JPH0363559B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel N-acyl-polypeptides and methods for their production. The N-acyl polypeptides of the present invention are useful as therapeutic agents for diabetes, vascular diseases, acromegaly, gastrointestinal diseases, etc., and particularly include peptides represented by the following formula and salts thereof. formula: [In the formula, _Acyl is a group represented by (1)R〓CO- (herein _{, R〓 is C1-20 alkyl} , phenyl, or phenyl substituted with _C1-4 alkyl), ( ₂ )R〓SO2 _{A group represented by - (where R〓 is phenyl substituted with C 1-10 alkyl, phenyl or C 1-4 alkyl ) ,}
(3) A group represented by R〓NHCO- (where R〓 is C ₁ to ₁₀
alkyl) or (4) biotinyl, B is a phenylalanine residue whose benzene ring may be substituted with NO ₂ , F is a group represented by -NH-CH(R ₅ )-X (where R ₅ is −CH ₂ OH, −CCH ₂ ) ₂ OH, −(CH ₂ ) ₃ OH,
-CH( _CH3 )OH, , isobutyl or benzyl,
X is a group represented by _-COOR1 , _-CH2OH or _-CONH2 , _R1 is _C1-3 alkyl), _Y1 and _Y2 are each _hydrogen or both are a direct bond, Z is _{C3- 4} alkyl or benzyl in which the benzene ring may be substituted with NO ₂ . In addition, 1st, 2nd, 4th, 6th, 7th and 8th place
The residue at position has the (D)- or (L)-configuration. ] In this specification, amino acid residues represented by abbreviations such as Phe, Cys, etc. should be understood to have L-configuration unless otherwise specified. Among the N-acyl polypeptides of the invention, compounds in which Mcyl is an acyl residue containing an aliphatic moiety of at least 7 (preferably at least 8) carbon atoms are a particularly interesting class of compounds. This type of compound (hereinafter referred to as N-acyl-polypeptides/T-type) has the property of maintaining its activity for a longer period of time when administered subcutaneously. N-acyl-polypeptides/T-type are:
A group in which Acyl is R〓CO- _{or R〓SO2} (especially _R〓CO- ), where R〓 is _C7-15E alkyl (preferably _{C7-10 alkyl), especially C8-15 alkyl} (preferably _C8-10 alkyl) _, R〓 is _C7-10 alkyl, especially _{C8 - C10} alkyl). Among N-acyl polypeptides/T-type, the remaining groups in formula [] are the above (6) to (15)
Particularly preferred are compounds which are groups having the meanings described in . The N-acyl-polypeptides of the invention can exist as their salts. Oxidative salts can be prepared with organic, polymeric or inorganic acids. Such acid addition salts include, for example, hydrochlorides and acetates. The present invention can provide a method for producing compound []. These compounds [] can be produced by conventional operations in the field of peptide chemistry or chemically equivalent operations thereof, such as a method comprising the following reaction steps: (a) Sequence represented by the above formula [] (b) removing one or more protecting groups from a protected N-acyl-polypeptide having Protected or unprotected N having the sequence shown by the formula []
-Acyl-Polypeptide is obtained by bonding two peptide units with each other through an amide bond, performing the above step (a) as necessary, and (c) producing a protected compound having a sequence represented by the formula []. (d) Y ₁ and Y ₂ are each hydrogen N-acyl-
The polypeptide [] is oxidized and Y ₁ and Y ₂ are combined to produce an N-acyl polypeptide [] which is a direct bonding target, and the resulting N-acyl polypeptide [] is A process of recovery in free base form or salt form. The above method can be carried out, for example, in a manner similar to the examples described below. If the method for preparing the starting material is not stated, the compound is known or can be prepared and purified by known methods. In the examples, the [α] ²⁰ _D value is an uncorrected value. The meanings of the abbreviations in the examples are listed below. AcOH = acetic acid AcOEt = ethyl acetate BOC = tert-butoxycarbonyl BTFA = boron tristrifluoroacetate, DCCI = dicyclohexylcarbodiimide DMF = N,N-dimethylformamide HOBT = N-hydroxybenzotriazole Leu-ol = leucinol residue: MBzl = p-methoxybenzyl Me = methyl MeOH = methanol NEt ₃ = triethylamine ONP = 4-nitrophenoxy Phe (pNO ₂ ) = 4-NO ₂ -phenylalanine TFA = trifluoroacetic acid THF = tetrahydrofuran Thr-ol = threoninol residue : z=benzyloxycarbonyl Example 1 CH ₃ − (CH ₂ ) ₈ −CO−(D)Phe−Cys− (MBzl)−
Phe−(D)−Trp−Lys(z)−Thr−Cys−(MBzl)
-Thr-ol 7.1g and thioanisole 50ml were added to TFA (0
°C) Dissolve in 120ml. Cool the solution to −10°C,
Add 92 ml of approximately 2 M BTFA in TFA and bring the solution to −10 °C.
Stir at ~-5°C for 1.5 hours. This mixture is anhydrous
Add to 400ml of MeOH (-70℃) with stirring,
After 5 minutes, hydrochloric acid/ethyl ether (approximately 5N)
Stir with a mixture of 20 ml and 9000 ml of anhydrous ethyl ether. Take the precipitated product and wash it with ethyl ether.
Quickly dissolve in 16000 ml of dioxane/water (7:3). While stirring, add 4N ammonium hydroxide until the pH reaches 7-7.5, and pour this solution into an open container for -SH group testing (e.g. Ellmann method).
Stir until it is negative. The pH is adjusted to 3-4 by adding dilute hydrochloric acid, and the solution is concentrated under reduced pressure and freeze-dried. The crude product was purified on silica gel with chloroform/MeOH/
Purify by chromatography using an AcOH/water mixture. Fractions containing the desired product were combined, diluted with water, concentrated and lyophilized to give the title compound as pure. [α] ²⁰ _D = −43.7° (in 95% AcOH,
c=0.92). The starting material was produced by the following method. (a) Production of BOC-Cys(MBzl)-Thr-ol: -N
-Methylmorpholine 2.1ml BOC-Cys(MBzl)
A solution of 6.3 g of -OH in 50 ml of THF is added with stirring, and then 2.4 ml of chloroformic acid isobutyl ester are added dropwise at -15°C. After stirring at -15°C for 5 minutes, a solution of 3.3 g of H-Thr-ol hydrochloride and 4.1 ml of N-methylmorpholine in 30 ml of DMF (previously -
(cooled to 10℃). Mixture at 0℃
Stir at room temperature for 2 hours and then at room temperature for 2 hours. to this
Add 50 ml of 10% potassium bicarbonate solution and concentrate the whole under reduced pressure. After dilution with AcOEt, 2N
Washed three times with citric acid, three times with potassium bicarbonate solution and once with sodium chloride solution, the organic layer was dried over sodium sulphate and evaporated under reduced pressure to give the title compound. [α] ²⁰ _D = −30° (DMF
Medium, c = 1.3). (b) Production of H-Cys(MBzl)-Thr-ol trifluoroacetate: - 7.1 g of BOC-Cys(MBzl)-Thr-ol and 5 ml of thioanisole are dissolved in 25 ml of methylene chloride.
Add the solution to 50 ml of TFA and leave at room temperature for 20 minutes. The mixture was diluted with approximately 1500 ml of ethyl ether and the precipitated product was collected and dried to give the title compound. [α] ²⁰ _D = 8.3° (in 95% AcOH, c = 1.1);
MP=152℃ (c) Production of BOC−Thr−Cys(MBzl)−Thr−ol:
- 5.9 ml of chloroformacetic acid isobutyl ester,
BOC−Thr− pre-cooled to −25℃
9.7g of OH and 9.4ml of N-methylmorpholine
Add dropwise to 50ml THF solution. This solution was heated at −15℃
Stir for 5 minutes at
Add ml solution. The mixture is stirred at 0° C. for 2 hours and at room temperature for 2 hours. 20ml of 10% potassium bicarbonate
is added and the mixture is concentrated under reduced pressure. the product
Dilute with AcOEt and wash with 2N citric acid, 10% potassium bicarbonate solution, then 30% sodium chloride solution. The AcOEt layer was dried with sodium sulfate and evaporated under reduced pressure, and the residue was purified with MeOH/
Recrystallize from AcOEt/hexane. After subsequent washing with ether and drying, the title compound was obtained. [α] ²⁰ _D = -23° (in DMF, c = 1);
MP=117℃. (d) Production of H-Thr-Cys (MBzl)-Thr-ol trifluoroacetic acid: - 150 ml of TFA was cooled to 0°C in advance.
Add 16 g of BOC-Thr-Cys(MBzl)-Thr-ol and 17 ml of thioanisole to 100 ml of methylene chloride solution. The whole is left at room temperature for 20 minutes and stirred in ethyl ether. The precipitated product was collected, washed with ethyl ether, and dried to obtain the title compound. [α] ²⁰ _D = +0.6° (c = 1 in 95% AcOH);
MP=72℃. (e) Production of BOC−(D)TrP−Lys(Z)−OMe: −
H-Lys(Z)-OMe hydrochloride 23.3 in 300ml DMF
g, NEt ₃ 9.8ml, HOBT11.8g and BOC-
(D) Add 21.3g of Trp-OH and mix the solution at -15°C.
Cool to Add 15.6g of DCCI in 50ml of DMF,
The mixture is stirred at 0° C. for about 16 hours and then at room temperature for 2 hours. The reaction mixture is diluted with AcOEt/ethyl ether and the dicyclohexyl urea is separated off. Wash the solution with 2N citric acid, water, 10% potassium bicarbonate solution and 30% sodium chloride solution. Dry the organic layer over sodium sulfate and concentrate vigorously under reduced pressure. Add ethyl ether/
Recrystallization was performed by adding hexane, and the crystalline product was collected, washed with ethyl ether/hexane, and dried to obtain the title compound. [α] ²⁰ _D = −12.6° (DMF
Medium, c=1); MP=140°C. (f) Production of H-(D)Trp-Lys(z)-OMe hydrochloride: - 150ml of TFA was cooled to 0℃ in advance.
Add to a mixture of 30 g of BOC-(D)Trp-Lys(Z)-OMe and 150 ml of methylene chloride. 40 whole at room temperature
Stir for a minute, then add hydrochloric acid/ethyl ether (approximately
5N) and 4000 ml of ethyl ether. After thorough stirring, the precipitate was collected, washed with ethyl ether, and dried to obtain the title compound. [α] ²⁰ _D = -44° (c = 1 in 95% AcOH);
MP=101℃. (g) Production of BOC-Phe-(D)Trp-Lys(Z)-OMe:- NEt ₃ 9.5ml, HOBT13g and BOC-Phe-
17g of OH to H-(D)Trp-Lys(Z)-OMe hydrochloride
Add to a mixture of 35g and 350ml DMF. This solution is cooled to -20°C and a solution of 14.5 g of DCCI in 50 ml of DMF is added. The mixture is stirred at 0° C. for about 18 hours and then at room temperature for 1 day. Separate the precipitated dicyclohexylurea, concentrate the solution, dilute with methanol, and add water until a precipitate forms. After filtration, the residue was washed with MeOH/water (4:1) and
Drying gave the title compound. [α] ²⁰ _D = +1.1° (c = 1 in DMF); MP = 180°C (h) Production of BOC-Phe-(D)Trp-Lys(Z)-OH: - 26 ml of 1N sodium hydroxide , BOC−Phe−
(D)Trp-Lys(Z)-OMe was added to a suspension of 16 g and dioxane/water (8:2), and the mixture was stirred at room temperature for 1.5 g.
Stir for an hour. Add water to the resulting solution to approx.
Dilute to 500 ml and then concentrate under reduced pressure. While stirring, 1N hydrochloric acid was added to adjust the pH to 1.5-2, and the precipitated product was collected, washed with water until neutral, and dried to obtain the title compound. [α] ²⁰ _D = +
7.6° (c=1 in DMF); Decomposes at 85-90°C. (i) BOC−Phe−(D)Trp−Lys(Z)−Thr−Cys
Production of (MBzl)-Thr-ol:- NEt ₃ 2.5ml, BOC-Phe-(D)TrP-Lys(Z)
-OH11.2g and HOBT4g, H-Thr-
Cys(MBzl)-Thr-ol trifluoroacetate 9
g and 100 ml of DMF. A mixture of 13.7 g of DCC and 10 ml of DMF is added to this solution (-20°C) and the whole is stirred at 0°C for about 18 hours and at room temperature for 2 hours. The precipitated dicyclohexyl urea is separated off, the solution is concentrated under reduced pressure, and diluted with methanol. Water was added until the product precipitated, the precipitate was collected, washed with MeOH/water (4:1) and dried to give the title compound. [α] ²⁰ _D = −14° (DMF
Medium, c=1); MP=135°C. (j) H-Phe-(D)Trp-Lys(Z)-Thr-Cys
(MBzl)-Thr-ol・Production of hydrochloride:- BOC-Phe-(D)Trp-Lys(Z)-Thr-Cys
(MBzl)-Thr-ol 13g cold TFA/water (15:
1) Dissolve in 60ml and leave the solution at room temperature for 30 minutes. This mixture was stirred in a mixture of 3000 ml of ethyl ether and 20 ml of hydrochloric acid/ethyl ether (approximately 5N) and the precipitated product was collected, washed with ethyl ether and dried to give the title compound. [α] ²⁰ _D = -3° (in DMF, c = 1):
Decomposes at 110℃. (k) Production of BOC−(D)Phe−Cys(MBzl)−OH: −
BOC-(D)Phe-ONP7.73g as H-Cys(MBzl)
-OH4.83g dioxane/water (7:3) 100ml
and 20ml of 1N sodium hydroxide solution,
This mixture is stirred at room temperature for 20 hours. The reaction mixture is diluted with water and the dioxane is removed under reduced pressure.
Wash the aqueous layer with ether and adjust the pH to 2 by adding hydrochloric acid. The precipitated product was extracted with AcOEt/ethyl ether, the organic layer was washed with water, dried over sodium sulfate and evaporated under reduced pressure to give the title compound as a foam. [α] ²⁰ _D = −17° (DMF
Medium, c = 0.9). (l) Production of H-(D)Phe-Cys(MBzl)-OH/hydrochloride: - 5g of BOC-(D)Phe-Cys(MBzl)-OH
Dissolve in 80 ml of TFA and 10 ml of water and leave at room temperature for 45 minutes. Dilute this solution with ethyl ether and
Add 20ml of hydrochloric acid/ethyl ether (approximately 5N), collect the precipitate, wash with ethyl ether,
Drying gave the title compound. [α] ²⁰ _D = -38.5° (in 95% AcOH, c = 0.93); MP = 189°C. (m) CH ₃ − (CH ₂ ) ₈ −CO−(D)Phe−Cys
Production of (MBzl)-OH: - 23.3 ml of 1N sodium hydroxide was added to H-(D)Phe-
Cys(MBzl)-OH/hydrochloride 10.0g in dioxane
Add to 100ml of mixture. Add to this while stirring.
17 ml of CH ₃ (CH ₂ ) ₈ COC are added dropwise and the pH is maintained at 8 by simultaneously adding 1N sodium hydroxide. The resulting mixture is stirred at room temperature for 20 hours.
The pH is adjusted to 2 by adding 4N hydrochloric acid, concentrated under reduced pressure, diluted with water, and extracted with AcOEt.
The organic layer is washed with water, dried over sodium sulphate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using methylene chloride/MeOH as eluent to give the title compound.
[α] ²⁰ _D = −19.3° (in DMF, c = 2.6); MP124
℃. (n) _CH3- ( _CH2 ) ₈ -CO-(D)Phe-Cys
(MBzl)−Phe−(D)Trp−(Lys(Z)−Thr−Cys
Production of (MBzl)-Thr-ol:- 0.7 ml of N-methylmorpholine was added to H-Phe-(D)
Trp−Lys(Z)−Thr−Cys(MBzl)−Thr−
ol・hydrochloride 5.7g, HOBT0.8g and CH ₃ −
(CH ₂ ) ₈ −CO−((D)Phe−Cys(MBzl)−OH2.7
Add to 60 ml of DMF solution of g. This solution is −15
Cool to 0.degree. C., add 1.20 g of DCCI in 10 ml of DMF and stir the mixture at 0-4.degree. C. for 70 hours. Separate the precipitated dicyclohexyl urea and drain the liquid.
Dilute with MeOH and add water while stirring until the product precipitates. After about 2 hours, the filtered residue was washed with MeOH/water (2:1) and dried under reduced pressure to give the title compound. [α] ²⁰ _D = −20.5° (in DMF, c = 0.8). Examples 2 to 19 Compounds represented by the following formulas were obtained in the same manner as in Example 1 (all compounds are of the acetate type. The specific optical rotations of the respective acyl groups and their products are shown in the table below. )

【table】

【table】

[Table] Example 20 Linear N-acyl-polypeptides corresponding to the monocyclic polypeptides (i.e., positions 2 and 7) treated as in Examples 1 to 19, without final oxidation A product in which the -Cys- moiety was not bonded was obtained. For example, the product obtained from the operation of Example 1, excluding the final oxidation step, is as follows. _CH3− ( _CH2 ) ₈ −CO−(D)Phe−Cys−Phe−(D)
TrP-Lys- Thr-Cys-Thr-ol (acetate) [α] ²⁰ _D = -33.8° (c = 0.42% in 95% AcOH). The N-acyl-polypeptides of the invention and their pharmacologically acceptable salts exhibit useful pharmacological properties as demonstrated in animal studies. These active compounds can be found, for example, in serum in rats.
It exhibits GH-secretion inhibiting activity as shown by suppressing GH-concentrations. This test (Test) is performed on male rats under Nembutal anesthesia. At least four rats are used per dose group and the test compound is administered at doses that are staggered on a logarithmic scale. Sixty minutes after administration, the rats are decapitated to collect blood, and the serum GH concentration is measured by radioimmunoassay. In this test, the N-acyl polypeptides according to the invention have activity when administered at subcutaneous doses of 0.1 to 1000 μg/Kg. The above test can be applied to measure effects over long periods of time. For example, from 6 to 6 days after administration
It can be applied to measure its effectiveness by decapitating it after 18 hours. The N-acyl-polypeptides, T-form, are of particular interest in that they have activity for extended periods of time, not exceeding 18 hours, when administered at doses within the range of the tests described above. Therefore, the N-acyl-polypeptides and salts thereof of the invention can be used in the treatment of diseases consisting of or accompanied by excessive GH secretion, such as diabetes and vascular diseases and acromegaly. It is pointed out that it can be done. N-acyl-polypeptides and their salts also inhibit gastric and pancreatic juice secretion as shown by standard animal tests, such as the following test for gastric secretion. Male rats (220-289 g each) were kept in individual cages in the laboratory and fasted for 48 hours immediately before testing (but with free access to water) for several days (14 g per day).
hours of daylight irradiation). At the beginning of the test, the pylorus was tied with ether anesthesia and 100μ of pentagastrin was administered.
g/Kg subcutaneously to stimulate gastric secretion. At the same time, the test compound is injected intramuscularly and 30 minutes later the animals are sacrificed and the gastric fluid volume is measured and the gastric acid concentration is estimated (titrated with thymol blue). Calculate the amount of gastric acid secretion and calculate its % inhibition compared to the untreated control group. The N-acyl-polypeptides of the invention are active in the above tests when administered intramuscularly at doses of 0.1 to 1000 μg/Kg. The N-acyl-polypeptides of the invention and their salts are therefore indicated for use in the treatment of gastrointestinal diseases, such as gastric ulcers, gastrointestinal bleeding and acute pancreatitis. Pharmacologically acceptable salts of the polypeptides of the present invention exhibit activity comparable to that of the free compound in the above test methods. The daily dosage for the above uses is about 0.01 to 100 mg of the N-acyl-polypeptide, administered in 2 to 4 divided doses or as a sustained release form. In a suitable dosage unit form, about 0.0025 N-acyl-polypeptides of the invention
~50 mg or a pharmacologically acceptable salt equivalent thereof, together with a solid or liquid pharmacological diluent or carrier. It will be appreciated that the appropriate daily dosage for a particular compound in the present invention will depend on a number of factors, including relative activity. A preferred compound of the invention is the compound of Example 1, namely the formula: This is a compound represented by This compound, when administered in its acetate salt, has, for example, a test ID ₅₀ of 13.0 μg/Kg (s.c.) for activity at 60 minutes and 16.0 μg/Kg (s.c.) for activity at 6 hours. ),
28 μg/Kg (s.c.) in activity at 18 hours; ID ₅₀ value tested
At acid concentrations of 4.5 μg/Kg (intramuscular administration) or more
(Each ID ₅₀ measurement represents the amount of compound required to exhibit a 50% inhibitory effect on the measured parameter compared to the untreated control group.)
Known compound: Somatostatin has an ID ₅₀ measurement of 93 μg/Kg of activity at 60 minutes of the test.
(subcutaneous administration); 55 μg/kg in the gastric juice content of the test
Kg (intramuscular administration), 35μg/Kg in gastric acid concentration
(intramuscular administration). Therefore, the required daily dosage of the compound of Example 1 is approximately 0.5 to 500 μg/Kg (subcutaneous administration) when used as a GH secretion inhibitor.
When used as an inhibitor of gastric juice secretion or pancreatic juice secretion, the dose is about 0.15 to 150 μg/Kg (intramuscular administration). As described above, the present invention can provide the following therapeutic methods and pharmacological compositions for use therein: (1) The N-acyl-polypeptide of the present invention or a pharmacologically acceptable salt thereof. an effective amount of
Excess GH in patients who need to be treated, which consists of administering to patients who need to administer
- Treatment of diseases with etiologies involving or associated with excessive GH secretion (e.g. diabetes, vascular disease and acromegaly) and treatment of gastrointestinal diseases (e.g. gastric ulcers, gastrointestinal bleeding and acute pancreatitis) (2) The present invention A pharmaceutical composition comprising an N-acyl polypeptide or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable diluent or carrier therefor.

Claims (1)

[Claims] 1 Formula: N-acyl polypeptides and their _salts represented by [Formula, Acyl is _a group represented by (1)R〓CO- (where R〓 is _C1-20 alkyl, phenyl or _C1-4 alkyl] (2) a group represented _{by R〓SO2-} (where R〓 is phenyl substituted with _C1-10 alkyl, _phenyl or _C1-4 alkyl),
(3) A group represented by R〓NHCO- (where R〓 is C ₁ to ₁₀
alkyl) or (4) biotinyl, B is a phenylalanine residue whose benzene ring may be substituted with NO ₂ , F is a group represented by -NH-CH(R ₅ )-X (where R ₅ is −CH ₂ OH, −(CH ₂ ) ₂ OH, −(CH ₂ ) ₃ OH,
-CH( _CH3 )OH, isobutyl or benzyl,
X is a group represented by _-COOR1 , _-CH2OH or _-CONH2 , _R1 is _C1-3 alkyl), _Y1 and _Y2 are each _hydrogen or both are a direct bond, Z is _{C3- 4} alkyl or benzyl in which the benzene ring may be substituted with NO ₂ . In addition, 1st, 2nd, 4th, 6th, 7th and 8th place
The residue at position is (D). coordination or (L)-coordination. ] 2. N-acyl polypeptides and salts thereof according to item 1, wherein Acyl is a group represented by R〓CO- or _R〓SO2- . 3. The N-acyl polypeptides and salts thereof according to item 2, wherein R〓 is _C7-15 alkyl and _{R〓 is C7-10 alkyl .} 4. N-acyl polypeptides and salts thereof according to item 1, wherein Acyl is a group represented by R〓CO-. 5 formula The N-acyl-polypeptides and salts thereof according to item 1, represented by: