KR101407044B1 - Pharmaceutical Composition for Preventing or Treating Aging-related Diseases Comprising Blocker of Progerin and Lamin A Binding and Screening Method Thereof - Google Patents

Abstract

The present invention relates to a novel compound for inhibiting the binding between progeline and lamin A, a pharmaceutical composition for preventing or treating an aging-related disease comprising the novel compound as an active ingredient, and a screening of a binding inhibitor for inhibiting binding between progeline and lamin A The pharmaceutical composition is effective for the treatment or prevention of a disease caused by progerin, particularly, a disease caused by an aged individual such as kidney cancer, leukemia, prostate cancer, progeny, etc., A drug that inhibits the binding between progesterone and lamin A can be specifically selected to effectively develop a therapeutic agent for a disease caused by aging individuals such as kidney cancer, leukemia, prostate cancer, and progeny.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for preventing or treating aging-related diseases, which comprises an inhibitor of progesterone and lamin A as an active ingredient, and a screening method for such a binding inhibitor Screening Method Thereof}

The present invention relates to a novel compound for inhibiting the binding between progeline and lamin A, a pharmaceutical composition for preventing or treating an aging-related disease comprising the novel compound as an active ingredient, and a screening of a binding inhibitor for inhibiting binding between progeline and lamin A ≪ / RTI >

As the lifespan of humans increases, interest in the process of aging is actively being raised, but there are still many things that are not clearly understood. Related studies have been conducted mainly on genetic and molecular aging mechanisms for human progeria. Well known progeria is Hutchinson 's Gilford progria syndrome (HGPS), a very rare autosomal dominant genetic disease caused by silent mutation of G608G of Lamin A (LMN A). The mutation produces a progeny (progerin, Prg), a selective cleavage site product that produces a new cleavage donor site and deletes 50 amino acids of the C-terminal domain of lamin A.

Expression of progerin causes morphological changes such as nuclear irregularity or a decrease in nuclear-cytoplasmic Ramin A In addition, inhibition of pro-gerin expression leads to reduction of nuclear degeneration, and progerin has been known as a major factor of HGPS. Progerin has also been reported to accumulate in senescent normal fibroblasts and cause nuclear deformation. In iPSCs (inducible pluripotent stem cells) of HGPS patients, the expression of progerin and lamin A / C is reduced and nuclear deformation, cellular senescence markers such as H3K9me3 and SA-beta-gal are restored. On the other hand, the differentiated HGPS cells re-express the aging markers according to the expression of progerin.

The onset and progression of HGPS and the aging phenomenon are similar to the general aging process. The inventors of the present invention have studied the action and effects of progesterone in the cells through the study on HGPS. During the study, it was confirmed that the aging effect was achieved by the interaction of progerin and lamin A, and the factors for inhibiting the interaction were identified, and it was confirmed that the agent was effective in the treatment of aging-related diseases.

Therefore, the present invention aims to provide a novel compound which inhibits the binding between progerin and lamin A.

The present invention also provides a pharmaceutical composition for preventing or treating an aging-related disease comprising the novel compound as an active ingredient.

The present invention also provides a method for screening a therapeutic agent for aging-related diseases, which comprises the step of selecting a candidate drug that inhibits binding between progerin and lamin A.

In order to solve the above problems, the present invention provides a compound represented by the following general formula (1) or (2): or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

 (2)

In Formula 1 and Formula 2, wherein R ₁ is

,

또는

,

or

이고,

ego,

R ₂ and R ₃ each independently is hydrogen or C _{1 -4} alkyl.

In one embodiment of the present invention, in the compound represented by Formula 1,

이며, R₂ 및 R₃는 메틸일 수 있다.
R ₁ is

And R < ₂ > and R < ₃ > may be methyl.

The compounds of formula (I) or (II) may be used in the form of a pharmaceutically acceptable salt. The acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid and arpartic acid. Preferably, hydrochloric acid is used as the inorganic acid, and methanesulfonic acid is used as the organic acid.

In addition, the compound represented by the formula (1) or (2) of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by a conventional method.

As can be seen from the following examples, the compound represented by the formula (1) or (2) of the present invention is excellent in the binding inhibition between progerin and lamin A and is useful for prevention and treatment of aging-related diseases caused by progerin . ≪ / RTI > The inventors of the present invention have investigated the aging mechanism induced by progerin, confirmed that aging is caused by the interaction of progerin and lamin A, and the factor for inhibiting the interaction is discovered, It is finished.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating an aging-related disease comprising a compound represented by the following general formula (1) or (2) or a pharmaceutically acceptable salt thereof as an active ingredient.

 [Chemical Formula 1]

 (2)

In Formula 1 and Formula 2, wherein R ₁ is

,

또는

,

or

이고,

ego,

R ₂ and R ₃ each independently is hydrogen or C _{1 -4} alkyl.

More specifically, in Formula 1,

이며, R₂ 및 R₃는 메틸일 수 있다. R ₁ is

And R < ₂ > and R < ₃ > may be methyl.

In one embodiment of the present invention, the compound represented by Formula 1 or Formula 2 can prevent or treat aging-related diseases through a mechanism of inhibiting binding between progerin and lamin A.

As shown in the following examples, the compound represented by the formula (I) or the formula (II) according to the present invention or a pharmaceutically acceptable salt thereof effectively inhibits the binding of progerin to lamin A, Can be usefully used for the treatment of diseases associated with aging caused by binding.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating an aging-related disease comprising a compound represented by the formula (1) or (2) or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutical composition for preventing or treating an aging- Or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a compound represented by formula (1) or (2), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of aging-related diseases ≪ / RTI >

In one embodiment of the present invention, the aging-related disease may be a disease selected from the group consisting of cancer disease and progeny, and the cancer disease may be cancer disease selected from kidney cancer, leukemia or prostate cancer, Syndrome or Hutchison's Guildford syndrome.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating an aging-related disease comprising the compound represented by the above formula (1) or (2) or a pharmaceutically acceptable salt thereof as an active ingredient, 1 to 90 parts by weight, or 10 to 90 parts by weight of the compound represented by the formula (I) or (II) or a pharmaceutically acceptable salt thereof, , The condition of the patient, the type of disease and the degree of progression.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating aging-related diseases comprising the compound represented by Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient may be a carrier, an excipient, a disintegrant, And one or more auxiliaries selected from the group consisting of sweeteners, coating agents, swelling agents, lubricants, lubricants, flavors, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating an aging-related disease comprising the compound represented by the above-mentioned formula (1) or (2) or a pharmaceutically acceptable salt thereof as an active ingredient is a granule, a powder, And may be selected from the group consisting of tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, drops or solutions.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, ≪ / RTI > can be administered to the subject in a conventional manner.

The preferred dosage of the pharmaceutical composition for preventing or treating aging-related diseases comprising the compound represented by the above-mentioned formula (1) or (2) or a pharmaceutically acceptable salt thereof as an active ingredient depends on the condition and body weight of the patient, The mode of administration, the route of administration and the period of time, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 1,000 mg / kg, specifically 0.1 to 1,000 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

The present invention also provides a method for screening a therapeutic agent for aging-related diseases, which comprises the step of selecting a candidate drug that inhibits the binding between progeline and lamin A.

In one embodiment of the invention, the screening method comprises

Progeline, fluorescent protein labeled-lamin A and candidate drug; And analyzing the level of binding of the pro-gerin and the fluorescent protein to select a candidate drug that inhibits the binding between pro-gerin and lamin A,

In another embodiment of the present invention, the aging-related disease may be a disease selected from the group consisting of cancer disease and progeria, and the cancer disease may be cancer disease selected from kidney cancer, leukemia or prostate cancer, Syndrome or Hutchison's Guildford syndrome.

The pharmaceutical composition according to the present invention inhibits the binding between progerin and lamin A and is useful for the treatment or prevention of diseases caused by progerin, particularly diseases caused by aged individuals such as kidney cancer, leukemia, prostate cancer, The above-mentioned screening method can be used to specifically select drugs that inhibit the binding between progerin and lamin A, thereby effectively developing a therapeutic agent for diseases caused by aged individuals such as kidney cancer, leukemia, prostate cancer, progeny, etc. .

FIG. 1 shows experimental results of Example 1 showing the binding relationship between progerin and lamin A / C (LMN A / C) and the effect thereof.
Fig. 2 shows the experimental results of Example 1 showing that proerger and lamin A / C are bound but not lamin B;
FIGS. 3 and 4 are experimental results of Example 2 and Example 3 showing the progenic inhibitor screening process and results.
5 to 9 show experimental results of Example 4 on the effect of the JH compound and the molecular mechanism according to one embodiment of the present invention.
FIGS. 10 to 12 are experimental results of Example 5 in which the anti-aging effect of JH compounds according to one embodiment of the present invention in HGPS cells and normal cells was examined.
FIG. 13 shows experimental results of Example 5 in which the effect of suppressing nuclear membrane irregularity of a JH compound according to an embodiment of the present invention in kidney cancer (RCC) was examined.
14 is a graphical representation of the in vivo toxicity test results.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Cell culture and reagent preparation

The cell line (293; DMEM, A549, HCT116; RPMI-1640) used in the present invention was purchased from ATCC, and other cell lines (UMRC2; C2, UMRC2V; C2V, CAKI2; Jung YJ (Pusan National University). Stable cell lines were obtained by the introduction of GFP-fused Lamin A or progerin vectors into HEK 293 cells. Cells were selected with G418 (200 ug / ml). All cell lines were maintained in a 37 [deg.] C growth chamber in liquid medium containing 10% FBS and 1% antibiotic.

Human fibroblasts obtained from HGPS patients (AG01972; women aged 14, AG03513; 15-year-old male), vasodilatory human fibroblasts obtained from ataxia patients (ATM; AG04405; 6-year-old male) and human fibroblasts obtained from normal (GM 00038; 9 year old female) was purchased from Coriell Cell Repositories and maintained in EMEM medium containing 15% FBS, 2 mM Glu (no antibiotic).

Common chemical inhibitors, including FTI (FTI-277), GGTI (GGTI-298), and callide were purchased from Sigma-Aldrich. In addition, the antibodies (sc-81611), lamin A / C antibody (sc-20680), lamin B antibody (sc-6216), GST antibody (Sc-16), p16 antibody (sc-759), DcR2 antibody (sc-169) The antibody (sc-8036), the antibody of DNA-PKcs (sc-9051) from Santa Cruz, the antibody of H3k9Me3 (ab-8858) from abcam, the antibody of p14 / ARF ) And Emerin were purchased from Novocastra.

2. Vector preparation and transfection

GFP-fusion progerin and GFP-fusion lamin A expression vectors were obtained from Scaffidi and Misteli (NCI). GRP-fused Lamin B and Lamin C were received from Lammerding J (Brigham and Women's Hospital / Harvard Medical School). The mutant form of lamin A was provided by Tesson F (University of Ottawa). Myc-ARF vectors and untagged progerin vectors were purchased from addgene. For mammalian expression of these vectors, transfection was carried out using Jetpei (polyplus). 1.5 [mu] g of vector was mixed with 1.5 [mu] l of Jetpei reagent dissolved in 150 nM NaCl solution. The reaction mixture was incubated at room temperature for 15 minutes, and then the reaction mixture was added to the cells. After 3 hours, the medium containing 10% FBS was replaced with serum-free medium.

3. Recombinant protein preparation and GST - Pulldown analysis

To investigate protein-protein interactions, recombinant proteins were prepared. The recombinant lamin AN-terminal region was prepared by cloning 300AA downstream of the initiation codon, and the lamin AC-terminal region (LC) and progerin-C-terminal region (Prog) Cloning. Total length P14 was prepared by PCR, cloned into pGEX vector and confirmed by DNA sequencing. Using the nickel column, the reprepressed protein was purified. For binding analysis, GST-bead-fused lamin AC or proergen-C was incubated for 2 hours at room temperature with 293 cell lysates obtained from the recombinant lamin AN terminal region, GFP-lamin A, lamin B or lamin C- Lt; / RTI &gt; After washing twice with PBS and once with RIPA, the precipitates were collected, transferred to SDS-PAGE, and subjected to Western blot analysis using anti-GFP, His and GST.

4. Immune sedimentation ( IP , Immunoprecipitation ) And Western Blot ( WB ) analysis

Whole cell lysates were prepared in RIPA buffer and the lysates were centrifuged at 14,000 rpm for 30 minutes. 20 [mu] g of cell extracts were separated via SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membrane was incubated with the appropriate primary antibody at 4 ° C for 1 hour to overnight and reacted with the secondary antibody at room temperature for 1 hour. Peroxidase activity was detected by chemiluminescence using an ECL kit (Intron) according to the manufacturer's instructions.

To investigate the interaction between lamin A and progerin, an antibody to GFP (2 g / sample) was added to the protein extract. After incubating at 4 ° C for 2 hours with stirring, Protein A and Protein G were added. After washing twice with PBS, the precipitate was dissolved in RIPA buffer and SDS sample buffer.

5. Fluorescent antibody method ( immunofluorescence staining ) And aging-specific acidic- Galactosidase  Active dyeing

Cells transfected with the vectors defined on the cover glass were fixed with 100% methanol for 10 minutes at 4 ° C and incubated with blocking buffer (PBS + anti-human Ab (1: 500)) for 1 hour. After washing twice with PBS, the anti-lamin A / C H3K9Me3, Or DNA-PKcs antibody and incubated for 2 hours with anti-goat Ab FITC or anti-rabbit Ab-rhodamine in blocking buffer (1: 1000) and mounted. Nuclei were stained with DAPI and immunofluorescence signals were analyzed using a fluorescence microscope (Zeizz).

For aging-specific acid- beta -galactosidase activity staining, cells were washed once with PBS (pH 7.2) and fixed with PBS containing 0.5% glutaraldehyde. After washing with PBS, cells were stained with X-gal solution (Senescence β-Galactosidase Staining kit; Cell Signaling Technology) overnight at 37 ° C.

6. RNA  Separation and RT - PCR

For RT-PCR, total cellular RNA was extracted using Qiagen RNA extraction kit. After measuring RNA concentration, 1 ㎍ of total RNA was reverse transcribed with cDNA using MMLV RT (Invitrogen) and random hexamer. RT-PCR was performed using the following specific primers:

3 '), reverse primer: SEQ ID NO: 2 (5'-TTTCTTTGGCTTCAAGCCCCC-3'), lamin A / C - forward primer: SEQ ID NO:

3 '(5'-ATCTTCCAGGAGCGAGATCCC-3'), reverse primer: SEQ ID NO: 4 (5'-AGTGAGCTTCCCGTTCAGCTC-3 ') GAPDH-forward primer: SEQ ID NO:

7. MTT  Analysis and cell proliferation

To measure cell viability, the cells were treated with the binding inhibitor of the present invention for 24 hours. For MTT analysis, cells were incubated with 0.5 mg / ml MTT solution for 4 hours at 37 ° C. After the excess solution was removed, the precipitate was dissolved in 200 μl of DMSO and quantitated by measuring the absorbance at 540 nm.

For cell counting, cells were harvested from the medium and stained with Trypan blue for 10 min at room temperature. The number of viable cells was measured using a hemocytometer.

8. ELISA

ELISA assays were performed to isolate the lamin A-proergen binding inhibitor. GST-Proline-C or LMN A-His was fixed in 96-well-plates using 0.5% paraformaldehyde. After drying and washing, the HEK 293 cell lysate transfected with GFP-lamin A, GST-lamin AC or GST-Progrin-C was incubated with the binding inhibitor of the invention at 0.1 mM (final concentration) for 1 hour. 96-well plates were washed with TBST and incubated with anti-GFP-Ab (1: 10,000, 45 min), anti-GST- Ab (1: 10,000, 45 min) and anti- 30 minutes). After washing twice, the plates were incubated with TMB solution (Calbiochem) and incubated with stop solution (1 NH ₂ SO ₄ ). Results were measured using an ELISA reader.

9. In vivo  analysis

C57Bl / 6J mice (n = 28) were divided into four groups, and DMSO or 20 mg / kg of JH compound according to one embodiment of the invention was injected intraperitoneally once a week for 6 weeks. The experiment was carried out in accordance with the regulations of the Animal Care Assessment and Accreditation Association of Pusan National University.

< Example  1> Progerin LMN  Combination with A induces aging.

The effect of progerin on the lamin family was investigated as the phenotype of HGPS was determined by proergen expression. Joint transcription of GFP-LMN A / B / C and GFP-proergene inhibited LMN A / C but not LMN B (see FIGS. 1A and 2A). In order to investigate the effect of progesterone (Prg) on intracellular LMN A / C, LMN A / C expression in untagged or GFP-tagged progerin infected cells was examined, Cdmf &lt; / RTI &gt; inhibition (see Figures 1B and 2B). Progherin expression also induced the expression of normal aging markers such as p16 and DcR2 (see FIG. 1B) and induces nuclear deformation typical of HGPS (see FIG. 1C). To determine whether LMN A was reduced by progerin, the effects of progerin on the expression of LMN A in HCT116 and Prg-stable cells were again determined. In both cases, LMN A / C was detected in progerin infected cells (see Figures 1D and 2C).

To confirm how progerin regulates LMN A / C, we performed binding assays and found that the C-terminal and LMN A of progerin were associated with LMN A / C, but not with LMN B ( See FIG. 1E), progerin showed a stronger affinity for LMN A / C than LMN A (see FIGS. 2D and 2E). On the other hand, progerin did not form a self-association (see FIG. 1F).

To find the binding domain, His-tagged LMN A N-terminal region (LMN AN; 1-300 AA) and LMN A intermediate region (LMN AM; 301-564 AA) were constructed and GST- LMN A or GST-proline. Through these experiments, it was found that the middle part of LMN A was involved in binding with progerin (see FIG. 2F).

For the measurement of binding strength, an ELISA system consisting of a fixed LMN AN terminal domain and a GST-fusion recombinant LMN A or proergin protein was constructed. Through the above analysis, it was found that the binding strength between LMN A and proergene was twice as strong as that of LMN A (see FIG. 1G).

< Example  2> Progerin  Screening of inhibitors

If the progesterone-induced aging is through LMN A binding, then inhibitors that interfere with the binding between the two will be able to relieve aging induced by progerin. To confirm this hypothesis, binding inhibitors between LMN A and proerger were uncovered. An ELISA-based screening system was used to identify the inhibitors. That is, the immobilized progerin was placed in a 96-well plate, the candidate substance selected in the chemical library and the GFP-LMN A were cultured, and the effect of the candidate substance was confirmed through monitoring of progerin and GFP protein binding (see FIG. 3A) ). (JH-0307809; JH1, JH-02051109; JH13, JH-03070708; JH4, and LG-02-089- 23; LG-02) were effective (see Figs. 3B and 4B). The chemical structure of the active chemicals was very similar and the cytotoxicity of the chemicals was measured via MTT analysis (see Figures 3B and 4C).

< Example  &Lt; 3 &gt; NMR  data

The production method of each compound JH1, JH4, JH13 and LG-02 is shown in the following Reaction Schemes 1 and 2.

[Reaction Scheme 1]

Reagents and conditions: (a) carboxylic acid, EDC, 4-DMAP, dry dichloromethane, 5-12 hours at ambient temperature; (b) BBr ₃ 1M solution, dry dichloromethane, dichloromethane at 0 ^o C → room temperature, 5 hr.

[Reaction Scheme 2]

시약 및 조건: (a) 카복실산, EDC, 4-DMAP, 건조 디클로로메탄, 12 시간, 상온; (b) 디클로로메탄에서 BBr₃ 1M 용액, 건조 디클로로메탄, 0 ^oC → 상온, 5 hr.

Reagents and conditions: (a) carboxylic acid, EDC, 4-DMAP, dry dichloromethane, 12 hours, room temperature; (b) BBr ₃ 1M solution, dry dichloromethane, dichloromethane at 0 ^o C → room temperature, 5 hr.

One. JH13 , JH1 Recipe

In anhydrous dichloromethane (S) - (+) - decursinol (1, 0.406 mmol, 1eq) appropriate carboxylic acid (0.609 mmol, 1.5eq) and the solution, N - (3- dimethylaminopropyl) - N '- ethyl carbodiimide (EDC, 0.812 mmol, 2eq) and 4- (dimethylamino) pyridine (4-DMAP, 0.162 mmol, 0.4 eq). The mixture was stirred at room temperature for 5-12 hours. Thereafter, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a decursin derivative (2a, b). (2a or 2b, 0.49 mmol, 1 eq) having 3,4-dimethoxyphenyl group in anhydrous dichloromethane (5 ml) was added to a solution of 1 M boron tribromide in dichloromethane (1.47 mmol , 3eq). The mixture was stirred at room temperature for 5 hours. The reaction solution was purified by silica gel short column chromatography to obtain a decanesin derivative having a 3,4-dihydroxyphenyl group (3a; JH13, 3b; JH1).

3a; JH13 compound [(7S) - (+) - 3- (3,4- Dihydroxy - phenyl) - acrylic acid, the yield of 8,8-dimethyl-2-oxo- 6,7-dihydro- 2H, 8H- pyrano [3,2- g] chromen-7-yl-ester)] is 93.2%, is obtained as a white solid Loses. The data are as follows.

mp: 115 [deg.] C, _Rf = 0.36 (1: 2 n- hexane-ethyl acetate); [α] 25 D +19.3 (c = 3, CHCl 3); ^{1 H NMR (400MHz, DMSO-} d 6): δ H 9.63 (1H, s, OH-7 '), 9.10 (1H, s, OH-6'), 7.90 (1H, d, J = 9.6Hz, H D, J = 15.2 Hz, H-3 '), 7.00 (1H, s, H-5'), 6.99 (1H, d, J = 8.4Hz, H-9 ' ), 6.81 (1H, s, H-10), 6.71 (1H, d, J = 8.4Hz, H-8'), 6.25 (1H, d, J = 9.6Hz, H-3), 6.22 (1H , d, J = 15.6Hz, H-2 '), 5.14 (1H, t, J = 4.0Hz, H-7), 3.24 (1H, dd, J = 4.0, 17.6Hz , H-6a), 2.88 ( 1H, dd, J = 4.0, 17.6Hz, H-6b), 1.35 (3H, s, CH 3 -8), 1.31 (3H, s, CH 3 -8); ¹³ C NMR (100 MHz, CDCl ₃ ):? _C 166.9 (C-1 '), 162.3 (C-2), 156.6 (C-6 '), 144.2 (C-3'), 143.9 (C-4), 128.8 ), 115.3 (C-8 '), 114.3 (C-2'), 114.1 (C-3), 112.8 -8), 70.0 (C-7 ), 27.8 (C-6), 24.8 (CH 3 -8), 23.3 (CH 3 -8); IT-TOF / MS : m / z = 409.1357 [M + H] &lt; ⁺ &gt;, 431.1134 [M + Na] &lt; ⁺ &gt; _.

3b; JH1 Compound [(7S) - (+) - 3- (3,4- Dihydroxyphenyl ) propionic acid, the yield of 8,8-dimethyl-2-oxo- 6,7-dihydro- 2H, 8H- pyrano [3,2- g] chromen-7-yl-ester] is 86.6%, is obtained as a brown solid . Related data are as follows.

mp: 87 [deg.] C, _Rf = 0.21 (1: 1 n- hexane-ethyl acetate); [[alpha]] D &lt; + &gt; 56.6 (c = 1, MeOH); ^{1 H NMR (400MHz, DMSO-} d 6): δ H 8.69 (1H, s, OH), 8.63 (1H, s, OH), 7.89 (1H, d, J = 9.6Hz, H-4), 7.39 ( (1H, s, H-5), 6.76 (1H, s, H-10), 6.54 (1H, d, J = 8.0 Hz, H- (1H, d, J = 8.4Hz , H-8 '), 6.24 (1H, d, J = 9.6Hz, H-3), 5.00 (1H, t, J = 4.2Hz, H-7), 3.14 ( 1H, dd, J = 4.4, 17.6Hz, H-6), 2.70 (1H, dd, J = 4.8, 17.6Hz, H-6), 2.61 (2H, t, J = 7.2Hz, H-3 ') , 2.490 (2H, t, J = 7.2Hz, H-2 '), 1.23 (3H, s, CH 3 -8), 1.22 (3H, s, CH 3 -8); ¹³ C NMR (100 MHz, DMSO-d ₆ ):? _C 171.8 (C-1 '), 160.2 (C-2), 155.7 , 144.1 (C-6 '), 143.8 (C-4), 130.9 (C-5), 129.5 5), 115.3 (C-8 '), 112.7 (C-4a), 112.5 (C-3), 103.4 -2 '), 29.7 (C- 3'), 27.0 (C-6), 24.3 (CH 3 -8), 23.2 (CH 3 -8); IT-TOF / MS: m / z = 411.1393 [M + H] &lt; ⁺ &gt;, 433.1283 [M + Na] &lt; ⁺ &gt; _.

2. JH4 Recipe

In anhydrous dichloromethane (S) - (+) - decursinol (1, 0.406 mmol, 1eq) solution of trans-3- (3-pyridyl) acrylic acid (0.609 mmol, 1.5eq), N - (3-dimethylamino It was added to the ethyl carbodiimide hydrochloride (EDC, 0.812 mmol, 2eq) and 4- (dimethylamino) pyridine (4-DMAP, 0.162 mmol, 0.4eq) - propyl) - N. The mixture was stirred at room temperature for 12 hours. Thereafter, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain a decursin derivative (2c, JH4).

2c; JH4 compound [(7S) - (+) - 3- (3- Pyridyl) - acrylic The yield of acid , 8,8- dimethyl- 2-oxo-6,7-dihydro- 2H, 8H- pyrano [ 3,2- g ] chromen-7-yl-ester was 96.7% . Related data are as follows.

mp: 105 [deg.] C, _Rf = 0.24 (1: 1 n- hexane-ethyl acetate); [α] 20 D +48.5 (c = 3, CHCl 3); _{^{1 H NMR (400MHz, CDCl 3}} ): δ H 8.72 (1H, d, J = 2.2Hz, H-5 '), 8.60 (1H, dd, J = 1.5, 4.9Hz, H-7'), 7.81 ( 1H, d, J = 8.4Hz, H-9 '), 7.67 (1H, d, J = 16.0Hz, H-3'), 7.58 (1H, d, J = 9.6Hz, H-4), 7.32 ( 1H, dd, J = 4.8, 8.0Hz, H-8 '), 7.18 (1H, s, H-5), 6.83 (1H, s, H-10), 6.49 (1H, d, J = 16.4Hz, H-2 '), 6.24 ( 1H, d, J = 9.6Hz, H-3), 5.21 (1H, t, J = 4.8Hz, H-7), 3.26 (1H, dd, J = 4.8, 17.2Hz , H-6a), 2.95 ( 1H, dd, J = 4.8, 17.2Hz, H-6b), 1.44 (3H, s, CH 3 -8), 1.39 (3H, s, CH 3 -8); ^{13 C NMR (100 MHz, CDCl} 3) δ C 165.6 (C-1 '), 161.2 (C-2), 156.3 (C-9a), 154.2 (C-10a), 151.2 (C-5'), 149.8 (C-7 '), 143.1 (C-4), 142.1 (C-3'), 134.2 ), 119.5 (C-2), 115.5 (C-5a), 113.4 (C-3), 112.9 7), 27.8 (C-6 ), 24.9 (CH 3 -8), 23.4 (CH 3 -8 IT-TOF / MS: m / z = 378.1325 [M + H] +, 400.1129 [M + Na] +.

3. LG -02 recipe

In anhydrous dichloromethane (S) - (+) - coumarin (4, 0.406 mmol, 1eq) solution of 3,4-methoxy bodies acid (0.609 mmol, 1.5eq), N - (3- dimethylaminopropyl) - It was added to the ethyl carbodiimide hydrochloride (EDC, 0.812 mmol, 2eq) and 4- (dimethylamino) pyridine (4-DMAP, 0.162 mmol, 0.4eq) - N '. The mixture was stirred at room temperature for 12 hours. Thereafter, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain coumarin derivatives (5). A solution of coumarin derivative (5, 0.49 mmol, 1 eq) having 3,4-dimethoxyphenyl group in anhydrous dichloromethane (5 ml) was added to a solution of 1 M boron tribromide in dichloromethane (1.47 mmol, 3 eq) . The mixture was stirred at room temperature for 5 hours. The reaction solution was purified by silica gel short column chromatography to obtain a coumarin derivative having a 3,4-dihydroxyphenyl group (6, LG-02).

6; LG -02 compound [(6S) - (-) - 3- (3,4- Dihydroxy - phenyl) - acrylic The yield of acid, 7,7-dimethyl-2- oxo-5,6-dihydro- 7H -pyrano [3,2- f] chromen-6-yl-ester] is 90.86%, is obtained as a white solid. Related data are as follows.

mp: 117 [deg.] C, _Rf = 0.15 (1: 1 n- hexane-ethyl acetate); [?] 20 D -140.82 (c = 2, CHCl ₃ ); ^{1 H NMR (400MHz, DMSO-} d 6): δ H 9.63 (1H, s, OH-7 '), 9.09 (1H, s, OH-6'), 8.09 (1H, d, J = 9.6Hz, H -4), 7.46 (1H, d , J = 16.0Hz, H-3 '), 7.21 (1H, d, J = 9.2Hz, H-10), 7.09 (1H, d, J = 9.2Hz, H- 9), 7.01 (1H, s , H-5 '), 7.00 (1H, d, J = 8.0Hz, H-9'), 6.72 (1H, d, J = 8.0Hz, H-8 '), 6.45 (1H, d, J = 10.0Hz , H-2 '), 6.24 (1H, d, J = 15.6Hz, H-3), 5.20 (1H, t, J = 4.6Hz, H-6), 3.32 ( 1H, dd, J = 4.8, 17.6Hz, H-5), 3.06 (1H, dd, J = 4.0, 18.0Hz, H-5), 1.32 (3H, s, CH 3 -7), 1.27 (3H, s, CH ₃ -7); ¹³ C NMR (100 MHz, CDCl ₃ ):? _C 166.9 (C-1 '), 161.5 (C-2), 149.1 (C-6 '), 144.0 (C-3'), 139.6 (C-4), 126.9 4a), 116.4 (C-4b), 116.1 (C-10), 115.3 (C-8 '), 114.4 -7), 69.8 (C-6 ), 25.4 (C-5), 24.2 (CH 3 -7), 22.8 (CH 3 -7); IT-TOF / MS : m / z = 409.1275 [M + H] &lt; ⁺ &gt;, 431.1090 [M + Na] &lt; ⁺ &gt; _.

In order to confirm the LMN A-proergin binding inhibitory activity of the binding inhibitors, in vitro binding assays were performed, and JH4 and JH13 were found to inhibit LMN A-proergene binding (see FIG. 3C). The inventors of the present invention also confirmed the binding inhibitory activity of JH4 and JH13 by GST-pulldown analysis using a cell lysate (see FIG. 3D). In the above results, JH4 was found to bind to LMN A self-binding or between LMN A and LMN B (Fig. 3E). The farensyl-transferase inhibitor (FTI-277) and rapamycin are known as mitigating factors for nuclear deformation, and the inventors of the present invention also investigated the effect of FTI on Prg-LMN A binding and found that FTI (See Fig. 3D). In order to confirm the specificity of the binding inhibitor, the effect on Prg-p14 / ARF binding was examined and it was confirmed that the JH compound inhibited the interaction of Prg-p14 / ARF (see FIG. 4D). However, the compound did not inhibit the interaction of p53-p14 / ARF (see Fig. 4E). These results suggest that the JH compound specifically inhibits the Prg-LMN A binding or the Prg-p14 / ARF interaction, and that the direct target of the JH compound is progenin. In order to confirm the in vivo cell effect, the compound was treated with HGPS fibroblasts for 24 hours and then subjected to IP using progester antibody, and the JH compound inhibited Prg and LMN A / C interactions as expected (Fig. 3F Reference). However, the JH compound did not inhibit the expression of Prg (see FIG. 3G).

< Example  4> JH  Investigation of the effects of compounds and molecular mechanisms

To investigate the effect and molecular mechanism of the JH compounds according to one embodiment of the present invention, IP analysis was again performed using GFP antibodies in 293 cells infected with progenin and GFP-LMN A. Experimental results showed that the progenin bound to LMN A was apparently reduced by the JH compound (see FIG. 5A). On the other hand, the bond between LMN A and LMN B or Emerin was not affected by the compound (see FIG. 5B). In addition, a decrease in p16 expression was detected by treatment of the JH compound (see FIG. 5A). Since LMN A / C dissociates to G2 / M group, coliseum (Col) was used as a positive control. The effect of FTI was also compared with the JH compound (see FIGS. 5A and B). As a result of the experiment, the JH compound appeared to inhibit nuclear deformation (see FIGS. 6A and 6B), and promoted the expression of LMN A / C (see FIG. 6C). In addition, p16 expression, which is increased by progerin infection, was suppressed (see Fig. 6C). To confirm the physiological effect, LMN A / C was examined in two kinds of HGPS cells cultured for 24 hours with a binding inhibitor, and it was confirmed that JH1, JH4 and JH13 suppress nuclear deformation (FIGS. 5C and 5D) 7). Among these compounds, JH4 showed a better effect (see FIG. 5D), which is consistent with the result that JH4 inhibits LMN A-Prg interaction more effectively in FIG. 3D. To determine whether inhibition of nuclear deformation is associated with the inhibitory effect of aging, histone 3 (H3K9Me3), a 3-methylated 9-lysine well known as an aging marker, was examined. JH4 and JH13 increased the expression of H3K9Me3 in HGPS cells (see Figures 5E and 7). Compared to FTI, JH4 and JH13 showed similar effects in inhibiting nuclear deformation and in promoting H3K9Me3 (see FIG. 8). The reduction of DNA-PK is known to be an important feature of HGPS cells, and its expression has also been investigated. As expected, the expression of DNA-PK was significantly increased by treatment with JH compounds (see Figures 5F and 9).

From the above results, it was found that inhibition of Prg-LMN A binding can inhibit nuclear degeneration and cell senescence.

< Example  5> JH  Promoting cell proliferation and molecular mechanism of compounds

The major features of cell senescence were cell viability through cell counting of HGPS and normal fibroblasts, which is a semi-permanent interruption of cell proliferation. As expected, JH compounds (especially JH4) promoted cell proliferation (see FIG. 10A). JH4 also promoted cell proliferation of normal fibroblasts (see Fig. 10B). It was confirmed that the JH compound (particularly JH4) suppresses SA-β-Gal in HGPS and normal cells through β-galactosidase staining (SA-β-Gal) specific for aging 11). JH compounds promoted LMN A / C expression and suppressed DcR2 expression in HGPS cells (see FIG. 10E). In order to confirm the effect of JH compounds on normal cells, normal fibroblasts were incubated for 4 days in a serum-free condition, and nuclear deformation was detected. However, as a result of treatment with the JH compound, LMN A / C expression was promoted and nuclear deformation was alleviated, and this result was not obtained in the FTI treatment group (see FIG. 10F). Since several mutations of LMN A have been reported to be associated with laminopathy, the inventors of the present invention also investigated the effect of JH compounds on LMN A mutations. As a result, it was found that JH4 did not have a positive effect on the mutation (see FIG. 12).

The inventors of the present invention also examined the effect of JH compounds on the basis of the fact that the nuclear membrane irregularity of kidney cancer (RCC) is induced by progerin and is mitigated by FTI, and JH compounds have nuclear membrane irregularities similar to FTI (See Fig. 13). These results suggest that nuclear deformation in HGPS cells and nuclear membrane irregularity in RCC cells are induced by proergen and can inhibit progerin-induced aging and nuclear deformation by inhibiting Prg-LMN A binding do. On the basis of the fact that the compound of the present invention showed excellent activity inhibiting aging induced by progerin and aging of normal cells, in We also examined the effect in vivo . As a result of the experiment, it was shown that the mice were not toxic to the liver and kidney, and had no weight change when the JH compound was injected for 6 weeks (see FIG. 14). The results show that the JH compound has no pronounced cytotoxicity (see FIGS. 3B and 4C), in vivo toxicity.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Pusan National University Industry-University Cooperation Foundation <120> Pharmaceutical composition for treating aging-related diseases          comprising inhibitor of progerin and screening method thereof <130> DP-2010-0744 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for lamin A / C <400> 1 aaggagatga cctgctccat c 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for lamin A / C <400> 2 tttctttggc ttcaagcccc c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 3 atcttccagg agcgagatcc c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 4 agtgagcttc ccgttcagct c 21

Claims (10)

delete delete A pharmaceutical composition for preventing or treating prostatic hyperplasia comprising a compound represented by the following formula (1) or (2) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

(2)

In Formula 1 and Formula 2, wherein R ₁ is

,

or

ego,
R ₂ and R ₃ are each independently hydrogen or C _1-4 alkyl. The method of claim 3,
Wherein the compound represented by formula (1) or (2) inhibits the binding between progerin and lamin A. The method of claim 3,
In Formula 1,
R ₁ is

Lt;
R ₂ and R ₃ are methyl. delete delete The method of claim 3,
Wherein the progeria is Winer syndrome or Hutchison's Guildford syndrome. Lt; RTI ID = 0.0 &gt; pro &lt; / RTI &gt; 10. The method of claim 9,
The screening method
Progeline, fluorescent protein labeled-lamin A and candidate drug; And
And analyzing the level of binding between the progesterone and the fluorescent protein to select a candidate drug that inhibits the binding between progesterone and lamin A, thereby screening for a therapeutic agent for anorexia nervosa.

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