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, alkylheteroaryl; or R₃、R₄Or R₇Form a fused cycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with a predominantly aromatic ring; and is

In one embodiment, Z is carbon. In another embodiment, R₁₀Is methyl. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-heterocycloalkyl, wherein n is 1-6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_nAminoalkyl, where n is 1 to 6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-dialkylamino, wherein n is 1-6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-N(CH₃)₂Wherein n is 1 to 6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-N(Et)₂Wherein n is 1 to 6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-aryl, wherein n is 1-6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-heteroaryl, wherein n is 1-6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-haloalkyl, wherein n is 1-6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-alkoxy, wherein n is 1-6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-ethoxy, wherein n is 1-6. In another embodiment, R₁，R₃、R₄，R₆，R₇And R₉Is- (CH)₂)_n-cycloalkyl, wherein n is 1-6.

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula III:

wherein

Z is carbon, nitrogen, phosphorus, arsenic, silicon or germanium;

r ', R "and R'" are independently the same or different and include hydrogen, alkyl, haloalkyl, alkylamino, phenyl, benzyl, alkyloxy, acetyl or benzoyl;

R₁₀Is absent, H, D, OH, halogen, oxo, nitro, CN, nitrilo-amido, amido-sulfide, amino, aldehyde,substituted ketones, -COOH, esters, trifluoromethyl, amides, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl, heteroarylalkyl, alkylheteroaryl;

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula IV:

wherein

R₁，R₃、R₄，R₆，R₇，R₉And R₁₀As defined above;

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula V:

wherein

R ', R ", R'" are independently the same or different and include hydrogen, alkyl, haloalkyl, phenyl, benzyl, alkyloxy, acetyl or benzoyl;

R₁'，R₃'，R₄'，R₆'，R₇' and R₉' same or different, including halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, monoalkylamino, dialkylamino, or arylamino; and R is₇As described above;

In one embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is a dialkylamino group. In another embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is dimethylamino. In another embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is diethylamino. In another embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is N-piperidinyl. In another embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is N-pyrrolidinyl. In another embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is N-piperazinyl. In another embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is N-piperazin-4-methyl. In another embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is N-morpholinyl. In another embodiment, R₁'，R₃'，R₄'，R₆'，R₇' and R₉' is ethoxy.

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula VI:

wherein R is₁'，R₃'，R₄'，R₆'，R₇' and R₉' same or different, including halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, monoalkylamino, dialkylamino, or arylamino; and R is₁₀As described above;

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula VII:

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula VIII:

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula IX:

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula X:

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula XI:

In one embodiment, the method of the present invention comprises using a triphenyl compound represented by the structure of formula XII:

In one embodiment, the method of the invention comprises using a triphenyl compound represented by the structure of formula XIII:

In another embodiment, the structure of formula I is represented by the structure of formula XIV:

In one embodiment, the term "alkyl" means a saturated aliphatic hydrocarbon group, including straight chain, branched chain, and cyclic alkyl groups. In one embodiment, the alkyl group has 1 to 12 carbon atoms. In another embodiment, the alkyl group has 1 to 7 carbon atoms. In another embodiment, the alkyl group has 1 to 6 carbon atoms. In another embodiment, the alkyl group has 1 to 7 carbon atoms. In another embodiment, the alkyl group has 2 to 6 carbon atoms. In another embodiment, the alkyl group has 1 to 7 carbon atoms. In another embodiment, the alkyl group has 2 to 8 carbon atoms. In another embodiment, the alkyl group has 3 to 6 carbon atoms. In another embodiment, the alkyl group has 3 to 7 carbon atoms. In another embodiment, the alkyl group has 1 to 4 carbon atoms. In another embodiment, the branched alkyl is an alkyl substituted with an alkyl side chain of 1 to 5 carbon atoms. In another embodiment, the branched alkyl is an alkyl substituted with a haloalkyl side chain of 1 to 5 carbon atoms. The alkyl group may be unsubstituted or substituted by halogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxy, thio and/or thioalkyl.

In one embodiment, "alkenyl" refers to unsaturated hydrocarbon groups, including straight, branched, and cyclic groups having one or more double bonds. The alkenyl group may have one double bond, two double bonds, three double bonds, and the like. In another embodiment, the alkenyl group has 2 to 12 carbons. In another embodiment, the alkenyl group has 2 to 6 carbons. In another embodiment, the alkenyl group has 2 to 4 carbons. In another embodiment, alkenyl is vinyl (-CH ═ CH)₂). Examples of alkenyl groups are ethenyl, propenyl, butenyl, cyclohexenyl and the like. The alkenyl group may be unsubstituted or substituted by halogen, hydroxy, alkoxy, carbonyl, acylamino, alkylamido, dialkylamido, nitroCyano, amino, monoalkylamino, dialkylamino, carboxy, thio and/or thioalkyl substituted.

In one embodiment, "alkynyl" refers to unsaturated hydrocarbon groups, including straight, branched, and cyclic groups having one or more triple bonds. Alkynyl groups can have one triple bond, two triple bonds, three triple bonds, and the like. In another embodiment, alkynyl has 2-12 carbons. In another embodiment, alkynyl has 2-6 carbons. In another embodiment, the alkenyl group has 2 to 4 carbons. In another embodiment, alkynyl is ethynyl (-CH ≡ CH)₂). Examples of alkynyl groups are ethynyl, propynyl, butynyl, cyclohexynyl, and the like. Alkynyl groups may be unsubstituted or substituted with halogen, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxy, thio and/or thioalkyl.

In another embodiment, "alkoxy" refers to an alkyl group as defined above attached to an oxygen. Examples of alkoxy groups are ethoxy, propoxy, tert-butoxy and the like.

In one embodiment, "haloalkyl" refers to an alkyl group as defined above substituted with one or more halogen atoms, e.g., F, Cl, Br, or I.

In another embodiment, "aryl" refers to an aromatic group having at least one aromatic carbocyclic or heterocyclic group, which may be unsubstituted or substituted with halogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxy, thio, or thioalkyl. In another embodiment, aryl is one or more 4-12 membered rings. In another embodiment, aryl is one or more 6-18 membered rings. In another embodiment, aryl is one or more 4-8 membered rings. In another embodiment, the aryl is a 6 membered ring. In another embodiment, aryl is a fused ring system consisting of 2 to 3 rings. Non-limiting examples of aromatic rings are phenyl, naphthyl, pyranyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridyl, furanyl, thienyl, thiazolyl, imidazolyl, isoeylAzole groups, and the like.

In another embodiment, "heteroaryl" means an aromatic group having at least one heterocyclic aromatic group, which may be unsubstituted or substituted with one or more groups selected from halo, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxy, or thio or thioalkyl. In another embodiment, the heteroaryl is a 4-12 membered ring. In another embodiment, the heteroaryl is a 6-18 membered ring. In another embodiment, the heteroaryl is a 4-8 membered ring. In another embodiment, the heteroaryl is a 6 membered ring. In another embodiment, aryl is a fused ring system consisting of 2 to 3 rings. Non-limiting examples of heteroaryl rings are pyrrolyl, thienyl, thiazolyl, benzothienyl, naphthothienyl, purinyl, isothiazolyl, furyl, furazanyl, isobenzofuryl, pyranyl, chromenyl, xanthenyl, phenoxyxanthenyl, indolyl, isoindolyl, indolizinyl, isoindolinyl, benzothienyl, and the like,Azolyl radical, isoOxazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl and the like.

In one embodiment, "hydroxy" means an OH group. In certain embodiments, when R of a compound of the invention₁，R₂Or R₃When OR, R is not OH.

In one embodiment, the term "halogen" means a halogen, such as F, Cl, Br or I.

In another embodiment, the term "phenol" means an alcohol (OH) derivative of benzene.

In one embodiment, "amino" means a nitrogen atom as described above attached by a single bond to a hydrogen atom, an alkyl, alkenyl, or aryl group as described above, or a combination thereof. A non-limiting example of an amino group is NH₂，N(Me)₂，N(Et)₂，N(Ph)₂And the like.

In one embodiment, a "cycloalkyl" is a non-aromatic monocyclic or polycyclic ring containing carbon and hydrogen atoms. Cycloalkyl groups may have one or more carbon-carbon double bonds in the ring, as long as their presence does not render the ring aromatic. Examples of cycloalkyl groups include, but are not limited to, (C)₃-C₇) Cycloalkyl radicals, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, and saturated cyclic and bicyclic terpenes and (C)₃-C₇) Cycloalkenyl groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl and cycloheptenyl, and unsaturated cyclic and bicyclic terpenes. Preferably, the cycloalkyl group is a monocyclic or bicyclic ring structure that, in addition to carbon atoms, contains sulfur, oxygen, nitrogen, or any combination thereof as part of the ring. In another embodiment, the cycloalkyl group is a 3-12 membered ring. In another embodiment, the cycloalkyl group is a 6 membered ring. In another embodiment, the cycloalkyl group is a 5-7 membered ring. In another embodiment, the cycloalkyl group is a 4-8 membered ring. In another embodiment, cycloalkyl groups may be unsubstituted or substituted with halogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO₂H. Amino, monoalkylamino, dialkylamino, carboxyl, thio and/or thioalkyl substituted.

In one embodiment, "heterocycloalkyl" refers to a non-aromatic mono-or polycyclic ring containing carbon, in addition to carbon, sulfur, phosphorus, oxygen, or nitrogen as part of the ring. The heterocycloalkyl group may have one or more double bonds in the ring, as long as they areIf present, the ring should not be an aromatic ring. Examples of heterocycloalkyl groups include, but are not limited to, piperidine, piperazine, pyran, morpholine. Preferably, the heterocycloalkyl group is a monocyclic or bicyclic ring structure that contains, in addition to carbon atoms, sulfur, oxygen, nitrogen, or any combination thereof as part of the ring. In another embodiment the heterocycloalkyl group is a 3 to 12 membered ring. In another embodiment, the heterocycloalkyl group is a 6 membered ring. In another embodiment, the heterocycloalkyl group is a 5-7 membered ring. In another embodiment, the heterocycloalkyl group is a 4-to 8-membered ring. In another embodiment, the heterocycloalkyl group can be unsubstituted or substituted with halo, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO₂H. Amino, monoalkylamino, dialkylamino, carboxyl, thio and/or thioalkyl substituted. In another embodiment, the heterocycloalkyl group is a cyclic urea, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrrolidinyl, or substituted heterocycloalkyl group,Azolinyl, isoAn azolinyl group,Oxazolidinyl group,Oxazolidone, isoOxazolidone group, pyrazoline group, pyrazolidinyl group, piperidyl group, piperazine group, morpholinyl group.

In one embodiment, the terms "alkylalkoxy," "alkylhaloalkyl," "alkylaryl," "alkylcycloalkyl," "alkylheterocycloalkyl," "alkylheteroaryl," and "alkylamino" are intended to refer to an alkoxy, haloalkyl, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, orAmino-linked alkyl as defined above. The alkoxy, haloalkyl, aryl, cycloalkyl, heterocycloalkyl, heteroaryl or amino group is as defined above. Examples include, but are not limited to CH₂-OEt，CH₂-N-piperidine, CH₂-N-piperazine, CH₂-N(Me)₂And the like.

In another embodiment, the heterocycloalkyl group of formulas I-IV fused with the primary aromatic ring forms a phenylpyrrolidinonyl group. In another embodiment, the aryl group of formulas I-IV fused to the primary aromatic ring forms a naphthyl group. In another embodiment, the fused heteroaryl groups of formulas I-IV fused to a predominantly aromatic ring form a quinoline or isoquinolinyl group.

In one embodiment, the present invention provides the use of a compound as described herein and/or an analog, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, hydrate, N-oxide, prodrug, polymorph, impurity or crystal thereof, or a combination thereof.

In one embodiment, the term "isomer" includes, but is not limited to, optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like.

In one embodiment, the term "isomer" is intended to include optical isomers of triphenyl compounds. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, which form has properties useful in the treatment of the diseases described herein involving telomerase expression and/or activity. In one embodiment, the triphenyl compound is the pure (R) -isomer. In another embodiment, the triphenyl compound is a pure (S) -isomer. In another embodiment, the triphenyl compound is a mixture of (R) and (S) isomers. In another embodiment, the triphenyl compound is a racemic mixture containing equal amounts of the (R) and (S) isomers. It is well known in the art how to prepare optically active forms (e.g., by resolution of racemic forms by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

In one embodiment, the invention includes "pharmaceutically acceptable salts" of the compounds of the invention that can be produced, forming alkali metal salts and forming addition salts of the free acids or free bases. Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention may be prepared from inorganic or organic acids. In one embodiment, examples of inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, and phosphoric acid. In one embodiment, the organic acid may be selected from the group consisting of aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic acid types of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, oxalic, p-toluenesulfonic, methanesulfonic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, pamoic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, sulfanilic, stearic, cyclohexylsulfamic, alginic, galacturonic. In one embodiment, suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc and organic salts made from N, N' -dibenzylethylenediamine, choline, chloroprocaine, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts can be prepared from the corresponding compounds in a conventional manner.

In other embodiments, pharmaceutically acceptable salts can be prepared from phenolic compounds by treatment with an inorganic base, such as sodium hydroxide. For example, in another embodiment, aliphatic and aromatic carboxylic acids may be used to prepare esters of phenolic compounds, such as acetates and benzoates.

The invention also includes the use of N-oxides of the amino substituents of the compounds described herein.

The present invention provides the use of derivatives of the compounds as described herein. In one embodiment, "derivatives" include, but are not limited to, ether derivatives, acid derivatives, amide derivatives, ester derivatives, and the like. In another embodiment, the present invention further includes the use of a hydrate of the compound as described herein. In one embodiment, "hydrate" includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate and the like.

In other embodiments, the invention provides the use of a metabolite of a compound as described herein. In one embodiment, "metabolite" means any substance produced by another substance through metabolism or a metabolic process.

In other embodiments, the invention provides the use of a pharmaceutical product of a compound as described herein. In other embodiments, the term "pharmaceutical product" means a composition (pharmaceutical composition) suitable for pharmaceutical use, e.g., as described herein.

In certain embodiments, the present invention provides compositions comprising or the use of a compound of the present invention in: increasing telomerase activity and/or expression in a cell or tissue; and/or treating a disease by increasing telomerase activity and/or expression in a cell or tissue of the subject; and/or treating epidermal acute or chronic diseases; and/or extended life; and/or treating age-and stress-related diseases of the vascular system, including atherosclerosis, calcification, thrombosis, hypertension or aneurysm; and/or treating age-related macular and/or age-related skin disorders; and/or treatment of age and stress related damage to the immune system; and/or to enhance the immune response to infection-resistant organisms.

In certain embodiments, the present invention provides compositions comprising a compound of the present invention and/or having the following properties: treating conditions associated with female infertility, including luteal phase deficiency or premature ovarian failure (major ovarian insufficiency or hypogonadotropic hypogonadism); and/or decreased granzyme activity; and/or treating diseases associated with male infertility, including impaired semen production or impaired semen transport; and/or as an aid to In Vitro Fertilization (IVF) techniques; and/or improving semen quality and/or ovum quality, e.g., and in certain embodiments, the compounds of the invention prolong blastocyst viability in ex vivo cultures, thereby improving implantation efficiency. In certain embodiments, treating the population with a compound as described herein makes them more receptive to other IVF therapeutics, or in certain embodiments, enables evaluation of combination therapies or novel compounds.

In one embodiment, the invention provides methods of treatment using the compounds of the invention or compositions comprising them as described herein. In certain embodiments, the invention provides for the use of compounds of the invention, and including compositions comprising them, in the treatment of the diseases, disorders, or conditions indicated.

In one embodiment, the invention provides a method of treating, preventing, inhibiting, lessening the severity of, reducing the incidence of, reducing the pathogenesis of, or delaying the onset of, in particular: a) alzheimer's disease; b) parkinson's disease; c) huntington's disease; d) stroke; e) nerve injury, motor neuron disease, Multiple Sclerosis (MS), peripheral and central nervous system injury, including spinal cord injury and cerebrovascular accidents; f) skin diseases associated with aging, such as dermal atrophy and thinning, elastolysis and skin wrinkling, sebaceous gland hyperplasia or hypoplasia, age-related lentigo, hyperpigmentation, graying and hair loss or thinning (alopecia, baldness) or chronic skin ulcers; g) degenerative joint disease; h) osteoporosis, osteoarthritis, and other degenerative diseases of the skeletal system; i) age and stress related diseases of the vascular system including atherosclerosis, calcification, thrombosis, hypertension and aneurysms; j) age-related macular degeneration; k) AIDS; 1) age and stress related damage to the immune system, including impaired tissue turnover following natural aging, cancer therapy, acute or chronic infection, degenerative inflammatory disease or following genetic disease leading to accelerated cell turnover, and related anemia and other degenerative diseases; m) healing of wounds, burns, abrasions or other acute or chronic disorders of the epidermis; n) congenital dyskeratosis; o) luteal phase defect; p) premature ovarian failure (major ovarian insufficiency or hypergonadotropic hypogonadism); q) impaired semen production or impaired semen transport; r) infection of an organism with resistance to infection by administering any of the compounds as described herein and optionally other therapeutic agents or compositions comprising them.

In one embodiment, the terms "treatment" or "treating" include prophylaxis and intermittent treatment of a disease. The terms "alleviate", "prevent" and "inhibit" have their commonly understood meaning of slowing or alleviating, in another embodiment, or delaying, in another embodiment, or reducing, in another embodiment, the incidence, severity or pathogenesis of a disease, disorder or condition. In embodiments, the term "treating" means delaying the development of, prolonging the elimination of, reducing the incidence of, or ameliorating the symptoms associated with a disease, disorder, or condition. In one embodiment, the terms "treat," "alleviate," "prevent," or "inhibit" refer to a reduction in morbidity, mortality, or a combination thereof associated with the indicated disease, disorder, or condition. In one embodiment, the term "progression" means an increase in scope or severity, progression, growth, or worsening. In another embodiment, the term "relapse" means recovery of the disease after resolution. In one embodiment, the treatment methods of the invention reduce the severity of the disease, or in another embodiment, reduce the symptoms associated with the disease, or in another embodiment, reduce the number of biomarkers expressed during the course of the disease.

In one embodiment, the term "treating" and aspects thereof which are encompassed, are intended to be administered to a subject having a disease, disorder or condition indicated, or in certain embodiments, to a subject susceptible to a disease, disorder or condition indicated. The term "susceptibility" is to be taken as specifically referring to a genetic trait or familial association associated with a statistical increase in the predisposition to, or incidence, severity, etc., of the indicated disease. In certain embodiments, the term "susceptible" is considered to specifically mean a lifestyle associated with an increased risk of the indicated disease. In certain embodiments, the term "susceptible" is considered to mean specifically the presence of a biomarker associated with the indicated disease, such as a biomarker in cancer, and the term "susceptible" means that cancer may comprise a precancerous precursor to the presence of the indicated cancer.

In certain embodiments, the term "reducing pathogenesis" is understood to include reducing tissue or organ damage associated with a particular disease, disorder or condition. In another embodiment, the term "reducing pathogenesis" is understood to include reducing the incidence or severity of the associated disease, disorder or condition. In another embodiment, the term "reducing pathogenesis" is understood to include reducing the number of said diseases, disorders or conditions having the symptoms described or associated therewith.

In another embodiment, the term "administering" means contacting a subject with a compound of the invention. Administration can be carried out in vitro, i.e. in a test tube, or in vivo, i.e. in cells or tissues of a living organism, e.g. a human. In one embodiment, the invention includes administering to a subject a compound of the invention.

In one embodiment, the methods of the invention utilize the use of contacting or binding a compound described herein to telomerase in an amount effective to increase telomerase activity and/or expression and thereby mediate the effect. In certain embodiments, the methods of the invention may comprise a preliminary step of identifying a cell or tissue in which increased telomerase activity and/or expression is desired. The cells may be in culture, i.e., in vitro or ex vivo or in a subject or patient. In one embodiment, increased telomerase expression and/or activity in a cell or tissue includes, for example, increased replication capacity and/or longevity of the contacted cell.

Pharmaceutical composition

In certain embodiments, the present invention provides methods of use comprising administering a composition comprising the compound. As used herein, "pharmaceutical composition" means a "therapeutically effective amount" of the active ingredient, i.e., a compound of the present invention, in combination with a pharmaceutically acceptable carrier or diluent. As used herein, "therapeutically effective amount" means an amount and dosage regimen that provides a therapeutic effect and treatment for the specified condition.

In certain embodiments, the present invention provides compositions comprising at least one compound of the present invention in any form or embodiment described herein. In certain embodiments, the terms "a" or "an" should be understood to include the singular or plural of the stated material. In certain embodiments, the terms "a" or "an" mean at least one.

In certain embodiments, any of the compositions of the present invention consist of a compound of the present invention in any of the forms or embodiments described herein. In certain embodiments, the compositions of the present invention consist essentially of a compound of the present invention in any of the forms or embodiments described herein.

In certain embodiments, the term "comprising" is intended to encompass the active agents shown, e.g., the compounds of the present invention, as well as other active agents and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc. well known in the pharmaceutical industry. In certain embodiments, any of the compositions of the present invention comprise a compound of formulas I-XVI in any of the forms or embodiments described herein. In certain embodiments, any of the compositions of the present invention consist of a compound of formulas I-XVI in any form or embodiment described herein. In certain embodiments, the compositions of the present invention consist essentially of a compound of the present invention in any of the forms or embodiments described herein. In certain embodiments, the term "comprising" means containing the indicated active agent, e.g., a compound of the present invention, and other active agents, as well as pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as known in the pharmaceutical industry. In some embodiments, the term "consisting essentially of … …" refers to a composition whose only active ingredient is the active ingredient in question, but may also include other compounds useful for formulation stabilization, preservation, etc., but not directly related to the therapeutic effect of the active ingredient in question. In certain embodiments, the term "consisting essentially of …" means a composition whose only active ingredients having substantially the same mode of action or substantially the same molecular target are the active ingredients shown, although other active ingredients may be incorporated, where such minor active ingredients act on different targets or have palliative capabilities. In certain embodiments, the term "consisting essentially of …" is intended to have an ingredient that facilitates the release of an active ingredient. In certain embodiments, the term "consisting of" means a composition comprising a compound as described herein as the sole active ingredient and a pharmaceutically acceptable carrier or excipient.

In another embodiment, the present invention provides a composition comprising a compound of the present invention, or a prodrug, analog, isomer, metabolite, derivative, pharmaceutically acceptable salt, pharmaceutical product, polymorph, crystal, impurity, N-oxide, ester, hydrate or any combination thereof, as described herein, and a suitable carrier or diluent.

The active ingredient may be formulated into the composition in the form of a neutralized pharmaceutically acceptable salt. Pharmaceutically acceptable salts include the acid addition salts formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or iron, or organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Pharmaceutical compositions comprising a compound of the invention may be administered to a subject by methods well known to those skilled in the art, for example, by oral, parenteral, intravascular, peri-cancerous (paracancearally), transmucosal, transdermal, intramuscular, intranasal, intravenous, intradermal, subcutaneous, sublingual, intraperitoneal, intraventricular, intracranial, intravaginal, by inhalation, rectal, intratumoral, or by any means of recombinant virus/composition that can be delivered to a tissue (e.g., needle or catheter). Alternatively, topical administration is to mucosal cells, as is required for dermal or ocular administration. Another method of administration is by inhalation or aerosol.

In one embodiment, the compositions of the present invention are formulated for oral delivery, wherein the active compound may be combined with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers. Tablets, dragees, pills, capsules and the like may also comprise the following components: binders, such as gum tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrating agents, such as corn starch, potato starch, alginic acid, and the like; lubricants, such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added, or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of an elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring (e.g., cherry or orange flavor). In addition, the active compounds may be incorporated into sustained release, pulsed release, controlled release or delayed release formulations or dosage forms.

In another embodiment, the compositions of the present invention comprise one or more pharmaceutically acceptable carrier substances.

In one embodiment, the carrier for these compositions is biocompatible and, in another embodiment, biodegradable. In other embodiments, the formulation provides a relatively constant level of release of an active ingredient. In yet other embodiments, a faster rate of release may be expected immediately after administration. In other embodiments, the release of the active compound may be event-triggered. The event that triggers the release of the active compound may be the same in one embodiment, or different in another embodiment. In one embodiment, the event that triggers the release of the active compound may be exposure to moisture, in another embodiment, a low pH, or in another embodiment, a temperature threshold. The preparation of these compositions is within the level of ordinary skill in the art using known techniques. Exemplary carriers for this aspect include microparticles of poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextrose, and the like. Other exemplary time release carriers include supramolecular biovectors comprising a non-liquid hydrophilic core (e.g., cross-linked polysaccharides or oligosaccharides) and an optional outer layer (comprising amphiphilic compounds such as phospholipids). In one embodiment, the amount of active compound included in a sustained release formulation depends on the site of administration, the rate and desired duration of release, and the nature of the disease to be treated, attenuated or inhibited.

In one embodiment, it is desirable to deliver the compounds described herein parenterally, intravenously, intramuscularly, or intraperitoneally. These routes are well known to those skilled in the art, some of which are further described, for example, in US patents 5543158; US 5641515 and US5399363, all of which are incorporated herein by reference in their entirety. In certain embodiments, solutions of the active compounds as free bases or pharmaceutically acceptable salts may be prepared by suitably mixing in water with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols or mixtures thereof and in oils. It must be stable under the conditions of preparation and storage and must avoid corrosion by the contaminating action of microorganisms such as bacteria and fungi.

In another embodiment, it is preferred to include isotonic agents, for example, sugars or sodium chloride. In other embodiments, it is desirable to prolong the absorption of the injectable composition. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

In certain embodiments, the parenteral carrier comprises sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, and fixed oils. Intravenous carriers include liquid and nutritional supplements, electrolyte supplements such as ringer's dextrose based supplements, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, calibrators, inert gases and the like.

In some embodiments, the compounds of the invention may be administered to a patient, in one embodiment, a human patient, at varying doses. In one embodiment, the compounds of the invention are administered at a dose of 0.1-200mg per day. In one embodiment, the compound of the invention is administered in a dose of 0.1 to 10mg, or in another embodiment, 0.1 to 25mg, or in another embodiment, 0.1 to 50mg, or in another embodiment, 0.3 to 15mg, or in another embodiment, 0.3 to 30mg, or in another embodiment, 0.5 to 25mg, or in another embodiment, 0.5 to 50mg, or in another embodiment, 0.75 to 15mg, or in another embodiment, 0.75 to 60mg, or in another embodiment, 1 to 5mg, or in another embodiment, 1 to 20mg, or in another embodiment, 3 to 15mg, or in another embodiment, is from 1 to 30mg, or in another embodiment, from 30 to 50mg, or in another embodiment, from 30 to 75mg, or in another embodiment, 100-2000 mg. In some embodiments, the compounds of the invention may be administered at different doses as a function of time or disease/symptom/condition severity, or age, or other factors that may be recognized by one of skill in the art.

The compounds of the invention may be administered in different doses. In one embodiment, the compound of the invention is administered at a dose of 1 mg. In another embodiment, the compound of the invention is administered in a dose of 5mg, or in another embodiment, 3mg, or in another embodiment, 10mg, or in another embodiment, 15mg, or in another embodiment, 20mg, or in another embodiment, 25mg, or in another embodiment, 30mg, or in another embodiment, 35mg, or in another embodiment, 40mg, or in another embodiment, 45mg, or in another embodiment, 50mg, or in another embodiment, 55mg, or in another embodiment, 60mg, or in another embodiment, 65mg, or in another embodiment, 70mg, or in another embodiment, is 75mg, or in another embodiment, 80mg, or in another embodiment, 85mg, or in another embodiment, 90mg, or in another embodiment, 95mg, or in another embodiment, 100 mg.

Although the compounds of the present invention may be administered as the sole active agent, they may also be used in combination with one or more other compounds, and/or in combination with other agents useful in the treatment and/or prevention of diseases, conditions and/or disorders, as will be appreciated by those skilled in the art. In another embodiment, the compounds of the present invention may be administered continuously with one or more such agents to provide sustained therapeutic and prophylactic effects. In another embodiment, the compounds may be administered by different routes, at different times, or a combination thereof.

In addition, the compounds of the present invention may be used alone or in combination with other means for the prevention or treatment of a disease, condition, or disorder. In some embodiments, these other treatment modalities include, but are not limited to, surgery, radiation therapy, hormone replacement therapy, dietary adjustments, wound debridement, and the like, as would be appropriate for the disease being treated. These treatment modalities may be performed continuously (e.g., treatment with a compound of the invention following surgery or radiation) or in combination (e.g., in addition to dietary therapy).

The other active agents may be used in therapeutic amounts generally as taught in physicicians' DESK REFERENCE (PDR) 53 th edition (1999) or in therapeutically useful amounts known to those of ordinary skill in the art. The compounds of the invention and other therapeutically active agents may be administered at the recommended maximum clinical dose or at lower doses. The dosage level of the active compound in the compositions of the invention may vary depending on the route of administration, the severity of the disease and the response of the patient to achieve the desired therapeutic response. The combination may be administered as separate compositions or as a single dosage form containing both drugs. When administered as a combination, the therapeutic agents may be formulated as separate compositions for administration at the same time or at different times, or the therapeutic agents may be administered as a single composition.

The pharmaceutical compositions of the invention may contain the compounds of the invention alone or in a pharmaceutically acceptable carrier, and may be in solid or liquid form, such as tablets, powders, capsules, pills, solutions, suspensions, elixirs, emulsions, gels, creams or suppositories (including rectal and urethral suppositories). Pharmaceutically acceptable carriers include gums, starches, sugars, cellulosic materials or mixtures thereof. Pharmaceutical formulations comprising a compound of the invention may be administered to a patient by, for example, subcutaneous implantation of a pill; in a further embodiment, the pill is provided for controlled release of the compound of the invention over a period of time. The formulations may also be administered by intravenous, intraarterial or intramuscular injection of liquid preparations, oral liquid or solid preparations or by topical administration. Administration may be accomplished by use of a rectal suppository or a urethral suppository. The pharmaceutical composition may also be a parenteral formulation; in one embodiment, the formulation comprises a liposome containing a complex of a compound of the invention.

The pharmaceutical compositions of the present invention may be prepared by known dissolution, mixing, granulation or tableting methods. For oral administration, the compounds of the invention or their physiologically tolerated derivatives, such as salts, esters, N-oxides, etc., are mixed with additives customary for this purpose, such as carriers, stabilizers or inert diluents, and are converted by customary methods into a form suitable for administration, such as tablets, coated tablets, hard or soft capsules, aqueous, alcoholic or oily solutions. Examples of suitable inert carriers are conventional tablet bases, for example lactose, sucrose or corn starch in combination with binders, for example acacia, corn starch, gelatin, or with disintegrating agents, for example corn starch, potato starch, alginic acid, or with lubricating agents, for example stearic acid or magnesium stearate. Examples of suitable oily carriers or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. The formulation may function as a dry or wet granulate. For parenteral administration (subcutaneous, intravenous, intraarterial or intramuscular injection), the compounds of the invention or their physiologically tolerated derivatives, such as salts, esters, N-oxides, etc., are converted, if desired together with customary and suitable substances for this purpose, such as solubilizers or other auxiliaries, into solutions, suspensions or emulsions. Examples are: sterile liquids, such as water and oils with or without the addition of surfactants and other pharmaceutically acceptable adjuvants. Exemplary oils are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

The formulation of pharmaceutical compositions comprising an active ingredient is well known in the art. Typically, these compositions are formulated as aerosols of the polypeptide for delivery to the nasopharynx, or as injectable liquid solutions or suspensions, although solid forms suitable for forming solutions or suspensions in liquid prior to injection may also be prepared. The formulation may also be emulsified. The active therapeutic ingredient is typically mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like or combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, or pH buffering agents to enhance the effectiveness of the active ingredient.

For topical administration to a body surface using, for example, creams, gels, drops, and the like, the compounds of the present invention or physiologically tolerated derivatives thereof, for example, salts, esters, N-oxides, and the like, may be prepared and used as solutions, suspensions, or emulsions in a physiologically acceptable diluent, with or without a pharmaceutical carrier.

In another embodiment, the active compound may be delivered in vesicles, particularly Liposomes (see Langer, Science 249: 1527-.

In one embodiment, the present invention provides a combined preparation. In one embodiment, the term "combined preparation" especially defines a "kit of parts" in the sense that the combined partners as defined above can be administered independently or by using different fixed combinations with a large number of combined partners, i.e. simultaneously, concurrently, separately or sequentially. In certain embodiments, for example, the parts of the kit of parts may then be administered simultaneously or sequentially staggered, at different time points and using the same or different time intervals for any part of the kit of parts. In certain embodiments, the proportions of the total amount of the co-formulations may be administered in a combined preparation. In one embodiment, the combined preparation may be varied, for example in order to comply with the requirements of a sub-population of patients to be treated or in order to comply with the requirements of a single patient having different requirements due to the specific disease, the severity of the disease, age, sex or weight, as can be readily carried out by a person skilled in the art.

It is to be understood that the present invention relates to compositions and combination therapies as described herein, if appropriate, for any disease, disorder or condition, as will be appreciated by those skilled in the art. The use of certain of such compositions and combination therapies for specific diseases, disorders, and conditions, which represent embodiments of the present invention, have been described above, and the present invention provides methods of treating such diseases, disorders, and conditions in a subject by administering a compound as described herein, either alone or as part of a combination therapy, or using compositions that represent additional embodiments of the present invention.

Methods of treating conditions or diseases capable of being affected by telomerase activation and/or expression

In certain embodiments, the present invention provides compounds and pharmaceutical compositions comprising them for use in the treatment of disorders and/or diseases capable of being affected by enhanced telomerase activation and/or expression. These compounds interact with telomerase and stimulate and/or increase telomerase expression and/or activity in tissues and cells of a subject. In certain embodiments, such reduced or absent activity results in the development or enhancement of a pathogenesis or symptom associated with the disease, disorder, or condition in the subject. Such diseases, disorders or conditions may include, inter alia: a) alzheimer's disease; b) parkinson's disease; c) huntington's disease; d) stroke; e) nerve injury, motor neuron disease, Multiple Sclerosis (MS), peripheral and central nervous system injury, including spinal cord injury and cerebrovascular accidents; f) skin diseases associated with aging, such as dermal atrophy and thinning, elastolysis and skin wrinkling, sebaceous gland hyperplasia or hypoplasia, age-related lentigo, pigmentation disorders, graying and hair loss or thinning (alopecia, baldness) or chronic skin ulcers; g) degenerative joint disease; h) osteoporosis, osteoarthritis, and other degenerative diseases of the skeletal system; i) age and stress related diseases of the vascular system including atherosclerosis, calcification, thrombosis, hypertension and aneurysms; j) age-related macular disease; k) AIDS; 1) age and stress related damage to the immune system, including impaired tissue turnover following natural aging, cancer therapy, acute or chronic infection, degenerative inflammatory disease or following genetic disease leading to accelerated cell turnover, and related anemia and other degenerative diseases; m) healing of wounds, burns, abrasions or other acute or chronic disorders of the epidermis; n) congenital dyskeratosis; o) sarcopenia and/or other muscle disease or disorder; p) luteal phase defect; q) premature ovarian failure (major ovarian insufficiency or hypergonadotropic hypogonadism); r) impaired semen production; s) impaired semen transport; and/or T) increased telomerase expression and/or activity in memory T cells, thereby potentiating an immune memory response and a response to a vaccine; and/or u) an increase in telomerase expression and/or activity in healthy tissue, thereby extending the life of a subject while maintaining a good health of the subject and/or other clinical treatment and/or diagnostic areas, including any embodiment encompassed by the term "treatment" as described herein.

In certain embodiments, the invention provides methods of increasing telomerase expression and/or activity in a cell or tissue by contacting the cell or tissue with a composition comprising a compound of the invention. In one embodiment, these methods may include the step of identifying a cell or tissue in which increased telomerase expression and/or activity is desired.

Telomerase is typically detected at low levels in normal somatic cells. Telomerase expression and/or activity is expressed at low levels in skin, lymphatic tissue, endometrial tissue, hair follicles and intestinal crypts, active mitotic cells and stem cells. Telomerase expression and/or activity is regulated at different molecular levels and by maturation and alteration of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC), including transcription, mRNA splicing,

In one embodiment, the compounds and compositions of the invention activate telomerase and thus the methods as described herein are useful.

The ability of a compound to increase telomerase expression and/or activity in a cell can be determined using a TRAP (telomere amplification) assay well known in the art (e.g., Kim et al, U.S. patent No. US 5629154; Harley et al, U.S. patent No. US 5891639). This activity is typically compared to that similarly measured in a control assay for such cells (e.g., telomerase activity is 50% greater than that observed in a solvent control). Cell lines suitable for use in this assay may include normal human fibroblasts (Now) or Normal Human Keratinocytes (NHK).

In certain embodiments, telomere length can be used as a useful indicator of telomerase expression and/or activity. In one embodiment, telomerase expression and/or activation is important in the treatment of cancer, premature or segmental premature aging, genetic abnormalities, and diseases associated with aging. Telomere length has different expression patterns in the development of a particular disease and is a function of its activity, and thus in certain embodiments it is valuable to determine such length as a function of therapeutic function with respect to prognosis for different diseases.

In one embodiment, telomere length can be determined by southern blotting, hybridization protection assays, fluorescence in situ hybridization, flow cytometry, in situ priming, quantitative-polymerase chain reaction, and single telomere length analysis, all of which are well known in the art (Kah-Wai Lin and Ju Yan, J.cell.mol.Med., 2005, Vol9, No.4,977-.

In certain embodiments, the present invention provides a method of treating a disease in a subject that can be affected by telomerase activation and/or expression, the method comprising administering to the subject an effective amount of a compound of the present invention, wherein the compound stimulates or enhances telomerase expression and/or activity in a cell or tissue of the subject.

In one embodiment, such diseases may include, for example, diseases associated with cell senescence or an increased proportion of cell proliferation in the absence of telomerase, resulting in accelerated deletion of telomere repeat sequences. The term "increased rate of proliferation" means that the rate of cell division is higher than normal cells in that type of cell or normal cells in other individuals of that type of cell. Aging of those cell populations at an abnormally early stage may ultimately lead to disease.

In certain embodiments, the disease may be treated by increasing telomerase expression and/or activity, which may include ex vivo cell therapy with compounds as described herein, using appropriately related cell types, as described further below. In one embodiment, the disease is alzheimer's disease, parkinson's disease, huntington's disease, stroke, nerve injury, motor neuron disease, Multiple Sclerosis (MS), or peripheral and central nervous system injury, including spinal cord injury and cerebrovascular accident; cells which may be used therein are cells of the central nervous system, including neurons and/or glial cells, such as astrocytes, endothelial cells and/or fibroblasts. In another embodiment, the disease is glutamate-induced apoptosis in the cerebellum.

In another embodiment, the disorder is an aging-related skin disorder, such as dermal atrophy and thinning, elastolysis and skin wrinkling, sebaceous gland hyperplasia or hypoplasia, age-related lentigines and/or hyperpigmentation, hair graying and loss or thinning (alopecia, baldness) or chronic skin ulcers; among the cells that may be used are fibroblasts, sebaceous gland cells, melanocytes, keratinocytes, langerhans cells, microvascular endothelial cells and/or hair follicle cells.

In another embodiment, the disease is degenerative joint disease; among the cells that may be used are cells of articular cartilage, such as chondrocytes and interstitial and/or synovial fibroblasts.

In another embodiment, the disease is osteoporosis, osteoarthritis, and/or other degenerative diseases of the skeletal system; cells which can be used therein are cells of the skeletal system, for example osteoblasts, bone marrow stromal cells or stromal cells and/or osteoprogenitor cells.

In another embodiment, the disease is sarcopenia and/or other degenerative diseases of muscle tissue; wherein cells that can be used are muscle cells or their progenitors or mesenchymal cells.

In another embodiment, the disease is an age and stress related disease of the vascular system, including atherosclerosis, calcification, thrombosis, hypertension, and aneurysm; among the cells that may be used are cells of the heart and vasculature, including endothelial cells, smooth muscle cells, and/or adventitial fibroblasts.

In another embodiment, the disease is age-related macular; cells which can be used therein are ocular cells, such as pigment epithelial cells and/or vascular endothelial cells.

In another embodiment, the disease is acquired immunodeficiency or AIDS; among the cells that may be used are T lymphocytes, such as CD4+ or CD8+ T cells. In another embodiment, the disease is an innate immune deficiency.

In another embodiment, the disease is anti-leukemia Graft (GVHD). In another embodiment, the disease is anti-leukemic graft disease (GVL).

In another embodiment, the disease is age and/or stress related damage to the immune system, including impaired tissue turnover following natural aging, cancer therapy, acute or chronic infection, degenerative inflammatory diseases or genetic diseases leading to accelerated cell turnover, and associated anemia and other degenerative diseases, wherein cells that may be used are other cells of the immune system, including cells in lymphoid, myeloid, erythroid lineages, such as B and T lymphocytes, monocytes, circulating and specialized tissue macrophages, neutrophils, eosinophils, basophils, NK cells and their corresponding progenitors.

In certain embodiments, the methods and/or compositions of the invention are applied to improve immune function in a diseased or healthy subject. In certain embodiments, the methods and/or compositions of the invention are applied to enhance T cell activity, e.g., cytotoxic T lymphocyte activity or responsiveness or resuscitation from anergy, which may be applied to treat a variety of diseases, e.g., infection or neoplasia. In certain embodiments, such methods and/or compositions are particularly useful for treating pathogens that are highly resistant to conventional therapies, particularly virulent organisms or organisms for which no other therapy is applicable, such as multi-drug resistant organisms.

In another embodiment, the disease is associated with female fertility, including luteal phase deficits, where there is a normal female menstrual cycle disorder and the body is unable to produce enough progesterone, resulting in delayed development of the uterine lining (endometrium); or premature ovarian failure (ovarian insufficiency or hypogonadotropic hypogonadism), in which the normal function of the ovaries is absent in women under 40 years of age.

In another embodiment, the disease is associated with male fertility, including impaired semen production or impaired semen transport.

In another embodiment, the cell types in which increased telomerase expression and/or activity may be therapeutically beneficial include, but are not limited to, cells of the liver, endocrine and exocrine glands, smooth muscle system, or skeletal muscle system.

In one embodiment, the AIDS disease is believed to be caused by early senescence of CD8+ cells. Such cell aging is not simply due to an abnormal amount of loss of telomere order in each cell doubling, but also to an increased proportion of cell replication, such that telomere loss is greater than normal for the group of cells. The present invention thus provides a method of treating an HIV-infected subject, and more specifically a method of reducing the early senescence of HIV-restricted CD8+ cells in an HIV-infected subject, comprising administering to the subject a compound of the present invention or a composition comprising the same.

In one embodiment, increased telomerase expression and/or activity may be beneficial in non-dividing and proliferating cells, for example in ischemia in diseases associated with increased sensitivity to stress-induced cell death, such as heart failure or stroke (Schneider, J.mol.cell.Cardiol34 (7): 717-24; Mattson, Exp Gerontol.35 (4): 489-. The present invention provides methods of reducing stress-or DNA damage-induced cell death in a subject (e.g., a subject experiencing a tissue ischemic disease resulting from heart failure or stroke) by increasing telomerase expression and/or activity in cells of the subject, comprising administering to the subject a composition comprising a compound of the invention.

In one embodiment, the invention provides a method of prolonging the lifespan of a subject, and the life thereof can be prolonged by augmenting the ability of the subject's cells to continue to replicate or resist stress-induced cell death. Examples of such cell populations are lymphocytes present in patients with down's syndrome. The present invention thus provides a method of enhancing the replicative capacity and/or longevity of lymphocytes present in a patient with down's syndrome by increasing telomerase expression and/or activity in cells of said patient, comprising administering to said subject a composition comprising a compound of the invention. These compositions can also be used to improve resistance to stress-induced cell death that occurs during normal aging.

In one embodiment, the present invention provides a method of increasing telomerase expression and/or activity and thereby promoting healing of wounds, burns, abrasions or other acute or chronic skin disorders, and in certain embodiments, particularly in the epidermis. The present invention thus provides a method of treating acute or chronic skin disorders by administering to the subject a compound as described herein and/or a composition comprising the same. In one embodiment, the composition is generally administered to the affected area.

In another embodiment, the acute or chronic skin disorder treated by the methods of the invention may include wounds, burns, abrasions, surgical incisions, donor graft sites and/or lesions due to infectious agents.

In another embodiment, the acute or chronic skin disorder may include chronic venous ulcers, diabetic ulcers, pressure ulcers, and mucosal surface ulcers or sores.

In another embodiment, the acute or chronic skin disorders may include persistent inflammatory diseases or surface lesions (e.g., abnormal halogen derivative formation and coagulation) caused by infection or genetic defect.

In one embodiment, a method of healing a wound, burn, abrasion or other acute or chronic condition of the skin comprises administering a composition comprising a compound as described herein so as to stimulate or promote cell proliferation or migration at the treatment site where the epithelial cell density is increased as a result of the application of therapy and thereby closing the wound if present or restoring normal physiological function.

In one embodiment, the present invention relates to skin manipulation for other purposes (e.g., cosmetic enhancement) and repair of any imperfections in the skin surface.

In another embodiment, the compounds and compositions as described herein may be used to protect the skin from UV radiation or for palliative treatment of damage thereby.

In another embodiment, the methods and compositions of the present invention may be used to treat hair disorders, preserve hair color, hair shine or quality. In certain embodiments, the methods and compositions of the present invention can be used to treat alopecia or to assist in treatment with hair replacement and alopecia treatment regimens.

In another embodiment, the methods and compositions of the present invention may be used in agricultural applications. In one embodiment, the compounds and compositions as described herein may be used to increase the yield of plants and crops, wherein in one embodiment, fruit yield is increased, and in another embodiment, fruit size is increased. In another embodiment, the compounds and compositions as described herein may be used to increase milk production in dairy cows, while in another embodiment, the size of the egg may be increased. In another embodiment, meat yield may be increased in domestic animals, including chickens, cattle and pigs, that are weighted for meat. In another embodiment, the compounds and compositions as described herein may be used in the racing industry to increase the reproductive capacity of herdshorses and mares and to increase muscle strength and vitality in order to achieve greater speed of progression.

In one embodiment, the methods and compositions of the invention may be used to increase the replicative capacity and/or longevity of cells in culture (e.g., ex vivo cell therapy or monoclonal antibody production) by increasing telomerase expression and/or activity in the cells. Increased telomerase expression and/or activity increases the replicative capacity of such cells by slowing down telomeric repeat deletions and/or improving resistance to stress-induced cell death during cell proliferation.

In one embodiment, the methods and compositions of the invention may be used for allogeneic cell therapy, which may include, in one embodiment, stem cell transplantation, donor cell transplantation, or cancer immunotherapy. In another embodiment, the methods and compositions of the present invention can be used to activate and/or mobilize stem cells. For example, and in certain embodiments, the methods and compositions of the invention can be used to increase stem cell survival in ex vivo cultures. In certain embodiments, the methods and compositions of the present invention may extend the culture time, thereby supporting expansion of the cell population, thereby improving transplantation efficiency, while in other embodiments, the methods and compositions of the present invention may increase cell viability and/or may increase the life of cells at the stem cell stage prior to cell differentiation. In certain embodiments, treating the population with a compound as described herein makes them more amenable to other stem cell manipulations, such as transformation or transduction.

In one embodiment, the stem cell may be an embryonic stem cell, while in another embodiment, the stem cell may be an adult stem cell. In one embodiment, the methods and compositions of the invention can be used to culture stem cells in vitro. In another embodiment, the methods and compositions of the invention can be used to culture non-stem cells in vitro. In one embodiment, the methods and compositions of the invention may be particularly useful for culturing pancreatic beta cells in vitro. In another embodiment, the methods and compositions of the present invention may be used in tissue engineering. In another embodiment, the stem cell may be a cancer stem cell. In one embodiment, the methods and compositions of the invention can be used to treat aplastic anemia.

In certain embodiments, the methods and compositions of the present invention may be used as an adjunct therapy in the treatment of cancer in combination with surgical, radiation, chemotherapy and immunotherapy methods/compounds and to enhance responsiveness to other cancer therapeutic agents. In certain embodiments, the compounds of the present invention inhibit cancer latency, i.e., maintenance of neoplastic cells in an active state, thereby making other compounds toxic to cancer cells more effective. In certain embodiments, the methods and compositions of the invention, when used in adjuvant therapy for cancer, can increase the likelihood of survival of a subject having cancer. In certain embodiments, the methods and compositions of the present invention can prevent damage to embryos from drug treatment, which in one embodiment can be anti-cancer drug treatment. In certain embodiments, the methods and compositions of the invention are used to develop cancer diagnostic agents. For example, and in certain embodiments, the compounds of the invention expand the population of cancer cells that are difficult to detect, thereby aiding their detection, and thus are a means/method of cancer diagnosis. In certain embodiments, the cancer stage may be a reflection of responsiveness to a compound as described herein.

In certain embodiments, the methods and compositions of the present invention are used to develop new treatment regimens. For example, and in certain embodiments, the compounds of the invention expand the population of cancer cell populations that are difficult to detect or treat, thereby enabling the design of anti-cancer compounds that are more suitable for treating a particular cancer cell population. In certain embodiments, treating the population with a compound as described herein makes them more susceptible to other anti-cancer therapeutic agents, or in certain embodiments, enables evaluation of combination therapies or novel compounds.

In certain embodiments, the methods and compositions of the invention are used for cancer stem cell expansion, and thus for the development of therapies and treatments for cancers comprising such cells. In certain embodiments, the methods and compositions of the invention are used for cancer stem cell expansion, cancer disease staging and diagnosis.

In one embodiment, the methods and compositions of the invention may be used to promote cell differentiation and metastasis, while in another embodiment they may be used for cell cloning and/or transduction or transformation. In another embodiment, the methods and compositions of the invention can be used to promote plasmid or naked DNA uptake, liposome uptake, etc., in cells, resulting in, in certain embodiments, increased efficiency of nucleic acid transformation.

In one embodiment, the present invention provides a method for increasing the replicative capacity and/or longevity of a cell by ex vivo administration, wherein a compound of the invention is added to explanted cells from a subject, thereby increasing the replicative capacity and/or longevity of the cell.

Explant cells may include, for example, stem cells, such as bone marrow stem cells (US patent US6,007,989); bone marrow stromal cells (Simonsen et al, Nat Biotechnol20 (6): 592-6, 2002) or adrenal cortex cells (Thomas et al, Nat Biotechnol18 (l): 39-42, 2000). Ex vivo cell-based therapies may also be performed on the disease conditions of the present invention. Examples include the use of myosatellite cells for the treatment of muscular dystrophy, osteoblasts for the treatment of osteoporosis, retinal pigment epithelium for the treatment of age-related macular, chondrocytes for the treatment of osteoarthritis, and the like.

In one embodiment, the present invention provides a method of treating congenital dyskeratosis comprising administering a compound or composition comprising a compound of the present invention.

Congenital Dyskeratosis (DKC), also known as jin-en-kosans syndrome, is a rare progressive bone marrow failure syndrome characterized by a triple combination of reticular skin hyperpigmentation, nail dystrophy and oral leukoplakia. The mutated gene in X-linked dkc (dkcl) encodes a highly conserved nucleolin known as "dystrophin". There is evidence of telomerase dysfunction, ribosome deficiency and protein synthesis dysfunction in this disease. Early mortality is often associated with bone marrow failure, infection, fatal pulmonary complications, or malignancy.

Patients with DKC had reduced telomerase expression and/or activation and abnormally short bundles of telomeric DNA compared to normal controls. Because telomeres play a role in maintaining chromosome stability, telomerase plays a key role in preventing cell senescence and cancer progression. DKC and a group of aplastic anemia are due to telomerase deficiency.

In one embodiment, the invention provides a method of stem cell proliferation in which a population of stem cells is treated with a compound of the invention and thereby the replicative capacity and/or lifespan of the population of cells is provided.

In certain embodiments, the present invention provides the use of compounds and/or compositions as described herein as therapeutic agents in preempting and reversing cellular aging, including, but not limited to, diseases associated with cellular aging. In certain embodiments, compounds and/or compositions as described herein may be used in cell or organ culture media in order to expand primary cell cultures, particularly for the purpose of diagnosis, analysis or vaccine production, or to maintain organ/cell viability at the time prior to transplantation (during ischemia, the time between interruption of blood supply and reconstitution). In certain embodiments, the invention provides the use of compounds and/or compositions as described herein for prolonging the survival of cells or tissues for transplantation, and/or in certain embodiments facilitating the addition of larger such implants, in certain embodiments such features are particularly useful in such applications as pancreatic islet or marrow cell transplantation, stem cell transplantation or tissue engineering.

In certain embodiments, the invention provides the use of a compound and/or composition as described herein as a stimulator of cells: (a) cells having replication-competent ability in the central nervous system, including astrocytes, endothelial cells and fibroblasts, which play a role in diseases associated with aging, such as alzheimer's disease, parkinson's disease, huntington's disease and stroke; or repair of nerve damage, motor neuron disease, peripheral and central nervous system injury, including spinal cord injury and cerebrovascular accident (CVI), such as stroke or diseases such as Multiple Sclerosis (MS); (b) cells with limited replication capacity in the epidermis, including fibroblasts, sebaceous gland cells, melanocytes, keratinocytes, langerhans cells and hair follicle cells, which may play a role in senescence-associated epidermal diseases such as dermal atrophy, elastolysis and skin wrinkling, sebaceous gland hyperplasia, senile lentigo, hair graying and loss or rarefaction (alopecia, baldness), chronic skin ulcers, keratoses and impaired wound healing associated with senescence; (c) cells with limited replication capacity in articular cartilage, such as chondrocytes and interstitial and synovial fibroblasts, which play a role in degenerative joint disease; (d) cells with limited replication capacity in bone, such as osteoblasts and osteoprogenitor cells, which play a role in osteoporosis; (e) cells with limited replication capacity in the immune system, such as B and T lymphocytes, monocytes, neutrophils, eosinophils, basophils, NK cells and their corresponding progenitors, which play a role in the impairment of the immune system associated with aging; (f) cells with limited replication capacity in the vascular system, including endothelial cells, smooth muscle cells and adventitial fibroblasts, which play a role in aging-related diseases of the vascular system, including atherosclerosis, calcification, thrombosis and aneurysms; (g) cells with limited replication capacity in body organs, including but not limited to liver, lung and pancreas (islet cells), which may play a role in liver disease (cirrhosis), lung disease or diabetes; (h) cells with limited replication capacity in the reproductive system, including follicular cells and luteal cells; (i) cells with limited replication capacity in the ear, including the inner and outer ciliated auditory cells of the spiral organ; and (j) cells with limited replication capacity in the eye, such as pigment epithelial cells and vascular endothelial cells, which play an important role in the age-related macula.

The following examples are provided to more fully illustrate preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

Examples

Example 1

Synthesis of Compound 77

A solution of l, l, l-tris (4-hydroxyphenyl) ethane (4g, 13mM), formaldehyde (3.6g,120mM) and 40% dimethylamine in water (15ml) was added to a solution of 50ml water and 60ml EtOH. The solution was refluxed for 2.5 hours. The solvent was partially evaporated and a white solid precipitated, filtered, washed with water and dried to give 7.85g of compound 77 as a white solid in 93% yield mp. ═ 169 °

NMR CDCl₃6.64(6H，s，ArH)，3.40(l2H，s，CH₂)，2.22(36H，s，N-CH₃)，2.06(3H，s，C-CH₃).

Example 2

Synthesis of Compound 84

Compound 84 was synthesized by the same method as described in example 1.

^---NMR CDCl₃6.71(6H，s，ArH)，3.58(12H，s，CH₂)，z.54(24H，q，J=7.0Hz)，1.04(24H，t，J=7.0Hz).

Example 3

Synthesis of Compound 78

L, l, l-tris (4-hydroxyphenyl) ethane (1.53g,5mM), formaldehyde (1.35gr, 45mM) and 1-methylpiperazine (2.5ml,50mM) were refluxed in 20ml water and 25ml EtOH for 3 hours. Evaporation gave a solid which was found by TLC and NMR to contain 2 products, not the starting material. Formaldehyde (0.75g,25mM) and 1-methylpiperazine (1.5ml, 30mM) were added to 5ml of water and 10ml of EtOH and the reaction was refluxed for 4 hours. Evaporation and work-up gave 3.3g of a pale yellow-white solid, 67% yield, mp.63 °. Is soluble in ethanol and has very good solubility in water.

NMR CDCl₃6.67(6H，s，ArH)，3.53(12H，s，CH₂) 2.44(49H, br. m, piperazine ring, 2.26(18H, s, N-CH)₃)，2.00(3H，s，C-CH₃).

Example 4

Synthesis of Compound 83

NMR CDCl₃87.93(3H，s，OH)，6.79(6H，s，Ar-H)，4.54(12H，s，Ar-CH₂)，3.55(12H，q，J=7.0Hz，CH₂)，2.05(3H，s，C-CH₃)，1.22(18H，t，J=7.0Hz，CH₃).

Example 5

Synthesis of Compound 81

Compound 81 was synthesized by the same method as described in example 1. A white solid was obtained, mp. ═ 135 °.

NMR CDCl₃6.68(6H，s，ArH)，3.61(l2H，s，CH₂)，2.51(24H，br.t，N-CH₂Cyclo), 2.03(3H, s, C-CH)₃)，1.76(24H，br.t，N-CH₂A ring).

Example 6

Synthesis of Compound 82

Compound 82 was synthesized by the same method as described in example 1. A white solid was obtained, mp. ═ 212 °.

NMR CDCl₃6.68(6H，s，ArH)，3.69(24H，t，J=4.5Hz，N-CH₂Cyclo), 3.52(12H, s, CH)₂)，2.45(24H，br.t，O-CH₂Cyclo), 2.03(3H, s, C-CH)₃).

Example 7

Synthesis of Compound 79

Step 1: compound 77(2.98g, 4.6mM), prepared by the method as described in example 1, was added to 20ml of acetic anhydride and heated to 100 ℃ for 4 hours. The mixture was cooled and water was added. The mixture was stirred at room temperature overnight and then with CH₂Cl₂And (4) extracting. Evaporation of the solvent gave the nona-acetate derivative as a yellow oil, which was further purified by chromatography (silica gel; 1% MeOH/CH)₂Cl₂) This gave 3.2g of a viscous yellow oil in 80% yield.

Step 2: aqueous KOH (4g) was added to a solution of the nonacetate from step 1 (2.5g) in 20ml EtOH. The mixture was stirred at room temperature for 20 hours. The mixture was acidified with HCl and CH₂Cl₂And (4) extracting. Evaporation of the solvent gave 2.2g of a yellow oil which was further purified by column chromatography (silica gel; 2% MeOH/CH)₂Cl₂) Recrystallization from toluene-hexane gave 1g of compound 79, 53% yield, as a white solid, mp78 °. TLC-Rf 0.55 in 5% MeOH/CH₂Cl₂In (1).

Example 8

Synthesis of l, l, l-tris (4-hydroxy-3, 5-dibromo-phenyl) -ethane (Compound 68)

Step 1: NaOH (1g, 25mM) in 10ml of water and dimethyl sulfate (5.1gr, 40mM) (1: 8 molar ratio) were added to 1,1, 1-tris (4-hydroxybenzene) in portions simultaneously over the course of 1 hourBase) -ethane (1.53g,5mM) in 20ml ethanol and 10ml water. The solution was then refluxed for 1 hour and stirred at room temperature for 70 hours. The white precipitate was filtered off, washed with water and dried to yield 1.74g1,1, 1-tris (4-methoxyphenyl) -ethane. Recrystallization from 50ml ethanol was carried out twice to give 1.15g of white crystals, 66% yield, m.p.160 °. TLC Rf 0.85 in CH₂Cl₂In (1).

NMR CDCl₃6.99，6.79(12H，AB_q，J_AB=8.8Hz)，3.78(9H，s，OCH₃)，2.11(3H，s，CH₃).

Step 2: to a solution of 1,1, 1-tris (4-methoxyphenyl) -ethane (0.49gr, 1.4mM) from step 1 in 22ml of 1, 2-dichloroethane was added a solution of bromine (1.65gr, 10.2) (7.3: 1 ratio) in 5ml of 1, 2-dichloroethane in portions. The solution was stirred at room temperature overnight and heated for 3 hours to 70 °. And worked up (sodium thiosulfate) to give 1.0g of crude product. TLC showed no starting material, while NMR showed a mixture indicating incomplete bromination (multiplet at 6.90ppm, and 4 methoxy groups). The solid was re-brominated with 1g of bromine and refluxed for 18 hours. The mixture was worked up as described above and triturated with hot ethanol to give 0.27g of a white solid, 23% yield, mp 160 °. TLC Rf 0.95 in CH₂Cl₂In (1).

NMR CDCl₃7.16(6H, s, ArH), 3.92, 3.91(6:4 ratio) (9H, 2s, OCH)₃) 2.04, (4:6 ratio) (3H, s, CH)₃).

Example 9

Synthesis of 1,1, 1-tris (4-hydroxy-3, 5-diiodo-phenyl) -ethane

To 1,1, 1-tris (4-hydroxyphenyl) -ethane (1.53g,5mM) in 40ml ethanol and 40ml water cooled in ice was added KOH (2.2g,39.2mM) followed by KI (5.8g, 34.8mM) and iodine (8.8g, 34.7 mM). The color changed from purple to brown. The reaction system is at room temperatureStirred for 3 hours. The mixture was added to crushed ice. Concentrated HCl is added to obtain an acidic pH and treated with thiosulfate solution and worked up. Evaporation gave 5.1g of a light brown solid, the hexaiodide product, which was subsequently triturated in ethanol to give 3g of a white solid in 61% yield, mp 230 °. RF ═ 0.8 (in 5% MeOH-CH)₂Cl₂In (1).

NMR CDC1₃7.3(6H,s),S.77(br.s,OH),1.97(3H,s,CH₃).

Compound 83 was synthesized by the same method as described in example 1. A white solid was obtained, mp. ═ 178 °.

NMR CDCl₃6.68(6H，s，AfH)，3.55(12H，s，CH₂)，2.51(24H，br.t，N-CH₂Ring, 2.03(3H, s, C-CH)₃)，1.55(24H，br.t，N，CH₂Ring), 1.42(12H, br.s).

Example 10

Repair of double stranded DNA fragments using telomerase

To assess telomerase activity and/or expression, DNA repair assays were performed. The generation of DNA fragments by ion irradiation, all compounds increased repair. These results indicate that telomerase activity and/or expression provides stability in stress situations.

Example 11

Effect of Triphenyl telomerase activating Compounds on Life extension Using nematodes

Nematodes (c. elegans) were dosed with the triphenyl telomerase activating compounds provided in the table below, and telomerase activity and/or expression was determined in a manner that extends nematode longevity. Nematodes were grown with 50 micromolar concentrations of activator and the mean, maximum and half-life of two representative molecules, compound 68 and compound 77, were determined compared to the control group (figure 8). The values represent the average lifetime.

TABLE 1

Compound #	R	x	The service life is prolonged%
				60	OH	P	-2
61	OH	P=O	30
				62	OCH₃	P	29
63	OCH₃	P=O	0
				64	H	P	19
65	CH₃	P	42
				66	Br	N	19

Example 12

Action of triphenyl telomerase activating compound on human glioblastoma cellsBy using

Compounds 1, 62, 68, 77 and 79 (referred to in the table above) were tested for telomerase activation and/or expression in glioblastoma cells. Compounds 79 and 68 were synthesized as described above. Compounds 1 and 62 are commercially available. mu.M of nuclear cell protein (as a source of telomerase) was added to a telomerase specific reaction mixture and Telomere Repeat Amplification (TRAP) assays were performed in the presence or absence of telomerase activator (compounds 79, 68, 1 and 62). The reaction products were then analyzed by autoradiography on PAGE. Telomerase activity/expression% was calculated.

U-251 glioblastoma cells were treated with varying concentrations of the compound of the invention for 1 or 3 hours. The medium was removed and the cells were washed several times with PBS. Chaps nuclear extraction was performed by standard methods and telomerase activity was measured by TRAP using radioactive nucleotides. Representative photographs of several experiments showing the products of telomerase activity observed by TRAP assay are shown in figure 1A. Cells were treated with 1 (lanes 2-3) or 0.25 μ M (lanes 3 and 5) of compound 68 for 1 (lanes 2-3) or 3 (lanes 4-5), respectively, hours. Lane 1 shows vehicle treatment only. Telomerase activity was quantified for multiple experiments by measuring the total radioactive label of the telomerase product using a beta-scintillation counter, and the telomerase activity in each sample was calculated as compared to the internal standard. The percent telomerase activity of the compounds of the invention (derived from vehicle-only treated control cells) was determined (fig. 1B). The compounds of the invention significantly increase telomerase activity in treated cells, and the level of activation depends on the nature, concentration of the compound, and the time the cells are exposed to the compound of the invention.

The effect of the compounds of the invention on telomerase protein levels and the effect of a single dose of compound 68 on telomerase long term treatment was tested by contacting cells of different cycles (3-24h) with 1 μ M of the compound and detecting telomerase protein levels by western blot analysis using anti-telomerase antibodies. Compound 68 increased telomerase protein levels in treated cells (fig. 2A). An increase in telomerase was observed over time, with a peak in activity at 1-3h of treatment, then a gradual decline. Telomerase activity levels were only slightly higher than those determined in vehicle-treated cells after 24h (fig. 2B). No change in β -actin (lower lane) levels, or other ribozymes such as topoisomerase I were observed, suggesting that a single dose resulted in rapid specific activation of telomerase, however, this activation was transient and telomerase levels returned to their basal values 24h after treatment, indicating the ability of the compounds of the invention to control telomerase activation.

Experiments were also performed to determine the effect of the compounds of the invention on telomerase mRNA levels (including splice variant terminal-mRNAs). Cells were treated with single doses of 1mM compound 68 for different cycles (1-24 h). Total RNA extracts were prepared and analyzed by northern blotting for equivalent RNA concentrations to a specific hTERT probe. FIG. 3A shows the telomerase full length and splice variant mRNAs in control untreated cells (lanes 1, 2). The levels of full-length mRNA and splice variant RNAs increased significantly over time in cells treated with compounds of the invention (up to 4.5-fold over control) (fig. 3B, lanes 3-7). The increased expression of telomerase is specific, as no effect on GAPDH mRNA was observed (lower lane). Addition of actinomycin D (RNA transcription inhibitor) (3h) during treatment with the compounds of the invention abolished activation of telomerase expression (fig. 3A and B, compare lanes 8 to 4), suggesting that the compounds of the invention activate telomerase gene expression.

Example 13

Effect of Compounds of the invention on human mesenchymal Stem cells

Human mesenchymal stem cells were isolated by iliac crest aspiration and grown in cell culture medium for 6 months (passage 19), after which a significant decrease in cell growth was observed. The compounds of the invention were added directly to the medium daily for 3 days. Significant survival and proliferation of hmscs was observed upon treatment with various compounds of the invention (fig. 4).

By placing on a slide in a tissue culture chamber 17x10³Cell/well culture of hmscs (passage 3) 60h test the possibility that compounds of the invention promote hMSC survival and proliferation by activating TERT. The compound of the invention was added with fresh medium for 6 h. Immunofluorescence using an anti-hTERT antibody (primary antibody) and a cy3 fluorescent secondary antibody was performed. Nuclei were stained with DAPI. The compounds of the invention activated telomerase expression in hmscs (fig. 5). Telomerase protein expression was observed in the cytoplasm and nucleus. The degree of activation of telomerase expression is Compound 68>79>77. Telomerase activity in hmscs was quantified by real-time PCR (quantitative telomerase assay kit) in nuclear extracts derived from hmscs 6 or 24h treated with compound 79(250 nM). Compound 79 enhanced telomerase activity in hmscs 12-or 6-fold in a time-dependent manner (fig. 5B).

Example 14

Effect of the Compounds of the invention on human keratinocytes

Human keratinocytes (at their 5 th generation) that had lost their proliferative capacity were obtained from a local skin bank and treated with compound 68 for 24 h. Cell viability, cell number and morphology were examined. Cells treated with the compounds of the invention showed significant (2-4 fold) cell proliferation and viable cells were observed (figure 6). Compound 79 exhibited a cell survival effect superior to that of compound 68. These results support the role of the compounds of the invention in skin grafting, wound healing, chronic skin ulcers and other skin disorders.

Example 15

Effect of the Compounds of the present invention on human retinal pigment epithelial cells

Chronic exposure of human Retinal Pigment Epithelial (RPE) cells to oxidative stress predisposes them to age-related macular degeneration (AMD). By using H₂O₂(1mM) for 24 hours and H₂O₂(0.5mM) treatment was continued for 24 hours to expose human RPE cells to oxidative stress. Then daily with the indicated compound in the absence of H₂O₂Cells were treated in the presence for 2 days. An increase in cell growth was observed as a result of treatment with the compound (fig. 7A). Telomerase activity was increased 30-and 50-fold in RPE cells treated with compounds 68 and 79, respectively (fig. 7B). These results suggest a role for the compounds of the present invention in the treatment of macular degeneration and other retinal diseases.

Example 16

Prolongation of survival in nematode c

The survival of nematodes c, elegans, which generally survive 12-14 days, represents an acceptable model for studying longevity. Nematode triphenyl telomerase activating compounds were administered and their effect on nematode longevity prolongation was determined. Nematodes were grown with activator at a concentration of 50 micromolar and the mean, maximum and half-life were determined compared to untreated controls. The lifetime increased by 13-43% (table 2) and the average lifetime increased by about 2-fold (from 12 days to 22 days, as shown in figure 8).

Table 2: prolongation of nematode C

Number of Compounds	Life extension (%)
		61	30
62	29
		68	13
77	43
		78	36
79	16

Example 17

Activation of telomerase expression in rat endometrial cells by Compounds of the invention

Estrus female rats (the stage in which the epithelial layer (EP layer) in the rat endometrium is degraded) were injected subcutaneously with 6mg/kg of compound 68 or vehicle. After 24h the rats were sacrificed and endometrial sections were prepared from the uterine horns of treated and untreated rats and analyzed by histological methods and immunohistochemical staining. Endometrial sections were stained with hematoxylin-eosin. Treatment with the compounds of the present invention prevents degradation of the epithelial layer of the endometrium. Furthermore, morphologically, endometrial tissue was similar to the results of the proliferative phase (pro-estrus and estrus) (fig. 9A, B). Activation of telomerase expression in endometrial cells of treated rats was examined by immunohistochemistry using anti-telomerase antibodies (fig. 9C-F). Telomerase staining was significantly increased in rat endometrial tissues injected with the compounds of the invention, especially in the endometrial cavity epithelial layer and secretory glandular epithelial layer (compare figures 9C and D, E and F). In addition, telomerase activity was measured in nuclear extracts of endometrium from different treatment groups. A 4-8 fold increase in telomerase activity was observed in rats injected with the compound of the invention (fig. 9G), indicating that the compound of the invention injected subcutaneously into the neck reached endometrial tissue, activating telomerase and causing tissue proliferation. These data support the effect of the compounds of the invention in activating telomerase and affecting tissue proliferation in vivo in animals. In certain embodiments of the invention, the data supports the effects of using the compounds of the invention for the treatment of endometriosis.

Example 18

Telomerase activation of rat cerebral cortical cells by the compounds of the invention

The effect of the compounds of the present invention injected subcutaneously into female rats and adult male mice on telomerase expression in different regions of the brain was examined. Co-localization of telomerase and the DNA-binding fluorescent dye DAPI (DAPI or 4', 6-diamidino-2-phenylindole) indicates activation and/or expression in these cells as a result of treatment. Very low telomerase expression was observed in specific cells in different regions of the CNS (fig. 10 and 11). Injection of compounds 79 and 77 into rats significantly activated telomerase expression in rat cortex, but compound 68 did not, indicating that compounds 79 and 77 (but not compound 68) can cross the Blood Brain Barrier (BBB) (fig. 10).

Compound 79 was injected subcutaneously into three-month-old male mice. Mice were sacrificed 24h later and cortex, cerebellum, hippocampus, hypothalamus, brainstem and olfactory bulb were isolated and immunofluorescence was performed. Low telomerase expression was observed in most of the brain regions examined except for Purkinje cells in the cerebellum (fig. 11). Injection of the compounds of the invention significantly and significantly increased telomerase expression in different brain regions. This activation appears to be specific for certain neuronal cells. For example, compound 79 increased telomerase expression in motor neuron cells present in the brain stem.

Confirmation of immunofluorescence results was obtained by examining the level of TERT protease in a nucleoprotein extract derived from the brain of rats treated or untreated with the compounds of the invention. Western blot analysis was performed using specific anti-hTERT antibody and telomerase activity (%) was determined and calculated compared to the results obtained using vehicle. A significant increase (9-13 fold) in telomerase protein was observed in rat brain cortical cells treated with the compounds of the invention (fig. 12), supporting the role of the compounds of the invention as a utility in the treatment of brain-related diseases.

Example 19

Prevention of glutamate-induced apoptosis in the cerebellum of mice

The ability of the compounds of the present invention to prevent glutamate-induced apoptosis in the cerebellum of mice was examined. Mice were injected with 6mg/kg of compound 79 or vehicle alone. Mice were sacrificed 24h after treatment and their brains removed. Cerebellum sections were prepared and subjected to glutamate treatment at different concentrations for 30 min. Sections were stained with propidium iodide and the number of apoptotic cells was counted and calculated using special software (Jimage). Prevention of apoptosis in mice treated with the compounds of the present invention was calculated as a percentage compared to control mice (fig. 13). This supports the effect of the compounds of the invention in the treatment of seizure disorders, stroke, alzheimer's disease, epilepsy, schizophrenia, and alcohol and opiate addiction.

Example 20

Telomerase activation of mouse heart cells by the compounds of the invention

The activation of telomerase in heart protein extracts derived from mice by the compounds of the invention was determined (figure 14). Cardiac telomerase expression was significantly increased following injection of compound 79. This supports the role of the compounds of the invention in the treatment of infarction, ischemia, myocarditis, etc.

Example 21

Prevention of drug-damaged embryos by the compounds of the invention

The ability of the compounds of the invention to prevent the effects of drugs on embryonic development damage was examined in rats. Female rats were treated with Camptothecin (CPT) (5mg/kg), an anti-cancer drug, and then injected with compound 68(6mg/kg) or vehicle (0.1%). Embryos were removed at day 14-15 of gestation and examined for lesions. Embryos treated with CPT and compound 68 developed normally, while embryos treated with CPT alone showed signs of dysplasia (fig. 15).

Example 22

Effect of the Compounds of the invention on BCLl, tumorigenicity

(BALB/C x C57BL/6) F1 mice were inoculated with 10⁵Murine B-cell leukemia lymphoma (BCLl) cells. Splenomegaly and lymphocytosis (leukemia) occurred in all control groups and mice treated with telomerase activating compounds (40 or 80 mg/kg).

TABLE 3

Experimental group	Survival mean (range)
		BCL1 only	29(21-35)
BCL1+ Compound 7710mM	26(22-41)

Telomerase-activating compounds do not promote leukemia progression and do not shorten the survival of leukemic vaccinated mice. The telomerase-activating compounds of the present invention do not promote the development of existing cancers and chronic administration of the compounds does not result in the development of cancer.

Example 23

Effect of the Compound of the present invention on graft-versus-leukemia (GVHD) as a representative T cell function

(BALB/C x C57BL/6) F1 mice were inoculated with 30x10⁶C57BL/6 splenocytes to induce GVHD. Mice treated with compound 77 exhibited more severe signs of acute GVHD as evidenced by weight loss compared to untreated controls. These results suggest that the compounds of the present invention can be used as potent adjuvants.

Example 24

Effect of the Compound of the present invention on graft-versus-leukemia (GVL) as representative of T cell immunotherapy Examples of the invention

F1 mice (BALB/cx C57BL/6) were inoculated with 10 after a total systemic irradiation of 400cGy⁵BCLl to allow graft engraftment of allogenic lymphocytes, inducing 30x10⁶C57BL/6 splenocyte-mediated graft-versus-leukemia (GVL) effects. The survival of the untreated control group ranged from 21-35 days (mean 29 days). Mice treated with heterologous stem cells have survival ranging from 13-22 days because leukemia activates severe GVHD. In contrast, survival of mice treated with the allogeneic stem cells and the compound of the invention ranged from 37-74 days, with 1 mouse surviving 125 days. This supports the role of the compounds of the invention in the treatment of leukemia.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. Use of a compound represented by the structure of formula V or an isomer, a pharmaceutically acceptable salt, a pharmaceutical product, or any combination thereof, for the manufacture of an agent that stimulates or promotes telomerase expression, activity, or a combination thereof in a cell or tissue in vitro:

wherein

R ', R ", R'" is hydrogen;

R₁'，R₃'，R₄'，R₆'，R₇' and R₉' same or different are dialkylamino, N-piperidinyl, N-pyrrolidinyl, N-piperazinyl, N-piperazin-4-methyl, and N-morpholinyl; and is

R₁₀Is methyl.

2. The use of claim 1, wherein the compound is represented by formula VIII:

3. the use of claim 1, wherein the compound is represented by formula IX:

4. the use of claim 1, wherein the compound is represented by formula X:

5. the use of claim 1, wherein the compound is represented by formula XI:

6. the use of claim 1, wherein the compound is represented by formula XII:

7. the use of claim 1, wherein the cell or tissue is contacted with a pharmaceutical composition comprising the compound.

8. The use of claim 1, wherein the cell or tissue is isolated from or in vivo from a subject having: neurodegenerative diseases, nervous system injury, vascular diseases, diseases or disorders associated with aging, graying of hair or hair loss, degenerative joint diseases, degenerative diseases of the skeletal system, degenerative diseases of muscle tissue, macular degeneration, infections, immune system injury, cancer, degenerative inflammatory diseases, genetic diseases leading to accelerated cell turnover, anemia, or infertility in males or females.

9. Use of a compound represented by the structure of formula V or an isomer thereof, a pharmaceutically acceptable salt, a pharmaceutical product, or any combination thereof, for the manufacture of a medicament for treating a disease capable of being affected by telomerase expression, activity, or a combination thereof, in a subject, the disease selected from the group consisting of a neurodegenerative disease, a nervous system injury, a vascular disease, a disease or disorder associated with aging, graying of hair or alopecia, degenerative joint disease, a degenerative disease of the skeletal system, a degenerative disease of muscle tissue, macular degeneration, diabetes, infection, an injury of the immune system, cancer, a degenerative inflammatory disease, a genetic disease leading to accelerated cell turnover, anemia, or male or female infertility:

wherein

R ', R ", R'" is hydrogen;

R₁₀Is methyl.

10. The use of claim 9, wherein the cancer is leukemia.

11. The use of claim 9, wherein the compound is represented by formula VIII:

12. the use of claim 9, wherein the compound is represented by formula IX:

13. the use of claim 9, wherein the compound is represented by formula X:

14. the use of claim 9, wherein the compound is represented by formula XI:

15. the use of claim 9, wherein the compound is represented by formula XII:

16. the use of claim 9, wherein the compound is included as part of a pharmaceutical composition.

17. Use of a compound represented by the structure of formula V or an isomer, a pharmaceutically acceptable salt, a pharmaceutical product, or any combination thereof, in the manufacture of a medicament for treating an acute or chronic skin disorder:

wherein

R ', R ", R'" is hydrogen;

R₁₀Is methyl.

18. The use of claim 17, wherein the acute or chronic disease is a wound, burn, abrasion, incision, graft site, lesion caused by an infectious agent, chronic venous ulcer, diabetic ulcer, pressure ulcer, mucosal pain or ulcer, melanoma or keloid formation.

19. The use of claim 17, wherein the compound is represented by formula VIII:

20. the use of claim 17, wherein the compound is represented by formula IX:

21. the use of claim 17, wherein the compound is represented by formula X:

22. the use of claim 17, wherein said compound is represented by formula XI:

23. the use of claim 17, wherein the compound is represented by formula XII:

24. the use of claim 17, wherein the compound is part of a pharmaceutical composition.

25. The use according to any one of claims 19 to 24, wherein the acute or chronic disease is a wound, a burn, an abrasion, an incision, a graft site, a lesion caused by an infectious agent, a chronic venous ulcer, a diabetic ulcer, a pressure ulcer, a mucosal pain or ulcer, melanoma or keloid formation.

26. Use of a compound represented by the structure of formula V or an isomer, a pharmaceutically acceptable salt, a pharmaceutical product, or any combination thereof, in the manufacture of an agent for inhibiting, abrogating, or delaying cellular senescence in a cell or tissue of a subject in vitro:

wherein

R ', R ", R'" is hydrogen;

R₁₀Is methyl.

27. The use of claim 26, wherein the compound is represented by formula VIII:

28. the use of claim 26, wherein the compound is represented by formula IX:

29. the use of claim 26, wherein the compound is represented by formula X:

30. the use of claim 26, wherein said compound is represented by formula XI:

31. the use of claim 26, wherein the compound is represented by formula XII:

32. the use of claim 26, wherein the compound is part of a pharmaceutical composition.

33. The use of claim 26, wherein inhibiting, abrogating, or delaying cellular senescence in a cell or tissue of a subject extends the lifespan of the subject.

34. The use of claim 26, wherein said cell or tissue comprises a cell or tissue for transplantation in said subject.

35. The use of claim 26, wherein the cell or tissue is isolated from or in vivo from a subject having: neurodegenerative diseases, diseases or disorders associated with aging, graying of hair or alopecia, degenerative joint diseases, degenerative diseases of the skeletal system, degenerative diseases of muscle tissue, macular degeneration, degenerative inflammatory diseases or genetic diseases leading to accelerated cell turnover.

36. Use of a compound represented by the structure of formula V or an isomer, a pharmaceutically acceptable salt, a pharmaceutical product, or any combination thereof, in the manufacture of a medicament for reducing or eliminating the effects of aging in a subject:

wherein

R ', R ", R'" is hydrogen;

R₁'，R₃'，R₄'，R₆'，R₇' and R₉' same or different is dialkylamine-yl, N-piperidinyl, N-pyrrolidinyl, N-piperazinyl, N-piperazin-4-methyl and N-morpholinyl; and is

R₁₀Is methyl.

37. The use of claim 36, wherein the compound is represented by formula VIII:

38. the use of claim 36, wherein the compound is represented by formula IX:

39. the use of claim 36, wherein the compound is represented by formula X:

40. the use of claim 36, wherein said compound is represented by formula XI:

41. the use of claim 36, wherein the compound is represented by formula XII:

42. the use of claim 36, wherein the compound is part of a pharmaceutical composition.

43. The use of claim 36, wherein the aging effect comprises an effect on the skin, hair, eyes, muscle tissue, or bone of the subject.

44. A compound represented by the structure of formula V or an isomer, a pharmaceutically acceptable salt, a pharmaceutical product, or any combination thereof:

wherein

R ', R ", R'" is hydrogen;

R₁₀Is methyl.

45. The compound of claim 44, wherein said compound is represented by formula VIII:

46. the compound of claim 44, wherein said compound is represented by formula IX:

47. the compound of claim 44, wherein the compound is represented by formula X:

48. the compound of claim 44, wherein said compound is represented by formula XI:

49. the compound of claim 44, wherein the compound is represented by formula XII:

50. a pharmaceutical composition comprising a compound of any one of claims 44-49.