JPH0364516B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel vasotocin derivatives, namely vasotocin structures having
and/or vasotocin derivatives that differ from the natural hormone in that they are modified at the 8-position. The novel vasotocin derivatives of the present invention are race inhibitors that block the body's receptors, making it difficult for the body's endogenous hormones to exert their agonist effects. In particular, such new derivatives have an inhibitory effect on uterine contractions, and the inhibitory effect of these derivatives on uterine contractions induced by vasopressin or oxytocin has been tested in rats. Another important aspect of the invention is that the vasotocin derivatives of the invention also inhibit the natural contraction pattern of the uterus. The vasotocin derivative of the present invention has the following formula: (In the above formula, Mpa is a 3-mercaptopropionoyl residue (-S-CH ₂ -CH ₂ -CO-); A is D-tyrosine-O-ethyl ether (in other words, 4-ethoxy-D-phthalate); enylalanine,
Namely, D-Tyr (Et)), D-tyrosine (D-
Tyr) or D-tryptophan (D-Trp) peptide residue; Ile is isoleucine peptide residue; B is glutamine (Gln), threonine (Thr), or valine (Val) peptide residue; Asn is asparagine peptide residue Residue; Cys is a peptide residue of cysteine; Pro is a peptide residue of proline; C is a peptide residue of L- or D-arginine (Arg), ornithine (Orn), or citrulline (Cit); and Gly-NH ₂ is a glycinamide peptide residue). The vasotocin derivatives of the present invention can have L-arginine at position 8 like the natural hormone, but can also have D-arginine, L-ornithine or L-citrulline. The compound of the present invention can have a glutamine peptide residue or a threonine or valine peptide residue at the 4-position, similar to natural vasotocin. The deaminated cysteine at position 1 of vasotocin is shown as a 3-mercaptopropionyl residue (or _dNH2 ) in the compounds of the invention. The property of non-transducing agonists is believed to lie in the modification of the amino acid at position 2 of the natural hormone, which, like other posterior pituitary hormones, is crucial for biological activity. It is. If the side chain of a normal amino acid is modified at this position, the receptor will not accept the molecule as an agonist. Therefore, in order to obtain an antagonist, in the present invention, tyrosine present at the 2-position of natural vasotocin is replaced with D-tyrosine-O-ethyl ether (D-Tyr).
(Et)), D-tyrosine (D-Tyr) or D-tryptophan (D-Trp). Vasotocin hormone differs from oxytocin hormone simply in the amino acid at position 8. Oxytocin has a leucine at position 8, and vasotocin has a leucine at position 8.
It has arginine at the position. Among several other known oxytocin derivatives that have antagonistic effects on uterine contractions induced by oxytocin, 1-desamino-(2-O-ethyltyrosine)- according to U.S. Pat. No. 4,402,942; Oxytocin (hereinafter referred to as anthocin) can be mentioned as the closest compound to the novel vasotocin derivative of the present invention. Known drugs that can have an opposite effect on excessive muscle contractions in the uterus are β-receptors.
agonists, prostaglandin synthesis inhibitors, calcium antagonists, and ethanol. All these substances have non-specific effects and therefore produce side effects. The vasotocin derivative of the present invention can be used to treat premature birth or excessive uterine spasm [partus partus] in relation to childbirth and menstruation.
Of great clinical interest are cases of partus praematurs (preterm birth) or dysmenorrhea (dysmenorrhea).
Anthocin is also said to have such properties, and the vasotocin derivative of the present invention is 2 to 18 times more effective than anthocin (in tests conducted on rat uteruses in vivo). ). Therefore, in order to obtain the same effect, the vasotocin derivative of the present invention requires only 1/2 to about 1/18 of that of anthocin, which makes the vasotocin derivative of the present invention useful from a medical standpoint and economically. potentially extremely advantageous from an objective standpoint. The features of the invention will be apparent from the claims. The vasotocin derivatives of the present invention can be provided in the form of a formulation containing other pharmaceutically acceptable additives and/or diluents. Preferably, the vasotocin derivative is dissolved in physiological saline and administered by injection, infusion, or intraanal administration. The vasotocin derivatives of the present invention can be produced by methods similar to those well known in the peptide art. For example, the compound of the present invention can be used, for example, in Law, HB
and “Journal” by Du Vigneaud, V.
Zhuze, AL, in ``Of the American Chemical Society'' 82 , (1960) 4579-4581.
Jost, K., Kasafirek, E. and Rudinger, J. Collection of Ciekoslovak Chemical Communications 29 , (1963),
Technologies reported during 2648-2662 and
Larsson, L-E, Lindeberg, G., Melin, P.
and Pliska, V. Journal of Medicinal Chemistry 21 , (1978),
352-356, it can be prepared in a conventional manner by incremental coupling of amino acids to each other in the liquid phase. So-called peptides are formed by combining amino acids with each other. Such engagement also starts with the use of a solid phase (usually a resin) to which the C-terminal portion of the first amino acid is attached, and then the N-terminal portion of the first amino acid is attached to the C-terminal portion of the next amino acid.
- This can be done by, for example, joining the terminal parts. Finally, the formed peptide is released from the solid phase. The next embodiment of the present invention will be described. In the following examples, so-called solid phase technology was used. This technique is described by Merrifild, RB, “Journal
of the American Chemical Society” (1963) 85 , 2149, “Biochemistry”
(1964), 3 , 1385 and Konig, W., Geiger,
R., “Hemitssie Berichte” (1970) 103 , 788.
has been reported. For the synthesis, 1% cross-linked polystyrene resin (BioRad S-Xl, trade name) with a chloromethyl function loading capacity of 1.2 meq/g was used.
This resin was prepared in DMF (dimethylformamide) using KF (potassium fluoride) as a catalyst.
Coupling reaction with BocGly (Hosiki, K. et al., Chemistry Letters, (1978), 165-168) yielded a Bocgly-O resin with a chloromethyl function loading capacity of about 1 meq/g. All subsequent syntheses used this product as a starting material as described in the Examples below. All amino acids used are of the L form unless otherwise specified. Example 1 1-β-mercaptopropionic acid-2-(4-
ethoxy)-D-phenylalanine-8-arginine-vasotocin [X=H, A=D-Tyr(ET); B=Gln; C=
Arg] BocGly resin (3.0
g, 3 meq) was charged. Table 1 and 2
Peptides were produced in increments on the resin according to the table. Activation of amino acids was performed using 10 meq. of the appropriately protected amino acid, 15 meq. hydroxybenzotriazole and 10 meq. dicyclohexylcarbodiimide in DMF (70 meq.
ml) and then the mixture was allowed to stand at room temperature for 1 hour (asparagine and glutamine were dissolved at 0 °C).
(activated for 15 min), then separate the precipitate,
Thereafter, the solution was treated with activated amino acids as shown in Table 1 (7th step). The binding process is completed using the Kaiser method ("Analytical Biochemistry" 34 ,
595 (1970)), and then the cycle was completed (9th step). Test is positive (binding yield
(less than 99%), the cycle was repeated starting from step 7. If the test was negative, a shutdown process was performed as shown in Table 2. Once everything was bound, the resin was placed on a filter and washed repeatedly with methanol. The dried product was placed in a glass container, cooled in an ethanol-dryas bath, and suspended in methanol (approximately 100 ml). The mixture was then saturated with sodium-dry ammonia to a concentration of approximately 50%. This container was then placed inside a steel cylinder and heated at room temperature for 23 hours.
It was left undisturbed for a day. After releasing the pressure, the product was filtered and the residue was extracted with hot DMF (2 x 100ml) at about 100°C. The liquid and extract were combined and evaporated. The residue was dissolved in a small amount of hot DMF and methanol was added to cloud point. The precipitate was collected by filtration and washed with methanol on the filter. After vacuum drying, purity was confirmed by thin layer chromatography. The yield was about 2.8g. 100ml of 100mg of the above protected peptide
The mixture was placed in a round-bottomed flask and flushed with dry nitrogen for approximately 15 minutes. The protecting group was removed from the product by introducing 50 ml of sodium-dry ammonia and adding sodium until a blue color remained in the solution for 15 seconds. Excess sodium was destroyed by adding ammonium chloride. Ammonia was removed with a stream of nitrogen and the residue was dissolved in 1 part methanol. The pH of the solution was adjusted to about 4 with concentrated acetic acid, and the solution was then titrated with 0.1 mM iodine in methanol until a brown color. This mixture was stirred with Dowex® 50 x 2 chloride form ion exchanger for 10 minutes at room temperature. The ion exchange agent was removed by filtration and the liquid was evaporated to dryness. This residue was dissolved in 3 ml of 20% acetic acid and purified using Sephadex G-25 (trade name) and 20% acetic acid as the eluent. Final purification was performed by reverse phase HPCL. The purity of the product was determined using an HPCL column μ-Bondapak C-18 in 45% ethal and 55% 5mM trifluoroacetic acid in water. This column was supplied by Water Associates, Inc., Milford, Mass., USA. The purity of the product was also demonstrated by amino acid analysis results. Amino acid analysis results: Asn: 1.00, Glu: 1.01,
Pro: 0.98, Gly: 0.95, 1/2Cys: 1.06, Ile:
1.02, Tyr: 1.00, Arg: 0.98 By using a method similar to that described in Example 1, the compounds shown in the following Examples were synthesized. Example 2 1-β-mercaptopropionic acid-2-D-tryptophan-4-valine-8-arginine
Vasotocin [A=D-Trp; B=Val; C=Arg] Amino acid analysis results: Asp: 0.99, Val: 1.01,
Pro: 0.94, Gly: 1.00, 1/2Cys: 1.95, Ile:
0.80, Arg: 1.03 Example 3 1-β-mertaptopropionic acid-2-(4-
Ethoxy)-D-phenylalanine-4-valine-8-D-arginine Vasotocin [A=D-Tyr(Et); B=Val; C=D-Arg] Amino acid analysis results: Asp: 0.99, Pro: 0.90 ,
Gly: 1.00, 1/2Cys: 0.93, Val: 0.98, Ile:
0.81, Tyr: 0.84, Arg: 1.03 Example 4 1-β-mercaptopropionic acid-2-(4-
ethoxy)-D-phenylalanine-4-valine-8-ornithine vasotocin [A=D-Tyr(Et); B=Val; C=Orn] Amino acid analysis results: Asp: 0.99, Pro: 1.00,
Gly: 1.00, 1/2Cys: 1.00, Val: 0.98, Ile:
0.80, Tyr: 0.85, Orn: 0.94 Example 5 1-β-mercaptopropionic acid-2-(4-
ethoxy)-D-phenylalanine-4-threonine-8-ornithine vasotocin [A=D-Tyr(Et); B=Thr; C=Orn] Amino acid analysis results: Asp: 0.92, Thr: 0.93,
Pro: 0.94, Gly: 1.00, 1/2Cys: 0.92, Ile:
0.95, Tyr: 0.86, Orn: 1.01 Example 6 1-β-mercaptopropionic acid-2-D-tryptophan-4-threonine-8-D-arginine Vasotocin [A=D-Trp; B=Thr; C=D −Arg] Amino acid analysis results: Asp: 0.98, Thr: 0.98,
Pro: 0.99, Gly: 1.00, 1/2Cys: 1.08, Ile:
0.95, Trp: 0.64, Arg: 0.99 Example 7 1-β-mercaptopropionic acid-2-D-tyrosine-4-valine-8-ornithine batocine [A=D-Tyr; B=Val; C=Orn] Amino acid Analysis results: Asp: 0.99, Pro: 0.91,
Gly: 1.00, 1/2Cys: 1.00, Val: 0.97, Ile:
0.80, Tyr: 0.95, Orn: 0.98 Compounds of Examples 14 and 15 below were also produced in the same manner as in Example 1. Example 8 1-β-mercaptopropionic acid-2-D-tryptophan-4-valine-8-ornithine
Vasotocin [A=D-Trp; B=Val; C=Orn] Amino acid analysis results: Asp: 1.03, Pro: 1.02,
Gly: 1.00, 1/2Cys: 1.06, Val: 1.02, Ile:
0.77, Orn: 0.99, Trp: 0.93. Example 9 1-β-Mercaptopropionic acid-2-D-tryptophan-4-threonine-8-ornithine Vasotocin [A=D-Trp; B=Thr; C=Orn] Amino acid analysis results: Asp: 1.05, Thr: 1.00,
Pro: 1.05, Gly: 1.00, 1/2Cys: 1.06, Ile:
0.94, Orn: 0.98, Trp: 0.80. [Table] [Table] Lomethane + acetic anhydride 2 Isopropanol 70 3 2
3 Dichloromethane 70 3 4
Compounds prepared by the methods described in the Examples above were maintained in a lyophilized state until used in the pharmacological studies described below. Pharmacological Tests Compounds of the invention were tested for uterine tonic effects on isolated rat uteruses using oxytocin as a standard. This sample was also used to evaluate the antagonist properties of the compounds of the present invention. In vitro studies were also performed using oxytocin as the agonist, and the results were compared with those obtained using anthocin. In vitro tests on rats Sprague Dawley during natural estrus
Dawley rats (weighing approximately 250 g) were selected by vaginal smear. Cut out a section approximately 20 mm long from the center of the horn-like protrusion of the uterus, and add the following composition (mM: NaCl153, KCl5.63, _CaCl2 0.541,
10 with _NaHCO3 5.95 and glucose 2.78)
ml of denaturing lock solution. The lock solution was treated with oxygen containing 5% CO ₂ at 30°C. Uterine contractions were allowed to remain unchanged for 30 minutes. Glass force transducer (Ft03)
The same magnitude was recorded using a static tension of 1.5 g. Antagonist effects of congeners were calculated as _pA2 values (Rudinger, J. Krejci, I. “Experientia”
18, (1962), 585-588). _pA2 is a measure of the inhibitory effect of a peptide, and was described by Schild, HO, British Journal of Pharmacology 2 , (1947), 189-206, as a measure of the effect of a given dose of an agonist. It is defined as the negative logarithm of the molar concentration of an antagonist that decreases to the effect of 1/2 of its dose. The agonist action of the antagonist is determined by the presence of up to 4 μg/ml in the bath containing the uterine sample.
was investigated by adding various amounts of peptide corresponding to . No agonist effect was observed in any of these cases. In Vivo Studies on Rats Spragi-Dauley rats (250 g) in natural heat were narcotized with Inactin (0.5 mg/100 g body weight, administered intraperitoneally). Myometrial activity was recorded by fixing a catheter filled with modified Locke fluid in the uterine cavity. The catheter was connected to a Statham P23d transducer and
Grass Polygraph (Model 7D)
recorded. Oxytocin (0.05μg/min/body weight
100g) was injected intravenously. If a regular contraction pattern is obtained, the antagonist (0.8-8.0μ
g/100 g body weight) was administered intravenously in a volume of 0.2 ml. The recorded curves were integrated over a 15 minute interval immediately before and after injection of antagonist. The inhibitory effect of oxytocin injection on the increase in the degree of uterine contractions was compared with that of anthocin. The inhibitory effect of anthosin was given a value of 1. [Table] All vasotocin derivatives tested were listed in Table 3.
As evidenced by the _pA2 value, it caused competitive inhibition of oxytocin action, similar to anthocin.
(It should be noted that pA ₂ values obtained in different laboratories cannot be compared as the test conditions are not exactly the same.) The antagonistic activity values obtained for pA ₂ has a larger value than anthosin, indicating that it is a more potent antagonist. In vivo tests show that the derivatives of the invention are antagonists with effects that are 2 to about 18 times more potent than anthocin. The vasotocin derivatives listed in Table 3 were able to completely inhibit the uterine contraction pattern in in vivo tests, ie, these derivatives inhibited the natural activity of the organ's muscles in addition to the activity produced by oxytocin. The dose was 2-20 μg/rat. Table 3 also shows the antidiuretic effect (AD) and the vasopressor effect (i.e. the effect on blood pressure), and since the values obtained are low in most cases, the vasotocin derivatives of the invention are used to inhibit uterine contractions. This indicates that there is no need to take these effects into consideration when administering the drug. Another important aspect is that all vasotocin derivatives of the present invention did not exhibit agonist activity when tested on ex vivo rat uterine samples. Therefore, it is thought that these derivatives do not cause any side effects. Example of drug production 0.5 mg of a vasotocin derivative was weighed and dissolved in distilled water together with 5 mg of mannitol. This solution was poured into ampoules, sealed, and lyophilized. After storage under lyophilization conditions, the contents of the ampoule could be diluted with isotonic saline to a concentration suitable for administration. The present invention relates to: (In the above formula, Mpa is a 3-mercaptopropionoyl residue group (-S-CH ₂ -CH ₂ -CO-); A is D-tyrosine-O-ethyl ether (in other words, 4-ethoxy-D- phenylalanine,
Namely, D-Tyr (Et)), D-tyrosine (D-
Tyr) or D-tryptophan (D-Trp) peptide residue; Ile is isoleucine peptide residue; B is glutamine (Gln), threonine (Thr), or valine (Val) peptide residue; Asn is asparagine peptide residue Residue; Cys is a peptide residue of cysteine; Pro is a peptide residue of proline; C is a peptide residue of L- or D-arginine (Arg), ornithine (Orn), or citrulline (Cit); and Gly-NH ₂ is a vasotocin derivative (denotes a glycinamide peptide residue). In the above formula, A is D-Tyr (Et), B is Thr, and C
indicates Orn, a vasotocin derivative. In the above formula, A is D-Tyr(Et), B is Val, and C
indicates Orn, a vasotocin derivative. In the above formula, A is D-Trp, B is Val, and C is
Vasotocin derivative showing Orn. A medicament comprising one or more of the above-mentioned vasotocin derivatives as an active ingredient in combination with pharmaceutically acceptable additives and/or diluents. Said drug wherein saline is present as a pharmaceutically acceptable diluent. A solution suitable for intravenous administration containing a vasotocin derivative as an active ingredient in physiological saline. A solution suitable for intraanal administration, which is prepared by dissolving the above-mentioned vasotocin derivative in physiological saline. A method of counteracting excessive muscular contractions of the uterus, comprising administering a drug containing an effective amount of one or more vasotocin derivatives. Vasotocin derivatives as described above for use in the treatment of excessive muscle contractions of the uterus.

Claims (1)

[Claims] Primary formula: (In the above formula, Mpa is a 3-mercaptopropionoyl residue (-S-CH ₂ -CH ₂ -CO-); A is D-tyrosine-O-ethyl ether (D-
Tyr (Et)), D-tyrosine (D-Tyr) or D
-Tryptophan (D-Trp) peptide residue; Ile is isoleucine peptide residue; B is glutamine (Gln), threonine (Thr) or valine (Val) peptide residue; Asn is asparagine peptide residue; Cys is a peptide residue of cysteine; Pro is a peptide residue of proline; C is a peptide residue of L- or D-arginine (Arg), ornithine (Orn), or citrulline (Cit); and Gly- _NH2 is a peptide residue of glycinamide. A vasotocin derivative characterized by being represented by (indicating a peptide residue).