WO1999008679A1

WO1999008679A1 - Telomerase inhibitor

Info

Publication number: WO1999008679A1
Application number: PCT/JP1998/003626
Authority: WO
Inventors: Naoki Kato; Kenji Yoshida; Masayuki Tsuchiya
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1997-08-15
Filing date: 1998-08-14
Publication date: 1999-02-25
Also published as: AU8648998A

Abstract

A telomerase inhibitor containing a compound of general formula (1) (wherein X is sulfur or the like; R1 and R2 are each hydrogen or the like; and R3 to R7 are each hydrogen, halogen, haloalkyl having one to three carbon atoms, or the like) or a pharmaceutically acceptable salt thereof.

Description

Specification

Telomerase inhibitor Technical field

The present invention relates to a telomerase inhibitor comprising 3-isothiazolone or a 3-isoxazolone derivative. Background art

Telomerase is known as an enzyme that catalyzes the elongation of the telomere terminal (terminal portion of a linear chromosome), and many studies have been made (G reider CW and Blackburn EH, (1987) Cell, 51, 8 87-898; Morin GB (1989) Cell, 59, 521-529) o

Telomerase activity is not detected in normal cells except for some cells such as hematopoietic stem cells. On the other hand, strong telomerase activity is detected in most cancer cells, suggesting that telomerase is involved in immortalization of cells and maintenance of infinite growth of cancer cells. Therefore, telomerase inhibitors can be expected to be anticancer agents with few side effects on normal cells or inhibitors of the growth of immortalized cells containing telomerase (Counter CM eta 1. , (1 989) EMBO J., 11, 1921-1929; Counter CM eta 1., (1994) Proc. Natl. Ac ad. Sci. USA, 91, 2900-2904 Ch adeneau C. eta 1., (199 5) Cancer Re s., 55, 2533-2536; Hiyama E. eta 1., (1995) Nature Med., 249—255, Shay J. W. eta 1., (1995) Mo 1. Cell. Biol., 15, 425-432).

At present, as a compound that inhibits telomerase, a nucleic acid-based antiviral agent such as azidothymidine is known, but it is said that there is a problem in terms of specificity (Y e gorov YE, Chernov DN, Akimov S. S., S., eta 1. (1996) FEBS Lett. 389, 115-118; Yasuo Kagawa, (1997) The latest medicine, 52, 306-312) . Recently, it has been reported that 2,6-diamidanthraquinone compounds inhibit telomerase (Sun D., Thompson B., eta 1., (1997) J. Med. Chem. , 40, 2113—2116). However, its inhibitory activity is not satisfactory, and a drug having better telomerase inhibitory activity is required. By the way, 3-isothiazolone and 3-isoxazolone derivatives have many known substances, and their uses are known to be antibacterial, antifungal, photographic photosensitive material, anti-inflammatory, analgesic, anti-osteoarthritis, etc. . These derivatives are not known at all as telomerase inhibitors, anticancer agents, or growth inhibitors of telomerase-containing immortalized cells. Disclosure of the invention

An object of the present invention is to provide a telomerase inhibitor containing 3-isothiazolone or a 3-isoxazolone derivative.

Under such circumstances, the present inventors have conducted intensive studies for the purpose of developing a compound having an excellent telomerase inhibitory action, and as a result, have found that 3-isothiazolone or 3- The inventors have found that an isoxazolone derivative has an excellent telomerase inhibitory action, and completed the present invention based on this finding.

That is, the present invention relates to the general formula (1)

(Wherein, X represents a sulfur atom or an oxygen atom; and R ₂ each independently represents a hydrogen atom, a halogen atom, or a linear or branched alkyl group having 1 to 3 carbon atoms; R ₃ , R ₄ , R ₅ , R ₆ and R ₇ are each independently a hydrogen atom, a halogen atom, a haloalkyl group having 1 to 3 carbon atoms, a cyano group, —COOR ₈ , one SR ₉ , or —OR _10, a _{_{represents;. R 8, R 9,}} R 10 are each independently contain a compound represented by the representative) a linear or branched alkyl group having a carbon number of 1-3, a carcinostatic And a telomerase inhibitor or a telomerase-containing immortalized cell growth inhibitor.

In the definition of the compound represented by the general formula (1), X has the above definition, but is preferably a sulfur atom.

The halogen atom in 1 ^ and R ₂ represents a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

The linear or branched alkyl group having 1 to 3 carbon atoms in 1 ^ and R ₂ means a methyl group, an ethyl group, a propyl group or an isopropyl group.

And R ₂ may be the same or different, but Ri is preferably a hydrogen atom, and R ₂ is preferably a hydrogen atom.

The halogen atom in R ₃ , R ₄ , R ₅ , R ₆ and R ₇ has the same meaning as described above, and is preferably a fluorine atom.

The alkyl group of the haloalkyl group having 1 to 3 carbon atoms in R ₃ , R ₄ , R ₅ , R ₆ and R ₇ has the same meaning as described above, and is preferably a methyl group.

The halogen atom of a haloalkyl group having 1 to 3 carbon atoms in R ₃ , R ₄ , R ₅ , R ₆ and R ₇ has the same meaning as described above, and is replaced by one or more halogen atoms. When two or more substituents are used, they may be the same halogen atom or different halogen atoms.

As the haloalkyl group having 1 to 3 carbon atoms in R ₃ , R ₄ , R ₅ , R ₆ and R ₇ , a trifluoromethyl group is preferable.

The linear or branched alkyl group having 1 to 3 carbon atoms in R ₈ , R ₉ and R _{1 ()} has the same meaning as described above, and is preferably a methyl group or an ethyl group. As COOR ₈ in R ₃ , R ₄ , R ₅ , R ₆ and R ₇ , it is preferable that R ₈ is an ethyl group.

In R ₃ , R ₄ , R ₅ , R ₆ and R ₇ , _SR ₉ is preferably a case where R ₉ is a methyl group.

As one OR ₁₀ in R ₃ , R ₄ , R ₅ , R ₆ and R ₇ , the case where R ₁₀ is a methyl group is preferred.

R ₃ , R ₄ , R ₅ , R ₆ and R ₇ may be the same or different. Further, among these, it is preferable that one to three of the above substituents are the above-mentioned substituents other than a hydrogen atom.

The _{_{_{R 3, R 4, R 5}}} , R 6 and R _7, a hydrogen atom, a fluorine atom, Torifuruo Romechiru group, an ethoxycarbonyl group, Shiano group, the substituent of each methylthio group is _{preferable. 9} definition as above However, as the compound represented by the general formula (1),

2- (3-chloro-1-4-fluorophenyl) -14-isothiazoline-13-one,

2- (3-chloro-1--2-fluorophenyl) -1-4-isothiazoline-1-3-

4-bromo-2- (3-chloro-4-fluorophenyl) -14-isothiazoline-3-one,

2- (3-trifluoromethylphenyl) -1-isothiazoline-13-one, 2- (2,3,4-trifluorophenyl) -14-isothiazoline-13-one,

2- (4-ethoxycarbonyldiphenyl) 4-isothiazolin-1-one, 2- (4-cyano-3-methylthiophenidyl) 4-sothiazoline-1-one,

Is preferred,

2- (3-chloro-1-4-fluorophenyl) -14-isothiazoline-13-one, 2-(3-chloro-2-fluorophenyl) 1-41-isothiazoline-3-one,

4-bromo-1- (3-chloro-4-fluorophenyl) -14-isothiazoline-1-one,

2- (4-ethoxycarbonyldiphenyl) -14-isothiazoline-13-one is particularly preferred.

The pharmaceutically acceptable salts in the present invention include, for example, salts of inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid and sulfuric acid, or acetic acid and ^ -uric acid Organic salts such as maleic acid, fumaric acid, citric acid, tartaric acid, etc., but are not limited to these examples.

Among the compounds in the present invention, 2- (3-chloro-4-fluorophenyl) -14-isothiazoline-13-one, 2- (3-chloro-1-fluorophenyl) -14-isothiazoline-3-one 4-Bromo-2- (3-chloro-1-fluorophenyl) -1-isothiazoline-13-one, 2- (3-trifluoromethylphenyl) -1-isothiazoline-13-one, and 2- (4-cyano) 3-Methylthiophenidyl) 1-4-isothiazolin-13-one is a compound that has not been described in the literature and is not known as a drug. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a plot of concentration versus human telomerase inhibitory activity (TRAP-SPA method) for compound 1.

FIG. 2 is a plot of concentration versus human telomerase inhibitory activity (TRAP-SPA method) for compound 2.

Figure 3 is a plot of concentration versus human telomerase inhibitory activity (TRAP-SPA method) for compound 3. Figure 4 shows the concentration-human telomerase inhibitory activity (TRAP-

5 P A method).

FIG. 5 shows the results of gel electrophoresis of human telomerase inhibitory activity (TRAP method) for Compounds 1-4. It is a turn. BEST MODE FOR CARRYING OUT THE INVENTION

The compound contained in the anticancer agent, telomerase inhibitor, or growth inhibitor of telomerase-containing immortalized cells of the present invention is a known compound and is commercially available, or It can be produced by a conventional method as described.

When the 3-isothiazolone derivative is produced from, for example, a 3,3'-dithiodipropionic acid amide derivative, for example, Lewis SN and Mil 1 er GA eta 1., (1971) J. Hetero cycl.Chem., 8, 571-580. When produced from a cis-1,3-thio-substituted acrylamide derivative, for example, C row

WD and Leona rd N.J., (1964) Tetrahe dron Lett., 23, 1477—1480, Crow. WD and Leona rd NJ, (1965) J. Org. Chem. , 30, 26

60-2665 or Harwood LM eta 1., (1994) J. Chem. Soc., Perk in Trans. 1, 16, 2245 -2251. When produced from a ketoamide derivative, it can be produced, for example, by the method described in S andris C. eta 1., (1987) Heterocycles, 25, 569-575. When producing from a derivative, it can be produced, for example, by the method described in Goerdeler J. and Mittler W., (1963) Chem. Ber., 96, 944-954. When the 4- and / or 5-position halogen-substituted 3-isothiazolone derivative is produced from the corresponding 3-isothiazolone derivative, for example, Lewis SN eta 1., (1971) J. Hetero cycl. Chem., 8, 571-580, or Miller GA eta 1., (1977) J. Heterocycl. Chem., 14, 627-630.

When the 3-isoxazolone derivative is produced from, for example, a hydroxyylamine derivative and a diketene or a 3-keto acid derivative, for example, Yone emitsu 0. (1982) Heterocycles, 19, 521-524, Jacobsen N (1984) Can. J. Chem., 62, 1940—1944 or Demote JP, (1980) J. Heterocyc 1. Chem., 17, 727—731. In the case of producing from a hydroxylamine derivative and a propiolic acid derivative, for example, Nakamura N. eta 1. (1966) Chem. Pharm. Bull., 14, 1277 — 1286, or can be produced by the method described in Ishida N. eta 1., (1971) Chem. Pharm. Bull., 19, 1 389-1394, and can be produced from a 3-hydroxysoxazolone derivative. When producing 2-substituted 1-3-isoxazolone derivatives, for example, Mu rak am i T. eta 1., (1979) Chem. Ph. Rm. Bu 1., 27, 2398-2404. Test example

Hereinafter, in order to show the usefulness of the present invention, test results on the tetra-merase inhibitory activity of representative compounds included in the present invention are shown.

Table 1 shows the chemical structural formulas of the corresponding compounds.

Compound i— (thing

No.XRR ₂ R ₃ R ₄ R ₅ R ₆ Ry

1 s — H — H — H — CF ₃ — H — H — H

2 s — H — H — F — F — F — H — H

3 s — H — H — H — H — COOEt — H — H

4 s — H — H — H — S e — CN — H — H

5 s — H — H — H — CI — F — H — H

6 s — H — H — F — CI — H — H — H

7 s —Br — H — H —CI — F — H — H

Test Example 1: Evaluation of human telomerase inhibitory activity of compounds by the TRAP-SPA method Materials and Methods

(1) Preparation of cell extract

Cell extract, a known method (K im NW eta 1. (1994 ) S ci en ce 206, 2011- 2015) to _c ie was prepared by adding some improvements, the human pre-leukemia cell lines the HL- 60 After culturing, cells in the logarithmic growth phase were centrifuged at 2,000 rpm for 5 minutes to collect the cells. Washed twice with ice-cold PBS and once with ice-cold RNase-free wash buffer (1 OmM Hepes pHT. 5, ImM MgC 12, 1 OmM KC 1, and ImM DTT) . The Perez bets cells, a solution buffer without RN ase (1 OmM T ris -HCI p H 7. 5, ImM Mg C 1 2, 1 mM EGTA, 0. ImM PM SF, 5mM / 3- mercaptoethanol, 5 x 10 ³ cells in 0.5% CHAPS (Choioami dop ropy l—dime t hy l—ammon io—1—propane sulfonate) and 10% glycerol) Suspended. After gently stirring and incubating on ice for 30 minutes, insolubles in the lysate were removed by centrifugation at 15,000 rpm for 30 minutes. The supernatant was aliquoted and stored at 180 ° C.

(2) Primer synthesis

The TS primer (sequence: 5'—AATCCGTCGAGCAGAGTT—3 ') and the non-piotinylated and biotinylated CX primer (sequence: 5'—CCCTTACCCTTACCCTTACCCTAA-3') were obtained from Saddy's Technology.

(3) Synthesis of peptide nucleic acid

The peptide nucleic acid (sequence: N—TAGGGTTAGACAA—C) was obtained from Nippon Perceptive Co., Ltd.

(4) Telomerase activity measurement

The telomerase activity is measured by a known method, the telomere repeat amplification protocol (hereinafter, referred to as “TRAP method”; Kim NW et al., 1994). S cinece 206, 201 1-2015; Piatysz ek eta l. (1995) Meth. Ce 11 Sci. 17, 11-15) and scintillati on prox imi tyass ay; Hereafter referred to as "SPA.") Some improvements to the system combining the technology (hereinafter referred to as "TRAP-SPA method"; a method described in Japanese Patent Application No. 8-17830). Was added. The TRAP-SPA method uses a tritium label incorporated when a primer extended by telomerase is amplified by the polymerase chain reaction. In this method, the amount of thymidine obtained is measured by a scintillation proximity technique developed by Amersham. Specifically, the following operations were performed.

The DNA synthesis reaction by telomerase was performed in a final reaction mixture (20 mM Tris—HC 1 pH 8.3, 1.5 mM Mg) containing a cell extract (corresponding to 1000-3000 cells) and an appropriate concentration of the test compound. _{C 1 2, 63mM KC l,} 0. 005% Twe en 20, 1 mM EGTA, d ATP respectively 50 M, dCTP and dGTP, dTTP in 2〃M, 1 ^ C i [Me - 3 H] dTTP (Ame rsh am, 114C iZmmo l), 0.1 g / β 1

B S A and 9 pmo 1 of TS primer) for 30 minutes at room temperature.

Next, in order to amplify the synthesized terrorist Mea oligonucleotide, 20mM Tr is-HC l pH 8. 3, 1. 5mM MgC l 2, 63mM KC 1, 0. 005% Twe en20, lmM EGTA, respectively 50 M d ATP of, dTTP of dCTP and dGTP, 2 // M, 1 C i. [Me - 3 H] dTT P (Am ersham Co., 114 C i / mmo 1) 0. 1 g / 1 BSA, 9 A 25 \ solution containing pmo 1 TS primer, 14 pmo 1 biotinylated CX primer, and 2 U TaQ DNA po 1 ymerase (Takara Shuzo) was added. The mixture was heated at 90 ° C for 90 seconds, followed by one cycle of 94 ° C for 15 seconds, 50 ° C for 30 seconds and 72 ° C for 45 seconds, and the polymerase chain reaction was performed in 25 cycles. . The polymerase chain reaction was performed using GenAmp PCR Systems 9600 manufactured by Perkin-Elma. The reaction product (201) was transferred to a 96-well plate (manufactured by Wa11ac) and 26.5 6.1 streptavidin-coated microparticle fluoromicrospheres were transferred.

(A solution of SmgZml in 0.4 M EDTA) was added, and the mixture was incubated at 37 ° C. for 10 minutes to bind the ³ H-labeled reaction product to streptavidin beads. Plates were counted on a MicroBeta scintillation counter (Wa11ac). (5) Inhibition of polymerase chain reaction

In the TRAP-SPA method described above, the system was replaced so that the DNA synthesis reaction by telomerase was performed in the absence of the compound, and the subsequent polymerase chain reaction was performed in the presence of the compound. Everything else was the same. The polymerase chain reaction inhibitory activity of the compound at 10 M was evaluated in comparison with the count of the reaction product by the polymerase chain reaction performed under the condition containing no compound (this 10 M was used in the telomerase inhibitory activity evaluation system). This corresponds to the final concentration in the polymerase chain reaction system when the compound is added at a concentration of 20 M). The results are shown in FIGS. 1 to 4 and Table 2. Figures 1 to 4 are plots of telomerase activity when various concentrations of compounds are included as relative activities, with the reaction product count when no compound was included as 100%. , N = 4, and the average is plotted).

Figure 1 is a plot of concentration 1 versus human telomerase inhibitory activity (TRAP-SPA method) for compound 1. FIG. 2 is a plot of the concentration-human telomerase inhibitory activity (TRAP-SPA method) for Compound 2. FIG. 3 is a plot of concentration-human telomerase inhibitory activity (TRAP-SPA method) for compound 3. Figure 4 is a plot of concentration versus human telomerase inhibitory activity (TRAP-SPA method) for compound 4.

Table 2 summarizes the 50% inhibitory concentrations (IC _{5 ()} ). Compound 1 to Compound 7 did not inhibit the polymerase chain reaction at a concentration of 1.

Table 2 Measurement results of human telomerase inhibitory activity

(TRAP - SPA method) Compound No. IC _5. (M)

0 3

2 0 5

3 0 6

4 6

5 0 2

6 0 3

7 0 3

Test Example 2: Method for evaluating compound inhibitory activity using ladder formation by the TRAP method as an index

The effect of the test compound was similarly evaluated from the inhibition of ladder formation by the TRAP method. That is, replace the biotinylated primer with a non-biotinylated CX primer, change the dCTP concentration to 20〃M, increase dTTP to 50〃M, and

The same operation as in Test Example 1 was performed, except that [Me— ³ H] dTTP was replaced by [a— ³² P] dCTP (3000 Ci / mmo 1 manufactured by Amersham). After completion of the polymerase chain reaction, the reaction products were separated by 10% polyacrylamide gel electrophoresis, and the reaction products were analyzed using FU JIX Bio 'Imaging Analyzer BAS 2000 (manufactured by Fuji Film Co., Ltd.). The compound was added at a concentration of 2.0 or 20 / M for measurement. Fig. 5 shows the results. Figure 5 shows human telomerase inhibition for compounds 1-4. Activity (TRAP method) This is a gel electrophoresis pattern. When added at a concentration of 20, all compounds almost completely inhibited ladder formation. At a concentration of 2 / M, in Test Example 1 (TRAP-SPA method), inhibition of ladder formation was observed for three compounds (Compounds 1 to 3) that had stronger telomerase inhibitory activity. Lane No. 11 shows the result of the TRAP reaction in a system containing no compound, and Lane No. 12 shows the result of the TRAP reaction without the cell extract. As positive controls for telomerase activity inhibition, lane numbers 9 and 10 show the effects of an antisense peptide nucleic acid (Peptide Nucleic Acid; PNA) on the type II portion of human telomerase RNA submit. Industrial applicability

As described above, the compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof has an action as an anticancer agent, a telomerase inhibitor, or a growth inhibitor of telomerase-containing immortalized cells. It is useful as a medicine.

Claims

-General formula (1)

Contract

of

(Wherein X represents a sulfur atom or an oxygen atom; and R ₂ each independently represents a hydrogen atom, a halogen atom, or a linear or branched alkyl group having 1 to 3 carbon atoms. R ₃ , R ₄ , R ₅ , R ₆ and R ₇ each independently represent a hydrogen atom, a halogen atom, a haloalkyl group having 1 to 3 carbon atoms, a cyano group, —COOR ₈ , —SR ₉ , or oR _10, _{_{represents;. R 8, R 9,}} R 1 () are each independently representing a linear or branched alkyl group having a carbon number of 1-3 indicated compound or by) its An anticancer agent containing a pharmaceutically acceptable salt.

2. General formula (1)

(In the formula, X and 1 ^ to 1 ^ ₁₀ represent the same meaning as defined in claim 1.) Or a telomerase inhibitor _c containing a pharmaceutically acceptable salt thereof

3. General formula (1)

(Wherein X and 1 ^ to 1 ^ _{1 ()} have the same meaning as defined in claim 1) or a telomerase-containing compound containing a pharmaceutically acceptable salt thereof. An inhibitor of growth of dead cells.

4. The compound is

2- (3-chloro-1--4-fluorophenyl) 1-4-isothiazoline-13-one, 2- (3-chloro-1-fluorophenyl) 1-4-isothiazoline-3-one, 4-bromo-1- ( 3-chloro-4- (4-fluorophenyl) -14-isothiazoline-13-one,

2- (3_Trifluoromethylphenyl) 4-isothiazoline-3-one, 2- (2,3,4-trifluorophenyl) 1-4-isothiazoline-3-one, 2- (4-ethoxy Carbonyl phenyl) 4-isothiazoline-3-one, or

2. The anticancer agent according to claim 1, which is 2- (4-cyano-3-methylthiophenidyl) 4-isothiazolin-13-one.

5. The compound is

2- (3-chloro-1-fluorophenyl) 1-4-isothiazoline-3-one, 2- (3-chloro-2-fluorophenyl) -1-isothiazoline-3-one, 4-bromo-1- ( 3-chloro-4-fluorophenyl) 1-41-isothiazoline — 3_ on,

2-(3-trifluoromethylphenyl) 1-4-isothiazoline-3-one, 2- (2,3,4-trifluorophenyl) 1-4-isothiazoline 1-one, 2-(4- (Ethokincarbonyl) 14-isothiazoline-13-one, or

3. The telomerase inhibitor according to claim 2, which is 2- (4-cyano-3-methylthiophenidyl) -1-isothiazolin-13-one.

6. The compound is

2- (3-chloro-1-4-fluorophenyl) 1-4-isothiazoline-3-one, 2- (3-chloro-2-fluorophenyl) -1-isothiazoline-3-one, 4-bromo-2- (3- Chloro-4-fluorophenyl) 1-4-isothiazoline-3-one,

2- (3-trifluoromethylphenyl) 1-4-isothiazoline-3-one, 2- (2,3,4-trifluorophenyl) -4 ^^ sothiazoline-1 3-one, 2-(4 1-ethoxycarbonyl) 1-4-isothiazolin-3-one, or

The telomerase-containing immortalized cell proliferation inhibitor according to claim 3, which is 2- (4-cyano-3-methylthiophenidyl) -14- ^ sothiazoline-13-one.

7.2 2- (3-Chloro-4-fluorophenyl) -1-4-isothiazoline-1-one,

2- (3-chloro-1--2-fluorophenyl) -1-isothiazolin-3-one, 4-bromo-1- (3-chloro-1-phenylphenyl) -1-isothiazoline-1-3-one,

Selected from the group consisting of 2- (3-trifluoromethylphenyl) 1-4-isothiazoline-13-one, 2- (4-cyano-13-methylthiophenyl) 1-4-isothiazoline-13-one Or a pharmaceutically acceptable salt thereof.

8. 2- (3-chloro-1-4-fluorophenyl) -1-isothiazoline-3-one, 2- (3-chloro-1-fluorophenyl) 1-41-isothiazolin-3-one, 4-bromo-12- (3-chloro-1-fluorophenyl) 1-4-isothiazoline 1-3-one,

2-(3-trifluoromethylphenyl) 1-4-isothiazoline 13-one, 2- (4-cyano 13-methylthiophenidyl) 1-4-isothiazoline 13-one, selected from the group consisting of Or a pharmaceutically acceptable salt thereof.