WO1999041261A1

WO1999041261A1 - Telomerase inhibitors

Info

Publication number: WO1999041261A1
Application number: PCT/JP1999/000611
Authority: WO
Inventors: Naoki Kato; Kenji Yoshida; Masayuki Tuchiya; Naoki Taka; Kazumi Morikawa; Masamitsu Honda
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1998-02-13
Filing date: 1999-02-12
Publication date: 1999-08-19
Also published as: AU2300499A

Abstract

Compounds represented by general formula (1) which have a telomerase inhibitory activity and are useful as drugs such as carcinostatics: In formula (I) R1 and R2 represent each hydrogen, etc.; R3 and R4 represent each hydrogen, etc.; R5 and R6 represent each carboxy, etc.; and n is 0 or 1.

Description

Specification

Telomerase inhibitor Technical field

The present invention relates to a platinum complex having telomerase inhibitory activity and useful as a medicament. Background art

Telomerase is known as an enzyme that catalyzes the extension of the telomere terminal (terminal portion of a linear chromosome), and has been extensively studied (Greider CWandBlackburn EH, (1987) Cell , 51, 8 87-898; Morin GB (1989) C e 11, 59, 521-5 29) o

Telomerase activity is not detected in normal cells except in some cells, such as germline cells and 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 drugs with few side effects on normal cells, or as inhibitors of the growth of immortalized cells containing telomerase (Counter CM eta 1., (1989 EMBO J., 11, 1921-1929: Counter CM eta 1., (1994) Pro Natl. Ac ad. Sci. USA, 91, 2900-2904; Chadeneau C. eta 1., (1995) Cancer Res., 55, 2533-2536; Hiy am a E. eta 1., (1995) Nature Med., 249-255, Shay J. W. eta 1., ( 1995) MoI. 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 specificity (Yegorov YE, Chernov) DN, A kimov S. S., eta 1. (1996) FEBS Lett. 389, 115-118; Kagawa Yasuo. (1997) Modern Medicine, 52, 306-312). Recently, it has been reported that 2,6-diamidanthraquinone compounds inhibit telomerase (Sun D., Thompson on B., eta 1., (1997) J. Med. Chem. , 40, 2113—2116). The inhibitory activity is not satisfactory, and a drug having better telomerase inhibitory activity is required.

—On the other hand, platinum complexes such as cisplatin and carboblatin are currently used as first-line drugs as anticancer drugs, but they still have problems such as side effects and drug resistance. In addition, there has been no discussion about the presence or absence of telomerase inhibitory activity in platinum complexes. Disclosure of the invention

An object of the present invention is to provide a medicament having telomerase inhibitory activity and useful as a therapeutic agent for cancer and the like, more specifically, a platinum complex having telomerase inhibitory activity.

The present inventors have conducted intensive studies to obtain a compound having telomerase inhibitory activity, and as a result, have found that known platinum complexes such as carboblatin and cisbratin have substantially no telomerase inhibitory activity. The inventors have found that the complex has telomerase inhibitory activity and exhibits an excellent effect as an anticancer agent, and have completed the present invention based on this finding. That is, the present invention relates to the general formula (1)

(1)

(Wherein 1 ぉ and 1 ^ ₂ are the same or different and represent a hydrogen atom or a methyl group; R ₃ and R ₄ are the same or different and represent a hydrogen atom or a methyl group; R ₅ and R _e is, identical or different, represent a hydrogen atom or a carboxyl group; n is provided compounds represented by the representative) 0 or 1, medicaments containing such compounds, and telomerase inhibitors containing such compounds. I do. Further, the present invention provides a carcinostatic agent based on a telomerase inhibitory action containing such a compound as an active ingredient. In the present invention, “telomerase inhibition” refers to inhibiting an enzymatic reaction that adds a telomere sequence to the 3 ′ end of DNA. Specifically, for example, telomerase inhibitory activity is determined by the TRAP-SPA method described later. when measuring means that you inhibit Teromea extension reaction measured in addition to oligonucleotides [Me- ³ H] dTTP.

In the present invention, the definition of “cancer drug” includes a drug that suppresses the growth of cancer cells or kills cancer cells, and a drug that suppresses the growth of immortalized cells containing telomerase. Is also included.

In the definition of the compound represented by the general formula (1), it is preferable that either or both of and R ₂ be a hydrogen atom.

Preferably, one or both of R ₃ and R 4 are hydrogen atoms.

R ₅ and R _6, either one is a hydrogen atom, it is preferred the other is a carboxyl group.

As n, either 0 or 1 is preferable.

The following cases are preferable as a combination of Rh R ₂ , R ₃ , R ₄ and n.

(a) H H H H 0 (b) (H Me) H H 0 (c) H H H H 1 (d) H H (H Me)

Here, H is a hydrogen atom, Me is a methyl group, and (H Me) indicates that one is a hydrogen atom and the other is a methyl group. same as below.

As the combination of R ₂ , R ₃ , R ₄ , R ₅ , R _e , and n, the following cases are preferable. Table 2

R ₄ R, n

(A) H H H H (H COOH) 0

(B) (H Me) H H H H 0

(C) (H Me) H H (H COOH) 0

(D) H H H H (H COOH)

(E) H H (H Me) (H COOH)

Here, (HCOOH) indicates that one is a hydrogen atom and the other is a carboxyl group.

—Specific examples of the compound represented by the general formula (1) include:

(2-aminomethylpyridine) hemimelitate platinum (Π), (2- (2-aminoethyl) pyridine) hemimelitrate platinum (Π), (2- (1-aminoethyl) pyridine) phthalate platinum (II),

Preference is given to (2- (1-aminoethyl) pyridine) hemimelitrate platinum (II) and (2- (2-aminoethyl) -1-N-methylpyridine) hemimeric platinum (Π). BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a plot of concentration-telomerase inhibitory activity (TRAP-SPA method) for compound 2.

Figure 2 is a plot of concentration-telomerase inhibitory activity (TRAP-SPA method) for compound 3.

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

Figure 4 shows a plot of concentration versus telomerase inhibitory activity (TRAP-SPA method) for compound 5.

Figure 5 is a plot of concentration-telomerase inhibitory activity (TRAP-SPA method) for compound 7. BEST MODE FOR CARRYING OUT THE INVENTION

The compound represented by the general formula (1) can be obtained, for example, by reacting platinum (II) botasium chloride with an aromatic heterocyclic amine, 2-aminoalkylpyridine, to give (2-aminoalkylpyridine) dichloroplatinum (II) Then, it is reacted with silver nitrate to obtain (2-aminoalkylpyridine) dinitrataplatinum (II), and then reacted with dicarboxylic acids.

The above manufacturing steps are shown below using chemical formulas.

Platinum (II) potassium chloride, aromatic heterocyclic amine and dicarboxylic acids, which are raw materials for producing the general formula (1), are commercially available or, for example, It can be produced by a conventional method as described in magazines, 108 (4), 317-324, (1988).

Compounds of general formula '(1) may comprise one or more pharmaceutically acceptable diluents, wetting agents, emulsifiers, dispersants, auxiliary agents, preservatives, buffers, binders, stabilizers And the like can be administered in any appropriate form depending on the intended administration route. For the compound represented by the general formula (1), a parenteral administration route, particularly an intravenous route, is a preferred administration route. Parenterally, it can be administered in the form of an aqueous or non-aqueous solution, suspension, or emulsion. The aseptic preparation can be performed by a conventional method, for example, by aseptic filtration.

The dose of the compound represented by the general formula (1) can be appropriately selected depending on the patient's body type, age, physical condition, degree of disease, elapsed time after onset, and the like. Example

Hereinafter, the compound of the present invention will be described in more detail with reference to Examples, but the present invention is not limited by these Examples.

Further, in order to show the usefulness of the present invention, test results on the telomerase inhibitory action of the compounds included in the present invention are shown in Test Examples.

Table 3 shows the chemical structural formulas of the corresponding compounds. Table 3 Example compounds

Compound R ₂ R ₃ R _{4 5} Re n

1 H H H H H H 0

2 H H H H (H COOH) 0

3 H H H H (H COOH) 1

4 (H Me) H H H H 0

5 (H Me) H H (H COOH) 0

6 H H H H H H 1

7 H H (H Me) (H COOH) 1 In the table, (H Me) means that one of them is H and the other is Me.

(H COOH) is, _c indicating that one is H, the other is COOH

Examples]: (2-aminomethylpyridine) phthalate platinum (II)

Synthesis of (Compound 1)

(2-aminomethylpyridine) dinitratoplatinum (Π) 427 mg water 4 The solution was dissolved in 0 ml, and 4 ml of an aqueous solution of 21 Omg of disodium phthalate was added, followed by stirring at room temperature for 18 hours. The precipitated precipitate was collected by filtration, washed with water and methanol, and dried under reduced pressure to obtain 22 Omg of (2-aminomethylviridine) phthalate platinum (II) as a colorless powder.

Melting point: 300 ° C or more (decomposition), IR: 3430, 3200 (N-H), 1610 (C = 0)

_{_{_{_{Calcd: 4 Ηι 2 Ν 2 0 4}}}} Ρΐ · 2H 2 0: C 33.39; H 2.40; N 5.56;

Measurement values: C 33.17; H 2.50; N 5.88 Example 2: (2-aminomethylpyridine) hemimelite toplatinum (Π)

Synthesis of (Compound 2)

In the same manner as in Example 1, hemimellitate platinum (II) was obtained in the same manner as in Example 1 except that the dicarboxylic acid was replaced with hemimelitic acid.

Melting point: 230-290 ° C (decomposition), IR: 3440, 3200 (N-H), 1710, 1615 (00) Example 3: (2- (2-aminoethyl) pyridine)

(II) Synthesis of (Compound 3)

In the same manner as in Example 1, the platinum compound was replaced with (2_ (2-aminoethyl) pyridine) dinitratoplatinum (II), and the dicarboxylic acid was replaced with hemimelitic acid. Hemimelitate Platinum (ナム) was obtained.

Melting point: 255 ° C (decomposition), IR: 3420, 3200, 3060 (N-H), 1705, 1600 (C = 0) Example 4: (2- (1-aminoethyl) pyridine) phthalate platinum (II)

Synthesis of (Compound 4)

In the same manner as in Example 1, the platinum compound was replaced with (2- (1-aminoethyl) pyridine) dinitratoplatinum (Π) and (2- (1-aminoethyl) pyridine) phthalene platinum (Π ).

Melting point: 220-240 ° C (decomposition), IR: 3420, 3180, 3060 (NH), 1600 (00) Example: Synthesis of (2- (1-aminoethyl) pyridine) hemimellitate platinum (II) (compound 5)

In the same manner as in Example 1, the platinum compound was replaced with (2- (1-aminoethyl) pyridine) dinitra toplatinum (Π), and the dicarboxylic acid was replaced with hemimelitic acid. (2- (1-aminoethyl) Pyridine) was obtained.

Melting point: 275-300 ° C (decomposition), IR: 3430, 3200 (N-H), 1600 (C = 0) Example 6: (2- (2-aminoethyl) pyridine) phthalate platinum (II)

Synthesis of (Compound 6)

In the same manner as in Example 1, the platinum compound was replaced with (2- (2-aminoethyl) pyridine) dinitra toplatinum (II), and (2- (2-aminoethyl) pyridine) phthalate platinum (II) I got

Melting point: 230 ° C (decomposition), I: 3400, 3300, 3070 (NH), 1610 (00) Example 7: (2- (2-aminoethyl) -N-methylpyridine) hemimelitrate platinum (II) Synthesis of (Compound 7)

In the same manner as in Example 1, the platinum compound was replaced with (2- (2-aminoethyl) -N-methylpyridine) dinitrataplatinum (II), and the dicarboxylic acid was replaced with hemimelitic acid. —Aminoethyl) -N-methylpyridine) hemimeryte toplatinum (II) was obtained.

Melting point: 260 ° C (decomposition), IR: 3420. 3100 (N-H), 1715, 1615 (C-0) Test Example 1: Evaluation of human telomerase inhibitory activity of compounds by the TRAP-SPA method (materials and methods)

(1) Preparation of cell extract

The cell extract was prepared by a known method (Kim NWeta 1. (1994) Science 206, 2011-2015) with some modifications. That is, the human proleukemia cell line HL-60 was cultured, and cells in the logarithmic growth phase were The cells were collected by centrifugation at 000 rpm for 5 minutes. Wash twice with ice-cold PBS and once with ice-cold RNase-free wash buffer (1 OmM Hepes pH 7.5, ImM MgC 12, 1 OmM KC 1, and ImM DTT). Was. The pellet of the cells was transferred to a solution buffer containing no RNase (1 OmM Tris-HC 1 H7.5, ImM MgC12 ₍ ImM EGTA, 0. ImM PMSF, 5 mM / 3-mercaptoethanol, 0.5 % CHAPS (Chio 1 o am i dop ropy 1-dimethhy 1-ammonio-1-ropanesulfonate (chloroamidopropyl-dimethyl-ammonio-1-propanesulfonate)) and 10% glycerol and resuspended at 5 X 10 ³ cells / 1. gently stirred, after Inkyube and Bok 30 minutes on ice, 15, 000, except taken insolubles lysate by r pm by centrifugation for 30 minutes The supernatant was aliquoted and stored at -80 ° C.

(2) Primer synthesis

The TS primer (sequence: 5′—AATCCGTCGAGCAGAGTT—3 ′) and the biotinylated CX primer (sequence: 5′—CCCTTACCCTTACCCT TACCCTAA—3 ′) were obtained from Saddy Technology.

(3) Telomerase activity measurement

The telomerase activity measurement is a known method, the telomere repeat amplification protocol (telome ricreeata mp 1 verification protocol, hereinafter, referred to as the "TRAP method"); Kim NW eta 1. (1994) S cinece 206, 201 1-2015; Piatyszeketa 1. (1995) Meth.Ce 11 Sci. 17, 1—15) and scintillation on atxy (scintillati on proxy imi tyassy); ). A system that combines technology (hereinafter referred to as the “TRAP-SPA method”; a method described in Japanese Patent Application No. Hei 8-17830) with some improvements. Was. The TRAP-SPA method uses a tritium label that is 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 procedure is performed to perform the DNA synthesis reaction by telomerase. The DNA synthesis reaction is carried out in a final reaction mixture of 25 1 (20 mM) containing a cell extract (equivalent to 1000 to 3000 cells) and an appropriate concentration of a test compound. tr is- HC 1 pH8. 3, 1. 5mM Mg C 1 2, 63mM KCI, 0. 005% Twe en 20, 1 mM EGTA, d ATP respectively 50 // M, dCTP and dGTP, 2〃M of dTTP , 1〃 C i [Me- ³ H] dTTP (Armash am, 114 C i Zmmo 1), 0.1 g / β 1

BSA, and 9 pmo 1 of TS primer) for 30 minutes at room temperature.

Next, to amplify the synthesized telomere oligonucleotide, 20 mM Tris—HC 1 H 8.3, 1.5 mM MgCl ₂ , 63 mM KC 1, 0.005% Tween 20, lmM EGTA, d ATP respectively 50〃 M, dCTP and dGTP, dTTP in 2〃M, 1 / zC i [Me - 3 H] dTT P (Ame rsh am Inc., 114 C i / mm o 1 ), 0. 1 g A solution of 251 containing / 1 BSA, 9 pmo 1 TS primer, 14 pmo 1 biotinylated CX primer, and 2 U Taq DNA po 1 ymerase (Takara Shuzo Co., Ltd.) was added. The mixture was heated at 90 ° C for 90 seconds, followed by polymerase chain reaction in 25 cycles of 15 seconds at 94 ° C, 30 seconds at 50 ° C and 45 seconds at 72 ° C. . Polymerase chain reaction was performed using GeneAmp PCR System 9600 manufactured by Perkin-Elma. The reaction product (201) was transferred to a 96 l plate (manufactured by Wa11 ac), and 26.5〃1 streptavidin-coated microparticle fluoromicrospheres (0.4 M in EDTA) were used. , SmgZml solution), and incubated at 37 ° C for 10 minutes to bind the ³ H-labeled reaction product to streptavidin beads. Plates were counted on a Micro Beta scintillation counter (Wa 11 ac). (4) Inhibition of polymerase chain reaction

In the above TRAP-SPA method, 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 without the compound (this 10 M was used to evaluate the telomerase inhibitory activity). In the system, this corresponds to the final concentration in the polymerase chain reaction system when the compound was added at a concentration of 20 M).

(Result)

The results are shown in FIGS. 1 to 5 and Table 4. Figures 1 to 5 are plots of telomerase activity with various concentrations of compound as relative activities, with the reaction product count without compound being 100% (relative activity). In the measurement, n = 3 or 2, and the average was plotted.) Table 4 shows the 50% inhibitory concentration

(I Cso). Compounds 2, 3, 4, 5, and 7 did not inhibit the polymerase chain reaction at a concentration of 10 M. Table 4 Measurement results of telomerase inhibitory activity

Compound I Cso (βΜ)

23.2

3.1.2

43.1

5 1. 3

7 1.2 Industrial applicability

The present invention provides a platinum complex having telomerase inhibitory activity, and is useful as a drug such as an anticancer drug.

Claims

Scope of claim General formula (1)

(Wherein, and R ₂ are the same or different and represent a hydrogen atom or a methyl group; R ₃ and R ₄ are the same or different and represent a hydrogen atom or a methyl group; R ₅ and R _e are the same Or differently, represents a hydrogen atom or a carboxyl group; n represents 0 or 1.)

2. The compound is

(2-Aminomethylpyridine) Hemime Retinate Platinum (II),

(2- (2-Aminoethyl) pyridine) Hemimetrate platinum (II), (2- (1-aminoethyl) pyridine) phthalate platinum (II),

(2- (1-aminoethyl) pyridine) hemimeric platinum (II), and (2- (2-aminoethyl) -1-N-methylpyridine) hemimeric platinum (II), 2. The compound according to claim 1, which is

3. The compound according to any one of claims 1 or 2, which has telomerase inhibitory activity.

4. A medicament containing the compound according to any one of claims 1 and 2.

5. A telomerase inhibitor comprising the compound according to claim 1 or 2.

6. An anticancer agent based on telomerase inhibitory activity, comprising the compound according to claim 1 as an active ingredient.