JP3222599B2 - Benzodiazepine derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to benzodiazepine derivatives useful as selective antagonists for gastrin receptors.

[0002]

BACKGROUND OF THE INVENTION Gastrin is cholecystokinin (CCK)
Together, they belong to the so-called gastrin group gastrointestinal peptide hormones. The gastrin receptor is also present in the entire upper gastrointestinal tract, pancreas, liver, biliary tract, etc., but is mainly present in fundic gland wall cells, and regulates gastric acid secretion. On the other hand, CCK receptors are present in the periphery such as the digestive tract (called CCK-A receptors) and centrally located in the brain (CCCK-A receptors).
Known as KB receptors) and are involved in the regulation of gastrointestinal motility, pancreatic juice secretion and central action, appetite and the like. Thus, antagonists to these receptors have been used to treat the respective peptide hormone-related diseases in the gastrointestinal and central nervous systems of various animals, including humans,
For example, antineoplastic drugs or drugs for treating pancreatitis, gallbladder, reducing gallstone attacks, improving appetite, irritable bowel syndrome
Syndrome) is considered to be useful. Studies on receptors in the gastrointestinal and central tracts have also revealed the importance of these hormones as bioactive substances ("Brain and peptide" metabolism, Vol. 18, No. 10, 33
-44 (1981) and JP-A-63-238069). These hormones are closely related chemically.
That is, gastrin and the CCK peptide have the same C-terminal 5 amino acid residues, and high homology is recognized between the gastrin receptor and the CCK receptor, particularly the CCK-B receptor. Therefore, gastrin receptor antagonist and CCK
Cross-reactivity with -B receptor antagonists is problematic. For example, gastrin receptor antagonists may be useful in the treatment of gastrin-related diseases such as gastrointestinal ulcer, Zollinger-Ellison syndrome, sinus C cell hyperplasia, and decreased gastrin activity, but cross-reactivity with CCK-B receptor Therefore, there was a risk of central side effects, and it was difficult to sufficiently achieve the therapeutic purpose.

[0003]

As a matter of course, in the treatment of gastrin-related diseases, it is desirable to use a substance that specifically antagonizes the gastrin receptor in order to avoid central side effects. For example, the utility of gastrin receptor-specific antagonists in the treatment of gastric ulcer has been pointed out (metabolic 29
/ 7, 1992). Although an anti-gastric ulcer agent having a gastrin receptor antagonistic action has already been reported (JP-A-63-238069, etc.), as described above, it has been selected only for gastrin receptor due to its high homology with CCK receptor. Substances that act chemically are not yet known.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies for the purpose of providing an antagonist which selectively acts on a gastrin receptor, and as a result, a certain benzodiazepine derivative has an excellent selective gastrin. They have found that they have receptor antagonism, and have completed the present invention.

That is, the present invention provides a compound of the formula I:

Embedded image Wherein R ¹ is carboxy, CHR ² R ³ , provided that R ² and R ³ are independently hydrogen, alkoxy, amino, azide,
OC (O) NH (CH ₂ ) ₃ COOR ⁴ , SCH ₂ COOR ⁴ (R
⁴ is hydrogen or lower alkyl), or CONR ⁵ R ⁶ ,
R ⁵ and R ⁶ independently represent hydrogen, (CH ₂ ) ₃ (N-substituted) sulfamoylphenyl, or together with the attached nitrogen atom, (CH ₂ ) ₅ C (CH ₃ ) ₂ OR ⁷ (R ⁷ is hydrogen or C (O) NH (CH ₂ ) ₃ COOR ⁴ ) or (C
H ₂₎ may form a substituted piperazine by ₃ COOH. A benzodiazepine derivative or an addition salt thereof.

Of the compounds of formula I, particularly preferred are R
¹ is carboxy; CHR ² R ³ , wherein R ² and R ³ are ethoxy, or one of them is hydrogen and the other is amino, azide, OC (O) NH (CH ₂ ) ₃ COOCH ₃
Or SCH ₂ COOR ⁴ (R ⁴ is hydrogen or ethyl); or any of the following groups:

Embedded image ).

In the above formula, lower alkyl means C ₁
A straight-chain or branched hydrocarbon group of -C ₆ , wherein methyl, ethyl, propyl, isopropyl, butyl, t-
Refers to butyl and the like. The benzodiazepine addition salt of the present invention means a pharmaceutically acceptable salt, preferably,
Organic salts such as hydrochloride, oxalate and sulfonate. The compound I of the present invention can be produced by any method known to those skilled in the art. For example, in the following Examples and Production Examples, a benzodiazepine compound (III-1; 5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one) is used as a starting material, and the structure of a target compound is described. The production process using a known reaction is described. However, these show particularly preferred production methods and reaction conditions, and compounds of the formula I produced by other methods known to those skilled in the art are also included in the scope of the present invention. The starting material (compound II) in the process described herein
I-1) is described in the literature (The Jounal of Organic Chemisitry,
26, 4936 (1991)).

The compounds of the present invention, as evident from formula I,
It also exists as an optically active isomer. However, as shown in the experimental examples described later, the racemic form and the optically active isomer have similar gastrin receptor selective antagonism, and both are useful for achieving the object of the present invention. Therefore, this specification mainly describes the racemic form. The specific isomer can be produced by optically resolving a racemic compound by a method known to those skilled in the art, or by optically resolving a starting material or an intermediate in a production process.

For example, the optically active compound of formula I wherein R ¹ is a carboxyl group is an optically active isomer (+ -III-14) obtained by optically resolving the intermediate (Compound III-14) in Production Example 1. ) Can be used (see Production Example 2). In Production Example 2, N-
Although the optical resolution is performed using Boc-phenylalanine, the present invention is not limited to this and can be arbitrarily selected from resolving agents used for optical resolution of amines. CC of the compound of the present invention
And antagonism of K-B receptor, was compared with the antagonism of gastrin receptor, the ratio IC ₅₀ values as high as about 15 to 54, it is that these compounds are selective gastrin receptor antagonist It was revealed. Hereinafter, the present invention will be described in detail with reference to examples.

Production Example 1 (2-hydroxyethyl) -3
-(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-17)

Embedded image (1) 1- (2-acetoxyethyl) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-12) Under a nitrogen atmosphere, dry dimethylformamide (310 ml)
5-phenyl-1,3-dihydro-2H-1,4-
Benzodiazepin-2-one (Compound III-1; The
Jounal of Organic Chemisitry, 26, 4936 (1991)) (1
A mixture of 6.1 g) and 60% sodium hydride (3.0 g) is stirred under ice cooling. Stir the mixture at 0 ° C. for 30 minutes. 2-bromoethyl acetate (12.5 g) in dimethylformamide (5 ml) is added in one portion. The mixture is stirred at 0 ° C. for 30 minutes until a clear solution, at room temperature for 3 hours and at 50 ° C. for 2 hours, added to ice-water and extracted with ethyl acetate. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to flash chromatography using silica gel (150 g) (hexane: ethyl acetate =
1: 1 and 1: 2) and recrystallized from a benzene: hexane mixture (17.08 g) (77.8%). mp
= 85-86 ° C.

(2) 1- (2-hydroxyethyl) -3
-Hydroxyimino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (Compound III-13) Compound (III-12) obtained in (1) was converted into iAmONO
(Isopentyl amyl nitrite) / t-BuOK)
To give the compound III-13
Was obtained (yield: 73.5%). (3) 1- (2-hydroxyethyl) -3-amino-5
-Phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (compound III-14) Oxime (compound III-) in methanol (200 ml)
13, 14.13 g) and 5% ruthenium / carbon (3.
0 g) of the mixture in an autoclave (12 kg / cm ² ).
Stir at 65-70 ° C for 20 hours. Eventually the pressure drops to 5.5 kg / cm ² . The catalyst is filtered and washed with methanol. The filtrate is concentrated under reduced pressure. 12.38 g of residue
And used for the next step.

(4) 1- (2-hydroxyethyl) -3
-(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-17) Amine (III-14, 113 mg), m-tolyl isocyanate (60 mg) and triethylamine (0.2 mg)
ml) of dimethylformamide (3 ml) at room temperature.
Leave for a time. Add H ₂ O. The mixture is extracted with ethyl acetate. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was flash chromatographed on silica gel (15 g) (ethyl acetate: hexane =
2: 1, and ethyl acetate).

Production Example 2 1- (2-hydroxyethyl)
-3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (compound (+)-III-17)

Embedded image (1) 1- (2-hydroxyethyl) -3- (N-Bo
c-1-phenylalanylamino) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-15) Compound (III-15) is obtained by protecting the amino group by a coupling reaction between the compound produced in Production Example 1 (3) and N-Boc-phenylalanine.

(2) (3R, 2'S) -1- (2-hydroxyethyl) -3- (N- (anilinothiocarbonyl) phenylalanylamino) -5-phenyl-1,3
-Dihydro-2H-1,4-benzodiazepin-2-one (compound (+)-III-16) The above compound III-15 was hydrolyzed, and the obtained amine (compound III-10, 5.42 g), phenyl Isothiocyanate (1.82 g) in dichloromethane (20 m
l) Let the solution stand at room temperature for 30 minutes. The volatile components are removed under reduced pressure and the residue is subjected to flash chromatography (ethyl acetate) on silica gel (100 g) to give the thiourea product (yield 7.45 g).

(3) (3R) -1- (2-hydroxyethyl) -3-amino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (compound (+)- III-14) Thiourea ((+) in trifluoroacetic acid (25 ml)
-III-16, 4.75 g) The solution is stirred at 55 ° C. for 20 minutes and the volatile constituents are removed under reduced pressure. Ethyl acetate is added, the solution is extracted with dilute hydrochloric acid, the extract is washed with ethyl acetate, made alkaline with aqueous sodium carbonate and extracted with ethyl acetate. The extract is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the amine (1.32 g, 54.3%). This is used in the next step. (4) (3R) -1- (2-hydroxyethyl) -3-
(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound (+)-III-17) Amine obtained on ((+)-III-14, 113 mg)
To give the title optically active compound ((+)-III-17) in the same manner as in Production Example 1 (4).

Example 1 1- (carboxymethyl) -3
-(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-25)

Embedded image (1) 1- (formylmethyl) -3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-
2H-1,4-benzodiazepin-2-one (Compound III-24) Oxalyl chloride (0.23 ml) in dichloromethane (6 ml)
l) Add dimethyl sulfoxide (0.4m
A solution of l) in dichloromethane (1.3 ml) is added dropwise. The mixture is stirred at -70 ° C for 10 minutes. The alcohol compound (III-17, 0.9 in dichloromethane (15 ml))
9 g) are added dropwise. The mixture is stirred at -70 ° C for 1 hour. Add triethylamine (1.5 ml). The mixture is stirred at room temperature for 1 hour. Add water. Separating the organic phase,
The aqueous phase is extracted with dichloromethane. The organic phases are combined, washed with water, dried over anhydrous sodium sulphate and concentrated under reduced pressure. The residue is subjected to flash chromatography on silica gel (70 g) (ethyl acetate). The fraction having a large Rf value was crystallized from benzene-hexane to give the title compound 63.
5 mg (64.4%) are obtained. mp = 135-137 ° C.
231 mg of starting material was recovered from the fraction with the lower Rf value (23.3%).

(2) 1- (carboxymethyl) -3-
(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-25) A solution of sodium chloride (850 mg) and sodium dihydrogen phosphate (850 mg) in water (8.5 ml) was prepared by adding aldehyde (III-24, 433 mg) to t-butanol (27 ml) and Add dropwise to the solution in 2-methyl 2-butene (5 ml). The mixture is stirred at room temperature overnight and concentrated under reduced pressure. Dissolve the residue in ethyl acetate. The mixture is extracted with 10% sodium carbonate, acidified to pH 3 with concentrated hydrochloric acid and extracted with ethyl acetate. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was crystallized from methanol to give the carboxylic acid (III-25, 736 mg, yield 82.
6%). mp = 168-171 ° C.

[Α] _D +3.7 (c 1.135, CHCl
₃ ) IRν _max (Nujol): 3340, 1655, 16
04, 1555 cm ^-1 NMR (CD ₃ OD) δ: 2.28 (3H, s), 4.
55 (1H, d, J = 20 Hz), 4.65 (1H,
d, J = 20 Hz), 5.47 (1H, s), 6.81
(1H, d, J = 8 Hz), 7.05-7.7 (12
H) Elemental analysis (as C ₂₅ H ₂₂ N ₄ O ₂ .2H ₂ O) Found: C, 63.15; H, 5.15;
10 Calculated: C, 62.75; H, 5.48; N, 11.
71

(3) Alternatively, alcohol (III-
To a solution of 17,747 mg) in 10 ml of acetone was added dropwise Jones' reagent (3 M, 1 ml) under ice cooling. The mixture is stirred at room temperature for 30 minutes. Add Jones reagent (0.5 ml) again. The mixture is stirred at room temperature for 20 minutes. Add water. The mixture is extracted with ethyl acetate. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is subjected to flash chromatography on silica gel (30 g) (dichloromethane-methanol-acetic acid-water = 90: 10: 1: 1) to give the title compound III-25. Yield 541 mg (yield 70.1
%).

Example 2 1- (2- (3- (carbomethoxy) propylcarbamoyloxy) ethyl) -3-
(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-53)

Embedded image (1) 3- (Carbomethoxy) propyl isocyanate (Compound III-52) Thionyl chloride (22 ml) is added dropwise to glutaric acid monomethyl ester (22.0 g). Stir the solution at room temperature for 3 hours. Excess thionyl chloride was distilled off under reduced pressure, and the residue was distilled at 105 ° C. and 17 mmHg to obtain an acid chloride (22.3).
8 g) are obtained. A solution of the acid chloride (22.38 g) in acetone (50 ml) is added dropwise over 30 minutes to an ice-cold solution of sodium azide (10.6 g) in water (70 ml). The mixture is stirred at 0 ° C. for 1 hour. Water is added and the mixture is extracted with benzene. The extract is washed with water and dried over anhydrous sodium sulfate. Heat the solution slowly. About 70 ° C for nitrogen gas
Occurs in The solution is heated at the same temperature for 1 hour and the volatile substances are distilled off at normal pressure. The residue is 95-97 at 22 mmHg.
Distillation at C gives a colorless oil (17.41 g, 80.8% yield from half ester). IRν _max (film) 2280 cm ^-1

(2) 1- (2- (3- (carbomethoxy) propylcarbamoyloxy) ethyl) -3-
(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (compound III-53) Alcohol (compound II) in dry benzene (15 ml)
I-17, 1.0 g), 3- (methoxycarbonyl) propyl isocyanate (Compound III-52, 0.83
g) and triethylamine (0.5 ml) solution are heated at reflux for 20 hours. The solid is collected by filtration, washed with benzene and dried. The mother liquor is concentrated under reduced pressure. The residue and solid are combined and flash chromatographed on silica gel (30 g) (chloroform: methanol;
1) and crystallization from ethyl acetate to give the carbamate (1.33 g, 99.7% yield). mp = 195-
196 ° C.

IRν _max (Nujol): 3395, 3
320, 1736, 1718, 1641, 1614, 1
561 cm ^-1 NMR (CDCl ₃ ) δ: 1.61 (2H, m);
86 (1H, m), 2.24 (2H, t, J = 8H
z), 2.28 (3H, s), 2.75 (1H, m),
2.99 (1H, m), 3.67 (3H, s), 3.8
6 (2H, m), 4.20 (1H, m), 4.50 (1
H, t, J = 5 Hz), 4.77 (1H, m), 5.6
0 (1H, d, J = 8 Hz), 6.84 (1H, t, J
= 4Hz), 7.0-7.7 (12H) Elemental analysis (C ₃₁ H ₃₃ N ₅ as O ₆₎ Found: C, 65.04; H, 5.91 ; N, 12.
25 Calculated: C, 65.14; H, 5.82; N, 12.
25

Example 3 1- (3- (Carboxy) propylpiperazinocarbomethyl) -3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-
2H-1,4-benzodiazepin-2-one (Compound I
II-84)

Embedded image (1) Ethyl 4-piperazinobutyrate (Compound III-6
9) A mixture of ethyl 4-bromo-n-butyrate (13.5 g), piperazine dihydrochloride (11.0 g) and piperazine hexahydrate (13.42 g) in ethanol (70 ml) was added to a mixture of 4
Heat to reflux for hours. After cooling, the solid is collected by filtration.
The filtrate is concentrated under reduced pressure. Add potassium carbonate aqueous solution. The solution is extracted with chloroform. The extract is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was 0.2 mmHg, 1
Distillation at 05-106 ° C gives a colorless oil (7.07
g, 51.0%).

(2) 1- (3- (carbethoxy) propylpiperazinocarbomethyl) -3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-
2H-1,4-benzodiazepin-2-one (Compound I
II-83) First, compound III-69 was described in the literature (JCS (19)
(1986) 2404).
Compound (III-69, 480 mg), carboxylic acid (III-2
5,883 mg), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (461 mg) and 1-hydroxybenzotriazole (54 mg) in dimethylformamide (7 ml) were mixed with ice-cooled triethylamine (1 ml). Add. The mixture is stirred for 2 days at room temperature and then distilled off at 40 ° C. and 1 mmHg. Add water to the residue. The solid was filtered, washed with water and dried, and then silica gel (5
0g) (chloroform: methanol; 20: 1) using the title compound (III-8).
3) 862 mg (70.5%) are obtained. NMR (CDCl ₃ ) δ: 1.26 (3H, t, J = 7
Hz), 1.77 (2H, m), 2.28 (3H,
s), 2.2-2.4 (8H), 3.38 (2H, b
r. s), 3.53 (2H, br.s), 4.13 (2
H, q, J = 7 Hz), 4.66 (2H, s), 5.6
6 (1H, d, J = 8 Hz), 6.8-7.7 (13H
m) (+)-isomer: [α] _D +81.8 (c 1.0
90, CHCl ₃ )

(3) 1- (3- (carboxy) propylpiperazinocarbomethyl) -3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydrodo-2
H-1,4-benzodiazepin-2-one (Compound II
I-84) An aqueous solution of potassium carbonate (10-ml)
%, 3 ml). The solution is heated to reflux for 6 hours and concentrated under reduced pressure. Dilute hydrochloric acid is added to the residue. The solid is collected by filtration, washed with water, dried and dried with carboxylic acid (compound III-8).
4,669 mg, 79.7%).

Example 4 1-((6,6-dimethyl-6)
-(Carboxy) propylcarbamoyloxy) hexylpiperazinocarbomethyl) -3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-2
H-1,4-benzodiazepin-2-one (Compound II
I-97)

Embedded image (1) Methyl 6-tetrahydropyranyloxyhexinoate (Compound III-90) Solution of methyl 6-hydroxyhexanoate (III-89, 10.9 g) and dehydropyran (7.5 ml) in dichloromethane (130 ml) To this is added p-toluenesulfonic acid (235 mg). The solution is stirred at room temperature for 3 hours and poured into aqueous sodium hydrogen carbonate solution. Separate the organic layer and extract the aqueous layer with dichloromethane. The organic layers are combined, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was distilled at 25 mmHg, 130-131 ° C,
10.16 g (59.1%) of a colorless oil are obtained.

(2) 1,1-Dimethyl-6-tetrahydropyranyloxyhexanol (Compound III-91) A solution of methyl iodide (7.0 g) in ether (50 ml) was stirred under vigorous stirring with magnesium (1.20 g). Add to After the addition is complete, the mixture is heated at reflux for 5 minutes and cooled. Ester (III-90, in ether (30 ml)
5.1 g) are added dropwise with ice cooling. Mix the mixture at room temperature for 1 hour.
Stir for 0 min at reflux temperature for 30 min. Ammonium chloride is added dropwise to the cold mixture. Separate the organic layer and extract the aqueous layer with ether. The organic layers are combined, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. nmr indicates that the starting material was mixed in the residue, and this was mixed with 20 ml of ethanol.
Dissolve in Add 10% sodium hydroxide (10 ml). The solution is left overnight at room temperature and concentrated to half volume. Add water. The mixture is extracted with ether. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is distilled at 115-120 ° C and 0.2 mmHg to give 2.07 g (40.1%) of a colorless oil.

(3) 1,1-dimethylhexane-1,6
-Diol (compound III-92) tetrahydropyranyloxy compound (III-91,
2.10 g) and p-toluenesulfonic acid (0.05
A solution of g) in methanol (20 ml) is stirred at room temperature for 3 hours. Volatile substances are distilled off under reduced pressure. Dissolve the residue in ether. The solution is washed with aqueous sodium hydrogen carbonate solution and water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is distilled at 145 ° -147 ° C. at 15 mmHg to give 1.1 g (82.5%) of a colorless oil. (4) 1,1-Dimethyl-6-bromohexanol (compound III-93) Triphenylphosphine (1.96 g) in tetrahydrofuran (10 ml) was added to a diol (compound III-9).
2.09 g) and carbon tetrabromide (2.47 g) in dry tetrahydrofuran (25 ml) at 0 ° C. over 1 hour. The mixture is stirred at room temperature for 7 hours. Filter insoluble material and wash with ether. The filtrate is concentrated under reduced pressure. The residue was subjected to flash chromatography on silica gel (30 g) (hexane: ethyl acetate; 4: 1), distilled at 125-128 ° C. and the bromide (0.945).
g, 60.0%).

(5) 1,1-Dimethyl-6-piperidazinohexanol (Compound III-94) Compound (III-93) was prepared according to the method described in JCS (196).
1 year), 2404).
Converted to 4). (6) 1- (6,6-dimethyl-6-hydroxyhexylpiperazinocarbomethyl) -3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-2
H-1,4-benzodiazepin-2-one (Compound II
I-95) Compounds III-25 and II in the general manner described above.
I-94 is coupled to give the title compound. (7) 1- (6,6-dimethyl-6- (3- (carbomethoxy) propylcarbamoyloxy) hexylpiperazinocarbomethyl) -3- (N '-(m-tolyl) ureido) -5-phenyl -1,3-dihydro-2H-1,
4-benzodiazepin-2-one (Compound III-9
6) A solution of alcohol (III-95, 740 mg), bis- (tri-n-butyltin) oxide (243 mg) and isocyanate (III-52, 550 mg) in dry tetrahydrofuran (20 ml) was heated at reflux for 10 hours, Concentrate under reduced pressure. Dissolve the residue in chloroform. The solution is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. Flash chromatography of the residue on silica gel (50 g) (chloroform: methanol; 9: 1)
To give a viscous oil quantitatively. IRν _max (Nujol) 3330, 1727, 165
5, 1613, 1576 cm ^-1

(8) 1-((6,6-dimethyl-6-
(Carboxy) propylcarbamoyloxy) hexylpiperazinocarbomethyl) -3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-2
H-1,4-benzodiazepin-2-one (Compound II
I-97) To a solution of the ester (1.03 g) obtained in (7) above in methanol (6 ml) was added 10% aqueous potassium hydroxide (1.3 ml). The solution is left at room temperature for 24 hours and neutralized with dilute hydrochloric acid. The solid is collected by filtration, washed with water, dried and subjected to flash chromatography (chloroform: methanol; 4: 1) on silica gel (30 g). Add benzene and distill volatiles at 1 mmHg. Yield 343 mg (35.9% yield) IRν _max (Nujol) 3230, 1660, 155
5 cm ^-1 NMR (CDCl ₃ + CD ₃ OD) δ: 1.32 (4H,
m), 1.41 (6H, m), 1.55 (2H, m),
1.71 (2H, m), 1.78 (2H, qui, J =
7Hz), 2.30 (3H, s), 2.42 (2H,
t, J = 7 Hz), 2.56 (4H, m), 3.13
(2H, t, J = 7 Hz), 3.57 (2H, m),
3.64 (2H, m), 4.74 (2H, s), 5.6
0 (1H, s), 6.82 (1H, d, J = 7 Hz),
7.1-7.7 (11H, m) Elemental analysis _{(C 42 H 53 N 7 O} 7 · 1 / 6C 6 as H ₆₎ Found: C, 66.13; H, 6.97 ; N, 11 .
72 Calculated: C, 66.13; H, 6.97; N, 12.
56

Example 5 1- (2- (6,6-dimethyl-6-hydroxyhexylthio) ethyl) -3- (N '
-(M-tolyl) ureido) -5-phenyl-1,3-
Dihydro-2H-1,4-benzodiazepin-2-one (Compound III-101)

Embedded image (1) 1-((2-acetylthio) ethyl) -3-
(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-100) To a solution of alcohol (III-17, 201 mg) in dichloromethane (2 ml) and triethylamine (1 ml) was added-
At 20 ° C., a solution of methanesulfonyl chloride (70 mg) in dichloromethane (1 ml) is added dropwise. The mixture is stirred at −20 ° C. for 30 minutes. Add aqueous ammonium chloride solution.
Separate the organic layer and extract the aqueous layer with dichloromethane. The organic layers were combined, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give mesylate (compound III-99, 223 mg,
93.8%). A mixture of this mesylate and potassium thioacetate (100 mg) in acetonitrile (2 ml) is stirred under a nitrogen atmosphere for 2 days. Add water. The mixture is extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is subjected to flash chromatography on silica gel (15 g) (hexane: ethyl acetate; 1: 1) and crystallized from benzene to give thioacetate 170 mg (79.4% yield). mp =
198-200 ° C (rac.mp = 174-175)
° C).

(2) 1- (2- (6,6-dimethyl-6)
-Hydroxyhexylthio) ethyl) -3- (N'-
(M-tolyl) ureido) -1,3-dihydro-2H-
1,4-benzodiazepin-2-one (compound III-
101) Metallic sodium (0.10 g) was added to ethyl alcohol (3 m
Dissolve by heating to reflux in l). Add 1- (2-
(Acetylthio) ethyl) -3- (N ′-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-2
H-1,4-benzodiazepin-2-one (III-
100, 473 mg) in one portion. Mix at room temperature 3
Stir for hours. Bromide (III) in ethanol (2 ml)
(-93, 240 mg) solution is added dropwise. Mix the mixture at room temperature for 1 hour.
Stir for 8 hours. Water is added and the mixture is extracted with dichloromethane. The solution is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to flash chromatography on silica gel (25 g) (ethyl acetate: hexane; 2: 1) to give 203 mg of thioether (yield 3).
6.5%). IRν _max (Nujol): 3330, 1660, 16
10,1599,1555 cm ^-1 NMR (CDCl ₃ ) δ: 1.21 (3H, s),
22 (3H, s), 1.2-1.51 (8H), 2.2
9 (3H, s), 2.38 (2H, m), 2.65 (2
H, m), 3.84 (1H, m), 4.56 (1H,
m), 5.57 (1H, d, J = 8 Hz), 6.8-
7.7 (13H) Elemental analysis _{(C 33 H 45 N 4 O} 3 as S) Found: C, 69.16; H, 7.04 ; N, 10.1
0; S, 5.14 Calculated: C, 69.20; H, 7.04; N, 9.78.
S, 5.60

The alcohols (III-101, 198)
mg), isocyanate (III-52, 100 mg) and triethylamine (0.1 ml) in dry toluene (5 ml).
l) Heat the solution under reflux for 40 hours. The volatiles are distilled off under reduced pressure. The residue was subjected to flash chromatography (ethyl acetate) using silica gel (30 g) to give the adduct 1- (2- (6,6-dimethyl-6- (3- (carbomethoxy) propylcarbamoyloxy) hexylthio) ethyl) -3-. (N '-(m-tolyl) ureido)
72 mg of -1,3-dihydro-2H-1,4-benzodiazepin-2-one (III-102) (yield 29.1)
%).

Example 6 1- (2- (carbethoxymethylthio) ethyl) -3- (N '-(m-tolyl) ureido) -1,3-dihydro-2H-1,4-benzodiazepin-2-one (Compound III-103)

Embedded image Metallic sodium (66 mg) in ethyl alcohol (5 ml)
Dissolve by heating under reflux. Add the thioester (I
II-100, 303 mg) in one portion. The mixture is stirred at room temperature for 3 hours. A solution of ethyl bromoacetate (180 mg) in ethanol (2 ml) is added dropwise. The mixture is stirred at room temperature for 6 hours. Cold dilute hydrochloric acid is added and the solution is extracted with dichloromethane. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to flash chromatography on silica gel (15 g) (toluene: ethyl acetate; 2: 1) and recrystallized from toluene to give 186 mg of the ester (56.3% yield). mp = 165-
167 ° C. IRν _max (Nujol): 3350, 1737, 16
73, 1647, 1566 cm ^-1 NMR (CDCl ₃ ) δ: 1.21 (3H, t, J = t
Hz), 1.63 (3H, s), 2.30 (3H,
m), 2.73 (1H, m), 2.85 (1H, m),
2.95 (1H, d, J = 15 Hz), 3.10 (1
H, d, J = 15 Hz), 3.90 (1H, m), 4.
10 (2H, q, J = 7 Hz), 4.61 (1H,
m), 5.56 (1H, d, J = 8 Hz), 6.7-
6.9 (3H), 7.0-7.7 (10H ) Elemental analysis _{(C 29 H 30 N 4 O} 4 as S) Found: C, 65.85; H, 5.61 ; N, 10. 6
4; S, 5.88 Calculated: C, 65.64; H, 5.70; N, 10.5
6; S, 6.04

Example 7 1- (2- (carboxymethylthio) ethyl) -3- (N '-(m-tolyl) ureido) -1,3-dihydro-2H-1,4-benzodiazepin-2-one ( Compound III-104) An aqueous solution of sodium carbonate (85 mg, 1.2 ml) is added to a solution of the above ester (III-103) (55, 322 mg) in ethanol (7 ml). The mixture is heated at reflux for 5 hours. Dilute hydrochloric acid is added and the mixture is extracted with ethyl acetate. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is heated in benzene and cooled to room temperature. The solid is filtered off to give the acid (III-104) quantitatively.
mp = 130-131 [deg.] C. IRν _max (Nujol): 3320, 1678, 16
48, 1614, 1561 cm ^-1 NMR (CDCl ₃ + CD ₃ OD) δ: 2.31 (3H,
s), 2.77 (1H, m), 2.88 (1H, m),
3.00 (1H, d, J = 15 Hz), 3.13 (1
H, d, J = 15 Hz), 3.96 (1H, m), 4.
63 (1H, m), 5.53 (1H, s), 6.83
(1H, d, J = 6Hz ), 7.1-7.7 (12H) Elemental analysis _{(C 27 H 26 N 4 O} 4 S · 1 / 2H 2 O as) Found: C, 63.24; H , 5.14; N, 10.8
3: S, 6.00 Calculated: C, 63.39; H, 5.32; N, 10.9
5; S, 6.27

Example 8 1- (2-azidoethyl) -3
-(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-121)

Embedded image A mixture of crude mesylate (III-99) synthesized in the manner described above from alcohol (III-17, 1.15 g) and sodium azide (350 mg) in hexamethylphosphoric triamide (7 ml) was added at 50 ° C. for 3 hours. Stir for hours. After adding to water, extract with ethyl acetate. The extract is washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the title compound (III-121).

Example 9 1- (2-aminoethyl) -3
-(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-122) Triphenylphosphine (1.0 g) is added to a solution of the azide compound (III-121) in tetrahydrofuran (25 ml), and the mixture is left at room temperature overnight. Water (10m
After l) was added and the mixture was heated under reflux for 30 minutes, the solvent was distilled off under reduced pressure. The residue is extracted with ethyl acetate. The extract is washed with water, dried over sodium sulfate and concentrated. The residue was subjected to silica gel (30 g) chromatography (chloroform: methanol; 5: 1) to give 832 g of an amine (III-1).
7 yield 72.5%). mp = 140-14
2 ° C. IRν _max (Nujol): 3320, 1680, 16
45, 1615, 1563 cm ^-1 NMR (CDCl ₃ ) δ: 1.72 (4H, br.
s), 2.28 (3H, s), 2.80 (2H, m),
3.75 (1H, m), 4.36 (1H, m), 5.5
5 (1H, d, J = 8 Hz), 6.83 (1H, m),
6.9-7.7 (13H, m) Elemental analysis _{(C 25 H 25 N 5 O} 2 · 0.8H 2 O as) Found: C, 67.96; H, 6.02 ; N, 15. 9
1 Calculated: C, 67.95; H, 6.07; N, 15.8.
5

Example 10 1- (3- (4-Sulfamoylphenyl) propylpiperazino) carbomethyl)-
3- (N '-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (compound III-194)

Embedded image (1) 4- (3-Bromopropyl) phenylsulfonamide (Compound III-192) A solution of 3-phenylpropylbromide (10.85 g) in chloroform (50 ml) was treated with chlorosulfonic acid (18 ml) in chloroform (50 ml). The solution is added dropwise at 3 ° C. over 1 hour. The mixture is stirred at room temperature for 1 hour and heated at reflux for 1 hour. After cooling, the mixture is poured into ice water. Separate the organic layer and extract the aqueous layer with chloroform. The organic layers are combined, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. A mixture of the residue and phosphorus pentachloride (22.7 g) was added to 8
Heat at 0 ° C. for 3 hours. After cooling, the mixture is diluted with chloroform and poured into ice water. Separate the organic layer and extract the aqueous layer with chloroform. The organic layers are combined, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was placed in tetrahydrofuran (100 ml), and the resulting mixture was stirred under ice-cooling with stirring for 1 hour over a 28% aqueous ammonia solution (20 ml).
0 ml) is added dropwise. The mixture is stirred at room temperature for 3 hours. The mixture is extracted with ethyl acetate. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to flash chromatography on silica gel (70 g) (ethyl acetate: hexane; 1: 1) to give benzene-
Crystallized from hexane, compound III-192 (8.
66 g; yield from 3-phenylpropyl bromide 5
7.4%).

(2) p- (3-Piperazinopropyl) phenylsulfonamide (Compound III-193) As in the case of the compound (III-94) described in Example 4 (4), the literature (J.C. Compound (III-192) was converted by the method described in S. (1961), 2404). (3) 1- (3- (4-Sulfamoylphenyl) propylperazinocarbomethyl) -3- (N ′-(m-tolyl) ureido) -5-phenyl-1,3-dihydro-2
H-1,4-benzodiazepin-2-one (Compound II
I-194) Compound (III-191) and compound (III-25) are coupled as described above to obtain the title compound in a yield of 41.3%.

Example 11 1- (3- (4- (ethoxymethylenesulfamoyl) phenyl) propylcarbamoylmethyl) -3- (N '-(m-tolyl) ureido)
-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (compound III-203)

Embedded image (1) 4- (3-azidopropyl) phenylsulfonamide (compound III-197) A solution of bromide (III-192, 2.00 g) and sodium azide (1.0 g) in hexamethylphosphoric triamide (10 ml) Is stirred overnight at room temperature, poured into water and extracted with ether. The extract is washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is crystallized from benzene-hexane. The product contains a small amount of HMP
A remains.

(2) p- (3-Aminopropyl) benzenesulfonamide (Compound III-201) Triphenylphosphine (1.47 g) was added to a solution of azide (III-197; 1.23 g) in dry tetrahydrofuran (20 ml). Add. The solution is left at room temperature for 6 hours. Water (10 ml) was added and the solution was heated at reflux for 30 minutes,
Concentrate under reduced pressure to half the volume. Chloroform is added, the solid is filtered off, washed with water and chloroform and dried to give the amine (0.65 g). mp = 14
0-141 ° C.

(3) 1- (3- (4-sulfamoylphenyl) propylcarbamoylmethyl) -3- (N'-
(M-tolyl) ureido) -5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (compound III-202) Carboxylic acid (III-25) and amine (I
II-201) to give the title compound in 78.6% yield. (4) 1- (3- (4- (ethoxymethylenesulfamoyl) phenyl) propylcarbamoylmethyl) -3-
(N '-(m-tolyl) ureido) -5-phenyl-
1,3-dihydro-2H-1,4-benzodiazepine-
2-one (Compound III-203) sulfonamide (III-202, 0.71 g), triethyl orthoformate (1.7 g) and a small amount of p-
The mixture of toluenesulfonic acids is heated at 120 ° C. for 6 hours. The solid was filtered off, washed with ethanol and dried to give the title compound III-203 (574 mg, yield 76.1).
%). mp = 197-199 [deg.] C. IRν _max (Nujol): 3320, 3235, 16
93, 1670, 1644, 1600, 1543, 11
60 cm ^-1 NMR (CDCl ₃ + CD ₃ OD) δ: 1.34 (3H,
t, J = 7 Hz), 1.79 (2H, qui, J = 7H)
z), 2.30 (3H, s), 2.66 (2H, t, J
= 8 Hz), 3.26 (2H, m), 4.31 (2H,
q, J = 7 Hz), 4.34 (1H, d, J = 16H)
z), 4.66 (1H, d, J = 16 Hz), 5.59
(1H, s), 6.83 (1H, d, J = 7 Hz),
7.1-7.7 (10H), 7.76 (2H, d, J =
8 Hz), 8.39 (1 H, s) Elemental analysis (as C ₃₇ H ₃₇ N ₆ O ₆ S.H ₂ O) Found: C, 63.80; H, 5.55; N, 12.0
1; S, 4.55 Calculated: C, 63.78; H, 5.79; N, 12.0
6; S, 4.60

Example 12 1- (2,2-Diethoxyethyl) -3- (N '-(m-tolyl) ureido) -5
Phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (Compound III-188)

Embedded image A solution of III-24 (150 mg) and p-toluenesulfonic acid (1 mg) in ethanol (5 ml) is heated to reflux for 18 hours. The solid in aqueous sodium bicarbonate solution is collected by filtration, dried and chromatographed on silica gel (10 g) (hexane: ethyl acetate; 1: 1) to give the title compound. mp = 215-216 ° C. NMR (CDCl ₃ ) δ: 0.95 (3H, t, J = 7)
Hz), 1.08 (3H, t, J = 7 Hz), 2.30
(3H, s), 3.3-3.7 (4H, m), 3.84
(1H, dd, J = 5, 14 Hz), 4.23 (1H,
dd, J = 5, 14 Hz), 4.76 (1H, t, J =
5Hz), 5.59 (1H, d, J = 8Hz), 6.8
-7.1 (13H, m) Elemental analysis (C ₂₉ H ₃₂ N ₄ as O ₄₎ Found: C, 68.95; H, 6.30 ; N, 11.
07 calc: C, 69.58; H, 6.44; N, 11.
19

The pharmacological effects of the compounds prepared in the above Examples were evaluated by antagonizing gastrin receptors using guinea pig gastric mucosa and antagonizing CCK-B and CCK-A receptors using mouse cerebral cortex and pancreas. These three items were examined in vitro.

Experimental animals: Male Sprague Dawley rats (weight 220-270 g), male Hartley guinea pigs (weight 450-600 g) or male ddY mice (weight 24-30 g) were used.

(1) Preparation of In Vitro Gastrin Receptor Antagonizing Gastric Glands: Male Hartley guinea pigs (weight 450-600
g; 3 animals) after blood loss and sacrifice, immediately remove the stomach, cut off the greater curvature and wash the contents in cold water. 2. After washing with Krebs solution (ice cooled), 1M Na
The mucus is gently wiped off with absorbent cotton soaked in Cl (ice-cooled) and washed with Krebs solution. 3. The mucosal surface is gently wiped with absorbent cotton soaked with Krebs solution, the mucous membrane is peeled off using a slide glass, and added to Hank's solution containing 0.1% BSA for washing. 4. A 0.1% collagenase solution is added to the detached mucosa, and the mixture is incubated at 37 ° C for 20 minutes with intermittent gentle shaking. The supernatant is decanted, a new 0.1% collagenase solution is added, and the mixture is further incubated for 20 minutes. 5. Using a 20 ml syringe, repeat suction and discharge three to four times to separate the cells (5-10 cells are adhered gastric glands instead of isolated cells), and filtered (200 nylon mesh). .

After centrifugation at 6.270 × g (1000 rpm) for 3 minutes, the supernatant was discarded, and 0.2% neutral protease (EC
3.4.24.4) Add the solution, mix by pipetting several times with a Komagome pipette, and incubate at 37 ° C for 15 minutes. After centrifugation at 7.270 × g (1000 rpm) for 3 minutes, the supernatant is discarded, the incubation medium is added, and the cells are washed by pipetting similarly. Repeat three times. All the above operations are performed under 95% CO ₂ -5% O ₂ aeration. 8. The number of viable cells is counted by a Thoma calculator. An equivalent volume of 0.4% trypan blue is added to 50 μl of the cell solution, and the cells are shaken gently to stain the dead cells. Then, the number of viable cells in the middle compartment 5 is counted. Not counting). Since the number of counts becomes 10 ⁵ / ml, the cells are diluted with an incubation medium to 10 ⁵ cells / ml, sealed and kept on ice until use.

Displacement Assay: a) Preparation of test compound: Approximately 2 mg is weighed in a 20 ml glass vial and dissolved in a DMSO solution to make a 1 mM concentration. Then, using a tube, 50% DMSO
Dilute 250 μM (final concentration 10 μM) with the solution. 25nM for each 1/10 concentration (final concentration 1nM)
Make dilution up to.

[0049] b) creating a [125 _I] gastrin preparation of labeled compounds 5 nM (final concentration 0.2 nM). Calculation Example: Specific activity _{2000 Ci / mmol (100 μCi / ml) 100 μCi / ml × nmol / 2000 μCi = 0.05 nmol / ml = 50 nM When} using _{50 nM / 5 nM = 10} , dilute 10-fold with the incubation medium.

[0050] c) cold gastrin I _{(Cold Gastrin
I))} Preparation Human gastrin I is diluted at the time of use with an incubation medium to prepare a concentration of _{50 μM} (final concentration _{2 μM)} .

D) Incubate under the following conditions
Do._{Total [125 I] Gastrin 10 μl Bound (Bo) 50% DMSO 10 μl (3 Tube) 105 / ml Cell 230μl Non Specific [125 I] Gastrin 10 μl Bound (NS) Cold Gastrin 10μl (2 Tube) 105 / ml Cell 230 μl Test compound [125 I] Gastrin 10 μl Total Bound test compound dilution 10μl (1 Tube per point) 105 / ml Cell 230μl}  The reaction is started by the addition of cells. 30 minutes at 25 ° C
After incubation, centrifuge at 3000 rpm for 5 minutes
And remove the supernatant by suction. Ice-cold incubation
Add 0.5 ml of medium, mix gently, and immediately centrifuge.
Is removed by suction. Radioactivity measured by gamma counter
Measure.

[0052] e) Calculation of IC _50: IC _50; specific binding of test compound (Total, cpm-N
S, cpm).

(2) Preparation of in vitro CCK-A and -B receptor antagonist CCK receptor preparation: male ddY mouse (body weight 24 to
After decapitation and sacrifice of 30 g), the cerebral cortex (CCK-B) and pancreas (CCK-A) were immediately removed. Preparation of receptor specimen (crude membrane fraction): The mice are sacrificed by decapitation, the cerebrum and pancreas are immediately removed, washed with a pre-cooled PBS solution, and the cerebrum is separated from the cortex. 2. The cerebral cortex and pancreas are washed buffe
r; pH 7.4, cooling). 3. Each was weighed to 1.5-2.0 g / vial, and -80
Store in ° C (stable for 2 months).

4. Add a 10-fold volume of washing buffer, homogenize (7 strokes) with a Teflon-glass homogenizer, and further homogenize with a Polytron homogenizer.
Homogenize for 0 seconds. 5.40 minutes x 40,000 xg (Serval, 15,800
Centrifuge at (rpm) to obtain a pellet. 6. Add a 10-fold volume of washing buffer, homogenize with a Polytron homogenizer for 20 seconds, and centrifuge similarly to obtain a pellet (repeated twice, measuring the amount of protein). 7. Add 10 volumes of incubation buffer,
After homogenizing for 20 seconds with a Polytron homogenizer, the cerebral cortex was finally diluted 40 times and the pancreas was diluted 200 times with the same buffer, and CCK-B and CCK-A, respectively.
Use as a receptor specimen.

Displacement assay: a) Preparation of test compound Approximately 2 mg each was weighed into a 20 ml glass vial,
Dissolve in DMSO solution to make 1 mM concentration. Next, 250 μM (final concentration: 10 μM) is diluted with a 50% DMSO solution to prepare a tube. Further, dilution is made to 25 nM (final concentration 1 nM) for each 1/10 concentration. For CCK-B Assay, nM For CCK-A Assay, μM Test Compound Test Compound 25 (1) 2.5 (0.1) 250 (10) 25 (1) 2500 (100) 250 (10) 25000 (1000) (): Final concentration

B) Preparation of labeled compound: Prepare [3 _H ] CCK-8 at 50 nM (final concentration 1 nM). Calculation example: specific activity _{60 μCi / nmol (100 μCi / ml) 240 μCi / ml × nmol / 60 μCi = 4 nmol / ml = 4000 nM 4000 nM / 50 nM = 80,} use 80 times dilution with incubation buffer.

C) Preparation of cold CCK-8: 883
-S is diluted in incubation buffer at the time of use to make a concentration of 25 μM (final concentration 1 μM).

D) Incubate under the following conditions
Do._{Total [3 H] CCK-8 10 μl Bound (Bo) 50% DMSO 20μl (3 Tube) Receptor specimen 470μl Non Specicific [3 H] CCK-8 10 μl Bound (NS) Cold CCK-8 20μl (2 Tube) Receptor specimen 470 μl Test compound [3 H] CCK-8 10 μl Total Bound Test Compound Diluent 20μl (Each point 1 Tube) Receptor specimen 470μl}  The reaction is initiated by the addition of the receptor specimen. 90 at 25 ° C
After incubation for 5 minutes, the solution was filtered by suction (Whatman
 glass microfilter, GF / C), cooled washing buffer
Wash three times. 5ml Aquasol-2 Kakti
(Aqueasol-2cocktail) and leave for several hours after shaking
And count the radioactivity for 5 minutes.

E) IC ₅₀ IC ₅₀ : Specific binding of test compound (Total, dpm-N
S, dpm) was calculated. The results are shown in Table 1 below. In the table, B
/ G ratio indicates antagonism to gastrin receptor and CCK
The ratio of antagonistic action to -B receptor, which indicates the gastrin receptor specificity of the test compound.

[Table 1]

────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Yasunobu Ishihara 61 Nishikujo-harikoji-cho, Minami-ku, Kyoto-shi, Kyoto (58) Field surveyed (Int. Cl. ⁷ , DB name) C07D 243/24 A61K 31/55 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. Formula I: ## STR1 ## Wherein R ¹ is carboxy, CHR ² R ³ , provided that R ² and R ³ are independently hydrogen, alkoxy, amino, azide,
OC (O) NH (CH ₂ ) ₃ COOR ⁴ , SCH ₂ COOR ⁴ (R
⁴ is hydrogen or lower alkyl), or CONR ⁵ R ⁶ ,
R ⁵ and R ⁶ independently represent hydrogen, (CH ₂ ) ₃ (N-substituted) sulfamoylphenyl, or together with the attached nitrogen atom, (CH ₂ ) ₅ C (CH ₃ ) ₂ OR ⁷ (R ⁷ is hydrogen or C (O) NH (CH ₂ ) ₃ COOR ⁴ ) or (C
H ₂₎ may form a substituted piperazine by ₃ COOH. A benzodiazepine derivative or an addition salt thereof. 2. R ¹ is carboxy; CHR ² R ³ , wherein R ² and R ³ are ethoxy, or one of them is hydrogen and the other is amino, azide, OC (O) NH (C
H ₂ ) ₃ COOCH ₃ or SCH ₂ COOR ⁴ (R ⁴ is hydrogen or ethyl); or any of the following groups: The benzodiazepine derivative according to claim 1, which is represented by: