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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
  • C07C205/27—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups
  • C07C205/32—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups bound to acyclic carbon atoms and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
  • C07C205/01—Compounds containing nitro groups bound to a carbon skeleton having nitro groups bound to acyclic carbon atoms
  • C07C205/03—Compounds containing nitro groups bound to a carbon skeleton having nitro groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
  • C07C205/04—Compounds containing nitro groups bound to a carbon skeleton having nitro groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing six-membered aromatic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K31/04—Nitro compounds
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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
  • C07C205/07—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by halogen atoms
  • C07C205/08—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by halogen atoms having nitro groups bound to acyclic carbon atoms
  • C07C205/09—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by halogen atoms having nitro groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C205/00—Compounds containing nitro groups bound to a carbon skeleton
  • C07C205/39—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by esterified hydroxy groups
  • C07C205/42—Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by esterified hydroxy groups having nitro groups or esterified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
  • C07C309/63—Esters of sulfonic acids
  • C07C309/72—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
  • C07C309/73—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton to carbon atoms of non-condensed six-membered aromatic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
  • C07C309/63—Esters of sulfonic acids
  • C07C309/72—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
  • C07C309/76—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing nitrogen atoms, not being part of nitro or nitroso groups, bound to the carbon skeleton
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
  • C07C311/15—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings
  • C07C311/21—Sulfonamides having sulfur atoms of sulfonamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring

Definitions

• the present invention relates to novel beta-nitrostyrene compounds and pharmaceutically acceptable salts and hydrates thereof having anticancer activity, and a composition for ' inhibition of telomerase activity and an anticancer agent comprising the same.
• telomere Human chromosomes have a peculiar structure, called as telomere, having the repeating nucleotide sequence of TTAGGG at their ends. This structure protects the end part of the chromosome so that a part carrying genetic information is not lost.
• the final RNA primer produced in a replication fork upon DNA replication of chromosomes is removed. Therefore, as the cell division is repeated, the telomere is gradually lost, whereby the length of the chromosome is gradually reduced (a problem of the replication at the chromosomal end) and in the end, the genetic substances are lost and the cell is dead (Harley, C.B. et al., CoJ Spring Harb . Symp. Quant . Biol .
• telomerase an enzyme, called as telomerase, which continuously lengthens the length of the telomere by action as a reverse transcriptase using the RNA part of the repeated single nucleotide sequence of the telemere, that is its own part, as a primer and thus, they do not die even after cell division continuously occurs.
• the telomerase can immortalize the human cells having limited division performance and the continuous shortening of the telomere serves as a biological clock determining how many times the cell divides ⁇ Science, 279: 349-352, 1998).
• the length of the chromosomal end is associated with the life span of the cell and the immortality of the cancer cells is attributable to the telomerase capable of continuously maintaining the length of the chromosomal length.
• the telomerase activity is not found but about 85% of cancer- cells have telomerase activity (Kim, N.W. et al., Science, 266, ' 2011-2015, 1994). Therefore, scientists expect that replication of various cancer cells can be terminated by inhibiting the activity of the telomerase existing in the cancer cells.
• Telomerase inhibitors known so far are largely classified into four groups.
• the first one is oligonucleotides inhibiting telomerase by targeting RNA elements of the telomerase.
• the most representative method is to use antisense nucleotide complementary to RNA of the telomerase (Feng, J. et al . , EMBO J. , 14, 4240-4248, 1995).
• the antisense nucleotide complementarily binds to target RNA to inhibit translation of RNA.
• transfection reagents should be used.
• the nucleotides which have entered the cell are destroyed by various nucleic acid degrading agents.
• the nucleotide should be chemically modified by various methods, which ultimately reduce the binding force to a nucleotide sequence.
• PNAs peptide nucleic acids
• the peptide nucleic acids are homologues of RNA and DNA, in which the pentose-phosphorus structure is substituted by N- (2-aminoethyl) glycine, and have high affinity and specificity for RNA and strong resistance to various nucleic degrading agents.
• the second one is ribozyme which is a nucleic acid molecule ' capable of digesting the nucleic acid sequence of telomerase.
• the ribozyme also inhibits telomerase by targeting RNA elements of the telomerase. Yokoyama et al . confirmed that when the hammer head ribozyme is expressed in a cancer cell line, the telomerase activity is reduced and the telomerase length is decreased (Yokoyama, Y. et al., Cancer Res . , 58, 5406-5410, 1998)
• the third one is to inhibit telomerase by targeting hTERT which is a catalytic domain of telomerase. Since telomerase has reverse transcriptase activity by itself, various agents for inhibiting reverse transcriptase activity may be used as a telomerase inhibitor (Strahl, C. and Blackburn, E.H., Mil . Cell . Biol . , 1 6, 53-56, 1996; Melana, S. M. et al . , Cancer Res . , 4, 693-696, 1997; Murakami, J. et al . , Eur. J. Cancer, 35, 1027- 1034, 1998) .
• telomere stabilizing substances include porphyrin derivatives, acridine derivatives, 2,6-diamino anthraquinone and the like (Harrison et al . , Bioorg. Med.
• WO 9940087 discloses a use of compounds interacting with the G- quadruplex which are perylene compounds and carbocyanines containing at least 7 rings, including two heterocycles .
• WO0140218 discloses arylamine derivatives useful for stabilizing the G-quardruplex structure and a use thereof as an anti- telomerase agent.
• EP0938897 A and US2003/0022878 A disclose that catechine is useful as a telomerase inhibitor, WO02076975, WO03024997, WO0230932, WO0208193, WO0140218, WO0002581, WO9702279, US2002/0103169, US6362210, US6004939, US6054442, FR2819255 and the like disclose various compounds and peptides having telomerase inhibiting activity. Particularly, US6362210 discloses carboxyl amides capable of inhibiting telomerase.
• novel beta-nitrostyrene compounds showing telomerase inhibiting activity, which is represented by the following formula (I) :
• R 1 to R 5 are each independently any one selected from the group consisting of hydrogen, halogen, halogenated phenoxy and a radical of R 6 -Y-X-(-X- is -0- or -NH-, -Y- is -S0 2 - or -CO-, R ⁇ is benzene substituted with at least any one selected from the group consisting of hydrogen, alkyl, halogen and N0 2 ) , or R 3 may form a benzene ring along with R 4 ; provided that R 1 , R 2 , R 3 , R 4 and R 5 are not hydrogen at the same time, salts and hydrates thereof
• R 1 and R 3 are each independently hydrogen
• R 2 is hydrogen or dichlorophenoxy
• R 4 and R 5 are each independently hydrogen or chlorine.
• the preferred compounds are beta-nitrostyrene compounds selected from the group consisting of 3- (3, 5- dichlorophenoxy) -beta-nitrostyrene, 2, 3-dichloro-beta- nitrostyrene, 2- (2-nitro-vinyl) -naphthalene, 5-dimethylamino- naphthalene-1-sulfonic acid 3- (2-nitro-vinyl) -phenyl ester, toluene-4-sulfonic acid 3- (2-nitro-vinyl) -phenyl ester, benezenesulfonic acid 4- (2-nitro-vinyl) -phenyl ester, 4-chloro- 3-nitrobenezenesulfonic acid 3- (2-nitro-vinyl) -phenyl ester, 4- chlorobenezen
• composition for inhibiting telomerase activity comprising the beta-nitrostyrene compound, or pharmaceutically acceptable salt or hydrate thereof according to the present invention as an effective ingredient.
• a method for anticancer or prevention of cancer comprising using the compound according to the present invention or the composition comprising the same as an effective ingredient.
• the beta-nitrostyrene compounds according to the present invention are prepared by the following two methods.
• Method 1 is to prepare compounds which are not substituted with sulfonyl, acetyl or acyl (Example 1 to 4) and
• Method 2 is to attach sulfonyl, acetyl or acyl to the compounds prepared by Method 1 (Example 5 to 11) .
• the concrete description is presented in the Examples .
• Aldehyde (leq. wt . ) , nitromethane (4eq. wt . ) and NH 4 OAC (0.5 eq. wt.) are put into a 10 mL sealed reactor and subjected to high frequency reaction at 60 °C. The progress of the reaction is confirmed by TLC and the reaction is continued for 1 hour.. After completion of the reaction, the mixture is treated with H 2 0 and extracted with ethyl acetate.
• the beta-nitrostyrene compounds of the formula (I) can be prepared in the form of various pharmaceutically acceptable salts known to the art, for example, inorganic metal salts such as alkali metal salts (ex.: sodium salts, potassium salts, etc.) and alkali earth metal salts (ex. : magnesium salts, calcium salts, etc.), ammonium salts, organic base salts (ex.: trimethylamine salts, triethylamine salts, pyridine salts, picoline salts, etc.) according to a conventional method.
• the compounds according to the present invention can be in the form of solvates, particularly hydrates.
• the hydrates can be prepared by a conventional method or produced spontaneously due to hygroscopic property of the compounds as time goes on.
• a partially modified TRAP method (Kim, N.W. et al., Science, 266, 2011-2015, 1994) which is known as a traditional method for measurement of telomerase activity to effectively examine numerous small molecular compounds is used.
• telomerase fraction is modified.
• the telomerase activity is measured using crude extracts obtained from cancer cells by using dissolution buffer.
• the crude extracts is subjected to ultra centrifuge using the glycerol gradient to obtain fractions. Each fraction is measured for the telomerase activity. The fraction of the highest activity, that is, the purified telomerase fraction is then used to measure the inhibition level of each compound.
• telomerase in order to confirm the extended product produced by telomerase, PCR is performed using a radioactive isotope and the resulting product is confirmed by electrophoresis.
• the PCR product is reacted with PicoGreen which is a fluorescent dye specifically reacting with only double stranded DNA and the fluorescence is then measured. Therefore, it is possible to rapidly obtain a result without riskiness which may be caused by use of radioactive isotopes and inconvenience of electrophoresis .
• the compounds did not show cytotoxicity at a concentration inhibiting telomerase activity and specifically inhibited only telomerase while not affecting other similar nuleic acid polymerases such as Taq polymerase, DNA polymerase, RNA polymerase, reverse transcriptase and the like.
• telomere inhibiting activity in order for the beta-nitrostyrene compounds showing telomerase inhibiting activity according to the present invention to act as an anticancer agent, they should not only inhibit telomerase activity in vitro but also induce reduction in length of telomere of cancer cells and cellular senescence when they are administered into cancer cells having substantial telomerase activity.
• telomere repeating nucleotide fragment of about 300 bp length as a probe
• telomere length is returned to the original state (Fig. 4). Similar to this result, the telomerase activity of cells is reduced by the treatment of the compound while the activity is restored when the cells are cultured without the compound (Figs. 5a and 5b) . In practice, it was confirmed by the Senescence-Associated ⁇ -galactosidase assay that telomere length is reduced and the cell aging is induced, when cancer cells are treated with the beta-nitrostyrene compound according to the present invention for a long period of time (Fig. 6) .
• the beta-nitrostyrene compounds according to the present invention specifically inhibits telomerase activity without cytotoxicity on cancer cells, and significantly reduce telomere length and induce cell aging upon treatment on cancer cells for a long period of time. From these results, it is noted that the compounds according to the present invention are useful as an anticancer agent specifically killing cancer cells having telomerase activity without side effects on normal cells, as compared to the existing anticancer agents. Therefore, according to the present invention, there is provided a composition for inhibiting telomerase and anticancer comprising the beta-nitrostyrene compounds, or pharmaceutically acceptable salts or hydrates thereof, along with a pharmaceutically acceptable carrier. Meanwhile, the composition according to the present invention can be formulated into pharmaceutical formulations according to conventional methods.
• an active ingredient is preferably mixed with a carrier, excipient and diluent or diluted therein, and then sealed in capsule, sachet or other carriers in the form of a container.
• a carrier excipient and diluent or diluted therein
• Such formulations can be in the form of, for example, tablet, pill, granule, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft or hard gelatin capsule, sterilized injection, sterilized powder, ointment, patch and the like .
• Suitable carriers, excipients and diluents include water, physiological saline, lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, crude cellulose, polyvinyl pyrrolidone, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.
• the formulations may further comprise a filler, an anticoagulant, a lubricant, a wetting agent, fragrance, an emulsifying agent, a preservative and the like.
• the composition according to the present invention can be .
• US6362210 describes a formulation method of carboxyl amide having telomerase activity inhibiting effect and the method described therein may be used.
• the content of the beta-nitrostyrene compound in the composition is suitably about 20 to 95 wt%.
• telomerase inhibitor and anticancer agent composition according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous and intramuscular administration.
• a normal daily dose of the telomerase inhibitor and anticancer agent composition as an active compound is in the range of 0.01 to 100 mg/kg body weight, preferably 0.1 to 50 mg/kg body weight
• the actual dose of the pharmaceutical composition according to the present invention is determined by various factors such as disease to be treated, administration route, age, sex and body weight of a patient, and severity of disease and thus, the above dose is not intended to be limiting as to the scope of the invention in any way.
• DPNS (3, 5-dichlorophenoxy) -beta-nitrostyrene (DPNS) significantly concentration-dependently inhibits telomerase activity in vitro, and Fig. lb shows the concentration inhibiting telomerase activity by 50% (IC 50 ) which are obtained by quantification of the result of Fig. 1;
• Fig. 2a is a result of Lineweaver-Burk plots showing that 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene inhibits telomerase activity by acting as a mixed-type noncompetitive inhibitor on TS primer
• Fig. 2b is a result of Lineweaver-Burk plots showing that 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene inhibits telomerase activity by acting as a mixed-type noncompetitive inhibitor on dNTP
• Fig. 3 is a result of a southern blot confirming that when a cervical cancer cell line (HeLa cell line) is treated with 3-
• telomere activity is specifically inhibited according to the treatment time, whereby the telomere length is reduced;
• Fig. 4 is a result of a southern blot confirming that when a cervical cancer cell line is treated with 3- (3, 5- dichlorophenoxy) -beta-nitrostyrene for a long period of time, the telomere length is reduced while the length is restored when the treatment is stopped;
• Fig. 5a is a result of an electrophoresis showing that when a cervical cancer cell line is treated with 3- (3, 5- dichlorophenoxy) -beta-nitrostyrene, the activity of telomerase existing in the cells is inhibited, and Fig. 5b shows a graph obtained by quantification of the result of Fig. 5a; and
• Fig. 6 are microscopic photographs of cervical cancer cell lines stained by X-gal stain which is specific to senescent cells (the senescent cells are shown blue) , in which Fig. 6a is a control of a cervical cancer cell line treated with only DMSO and Fig. 6b is an experimental group treated with 3- (3, 5- dichlorophenoxy) -beta-nitrostyrene .
• Example 1 Examination of the effect on telomerase activity First, partial telomerase fractions were provided to examine an effective telomerase inhibitor. 1 10 8 HeLa cells
• the lysed cells were centrifuged at 15,000 rpm for 30 minutes. The supernatant was separated and loaded on 10 to 40 % glycerol gradient solution (10 mM Tris (pH 7.5), 1 mM magnesium chloride, 1 mM EGTA, 0.1 mM PMSF, 5 mM ⁇ -mercaptoethanol, 10 ⁇ 40 % glycerol). The solution was then ultra-centrifuged at 25,000 rpm at 4 ° C for 24 hours using Beckman SW-28 rotor to fractionate the protein gradients into a 96 well deep plate. Each fraction was examined for the telomerase activity according to the following method. The fraction showing the highest activity was used for the next inhibitor examination.
• TRAP Telomere Repeat Amplification Protocol: Kim, N.W. et al., Science, 266, 2011-2015, 1994
• TRAP Telomere Repeat Amplification Protocol: Kim, N.W. et al., Science, 266, 2011-2015, 1994
• Step 1 The fraction obtained as above (about 60 ng of protein) was mixed with reaction buffer (20 mM Tris (pH 8.3), 15 mM magnesium chloride, 63 mM potassium chloride, 0.005 % Tween- 20, 1 mM EGTA, 100 ⁇ M dNTPs, 50 nM TS primer (SEQ ID NO: 1)) and reacted at 37 ° C for 8 minutes. The reaction was quenched by heating at 94 ° C for 90 seconds. The treatment group was treated with 0.1 to 10 ⁇ M of the beta-nitrostyrene compound and the control was treated with dimethyl sulfoxide (DMSO) of the same amount to examine the effect of the compound on telomerase activity. The product extended by telomerase was then used as a template for amplification.
• reaction buffer 20 mM Tris (pH 8.3), 15 mM magnesium chloride, 63 mM potassium chloride, 0.005 % Tween- 20, 1 mM EGTA, 100
• Step 2 The telomerase product produced in Step 1 was amplified for 30 cycles of 30 seconds at 94 ° Q 60 ° C and 72 ° C' using TS primer and ACX primer (SEQ ID NO: 2).
• TSNT SEQ: ID NO: 3
• NT SEQ' ID NO: 4
• Step 3 According to the established TRAP, electrophoresis was used to confirm the amplified result.
• electrophoresis was used to confirm the amplified result.
• much time and labor are consumed to examine the compounds in a large number of compounds and cumbersome process is required owing to use of radioactive isotopes.
• PicoGreen Molecular Probes
• Molecular Probes a fluorescent dye which specifically reacts with only double stranded DNA and shows a quantitative result
• absorption was measured using a fluorescent spectrometer with exiting wave length of 485 nm and emitting wave length 535 nm.
• telomerase activity inhibiting effect of the compound was calculated based on the rate of the value measured under the compound (treatment group) to the value measured under DMSO
• IC 50 a concentration at which the compound inhibits telomerase activity by 50 % as compared to the control, was used.
• 3- (3, 5-dichlorophenoxy) - beta-nitrostyrene having IC 50 of 3 ⁇ M or less was selected from the compounds in a large number of compounds and synthesized into derivatives thereof, followed by examination of their inhibiting effects on telomerase (Table 1) .
• Fig. la The result is shown in Fig. la. Also, the result of Fig. la was quantified to determine the concentration inhibiting telomerase activity by 50 % (IC 50 ) and the result is shown in Fig. lb.
• Example 2 Pharmacokinetic assay of the inventive compound In order to investigate the inhibiting effect of 3- (3, 5- dichlorophenoxy) -beta-nitrostyrene on telomerase, effects of 3-
• (3, 5-dichlorophenoxy) -beta-nitrostyrene is a mixed-type noncompetitive inhibitor on TS primer (Fig. 2a) . Also, effects of 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene on TRAP with dNTP at various concentrations were examined by Lineweaver-Burk plots.
• telomerase inhibiting effect of 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene is a mixed-type noncompetitive inhibitor on dNTP, as well (Fig. 2b) .
• the cytotoxicity test was performed by investigating drug toxicity affecting cell growth for a short period of time. Cervical cancer call line HeLa (ATCC CCL-2) and breast cancer cell line MCF-7 (ATCC HTB-22) were used and the MTT method (Klemke, R.L. et al . , J. Cell Biol . , 127, 859-866, 1994, this experiment was performed using the CellTiter 96R Non-Radioactive Cell Proliferation Assay kit (Promega) and the experiment method also followed the method of Promega) was used. The cells were dispensed in a 96 well plate, each well containing 5 x 10 4 cells. At the next day, the telomerase inhibitor was administered at various concentrations.
• MTT (3- [4, 5-dimethyltiazol-2-l] -2, 5-diphenyl tetrazolume bromide) dye was dispensed. Then, the plate was stored in an incubator for 2 hours and the medium was removed. MTT dissolution buffer was dispensed to the plate and stored until the cells were completely dissolved. The absorption at 570 nm wavelength was measured using a microplate reader and the measured values are converted into % cell survival rate. From the average cell growth inhibition graph obtained by repeated experiments, the concentration showing 50% inhibition (IC 50 ) was calculated and is shown in Table 2.
• telomerase itself has reverse transcriptase activity the compounds were examined for effects on other polymerases similar to reverse transcriptase to know if they specifically act on telomerase. Therefore, in the present invention, effects on the four following enzymes were examined.
• This Taq polymerase activity experiment was performed by a conventional method (Nakamura, T.M. et al., Science, 277, 955- 959, 1997) adding the telomerase inhibitor at various concentrations (0.1 to 10 ⁇ M) under PCR conditions using K136 primer (SEQ ID NO : 5) and K137 primer (SEQ ID NO : 6) which amplify a 450 bp fragment of GAPDH gene.
• Taq polymerase was a product of Takara and reacted 25 cycles of 1 minute at 94 ° Q 55 ° Cand 72 ° C
• the reaction product was electrophoresed on 1.2 % agarose gel, stained by SYBRGreen (Moleculr Probes) , developed on LAS-1000 CCD system (Fujifilm) and quantified using an image gauge program (Fuji film).
• SYBRGreen Moleculr Probes
• the effect of 3-(3,5- dichlorophenoxy) -beta-nitrostyrene on Taq polymerase activity, as compared to PIPER (Calbiochem) and TMPyP 4 (Calbiochem) which are known as representative telomerase inhibitors, is shown in Table 3.
• 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene showed little inhibiting effect on Taq polymerase activity, as compared these inhibitors and thus, it was noted that the compound is specific to telomerase.
• plasmid 100 ng random hexamer oligo, 0.2 mM of deoxyadenoxine 5' -triphosphate (dATP) , deoxycytosine 5' -triphosphate (dCTP) and deoxyguanosine 5' -triphosphate (dGTP), 0.07 mM digoxigenine-11- 2' -deoxyuridine-5' -triphosphate (DIG-11-dUTP) and 1 unit of klenow fragment and reacted at 37 ° C for 1 hour.
• the reaction product was transferred to a nylon membrane using a slot blot apparatus (Amersham Pharmacia Biotech) and quantified using anti-digoxigenin-AP (Roche).
• reaction solution 40 mM Tris (pH 7.9), 6 mM magnesium chloride, 2 mM spermidine, 10 mM
• RNA polymerase mM ribonucleotide mixture (rNTP mix) , 40 units of T7 RNA polymerase, 100 units of RNase inhibitor) and reacted at 37 ° C for 2 hours. After completion of the reaction, the amount of the synthesized RNA was quantified by a fluorospectrometer using RiboGreen (Molecular Probes) . The result is shown in Table 5, as compared to those of PIPER and TMPyP . From the result, it was noted that 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene hardly affects on T7 RNA polymerase activity.
• hexamer oligo 0.2 mM deoxyadenosine 5' -triphosphate (dATP)', deoxycytosine 5' -triphosphate (dCTP) , deoxyguanosine 5';l- triphosphate (dGTP), 0.07 mM digoxigenine-11-2' -deoxyuridine-5' - triphosphate (DIG-11-dUTP) and 10 units of M-MLV reverse transcriptase and reacted at 37 ° C for 1 hour.
• the reaction production was transferred to a nylon membrane using a slot blot apparatus and quantified using digoxigenin antibody (anti- digoxigenin-AP) .
• 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene In order for 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene to act as an anticancer agent, it should not only inhibit the activity of telomerase in vitro but also induce reduction in length of telomere of cancer cells and cell senescence when it is administered to the cancer cells having telomerase activity.
• a probe was prepared ' by random priming using a repeating nucleotide sequence fragment (TTAGGG) 50 of about 300 bp as a template and southern blotting was performed.
• TTAGGG repeating nucleotide sequence fragment
• HeLa cells were cultured in a 10 cm plate containing DMEM with 10 % fetal bovine serum under conditions of 5 % C0 2 and 37 ° C while 100 nM 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene was added to the medium. Control was treated with a same volume of DMSO.
• genome DNA was obtained, re-suspended in 0.1* TE(1 mM Tris, pH 8.0, 0.1 mM EDTA) and stored at 4 ° C or -20 ° C
• the genome DNA obtained by this method was completely digested with restriction enzyme Hinf I, Rsa I, purified by serial phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) extraction and isopropanol precipitation and quantified by absorption spectroscopy.
• Hinf I, Rsa I restriction enzyme
• the probe of the hybridization was a DIG system (digoxygenin-labelling kit; produced by Roche) and prepared by random priming using a telomere repeating nucleotide sequence fragment of about 300 bp.
• telomere length was restored to the telomere length of the control treated with DMSO (Fig. 4, Lanes 1 and 4) .
• the cells which had been treated with 100 nM 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene after PD 130 and further cultured for PD 190 had the telomere length shortened as compared to the cells which had been treated with 100 nM 3- (3, 5-dichlorophenoxy) -beta-nitrostyrene for PD 130 (Fig. 4, lane 2 and 3) .
• Example 6 Measurement of telomerase activity based on cells Upon cultivation of HeLa cells, the control had 100 nM DMSO added to the medium and was cultured for PD 193 ("DMSO") and a treatment group had 100 nM 3- (3, 5-dichlorophenoxy) -beta- nitrostyrene added to the medium and was cultured for PD 190 ("DPNS"). Another treatment group had 100 nM 3- (3, 5- dichlorophenoxy) -beta-nitrostyrene added to the medium and cultured for PD 130, and then cultured further for PD 60 without the compound ("DPNS removal”). The cells were lysed in cold dissolution buffer used in Example 1 and protein amounts were quantified. The total extracts of the same amount were measured for telomerase activity by the modified TRAP method as described in Example 1 and electrophoresed. The quantified graphs are shown in Figs. 5a and 5b.
• telomerase activity of the cells was reduced by the treatment of the compound but restored when cultured without the compound.
• Example 7 Senescence-associated ⁇ -galactosidase assay HeLa cells which had been treated with 100 nM 3- (3, 5- dichlorophenoxy) -beta-nitrostyrene for PD 100 were as immobilized in 2 % formaldehyde, 0.2 % glutaraldehyde and reacted in 1 mg/ml X-gal solution (40 mM citric acid/sodium phosphate buffer (pH 6.0), 5 mM ferricyanide, 5 mM ferrocyanide, 150 mM calcium chloride, 2 mM magnesium chloride, 1 mg/ml X-gal) for 20 hours. Then, the cells were washed with PBS and the stained cells were observed under optical microscope (Axiovert, produced by Zeiss) .
• the beta-nitrostyrene compounds according to the present invention can specifically inhibit telomerase without any effect on other similar nucleic acid polymerase and have low cytotoxicity. Thus, they can be usefully used as an anticancer agent for any cancer cells having telomerase activity. While the present invention has been described with reference to the particular illustrative embodiments, it is not to be restricted by the embodiments but only by the appended claims. It is to be appreciated that those skilled in the art can change or modify the embodiments without departing from the scope and spirit of the present invention.

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• 2003
  • 2003-11-10 KR KR1020037015424A patent/KR100614422B1/ko not_active IP Right Cessation
  • 2003-11-10 AU AU2003277721A patent/AU2003277721A1/en not_active Abandoned
  • 2003-11-10 WO PCT/KR2003/002402 patent/WO2004046081A1/en not_active Application Discontinuation

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