JPH111477A - 1,4-benzodiazepine derivatives and uses thereof - Google Patents

Abstract

(57) [Problem] To provide a drug having excellent affinity for thrombopoietin receptor and having a platelet production regulating action. SOLUTION: The following general formula: Wherein R represents a phenyl group or an indolyl group, and n represents an integer of 2 to 6. The 1,4-benzodiazepine derivative or a pharmaceutically acceptable salt thereof is excellent in the thrombopoietin receptor. It has a high affinity and is extremely useful as a drug having a platelet production regulating action.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel 1,4-benzodiazepine derivative having an affinity for a thrombopoietin receptor deeply involved in megakaryopoiesis and platelet production and having a platelet production regulating action, or a pharmacologically acceptable derivative thereof. And the use thereof.

[0002]

2. Description of the Related Art Platelets are blood components that play a major role in hemostasis and thrombus formation in living organisms. Platelets are released from the bone marrow stem cells into the blood from megakaryocytes that have been differentiated and matured from the bone marrow from megakaryocyte progenitor cells and have a life span of about 10 days, a number that is known to remain constant over the long term. Had been. The gene for thrombopoietin, a major factor in the process of megakaryopoiesis, has recently been cloned [Nature, 369, 533 (1994)]. Thrombopoietin is a ligand for the protein (thrombopoietin receptor: MPL) encoded by c-mpl.
It has also been found that megakaryocyte progenitors stimulate proliferation and differentiation and maturation of megakaryocyte cells, and further increase platelet production [Nature, 369, 568 (1994)].

As a physiologically active substance that regulates platelet production via a thrombopoietin receptor, it has been known that not only thrombopoietin itself but also low-molecular peptides have an affinity for thrombopoietin receptor (WO96 / 40189, WO96). / 40750 specification).

[0004]

Physiologically active substances such as thrombopoietin and the above-mentioned low molecular weight peptide regulate platelet production via thrombopoietin receptor, and are excellent for various blood disease states accompanied by abnormal platelet count. Promising as a drug. However, thrombopoietin is a polypeptide cytokine consisting of 332 amino acids and is expected to be degraded in the gastrointestinal tract when used as a drug. Although it can be used as an injection, it is not practical for oral administration. Can be In addition, the possibility of oral administration of low molecular weight peptides having an affinity for thrombopoietin receptor is unknown, and it is hoped that low molecular weight non-peptide compounds that have excellent thrombopoietin receptor affinity and can be orally administered will be developed. It is rare.

An object of the present invention is to find a low molecular weight non-peptide compound which has an excellent affinity for thrombopoietin receptor and which can be orally administered, and is expected to have an excellent effect on various pathological conditions accompanied by abnormal platelet count. To provide medicine.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, the present invention has
The present inventors have found that a 4-benzodiazepine derivative or a pharmacologically acceptable salt thereof has excellent thrombopoietin receptor affinity, and have completed the present invention.

That is, the present invention provides the following general formula (I)

Embedded image (Wherein, R represents a phenyl group or an indolyl group, and n represents an integer of 2 to 6), or a novel 1,4-benzodiazepine derivative or a pharmaceutically acceptable salt thereof,
And uses thereof.

The 1,4-benzodiazepine derivatives of the present invention having a structure similar to that of the compound represented by the general formula (I) are disclosed in JP-A-61-63666 and JP-A-63-2380.
No. 69 and Journal of Medicinal Chemistry, 30, 12,
On page 29 (1987) and the like, they are disclosed as CCK antagonists and in WO95 / 14470 as therapeutic agents for arrhythmias by blocking potassium ions, but these documents show that the thrombopoietin receptor affinity according to the present invention is not disclosed. Is not mentioned at all.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the general formula (I) of the present invention, the phenyl group or indolyl group represented by R may be appropriately substituted, and the alkylene chain represented by (CH ₂ ) _n For example, ethylene, propylene, butylene, pentylene and hexylene chains can be mentioned. The compound of the present invention represented by the general formula (I) may have asymmetric isomers, and the present invention also includes these isomers and mixtures thereof.

The compound represented by the above general formula (I) of the present invention can be converted into a pharmacologically acceptable salt, or the resulting salt can be converted into a free base as required. The pharmacologically acceptable salts of the present invention include, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid,
Mineral acid salts such as phosphoric acid, or acetic acid, maleic acid, fumaric acid, citric acid, oxalic acid, succinic acid, tartaric acid, malic acid, mandelic acid, methanesulfonic acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, etc. And the like.

Preferred embodiments of the 1,4-benzodiazepine derivative of the present invention include the following compounds and pharmaceutically acceptable salts thereof, but the present invention is not limited to these examples. is not. (1) (±) -1- (2-aminoethyl) -1,3-dihydro-5-phenyl-3- (phenylmethyl) -2H
-1,4-benzodiazepin-2-one (2) (±) -1- (3-aminopropyl) -1,3-
Dihydro-5-phenyl-3- (phenylmethyl) -2
H-1,4-benzodiazepin-2-one (3) (±) -1- (4-aminobutyl) -1,3-dihydro-5-phenyl-3- (phenylmethyl) -2H
-1,4-benzodiazepin-2-one (4) (±) -1- (5-aminopentyl) -1,3-
Dihydro-5-phenyl-3- (phenylmethyl) -2
H-1,4-benzodiazepin-2-one (5) (±) -1- (6-aminohexyl) -1,3-
Dihydro-5-phenyl-3- (phenylmethyl) -2
H-1,4-benzodiazepin-2-one (6) (±) -1- (2-aminoethyl) -1,3-dihydro-3- (1H-indol-3-ylmethyl)-
5-phenyl-2H-1,4-benzodiazepine-2-
ON (7) (±) -1- (3-aminopropyl) -1,3-
Dihydro-3- (1H-indol-3-ylmethyl)
-5-phenyl-2H-1,4-benzodiazepine-2
-One (8) (±) -1- (4-aminobutyl) -1,3-dihydro-3- (1H-indol-3-ylmethyl)-
5-phenyl-2H-1,4-benzodiazepine-2-
ON (9) (±) -1- (5-aminopentyl) -1,3-
Dihydro-3- (1H-indol-3-ylmethyl)
-5-phenyl-2H-1,4-benzodiazepine-2
-One (10) (±) -1- (6-aminohexyl) -1,3-
Dihydro-3- (1H-indol-3-ylmethyl)
-5-phenyl-2H-1,4-benzodiazepine-2
-ON

The compound of the present invention represented by the general formula (I) can be produced by the following method, but the production method of the compound is not limited to this method.

[0013]

Embedded image (In the formula, R and n represent the same meaning as described above.)

That is, in the step 1, the process described in JP-A-61-6
No. 3666, JP-A-63-238069 and Journal of Medicinal Chemistry, Vol. 30, No. 12,
Compounds of general formula (II) disclosed on page 29 (1987) and the like, and the following general formula (IV)

Embedded image (Z represents a halogen atom such as a chlorine atom or a leaving group such as a mesyloxy group, and n represents the same meaning as described above) and an inert solvent such as N, N-dimethylformamide and tetrahydrofuran. By reacting in the presence of a base such as sodium hydride and lithium diisopropylamide in a medium at a temperature ranging from 0 ° C. to the reflux temperature of the solvent, the compound of the general formula (III) can be obtained.

In the step 2, the compound of the general formula (I) according to the present invention is obtained by reacting the compound of the general formula (III) with hydrazine hydrate or methylamine in a polar solvent such as ethanol. Can be.

The thus-produced pharmaceutical containing at least one novel 1,4-benzodiazepine derivative represented by the above general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient is: Usually, they are administered as oral preparations such as capsules, tablets, fine granules, granules, powders, syrups, or injections. These preparations can be produced by a conventional method by adding pharmacologically and pharmaceutically acceptable additives. That is, in the case of oral preparations, excipients (lactose, D-mannitol, corn starch, crystalline cellulose, etc.), disintegrants (carboxymethylcellulose, carboxymethylcellulose calcium, etc.), binders (hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl) Injectable components such as pyrrolidone), lubricants (magnesium stearate, talc, etc.), coating agents (hydroxypropylmethylcellulose, sucrose, titanium oxide, etc.), plasticizers (polyethylene glycol, etc.) Solubilizers or solubilizers that can constitute aqueous or ready-to-use dosage forms (distilled water for injection, physiological saline, propylene glycol, etc.), pH regulators (inorganic or organic acids or bases), isotonic agents (Salt, glucose, glycerin, etc.), stabilizer Formulation ingredients is used.

[0017]

The present invention will now be described by way of examples, but the invention is not limited to the specific details of these examples.

Example 1 (±) -N- [2- [2,3-dihydro-3- (1H-
(Indole-3-ylmethyl) -2-oxo-5-phenyl-1H-1,4-benzodiazepin-1-yl] ethyl] phthalimide (±) -1,3-dihydro-3- (1H-indole-
3-ylmethyl) -5-phenyl-2H-1,4-benzodiazepin-2-one 3.00 g, 60% sodium hydride 0.34 g and N, N-dimethylformamide 60 ml
After stirring for 1.5 hours under ice cooling, 4.60 g of N- (2-bromoethyl) phthalimide was added, and the mixture was stirred at room temperature for 16 hours. After 200 ml of water was added to the reaction mixture, suction filtration was performed to obtain a gummy solid. The gummy solid was dissolved in ethyl acetate, washed with water and saturated saline, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was subjected to column chromatography (silica gel, dichloromethane → dichloromethane:
Purified by methanol = 20: 1) to obtain a slightly yellow crystal 0.1.
94 g were obtained. A part of the crystals was recrystallized from a mixed solvent of dichloromethane: ethyl acetate (3: 1) to give a melting point of 22%.
Light yellow crystals of 4.5-228.5 ° C were obtained. Elemental analysis C ₃₄ H ₂₆ N ₄ O ₃ Theoretical C, 75.82; H, 4.87; N, 10.40 Experimental C, 75.72; H, 4.90; N, 10.36

Example 2 (±) -N- [3- [2,3-dihydro-3- (1H-
(Indole-3-ylmethyl) -2-oxo-5-phenyl-1H-1,4-benzodiazepin-1-yl] propyl] phthalimide (±) -1,3-dihydro-3- (1H-indole-
3-ylmethyl) -5-phenyl-2H-1,4-benzodiazepin-2-one 3.00 g, 60% sodium hydride 0.34 g and N, N-dimethylformamide 60 ml
Was stirred under ice-cooling for 1.5 hours, and 4.50 g of N- (3-bromopropyl) phthalimide was added.
Stirred for hours. After 200 ml of water was added to the reaction mixture, suction filtration was performed to obtain a gummy solid. The gummy solid was dissolved in ethyl acetate, washed with water and saturated saline, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was subjected to column chromatography (silica gel, dichloromethane: methanol = 2
0: 1) to give 3.36 g of slightly yellow crystals.
A part of the crystals was obtained by adding dichloromethane: ethyl acetate (2:
Recrystallized from the mixed solvent of 1), melting point: 213 to 216 ° C
To yield slightly yellow crystals. Elemental analysis C ₃₅ H ₂₈ N ₄ O ₃ Theoretical C, 76.07; H, 5.11; N, 10.14 Experimental C, 75.85; H, 4.88; N, 10.12

Example 3 (±) -N- [4- [2,3-dihydro-3- (1H-
(Indole-3-ylmethyl) -2-oxo-5-phenyl-1H-1,4-benzodiazepin-1-yl] butyl] phthalimide (±) -1,3-dihydro-3- (1H-indole-
1.50 g of 3-ylmethyl) -5-phenyl-2H-1,4-benzodiazepin-2-one, 0.17 g of 60% sodium hydride and 30 ml of N, N-dimethylformamide
After stirring the mixture for 1 hour under ice cooling, 2.48 g of N- (4-bromobutyl) phthalimide was added, and the mixture was stirred at room temperature for 16 hours. After 100 ml of water was added to the reaction mixture, suction filtration was performed to obtain a gummy solid. The gummy solid was dissolved in ethyl acetate, washed with water and saturated saline, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was subjected to column chromatography (silica gel, dichloromethane: methanol = 5).
0: 1) to give 2.13 g of a yellow amorphous solid. Elemental analysis C ₃₆ H ₃₀ N ₄ O ₃ Theoretical C, 76.31; H, 5.34; N, 9.89 Experimental C, 76.18; H, 5.16; N, 9.85

Example 4 (±) -N- [3- [2,3-dihydro-2-oxo-
5-phenyl-3- (phenylmethyl) -1H-1,4
-Benzodiazepin-1-yl] propyl] phthalimide (±) -1,3-dihydro-5-phenyl-3- (phenylmethyl) -2H-1,4-benzodiazepine-2-
A mixture of 3.00 g of ON, 0.40 g of 60% sodium hydride and 50 ml of N, N-dimethylformamide was added under ice-cooling for 1 hour.
After stirring for an hour, 5.00 g of N- (3-bromopropyl) phthalimide was added, and the mixture was stirred at room temperature for 2 hours. After 200 ml of water was added to the reaction mixture, suction filtration was performed to obtain a gummy solid. The gummy solid was dissolved in ethyl acetate, washed with water and saturated saline, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography (silica gel, hexane: ethyl acetate = 2: 1) to obtain 4.40 g of a colorless amorphous solid. Elemental analysis C ₃₃ H ₂₇ N ₃ O ₃ theory C, 77.17; H, 5.30; N, 8.18 Found C, 76.89; H, 5.63; N, 8.05

EXAMPLE 5 (±) -1- (2-aminoethyl) -1,3-dihydro-3- (1H-indol-3-ylmethyl) -5-phenyl-2H-1,4-benzodiazepine-2- ON (±) -N- [2- [2,3-dihydro-3- (1H-
Indole-3-ylmethyl) -2-oxo-5-phenyl-1H-1,4-benzodiazepin-1-yl] ethyl] phthalimide 1.02 g, hydrazine hydrate 0.1
A mixture of 0 ml and ethanol was heated at reflux for 10 hours.
After cooling, a 5% aqueous sodium hydroxide solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was subjected to column chromatography (silica gel, dichloromethane:
Purification by methanol = 9: 1) gave 0.15 g of a yellow-orange amorphous solid. IR spectrum ν (KBr) cm ^-1 : 3344, 1676
, 1604 NMR spectrum δ (CDCl ₃ ) ppm: 2.49 (2H, s), 2.
79 (1H, q, J = 6.5Hz), 2.89 (1H, q, J = 6.5Hz), 3.65 (1H, dd, J = 1
3,6Hz), 3.77-3.85 (3H, m), 4.39 (1H, td, J = 13,6.5Hz), 7.03
-7.63 (14H, m), 8.23 (1H, s) High Resolution Mass Spectrum: C ₂₆ H ₂₄ N ₄ O theoretical m / z: 408.1950 Found m / z: 408.1946

EXAMPLE 6 (±) -1- (3-Aminopropyl) -1,3-dihydro-3- (1H-indol-3-ylmethyl) -5
Phenyl-2H-1,4-benzodiazepin-2-one (±) -N- [3- [2,3-dihydro-3- (1H-
2.53 g of indole-3-ylmethyl) -2-oxo-5-phenyl-1H-1,4-benzodiazepin-1-yl] propyl] phthalimide, hydrazine hydrate 0.
A mixture of 24 ml and 55 ml of ethanol was heated at reflux for 23 hours. After allowing to cool, add a 5% aqueous sodium hydroxide solution,
Extracted with ethyl acetate. The organic layer was washed with water and saturated saline, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was recrystallized from acetone, mp 169.5-171.
1.00 g of colorless crystals at 5 ° C. were obtained. Elemental analysis C ₂₇ H ₂₆ N ₄ O Theoretical C, 76.75; H, 6.20; N, 13.26 Experimental C, 76.43; H, 5.89; N, 13.03

Example 7 (±) -1- (4-Aminobutyl) -1,3-dihydro-3- (1H-indol-3-ylmethyl) -5-phenyl-2H-1,4-benzodiazepine-2- ON (±) -N- [4- [2,3-dihydro-3- (1H-
1.50 g of indole-3-ylmethyl) -2-oxo-5-phenyl-1H-1,4-benzodiazepin-1-yl] butyl] phthalimide and hydrazine hydrate 0.1
A mixture of 4 ml and 20 ml of ethanol was heated at reflux for 5 hours. After cooling, a 5% aqueous sodium hydroxide solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was subjected to column chromatography (alumina, dichloromethane: methanol = 20: 1 → dichloromethane: methanol =
9: 1) to give 0.81 g of a slightly brown amorphous solid. IR spectrum ν (KBr) cm ^-1 : 3360, 1672
, 1602 NMR spectrum δ (CDCl ₃ ) ppm: 1.22-1.57 (4H,
m), 2.53 (2H, dd, J = 13.5,6.5Hz), 3.63-3.71 (2H, m), 3.78-
3.84 (2H, m), 4.44 (1H, td, J = 14,7Hz), 7.05-7.65 (14H, m),
8.01 (1H, s) High-resolution mass spectrum: C ₂₈ H ₂₈ N ₄ O Theoretical value m / z: 436.2263 Experimental value m / z: 436.2263

Example 8 (±) -1- (3-aminopropyl) -1,3-dihydro-5-phenyl-3- (phenylmethyl) -2H-
1,4-benzodiazepin-2-one (±) -N- [3- [2,3-dihydro-2-oxo-
5-phenyl-3- (phenylmethyl) -1H-1,4
A mixture of 3.70 g of -benzodiazepin-1-yl] propyl] phthalimide, 0.35 ml of hydrazine hydrate and 40 ml of ethanol was heated under reflux for 5.5 hours. After cooling down,
200 ml of a 5% aqueous sodium hydroxide solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography (alumina, dichloromethane: methanol = 10: 1) to obtain 0.99 g of a pale pink amorphous solid. IR spectrum ν (KBr) cm ^-1 : 3372, 1674
, 1604 NMR spectrum δ (CDCl ₃ ) ppm: 1.52-1.66 (2H,
m), 2.44-2.52 (2H, m), 3.60 (2H, d, J = 7Hz), 3.70 (1H, ddd, J =
14,7,5Hz), 3.79 (1H, t, J = 7Hz), 4.57 (1H, td, J = 14,7Hz), 7.
15-7.54 (14H, m) High-resolution mass spectrum: C ₂₅ H ₂₅ N ₃ O Theoretical value m / z: 383.1998 Experimental value m / z: 383.2003

Hereinafter, in order to confirm the excellent thrombopoietin receptor binding affinity of the compound of the present invention, a thrombopoietin receptor competition experiment between the thrombopoietin and a test compound was performed and evaluated.

Test Example 1 Human thrombopoietin receptor (MPL) expression plasmid
Construction of (1) First, by plaque hybridization method,
A phage clone retaining the entire region of the MPL cDNA was obtained.
For this purpose, a part of human MPL cDNA was obtained from human fetal liver cDNA (manufactured by CLONTECH) by PCR. Note that MPL cD
The start codon to stop codon of NA are registered in GenBank M90102, and the sequence upstream of the start codon is registered in EMBL X73551. The primer for PCR is the start codon A of MPL.
From primers 5'-GTGCGTCTCTTCTTTCCGCT-3 'based on a sequence of 331 to 350 bases counted from 1888
Antisense primer 5'-TCA based on 1907 base sequence
AGGCTGCTGCCAATAGC-3 'was used. PCR is Takara EX Ta
q (manufactured by Takara Shuzo) using the attached reaction buffer under normal conditions. After agarose gel electrophoresis, the PCR product was recovered from the gel using SUPREC-01 (Takara Shuzo) according to the attached protocol. The recovered PCR product, using the Rediprime DNA labeling system (Amersham), in accordance with the attached protocol [α- ³² P]
It was labeled with dCTP and used as a probe. Using this, Huma
n From the Fetal Liver 5'-STRETCH cDNA library (manufactured by CLONTECH), isolate a phage clone that retains the entire coding region of MPL cDNA and at least 60 bases upstream from the start codon according to the attached protocol. Prepared. (2) Next, a DNA encoding human MPL extracellular region cDNA (amino acid sequence of positions 1 to 491) was obtained by PCR. The phage obtained above was used as the template for PCR, and the primers were 17 nucleotides upstream of the base codon of MPL.
Sense primer 5'-CTAAGGCAGGC based on base sequence
ACACAG-3 'and antisense primer 5'-GGTGACCCAGGCGGTCTCGGT based on the amino acid sequence from 486 to 491
GGC-3 'was used. At this time, the MPL extracellular domain protein
A BstEII site was inserted so that the C-terminal region could be linked to human IgG Fc, and the reading frame was matched. In addition, the human IgG Fc region cDNA is described in the literature of BD Bennett et al. [BD Bennett et al., Journal of Biological Chemistry.
, 266, 23060-23067, 1991) with reference to the sense primer 5'-CGCGGTCACCGACAAAACTCA-3 'and the antisense primer 5'-GCACTCATTTACCCGGAGACAGGGA.
Using GA-3 ', QUICK-CLONE cDNA of human spleen (CLONTE
(Manufactured by CH Co.) as a material and obtained by the PCR method. The PCR product thus obtained was incorporated into pCR3 (manufactured by Invitrogen) according to the following steps to construct an MPL expression vector. (3) The MPL extracellular region cDNA and the human IgG Fc region cDNA obtained by PCR were inserted into a pCR3 mammalian cell expression vector using EUKARYOTIC TA CLONING KIT (manufactured by Invitrogen) according to the attached protocol. Was transformed. From the obtained transformants, a strain inserted in the correct direction in which expression was possible was selected, and this strain was cultured in a large amount according to a conventional method. A plasmid was prepared from this strain according to a standard method, and MPL was prepared.
They were named (B) -pCR3 and IgG Fc (B) -pCR3. (4) About 200 μg of MPL (B) -pCR3 was added to 0.64 units of BstEII.
After cutting with (Toyobo Co., Ltd.) and 200 units of ScaI (Takara Shuzo Co., Ltd.), this was subjected to agarose electrophoresis. A gel fragment containing a 3085 bp DNA fragment containing the MPL cDNA region was cut out from the plasmid, and DNA was extracted from the gel fragment by a conventional method. (5) About 20 μg of IgG Fc (B) -pCR3, 40 units of BstEII
(Toyobo) and 80 units of ScaI (Takara Shuzo), followed by dephosphorylation with alkaline phosphatase (Toyobo) and then subjected to agarose electrophoresis. A gel fragment containing a 4150 bp DNA fragment containing an IgG Fc region cDNA was cut out from the plasmid, and DNA was extracted from the gel fragment. (6) The DNA fragment (about 30 ng) obtained in (4) and the DNA fragment (about 20 ng) obtained in (5) were ligated with 4.6 units of T4 DNA ligase (manufactured by Toyobo). E. coli XL1-Blue (Stratagene) was transformed by electroporation. From the obtained transformants, a strain inserted in the correct direction in which expression was possible was selected, and this strain was cultured in a large amount according to a conventional method. A plasmid was prepared from this strain according to a standard method, and MP
It was named L-IgG Fc (B) / pCR3.

Test Example 2 Human IgG Fc region-fused human MPL protein (MPL-IgG)
Preparation of human fetal 293 cells with constant expression and purification of MPL-IgG Using MPL-IgG Fc (B) / pCR3, electroporation [Yoshinari Watanabe: Tissue Culture Techniques 3rd Edition [Application] (Japanese tissue culture) Society), 501-503, 1996]
293 cells were transformed. Transformed human embryo 293
After culturing the cells for about 2 days in DMEM medium containing 10% FCS,
0.4 mg / ml Geneticin (LIFE TECHNOLOGIES)
And cultured in DMEM containing 10% FCS for about 2 weeks to obtain a transformant. The transformant was cultured until it became about 50% confluent, replaced with a DMEM medium containing 1% Nutridoma (Boehringer Mannheim), and the culture was continued. The culture was continued for 3 to 4 weeks while changing the medium about every week. This medium was centrifuged, and the culture supernatant was collected, followed by filtration using VacuCap ^™ (manufactured by Gelman Sciences). From about 7 L of culture supernatant, HiTrap Protein
Using G (manufactured by Pharmacia), column chromatography was performed according to the attached protocol to purify MPL-IgG.

Test Example 3 Competition between Thrombopoietin and Test Compound Using ELISA Method
The combined microtiter plate wells were coated overnight at 4 ° C. with 10 ng of MPL-IgG diluted in 100 μl of PBS (−). The subject dissolves the test compound in DMSO, and then PBS (-) / 0.1% BSA
A 0.05% Tween 20 was used to mix with a thrombopoietin (R & D) solution (final concentration: 1 × 10 ⁻¹⁰ M) so that the final DMSO content was 5%. From the well
The MPL-IgG solution was removed, the subject was added, and coated at room temperature for 1 hour or more. Remove this solution and add 200 μl PBS
After washing the bottom of the well with (-) / 0.05% Tween 20, goats
Anti-human TPO Neutralizing Antibody (R & D)
And incubated at room temperature for 1 hour or more. 200 μl
After washing the wells with PBS (-) / 0.05% Tween 20, the horseradish peroxidase-labeled donkey anti-goat IgG antibody (Chem
icon International Co., Ltd.) for 1 hour or more at room temperature. PB containing 200 μl of 0.05% Tween 20
After washing the wells with S (-), 100 μl of TMB solution (DAKO
Was added and incubated at room temperature for 5 minutes. 100 μ
The 1M H ₂ SO ₄ (Wako Pure Chemical Industries) was added the reaction of l was stopped.
The optical density was analyzed at 450 nm, and the inhibition of thrombopoietin binding by the test compound was examined, with the binding of thrombopoietin when the test compound was not added taken as 100%, to evaluate the affinity for the thrombopoietin receptor. Figure 1 shows the results
Shown in As is clear from these results, the compounds of the present invention exhibited excellent affinity for the thrombopoietin receptor.

[0030]

The 1,4-benzodiazepine derivative of the present invention represented by the above general formula (I) or a pharmaceutically acceptable salt thereof has an excellent affinity for thrombopoietin receptor, It is extremely useful as a production regulator.

[0031]

[Brief description of the drawings]

FIG. 1 is a graph showing the affinity of a compound of the present invention for a thrombopoietin receptor by measuring the binding inhibitory effect of a compound of the present invention on thrombopoietin (TPO).

Continued on the front page (72) Inventor Nobuhiko Iwasaki 37-1, Inoguchi, Katsuyama-shi, Fukui Prefecture Inside Hokuriku Pharmaceutical Co., Ltd. (72) Inventor Yoshitaka Ikeda 1-1-1, Inoguchi, Katsuyama-shi, Fukui Prefecture Hokuriku Pharmaceutical Co., Ltd. In company

Claims (2)

[Claims] (1) The following general formula: (In the formula, R represents a phenyl group or an indolyl group, and n represents an integer of 2 to 6.) 1,4-benzodiazepine derivative or a pharmaceutically acceptable salt thereof. 2. A platelet production regulator comprising the 1,4-benzodiazepine derivative according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.