JP2001097948A - Compound having affinity for thrombopoietin receptor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a thrombocytopenia medicine, a platelet production control agent or a platelet production promoter which has affinity for thrombopoietin receptors and can orally be administered. SOLUTION: 3-Aminoacetyl-1-phenylsulfonylindole or its pharmacologically acceptable salt. The 3-aminoacetyl-1-phenylsulfonylindole or its pharmacologically acceptable salt is useful as an orally administrable thrombocytopenia medicine, platelet production control agent or platelet production promoter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a compound having an affinity for a thrombopoietin receptor and having a platelet production regulating action or a platelet production promoting action.

[0002]

2. Description of the Related Art Thrombopoietin (TPO) is c-Mp
1 is a ligand for the protein encoded by the l gene (thrombopoietin receptor) and was cloned in 1994 (de Sauveage, FJ et al, Nature 369, 533-538 (199
4); Bartyley, TD et al, Cell 77, 1117-1124 (199
Four)). TPO is a central humoral factor that stimulates megakaryocyte production, and acts on a wide range of megakaryocyte production from megakaryocyte precursor cells to mature megakaryocytes. Furthermore, it is known to be a major physiological regulator in platelet production, is expected as a therapeutic agent for thrombocytopenia, and is being developed.

[0003] It has an affinity for the thrombopoietin receptor,
As factors regulating platelet production, in addition to this TPO itself, low-molecular peptides (WO96 / 40189,
WO98 / 25965, JP-A-10-72492
No.), 1,4-benzodiazepine derivatives (Japanese Unexamined Patent Publication No.
No. 1477).

[0004]

In preclinical and clinical studies, TPO has been reported to have a platelet increasing effect and a platelet recovery promoting effect after bone marrow suppression by cancer chemotherapy and the like. However, in recent clinical trials, neutralizing antibodies appeared in some cancer patients who received TPO, and thrombocytopenia occurred in healthy individuals (platelet transfusion donors) who received TPO for the purpose of collecting platelets. (Neutralizing antibody positive), and in severe cases, conversely, the necessity of platelet transfusion has been reported.

[0005] Further, since TPO is a protein composed of 332 amino acids, it is degraded in the digestive tract and is difficult to administer orally. In clinical trials, it is administered subcutaneously as an injection. . Therefore, development of a therapeutic agent for thrombocytopenia, a platelet production regulator or a platelet production promoter which has an affinity for the thrombopoietin receptor and can be orally administered is desired.

[0006]

Means for Solving the Problems The present inventors have made intensive studies in order to solve the above-mentioned problems, and as a result, 3-aminoacetyl-1-phenylsulfonylindole or a pharmacologically acceptable salt thereof has been obtained. The present inventors have found that they have an affinity for thrombopoietin receptor and completed the present invention.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION 3-Aminoacetyl-1 of the present invention
-Phenylsulfonylindole can be converted, if necessary, to a pharmacologically acceptable salt or from the salt formed to a free base. Examples of pharmacologically acceptable salts include inorganic acid salts such as hydrochloride, phosphate and sulfate, and organic acid salts such as acetate, citrate and methanesulfonate.

The medicament comprising 3-aminoacetyl-1-phenylsulfonylindole or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient is usually used in tablets and capsules for mammals (including human patients). , Powders, fine granules, syrups and the like, oral administration, rectal administration, or injection. In addition, the compound of the present invention
It can be administered as a single therapeutic or as a mixture with other therapeutics. They may be administered alone, but are generally administered in the form of a pharmaceutical composition. These preparations can be produced by a conventional method by adding pharmacologically and pharmaceutically acceptable additives. That is, additives such as ordinary excipients, lubricants, binders, disintegrants, wetting agents, and coating agents can be used for oral preparations.
Oral solutions include aqueous or oily suspensions, solutions, emulsions,
It may be in the form of a syrup, elixir or the like, or it may be provided as a dry syrup prepared with water or another suitable solvent before use. The above-mentioned liquid medicine, a suspending agent,
Conventional additives such as fragrances, diluents or emulsifiers can be included. When administered rectally, it can be administered as a suppository. Suppositories are based on suitable substances such as cocoa butter, lauric fat, macrogol, glycerogelatin, witepsol, sodium stearate or a mixture thereof, and, if necessary, emulsifiers, suspending agents, and preservatives. Etc. can be added. Injectable preparations include distilled water for injection, physiological saline, and water-soluble or dissolvable dosage form.
% Glucose solution, propylene glycol and other solubilizers or solubilizers, pH regulators, isotonic agents, stabilizers and other formulation components are used.

The dose of the compound of the present invention when administered to humans varies depending on the age and condition of the patient. Usually, in the case of an adult, 1 mg to 1000 mg orally or rectally.
mg / person / day, 0.1 to 500 mg / person / injection
About a day. However, these numerical values are merely examples, and the dose may be appropriately adjusted according to various conditions such as the patient's symptoms.

[0010]

EXAMPLES The present invention will now be described specifically with reference to examples and test examples, but the present invention is not limited to these examples.

The 3-aminoacetyl-1-phenylsulfonylindole of the present invention can be synthesized by the following method. Scheme 1

Embedded image

Example 1 Preparation of 3-acetyl-1-phenylsulfonylindole (2) (Step 1) In a stream of argon and under ice-cooling, dichloromethane (250 ml) was added to ground anhydrous aluminum chloride (67.2 g, 504 mmol). ) Was added and acetic anhydride (23.7 ml, 252 mmol) was added dropwise.
After completely dissolving the anhydrous aluminum chloride, 1-phenylsulfonylindole (1) (21.6 g, 84.0 mmol)
Was added dropwise, followed by stirring for 10 minutes. After the reaction, the reaction solution was poured into ice water and extracted with dichloromethane. The organic layer was washed with a saturated aqueous solution of sodium hydrogen carbonate and then with a saturated saline solution. After drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to give pale blue crystals.
4.1g was obtained. The obtained crystals were recrystallized from methanol to give 3-acetyl-1-phenylsulfonylindole (2) (23.2 g, yield 92%) as colorless plate crystals (melting point 164-1).
64.5 ° C).

IRν _max cm ^-1 (Nujol): 1663 (C = O). ¹ H-NMR (in CDCl ₃ ) δ (ppm): 2.54 (3H, s, COCH ₃ ), 7.10
-8.40 (9H, aromatic-H), 8.15 (1H, s, indole C ² -H).

Example 2 Preparation of 3-bromoacetyl-1-phenylsulfonylindole (3) (Step 2) Ethyl acetate (300 ml) was added to copper bromide (18.90 g, 84.6 mmol) at room temperature in an argon stream. And then 3-acetyl-1-
Phenylsulfonyl indole (2) (12.60 g, 42.1 mm
ol) in ethyl acetate (380 ml) was added dropwise, and the mixture was stirred while heating under reflux for 50 minutes. After cooling, the precipitate of the inorganic substance was collected by suction filtration. After sufficiently washing the collected matter with ethyl acetate, the filtrate and the washing liquid were combined, and a saturated aqueous solution of sodium hydrogen carbonate was added.
Then, it was washed with saturated saline and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to give pale yellow crystals 15.38 g.
I got The obtained crystals were recrystallized from benzene-hexane to give 3-bromoacetyl-1-phenylsulfonylindole (3) (12.61 g, 79%) as colorless plate crystals. Further, recrystallization was partially performed from benzene-hexane to obtain colorless plate crystals (melting point: 130.5-131 ° C.).

Elemental analysis C ₁₆ H ₁₂ BrNO ₃ S Theoretical: C, 50.81; H, 3.20; N, 3.70. Experimental: C, 50.69; H, 3.16; N, 3.75. IR ν _max cm ^-1 (Nujol ): 1678 (C = O). ¹ H-NMR (in CDCl ₃ ) δ (ppm): 4.32 (2H, s, COCH ₂ Br), 7.10
-8.41 (9H, aromatic-H), 8.28 (1H, s, indole C ² -H). MS (EI) m / z: 379 (M ⁺ +2, 14%), 377 (M ⁺ , 13%) , 77 (B
P).

Example 3 Preparation of 3-aminoacetyl-1-phenylsulfonylindole hydrochloride (5) (Step 3-1) 3-Bromoacetyl-1-phenylsulfonylindole (3) (4.94 g, 13.1 mmol) Was added to a solution of hexamethylenetetramine (2.01 g, 14.3 mmol) in chloroform (40 ml), and the mixture was stirred at room temperature for 1 hour.
The crystals precipitated after the reaction were subjected to suction filtration to obtain 5.81 g of crude crystals of intermediate (4) (crude yield: 86%).

(Step 3-2) The obtained intermediate (4) (5.8
Concentrated hydrochloric acid (9.1 ml) was added to a mixed solvent solution of 1 g (11.2 mmol) of water (71 ml) and ethanol (35 ml) at room temperature, and the mixture was stirred at 65 ° C. for 1 hour, and evaporated to dryness under reduced pressure. Next, the following operation was performed to remove ammonium chloride and bromide ions as impurities. The pale yellow solid of the residue was suspended in ether, and a 5% aqueous sodium hydroxide solution was added and stirred in a separating funnel,
After confirming that the aqueous layer was alkaline, the ether layer was separated, the ether layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and the desiccant was filtered off. Dry hydrogen chloride gas was blown into the obtained ether layer, and the precipitated crystals were collected by suction filtration. This was reprecipitated from ethanol-ether to give 3-aminoacetyl-1-phenylsulfonylindole hydrochloride (5) (3.35 g) as pale red crystals (melting point: 215-218 ° C).

Elemental analysis C ₁₆ H ₁₅ ClN ₂ O ₃ S Theoretical: C, 54.78; H, 4.31; N, 7.99. Experimental: C, 54.37; H, 4.69; N, 7.51. IR ν _max cm ⁻¹ (Nujol): 3365 (N ⁺ H ₃ ), 1680 (C = O). ¹ H-NMR (in DMSO-d ₆ ) δ (ppm): 3.30-3.58 (3H, br,
^{_{COCH 2 N H 3 +),}} 4.58 (2H, brs, COC H 2 NH 3 +), 7.10-8.35 (9
H, m, aromatic-H), 9.00 (1H, s, indole C ² -H). MS (EI) m / z: 314 (M ⁺ , 14%), 284 (BP).

Test Example 1 Establishment of Thrombopoietin-Dependent Cell Line BaF / mpl (1) Preparation of pCR3-Uni / mpl The cDNA of c-mpl (thrombopoietin receptor gene) was RT-PC
Amplified by the R method. That is, human fetal hepatocyte-derived pol
1st-strand cDNA Synthesis Kit (CLO) using yA + mRNA (human fetal liver polyA + mRNA, CLONTECH) as a template
CDNA (manufactured by NTECH) according to the attached protocol
Was synthesized.

Using the data registered in Gene Bank M90102, EMBL X73551, PCR sense primer 5'-CG
GAGAAGATGCCCTCCTGG-3 'and antisense primer
5′-TCAAGGCTGCTGCCAATAGC-3 ′ was synthesized according to a conventional method. PC
Eukaryotic TA Cloning Kit which is an expression vector for R products
(Invitrogen) In order to integrate into the attached pCR3-Uni in the positive direction, only the sense primer is used in advance with the Kination Kit.
(Manufactured by TOYOBO) to phosphorylate the 5 'end. PCR reaction is Ex Taq (Ex Taq DNA polymerase, TAKARA)
And under normal conditions according to the attached protocol. The PCR product is subjected to electrophoresis and stained with ethidium bromide solution (Nippon Gene), followed by Gene Clean II
C-mpl cD from agarose gel using BIO 101
NA was extracted. The isolated cDNA was incorporated into the plasmid pCR3-Uni under ordinary conditions using the attached ligase buffer. Ligation product is Hanahan to E. coli TOP10F 'strain
(hanahan) method. The resulting transformants were confirmed for inserts by restriction enzyme analysis and sequence analysis, and prepared in large amounts according to a conventional method. Transfer the prepared plasmid to pCR3-Uni
Named / mpl.

(2) Preparation of Cells Used for Transformation Ba / F3 cells (RIKEN Cell Development Bank) are cells that proliferate in a murine interleukin-3 (m-IL3) -dependent manner. In order to maintain these cells, m-IL3 is required. Therefore, WEHI-3 secretes m-IL3
Cells (RIKEN Cell Development Bank) were supplemented with 10% fetal bovine serum (manufactured by JRH BIOSCIENCES) and 4 mM L-glutamine (GIBCO B
RL) and 50 μg / mL gentamicin (SIGMA) in RPMI1640 medium (Nissui Pharmaceutical), and the culture supernatant was collected. The culture supernatant is a membrane with a pore size of 0.22 μm (0.2
After filtration through 2 μm disporsabl filterware (manufactured by Nalgene), the mixture was refrigerated and stored. Ba / F3 cells were cultured by adding 10% dose of WEHI-3 culture supernatant to RPMI1640 medium.

(3) Transformation of Ba / F3 cells by DEAE-dextran method Ba / F3 cells in the logarithmic growth phase were seeded at a density of 5 × 10 ⁵ cells / mL, and cultured overnight in a CO ₂ incubator. . pCR3-
Uni / mpl was diluted with PBS (manufactured by Nissui Pharmaceutical) to a concentration of 10 μg / mL. The same amount of PBS was added to 85 μL of a DEAE-dextran solution (SIGMA) dissolved in physiological saline at a concentration of 2 mg / mL to dilute the mixture, and 170 μL of the above plasmid solution was added and mixed.

The Ba / F3 cells cultured overnight were washed twice with PBS, the number of cells was counted, and 2 × 10 ⁶ cells were collected. After removing the supernatant by centrifugation, a mixed solution of plasmid / DEAE-dextran was added to the cells and gently stirred. After treatment at room temperature for 15 minutes, the cells were washed twice with PBS, and cultured in a medium for Ba / F3 cells for 48 hours in a CO ₂ incubator.

(4) Selection of Transformed Cells by G418 and TPO Ba / F3 transformed cells were transformed in a Ba / F3 cell culture medium containing G418 (Geneticin, GIBCO BRL) at a concentration of 400 μg / mL.
Selection was made by culturing for a week. During this time, the medium was changed every four days.

The G418-resistant cells further contain a source of m-IL3, W
Cells capable of proliferating in a TPO-dependent manner were selected by culturing for 1 week in a medium containing 10 ng / mL of h-TPO (human-TPO, manufactured by Genzyme) instead of the EHI-3 cell culture supernatant. During this time, the medium was changed every two days. The finally obtained cells capable of proliferating in a TPO-dependent manner were designated as BaF / mpl cells.

Test Example 2 Test for measuring the effect of BaF / mpl cells on metabolic activity When a ligand binds to a receptor expressed on the cell membrane,
As a result of intracellular signaling, the metabolic activity of the cell increases, releasing hydrogen ions outside the cell. A site sensor (Cytosensor Microphysiometer, NMD) that detects this release of hydrogen ions as the rate of change of extracellular hydrogen ion concentration
Was used to measure the metabolic activity of BaF / mpl cells when the compound of the present invention was added.

(1) Preparation of cell cup BaF / mpl cells were prepared in the presence of 10 ng / mL human TPO (manufactured by PEPRO TECH) in RPMI1640 medium containing 10% FBS (Nihon Molecular Depot).
vices, NMD). Similarly, Ba / F3 cells were prepared from 10 ng / mL murine interleukin-3 (m-IL3) (PE
(PRO TECH) in the presence of RPMI 1640 medium containing 10% FBS. BaF / mpl or Ba / F3 cells in logarithmic growth phase are incubated overnight in RPMI medium containing 10% FBS,
All cells were synchronized in stationary phase. The next day, BaF / mpl cells or Ba / F3 cells were suspended in the medium to a concentration of 1.3 × 10 ⁷ cells / mL. 60 μL cell suspension and 20 agarose
Mix μL, and each cell cup of the site sensor (NMD)
Was seeded at the center of each of 8 μL. The outside of the cell cup was filled with 1 mL of medium and the plate was placed on ice until the agarose had set. After arranging a spacer (manufactured by NMD) at the bottom of the cell cup, a capsule insert (manufactured by NMD) was placed in each cup, and 1 mL of medium was added from above to submerge in the cell cup.

(2) Preparation of measurement medium RPMI medium for site sensor (manufactured by NMD) is dissolved in 1 L of tissue culture distilled water (manufactured by Cosmo Bio), adjusted to pH 7.4, and aspirated with a 0.45 μm filter. Filtered.

(3) Measurement by Site Sensor 20 ng each of TPO and 3-aminoacetyl-1-phenylsulfonylindole hydrochloride (compound of the present invention)
/ mL, dissolved in RPMI medium for cytosensor so as to have a concentration of 100 μM to obtain a sample for measurement. At that time, a DMSO solution of the compound of the present invention was used, and dissolved in a medium so that the final concentration of DMSO was 1%. The sample was set on the cytosensor under the condition where the metabolic rate was constant. In the cytosensor measurement, a change in the metabolic rate of the cells was measured after returning from the feeding of the measurement medium to the feeding of the sample. The sample is stimulated for 10 minutes,
Thereafter, the measurement medium was fed until the metabolic rate returned to the original rate before stimulation. In addition, the same test was conducted to confirm that 1% DMSO did not affect the metabolic rate of cells.

As a result, TPO was added to BaF / mpl cells at 0.8 ng / mL or less.
When added to 20 ng / mL, the metabolic activity increased in a dose-dependent manner at 4 ng / mL or more. Similarly, the compound of the present invention is converted to BaF / mp
When 4 μM to 100 μM was added to l-cells, a dose-dependent increase in metabolic activity was observed at 20 μM or more, and the compound of the present invention showed TP
It was found to have affinity for the O receptor. On the other hand, when the compound of the present invention was added from 4 μM to 100 μM to Ba / F3 cells, which are m-IL3-dependent cells, no increase in metabolic activity as observed in BaF / mpl cells was observed.

Test Example 3 Test for measuring thrombopoietin-like activity using BaF / mpl cell proliferation as an index BaF / mpl cells were subjected to 10% FBS (fetal calf serum, Sanko in the presence of human TPO (h-TPO) (PEPRO TECH)). RPMI164
The cells were cultured in a 0 (manufactured by Gibco) medium. Similarly, Ba / F3 cells were transformed with growth factor murine interleukin-3 (m-IL3) (PEPRO T
(Manufactured by ECH). BaF / mpl cells or Ba / F3 cells in logarithmic growth phase were collected by sedimentation by centrifugation, suspended in RPMI1640 medium containing 10% FBS in the absence of growth factors, and
The cells were cultured for 6 hours.

After culturing, BaF / mpl cells or Ba / F3 cells are
5 × 10 ^{5 in} RPMI 1640 medium containing 10% FBS in the absence of growth factors
cells / mL and suspended at 50μL / well (2.5 × 10 ⁴ cells /
(50 μL) was dispensed into culture microplates. Then, 3-aminoacetyl-1-phenylsulfonylindole hydrochloride (the compound of the present invention) prepared with DMSO (manufactured by Dojinsha) at a concentration of 10 mM was mixed with 0.1% BSA (manufactured by Sigma) / RPMI1.
Dilute to the working concentration in 640 medium (final concentration: 10 μM to 0.1 μM
/ 1% DMSO) and 50 µL / well, and cultured overnight. At the same time, the growth factor h-TPO was added to the BaF / mpl cells,
Wells in which growth factor m-IL3 was added to / F3 cells and wells in which final concentration of 1% DMSO was added as control were prepared and cultured in the same manner.

After the culture, the cell viability measuring kit Cell Titer
Cell viability was measured using the 96 AQueous Kit (Promega). Add A solution (10 mL of MTS solution and 0.5 mL of PMS solution) to BaF / mpl cells and Ba / F3 cells according to the package insert of the kit.
mL of the mixture) was added at 20 μL / well, and then at 37 ° C., 2-3
Cultured for hours. After the culture, the absorbance (wavelength 490 nm) was measured, and the cell survival rate was determined by the following formula. [ODa] = Absorbance when adding 20 ng / mL h-TPO to BaF / mpl cells [ODb] = When adding 2 ng / mL m-IL3 to Ba / F3 cells (absorbance does not change even if the concentration is higher than this) Absorbance [ODc] = 1% DM for BaF / mpl cells or Ba / F3 cells
Absorbance when adding SO [OD sample] = Absorbance when adding ligand (containing 1% DMSO at final concentration) to BaF / mpl cells or Ba / F3 cells Cell viability using BaF / mpl cells ( %) = 100 ×
([OD sample]-[ODc]) / ([ODa]-[ODc]) Cell viability (%) using Ba / F3 cells = 100 × ([OD
[Sample]-[ODc]) / ([ODb]-[ODc])

As a result, when 100 μM of the compound of the present invention was added to BaF / mpl cells, a cell viability of 76% was shown. On the other hand, when 100 μM of the compound of the present invention was added to Ba / F3 cells, the cell viability was 3%. The cell viability when adding 20 ng / mL h-TPO to BaF / mpl cells was 10
The result of 0% is shown in FIG.

[0035]

Industrial Applicability The 3-aminoacetyl-1-phenylsulfonylindole of the present invention or a pharmacologically acceptable salt thereof has an affinity for a thrombopoietin receptor, and thus can be administered orally to a therapeutic agent for thrombocytopenia. It is useful as a platelet production regulator or platelet production promoter.

[Brief description of the drawings]

FIG. 1 The abscissa is the log (concentration (M)) of the compound, and the ordinate is BaF
The figure shows the cell viability when the compound of the present invention is added, assuming that the cell viability when 20 ng / mL TPO is added to / mpl cells is 100%.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yukihisa Watanabe 1-2-1 Onodai, Midori-ku, Chiba-shi, Chiba Torii Pharmaceutical Co., Ltd. (72) Inventor Tetsuo Yokoyama 1-16 Momoyamadai, Tarumi-ku, Kobe-shi, Hyogo JCR COUNT 314 F term (reference) 4C086 AA01 AA02 AA03 BC13 MA04 NA14 ZA54 ZC41 4C204 BB01 CB03 DB16 EB02 FB32 GB01

Claims (3)

[Claims] (1) 3-aminoacetyl-1-phenylsulfonylindole or a pharmacologically acceptable salt thereof. 2. A pharmaceutical composition comprising the compound according to claim 1. 3. The pharmaceutical composition according to claim 2, which is a therapeutic agent for thrombocytopenia, an agent for regulating platelet production or an agent for promoting platelet production.