WO2002000688A1 - Peptide compound and pharmaceutical compositions and medicines containing the same as the active ingredient - Google Patents

Abstract

Peptide compound (I) or pharmacologically acceptable salts thereof exhibit selective antagonism against oxytocin receptor more than against vasopressin receptor and are effective in the prevention or treatment of threatened premature delivery.

Description

 Description Peptide compounds, pharmaceutical compositions and pharmaceuticals containing them as active ingredients

 The present invention relates to peptide compounds. More specifically, the present invention relates to a peptide compound having a selective oxytocin receptor antagonistic activity or a pharmacologically acceptable salt thereof, a pharmaceutical composition containing the same as an active ingredient, and a drug. The peptide compound provided by the present invention or a pharmacologically acceptable salt thereof is particularly useful for preventing or treating imminent preterm birth. Background art

 Imminent premature birth is a major cause of prenatal morbidity and mortality, but methods to prevent existing premature birth are not successful and can have significant side effects.

 One of the major causes of this imminent preterm birth is the uterine contractile effect of oxytocin. Since the action of oxytocin is thought to be mediated by the oxytocin receptor on the cell membrane, a substance that antagonizes the binding of oxytocin to its receptor is useful as an agent for preventing or treating imminent premature birth.

 In addition, oxotocin receptor antagonists often also antagonize the receptor for vasopressin, an antidiuretic hormone with a similar structure to oxytocin. Therefore, selective antagonism of the oxytocin receptor is an essential condition as a preventive or therapeutic agent for imminent premature birth with less side effects such as lowering of blood pressure.

Conventionally, several oxytocin receptor antagonists have been reported. Examples include Endocrology, si, 267 (1967), Endocrinology, loe, 81 (1980), J. Med.Chem., 26, 1607 (1983), and Endocrinology, 88, 173 (1981). Obs tet. And Gynecol. Scand., 64, 499 (1985) report a oxytocin antagonist. But However, these are not sufficient in terms of activity and selectivity for oxytocin receptors.

Further, Pept. 1994, Proc. Eur . Pept. Symp., In 23 ^rd (1995), although O key cytosine antagonists represented by the following chemical formula is published, the compounds provided in the present invention (I) and the C-terminal structure is different.

In addition, Japanese Patent Application Laid-Open No. H10-077995 reports a compound represented by the following chemical formula (Π) having an oxytocin receptor antagonistic activity equivalent to that of compound (I). It has glycinamide at the C-terminus and has a different structure from compound (I).

発明の開示

Disclosure of the invention

 The present inventors have conducted intensive studies to provide compounds having potent and selective antagonism against oxytocin receptors, and as a result, have found that certain peptide compounds or their pharmacologically acceptable They found a salt and completed the present invention.

 That is, a first aspect of the present invention provides a peptide compound represented by the following chemical formula (I) or a pharmacologically acceptable salt thereof.

本発明の第 2は、 本発明の第 1記載の化合物またはその薬理学的に許容される 塩を有効成分とするォキシトシン受容体拮抗剤を提供する。

A second aspect of the present invention provides an oxytocin receptor antagonist comprising, as an active ingredient, the compound according to the first aspect of the present invention or a pharmacologically acceptable salt thereof.

 A third aspect of the present invention provides a pharmaceutical composition for preventing or treating impending premature birth, comprising the compound according to the first aspect of the present invention or a pharmacologically acceptable salt thereof as an active ingredient. A fourth aspect of the present invention provides a prophylactic or therapeutic agent for imminent premature birth, comprising the compound according to the first aspect of the present invention or a pharmacologically acceptable salt thereof as an active ingredient.

 A fifth aspect of the present invention provides an oxytocin receptor antagonist which becomes a compound according to the first aspect of the present invention or a pharmacologically acceptable salt thereof in a living body.

 A sixth aspect of the present invention is to provide a pharmaceutical composition for preventing or treating imminent premature birth, comprising, as an active ingredient, the compound according to the first or a pharmacologically acceptable salt thereof in vivo. provide.

 A seventh aspect of the present invention provides a preventive or therapeutic agent for imminent premature birth, comprising the compound according to the first or a pharmacologically acceptable salt thereof as an active ingredient in a living body. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, embodiments of the present invention will be described.

 First, the method for producing compound (I) will be described using some abbreviations, and the contents of the abbreviations are as follows.

 (Optical isomer)

 When amino acids and the like used in the present specification have optical isomers, "D-" is added only when D-form is used, and L-form is shown unless otherwise specified.

 (Amino acid residue)

 Arg: arginine, Asn: asparagine, lie: isoleucine, G1n: glumin, G1y: glycine, Pro: proline, Trp: tryptophan, Pen: penicillamine

(Protecting group) Bo c: t-butoxycarponyl, MBz 1: 4-methoxybenzyl, Mt s: mesitylenesulfonyl

 (Dehydration condensation coupling reagent)

 HOB t: 1-hydroxybenzotriazole, DCC: dicyclohexyl carbodiimide

 (Solvent)

 DIEA: diisopropylethylamine, DCM: dichloromethane, TFA: trifluoroacetic acid, DMF: N, N-dimethylformamide

 (Other)

 EM: Ethyl mercaptan, PAM resin: Phenylacetamidomethylated resin, TPA (MB z 1): Compound having the following structure

 Compound (I) can be synthesized by a solid phase method or a liquid phase method which is a general peptide synthesis method. There are no general rules for selecting the solid phase method or the liquid phase method, and the synthesis method is selected based on the physical properties and required amount of the target peptide.

In the synthesis of compound (I) by the solid phase method, as shown in the Examples below, a protected amino acid is formed on a PAM resin by the active ester method or TBTU (2- (1H-benzotriazole-1-yl) _1,1). , 3,3-tetramethylperonium tetrafluoroborate) method can be repeated to construct peptide chains. After removing all protecting groups other than CHO (formyl) group on the side chain of D-Trp residue by liquid hydrogen fluoride (HF) treatment and dissociating the peptide from the resin, the CHO group was converted to ammonium acetate buffer ( It can be removed at pH 9.47) and cyclized with potassium ferricyanide. This The crude peptide thus obtained can be fractionated and purified using reverse phase HPLC to obtain compound (I).

 In the present invention, the “pharmacologically acceptable salt” of the compound (I) includes conventional non-toxic salts, that is, acid addition salts and salts with various bases. More specifically, inorganic acid salts such as hydrochloric acid, nitric acid, and sulfuric acid; organic acid salts such as acetic acid, citric acid, fumaric acid, and tartaric acid; sulfonic acid salts such as methanesulfonic acid and p-toluenesulfonic acid; and alanine and leucine Inorganic base salts such as glutamic acid and the like, alkali metal salts (eg, sodium salt, potassium salt, etc.), alkaline earth metal salts (eg, magnesium salt, calcium salt, etc.) and triethylamine salts, pyridine salts, Organic amine salts such as picolin salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N, N, dibenzylethylenediamine salt and the like. Compound (I) may be isolated as a hydrate or solvate or a polymorphic substance, and these are also included in the present invention.

 Since compound (I) has a strong antagonistic effect on oxytocin receptor, it can be used as an agent for preventing or treating imminent premature birth.

 Compound (I) may be administered parenterally by injection, nasal drop, eye drop, poultice, ointment, cream or suppository, or tablet, capsule, granule, powder, inhalant or the like. Oral administration using a syrup or the like can be mentioned. In preparing these formulations, they can be manufactured by a conventional method using a pharmaceutically acceptable carrier.

The dose of compound (I) as a drug is appropriately determined in consideration of the degree of symptoms, general condition of the patient, age, body weight, administration route, dosage form, and the like. In the case of parenteral administration, it is usually 2 g to 20 mg / kg, preferably 20 g to 2 mg / kg per day in terms of the amount of (I). In addition, it is usually 18 to 101] 18 / ¾: once, preferably 108 to 1111¾. In the case of oral administration, it is usually 10 zg to 100 mg per day. And preferably from 100 g to 10 mg / kg. In addition, the dose is usually 5 ^ g to 50mgZkg, preferably 50 / _ig to 5mg / kg. Example

 Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

 For compound (I), a peptide chain was constructed on a resin by a solid phase method using a Boc amino acid using a peptide synthesizer (430A type manufactured by ABI). The reverse phase HP LC analysis conditions at that time are shown below.

Column: TSK gel ODS-120T (4.6 X 250mm, Toso Ichisha) Eluent: TFA 0.1% aa. Soln./CH ₃ CN = 70/30 → 50/50 (v / v, 30 min. )

 Detection wavelength: 214 nm, Flow rate: 1. Oml / min.

 The carrier resin, protected amino acids and reagents used in the synthesis are shown below.

B oc -A rg (Mt s) — OCH ₂ — PAM resin (Bo c — Ar g (Mt s): 0.64 mm o 1 /, 0.5 mm o 1), Bo c — Pro — OH , Bo c— L— Pen (MB z 1) -OH, Bo c_As n -〇H, Bo c— G in -OH, B oc -I 1 e-OH, Bo c— D-Tr p (CHO) -OH, TP A (MB z 1)

 Further, an active ester method or a TBTU method was used for the condensation reaction. Each method is described below.

 (Active ester method using HOB t)

 The protected amino acid to be introduced, HOBt and DCC were all 2 mmol, and the condensation reaction time was set to 120 minutes.

(TBTU method) The protected amino acid to be introduced, HOBt and TBTU were all used at 2 mmol, DI EA was used at 4 mmol, and the condensation reaction time was set at 120 minutes. Furthermore, the completeness of the reaction when each amino acid was introduced was confirmed by the Kaiser test (Isatin test when Pen (MBz1) residue was introduced).

[1] Solid phase synthesis

(1) Boc-A rg (Mt s) — OCH ₂ — PAM resin (0.5 mmo 1) was added to the reaction vessel of the synthesizer, washed three times with 10 ml of DCM, left overnight in DCM, The resin was sufficiently swollen. After filtration, 10 ml of a 50% TFA_ / DCM solution was added to the above resin, and the mixture was stirred for about 2 minutes. After filtration, 10 ml of a 50% TFA / DCM solution was further added, and the mixture was stirred for 30 minutes to perform a Boc removal reaction. After completion of the reaction, the TFA solution was filtered, washed three times with 10 ml each of DCM, and filtered.

 (2) The remaining TFA was removed by neutralizing twice with 10 ml each of a 10% DI EAZDMF solution to release amino groups. Further, washing and filtration were repeated 6 times with 10 ml each of DMF, and the remaining DIEA was removed.

 (3) Next, Boc-Pro-OH was activated in parallel with the above operations. That is, Boc—Pro—OH (431 mg, 2 mmol) and HOB t (306 mg, 2 mmol) were dissolved in 4 ml of DMF, added to another reaction tank, and 4 ml of 0.5 M DCC / DCM solution was added. The mixture was added and stirred under a nitrogen stream for about 40 minutes to lead to an activated ester.

(4) The obtained activation solution was added to the reaction vessel containing the resin and stirred to start the condensation reaction. The reaction time was 120 minutes. After the completion of the condensation reaction, the resultant was washed three times with 10 ml each of DMF and further six times with 10 ml each of DCM, followed by filtration. After washing and filtration, a part of the resin was subjected to a Kaiser test. With the introduction of one amino acid, the condensation reaction was repeated until the Kaiser test (or Isatin test) became negative. In the second and subsequent condensation reactions (recoupling), the steps except for the above (1) are repeated, but in the case of the active ester method, 10% DI EA / D The washing of the MF solution was performed only once, and the above operation (2) was not performed in the TBTU method.

 After introducing all the amino acids, the obtained protected peptide resin was sufficiently washed with an appropriate amount of DCM, dried under reduced pressure (yield: 127 Omg), and subjected to HF treatment.

 The amounts of the protected amino acids and reagents used in this synthesis are shown in Table 1 below. Reaction time

1- -1 Boc-Pro-OH 2 Substitute ol HOBt 2 thigh ol DCC 2 listen ol 2hr

-2 Boc-Pro-OH 2 Marl ol HOBt 2 thigh ol DCC 2 Awakening ol 2hr

2- -1 Boc-Pen (MBzl)-OH 2 thigh ol HOBt 2 thigh ol DCC 2 marauder ol 2hr

-2 Boc-Pen (MBzl) -OH 2 Fuji ol HOBt 2 strokes 1 TBTU 2 Recommended ol awake 4 strokes 1 2hr

3- -1 Boc-Asn-OH 2 ol ol HOBt 2 hire ol DCC 2 mmol 2 hr

-2 Boc-Asn-OH 2 thigh ol HOBt 2 good ol DCC 2imol 2hr

4- -1 Boc- Gin-OH 2 Substitute ol HOBt 2 Marl ol DCC 2 Dragon ol 2 r

-2 Boc-Gin-OH 2 thigh ol HOBt 2 cough 1 DCC 2 舰 ol 2hr

5- -1 Boc - lie - OH · 1 / 2H 2 0 2 Hall ol HOBt 2 Yuzuru ol DCC 2 strokes ol 2 hr

-2 Boc-lie- OH1 / 2 H ₂ 0 2 orchid ol HOBt 2 sol ol DCC 2 dust ol 2 hr

-3 Boc-lie- OH l / 2¾0 5 thigh ol HOBt 5 strokes 1 DCC 2 marauder ol 70 r

6- -1 Boc-D-Trp (CHO) -OH 2 Customer ol HOBt 2 Restaurant 1 DCC 2 Marauder d 2hr

-2 Boc-D-Trp (CHO) -OH 2 Marl ol HOBt 2 dish ol DCC 2

-3 Boc-D-Trp (CHO) -OH 2 marl ol HOBt 2 dragon ol TBTU 2 substitu ol DIEA 4 舰 ol 2hr

7- -1 TPA (MBzl) 2 t ol HOBt 2 t ol DCC 2 ol 2 hr

-2 TPA (MBzl) 2iimol HOBt 2mmol DCC 2 丽 ol 6hr

-3 TPA (MBzl) 2mmol HOBt 2mmol TBTU 2 hall ol DIEA 4 excitation 1 2hr [2] liquid HF treatment

 Dissociation of the peptide from the resin and removal of all protecting groups except for the CHO group of the Trp (CHO) residue were performed by liquid HF treatment.

Liquid HF treatment: Add 127 Omg of the protected peptide resin obtained in [1] and a Teflon-coated magnet to an HF reaction vessel, add 1.3 ml of anisol and 3 ml of EMI. Leave for 30 minutes at room temperature under reduced pressure. did. The reaction vessel was cooled with a refrigerant, 13 ml of double distilled HF was added, and the mixture was stirred at 120 ° C. for 120 minutes. HF was distilled off under reduced pressure (1 hour with an aspirator, 2 hours with a vacuum pump), and 30 ml of getyl ether was added to the residue, followed by stirring until smooth. After suction filtration, it was washed three times with 30 ml each of getyl ether and dried under reduced pressure. .

 [3] Extraction and de-CHO treatment

 The peptide-resin mixture obtained in [2] was sufficiently added to 30 ml of 1M acetic acid which had been sufficiently degassed and cooled, and stirred to dissolve the peptide component. After suction filtration, the filtrate was quickly poured into 150 Oml of pure water. The filtered material on the glassfill was further washed three times with 30 ml each of cold 1 M acetic acid, and quickly added to the pure water. To this diluted aqueous solution, 13 ml of 28% aqueous ammonia was dropped to obtain ρΗ9.47. The mixture was stirred at 24.5 for about 24 hours to carry out a C C-C reaction.

 [4] cyclization reaction

After confirming the completion of the de-CHO reaction in [3], K ₃ [F e (CN) ₆ ] 25 Omg / Ί5 ml H ₂₀ was added dropwise to the reaction solution, followed by stirring at 24.5 ° C. for 1 hour. After confirming the disappearance of the raw materials, 15 g of a strong anion exchange resin “AG1-X2 resin CI type dry mesh 200-400” manufactured by BIO-RAD was added, and the mixture was further stirred for 1 hour. After confirming that the reaction solution had changed from yellow to colorless, 75 ml of 1M acetic acid was added to adjust the pH to 6.3. The resin was removed by suction filtration and washed three times with 50 ml each of pure water.

 [5] desalination

The filtrate and washings obtained in [4] were added to a reverse-phase ODS column, and TFA0. It was washed with 1% ad. Soln. And desalted. Further, elution was performed with TFAO. 1% a. Soln./CH ₃ CN (vZv, 50/50), and the eluted fraction was collected. After CH ₃ CN was distilled off under reduced pressure, the residue was freeze-dried to obtain 124 mg of a crude peptide.

 [6] Purification by reversed-phase HP LC

 1 Omg of the crude peptide obtained in [5] was dissolved in TFAO. 1% aa. Soln. 3001, added to a reversed-phase column, and eluted under the following conditions to separate the fraction containing compound (I). I took it. The conditions of HP LC are described below.

 Column: TSK gel ODS-120T (21.5 X 300mm, manufactured by Tosoh Corporation)

Eluent:... TFA0 1% aQ soln / CH 3 CN = 65 / / 35 (v / v, 30m in.)

 Detection wavelength: 214 nm, Flow rate: 8.0 m 1 / min.

The above procedure was repeated a total of eight times. The fractions collected were collected, CH ₃ CN was distilled off under reduced pressure, and the residue was lyophilized and purified to obtain the desired purified peptide (N-[(4-mercaptocyan-4-yl) acetyl] 1-D-Tributyryl-1 L-1 Isoleucyl-1 L-Gluminyl-1 L-asparaginyl-1-3-mercapto-L-valyl-L-prolyl-L-arginine (S, S) disulfide 47.5 mg (Yield) : 59.4%).

MSm / z: 1 1 17 ((3 _{49 3} 0 丄 S ₃ + H)

 Amino acid composition analysis (6 N HC1, 110 ° C, 24 hours): Asp (1) 1.02, Pro (1) 0.97, lie (1) 0.97, Arg (1) 1 . 01

(Test example)

 1. Effect on oxytocin-induced uterine contractile response in estrous rat uterine muscle [Effect on oxytocin (OT) receptor]

Estradiol lmg / kg was subcutaneously administered to SD female rats. Twenty-four hours later, the uterine horn was removed, a small section was prepared, and immediately suspended on a Magnus apparatus. Organ '

(. Munsick soln) nutrient solution of 10 m 1 to the bus meets the 95% 0 ₂ at 37 ° C - lasted bubbled 5% C0 ₂ gas mixture. The specimen subjected to suspended load 1 g, allowed to stand 1 one 2 hour, was administered 10_ ⁹ M OT in organ bath. After observing stable contraction waveform by OT, Compound (I) or the compound as a control compound (II) obtained by accumulating administered final concentration 10_ ^1Q ~10- ⁶ M respectively. After administration of the drug, a contraction waveform was observed for 5 minutes, and the amplitude and frequency for 5 minutes were measured. Table 1 shows the results of compound (I) and compound (II). Compound (I) inhibited oxytocin-induced uterine contractile response in a dose-dependent manner, with IC ₅ . Value (a value representing the concentration of antagonist that inhibits 50% of the reaction by § Gore two strike alone) was 3. 18 X 10_ ^S M.

2. Action on vasoconstriction by vasopressin stimulation using rat extirpated aorta [Action on vasopressin VIa receptor (hereinafter abbreviated as VIa receptor)] 'Rat abdominal aorta was isolated Later, a ring specimen was prepared and immediately suspended on a Magnus device. (. Munsick soln) Organ nutrient solution buses 1 Om 1 meet, 37 ° C with 95% 0 ₂ - lasted bubbled 5% C0 ₂ gas mixture. Applying a suspension load of 0. 5 g is the specimen, administered 10- ⁸ M arginine vasopressin the (AVP) in organ bath, were observed vasoconstriction. Compound (I) or a control compound was cumulatively administered to a final concentration of 1 0 one ⁹ ~ 10_ ⁵ M, respectively. With respect to the vasoconstriction response by AVP, the relaxation response by each drug was measured. Table 2 shows the results of compound (I) and compound (II). Compound (I) dose-dependently inhibited AVP-stimulated vasoconstriction, with IC ₅ . The value was 8. 70X10 one ⁷ M. Table 2

産業上の利用可能性

Industrial applicability

 As described above, Compound (I) has a selective antagonism of oxytocin receptor over vasopressin receptor as compared with conventional compounds. Can be provided.

 (This application was filed with a priority claim based on Japanese Patent Application Number: Japanese Patent Application No. 2000-195332.)

Claims

The scope of the claims 1. Peptide compounds represented by the following chemical formula (I) or pharmacologically acceptable ■ Ίπτ.

 2. An oxytocin receptor antagonist comprising the compound according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient.  3. A pharmaceutical composition for preventing or treating impending premature birth, comprising the compound according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient.  4. A prophylactic or therapeutic agent for imminent preterm birth comprising the compound according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient.  5. An oxytocin receptor antagonist which is a compound according to claim 1 or a pharmacologically acceptable salt thereof in a living body.  6. A pharmaceutical composition for preventing or treating impending premature birth, comprising as an active ingredient the compound according to claim 1 or a compound that is a pharmacologically acceptable salt thereof in a living body.  7. A preventive or therapeutic agent for imminent premature birth comprising the compound according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient in vivo.