EP0247033A1

EP0247033A1 - Conformationally constrained oxytocin antagonists with prolonged biological activities

Info

Publication number: EP0247033A1
Application number: EP19850905687
Authority: EP
Inventors: Victor J. Hruby; Todd W. Rockway
Original assignee: Gibson-Stephens Neuropharmaceuticals Inc
Current assignee: Gibson-Stephens Neuropharmaceuticals Inc
Priority date: 1985-10-25
Filing date: 1985-10-25
Publication date: 1987-12-02
Also published as: WO1987002676A1

Abstract

Polypeptides de formule (I) où X est hydrogène ou -NH2; Y et Z sont indépendamment soufre ou CH2, R1 et R2 sont hydrogène, méthyle, cyclopentaméthylène ou un groupe alkyle possédant de 1 à 5 atomes de carbone; R3 est Phe, D-Phe, Tyr, D-Tyr, Tyr(OMe), D-Tyr(OMe), Tyr(OEt), D-Tyr(OEt), Phe(4-Me), D-Phe(4-Me), Phe(4-Et) or D-Phe(4-Et); R4 est Gln, Thr, Ser, Asn, ou Val; et R5 est Orn, Arg, Lys, Leu, ou Ile; à condition toutefois que lorsque X est hydrogène et R3 est Tyr(OMe), R4 ne puisse pas être Thr ou lorsque X est hydrogène, R3 est Phe et R4 est Asn, R5 ne puisse pas être Orn. Ces polypeptides sont des antagonistes de l'oxytocine et sont utiles en tant qu'inhibiteurs des contractions induites par l'oxytocine chez les mammifères.Polypeptides of formula (I) where X is hydrogen or -NH2; Y and Z are independently sulfur or CH2, R1 and R2 are hydrogen, methyl, cyclopentamethylene or an alkyl group having 1 to 5 carbon atoms; R3 is Phe, D-Phe, Tyr, D-Tyr, Tyr (OMe), D-Tyr (OMe), Tyr (OEt), D-Tyr (OEt), Phe (4-Me), D-Phe (4 -Me), Phe (4-Et) or D-Phe (4-Et); R4 is Gln, Thr, Ser, Asn, or Val; and R5 is Orn, Arg, Lys, Leu, or Ile; provided, however, that when X is hydrogen and R3 is Tyr (OMe), R4 cannot be Thr or when X is hydrogen, R3 is Phe and R4 is Asn, R5 cannot be Orn. These polypeptides are oxytocin antagonists and are useful as inhibitors of oxytocin-induced contractions in mammals.

Description

CONFORMATIONALLY CONSTRAINED OXYTOCIN ANTAGONISTS WITH PROLONGED BIOLOGICAL ACTIVITIES This invention relates to polypeptides which are analogs of the hormone oxytocin. The polypeptides are biologi¬ cally long-acting, oxytocin antagonists which inhibit oxytocin induced uterine contractions.

The government has rights in this invention pursuant to National Science Foundation Grant No. 7807222 and United States Public Health Service Grant No. AM17420.

BACKGROUND OF THE INVENTION Naturally occurring oxytocin is the hormone believed to be responsible for the induction of labor and uterine contrac¬ tions in mammals. Oxytocin has the following formula:

1 1

(H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂) .

Most peptide hormones and neurotransmitters, like oxytocin, display their biological activities via membrane-bound receptors on a cell surface. A receptor is that entity, on a cell, which recognizes and binds a chemical substance. Once a chemical substance, such as a drug, binds to the receptor, it may act to initiate or block various biochemical an physiological se¬ quences. Such initiation or blockage is known as transduction.

It is believed that binding and transduction each in¬ volve different hormone-receptor interactions. For example, com¬ petitive antagonists, or inhibitors, are molecules that bind to the receptor but do not transduce the biological system to pro¬ duce a response. Thus, in other words, antagonists block the action of the native hormone. It is, therefore, critical in drug design to develop a drug which has a structure and conformation specific to a particular receptor and specific for either binding or transduction. For example, a hormone antagonist must have a structure and conformation which enables it to bind to the corre¬ sponding hormone receptor and at the same time not transduce.

Oxytocin antagonists have been developed and studied. See generally, Hruby, V.J., "Topics in Molecular Pharmacology", Burgen, A.S.V. and Roberts, G.C.K., eds. , Elsevier/North Holland Biomedical Press, Amsterdam, pp. 99-126 (1981); Hruby, V.J. and Mosberg, H.I. in "Hormone Antagonists," Agarwal, M.K., ed., Walter de Gruyter and Co., Berlin, pp. 433-474 (1982); Manning, M. and Sawyer, W.H. in "Conference on the Neurohypophysis, " Moses, A., ed., S. Karger, Basel, pp. 9-21 (1977) and Sawyer, W.H. , Grzonka, Z. and Manning, M. , Molec. Cell Biochem. , 22, 117-134 (1982) which are herein specifically incorporated by ref¬ erence.

Examples of previously synthesized oxytocin antagonists include [dPen , Tyr(OMe) , Thr ] oxytocin, and [dPen , Phe , Asn 4, Orn8] oxytocin which are disclosed by Sawyer, W.H., Holdar,

J. , Grazis, D., Seto, J. , Barkowski, K. , Lowbridge, J. , Turan, A. and Manning, M. in Endocrinology, 106, 81-91 (1980) which is herein specifically incorporated by reference.

Before proceeding further, brief explanation of the terminology used to describe polypeptides. Peptides are identi¬ fied by a ino acid sequence using established abbreviations. For example, as used herein, "Gly" stands for glycine, "Leu" stands for leucine, '-'Tyr" stands for tyrosine, "Pen" stands for penicillamine, "dPen" stands for 1-deaminopenicillamine, "Cys" stands for cysteine, "Phe" stands for phenylalanine, "Phe(4-Me)" stands for 4-methyl phenylalanine, "Thr" stands for threonine, "Gin" stands for glutamine, "Ser" stands for serine, "Val" stands for valine, "Asn" stands for asparagine, "Orn" stands for ornithine, "lie" stands for isoleucine and "Pro" stands for proline.

Polypeptide derivatives in which one or more of the amino acids have been replaced by another amino acid are often described by reference to the basic compound and the position and nature of the substitution. The position of substitution is usu¬ ally identified by reference to the number of the amino acid in the sequence starting with the amino acid at the amino terminus of the peptide chain. F ] oxytocin stands for H-dP -Gly- NH„. Additionally, amino acids may exist as stereoisomers in both L and D configurations.

One problem associated with the development of oxytocin antagonists is that such compounds have exhibited only short du¬ rations of biological acitivity or have not exhibited sufficient biological activity in vivo.

SUMMARY OF THE INVENTION

The present invention provides novel polypeptide com¬ pounds which are long acting antagonists of oxytocin and which exhibit prolonged in vitro and in vivo biological activity. The compounds are a series of cyclic, conformationally constrained analogs of the natural hormone, oxytocin, which are especially effective in inhibiting oxytocin induced uterine contractions in mammals.

In accordance with the present invention, there are provided polypeptides of the formula:

wherein

X is hydrogen or -NH„ ;

Y and Z are independently sulfur or -CH ;

1 2 R and R , which may be the same or different, are hy¬ drogen, methyl, cyclopentamethylene or lower alkyl having 1 to 5 carbon atoms;

3 . R is Phe, D-Phe, Tyr, D-Tyr, Tyr(OMe), D-Tyr(OMe),

Tyr(OEt), D-Tyr(OEt), Phe(4-Me), D-Phe(4-Me), Phe(4-Et), and

D-Phe(4-Et) ; 4 R is Gin, Thr, Ser, Asn, or Val; and

R is Orn, Arg, Lys, Leu, or lie; provided, however, that when X is hydrogen and R is Phe and R is Asn, R⁵ may not be Orn.

Particularly preferred compounds include:

[Pen , Tyr(OMe) , Thr⁴, Orn⁸] oxytocin. [Pen , D-Phe(4-Me) , Thr , Orn ] oxytocin,

[Pen , D-Phe(4-Et) , Thr , Orn ] oxytocin, and

[Pen , Phe(4-Me) , Thr , Orn ] oxytocin.

In accordance with the present invention there is pro¬ vided a process for inhibiting oxytocin induced contractions in mannals by administering a safe and effective amount of the oxytocin antagonist described above.

Additional objects and advantages of the invention will be set fortlx in part in the description which follows, and in part will be obvious from the description, or may be learned from the practice of the invention. The objects and advantages may be realized and attained by means of the instrumentalities and com¬ binations particularly pointed out in the appended claims. DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the presently preferred embodiments of the invention, which together with the following examples, serve to explain the principles of the inven¬ tion.

As noted above, the present invention relates to poly¬ peptides of the formula:

wherein

X is hydrogen or -NH_;

Y and Z are sulfur or CH„;

R 1 and R2, which may be the same or different, are hy¬ drogen, methyl, cyclopentamethylene or lower alkyl having 1 to 5 carbon atoms;

R is Phe, D-Phe, Tyr, D-Tyr, Tyr(OMe), D-Tyr(OMe^*), Tyr(OEt), D-Tyr(OEt), Phe(4-Me), D-Phe(4-Me), Phe(4-Et), or

D-Phe(4-Et) ;

4 .

R is Gin, Thr, Ser, Asn, or Val; and

R 5 i.s Orn, Arg, Lys, Leu, or lie; provided, however, that when X is hydrogen and R is

Tyr(OMe), R may not be Thr or when X is hydrogen, R is Phe, and R 4 i.s Asn, R5 may not be Orn. All amino acid residues which have chiral centers are of the L configuration except for those in position 2 which can be either the L or D configuration. When R 1 and R2 are both methyl groups the ammo acid residue in the 1 position is penicillamine, "Pen". When an alkyl group is in one or both of these positions the molecule is fur¬ ther restrained which accounts in part for the prolonged biologi¬ cal activity of the compounds.

Particularly preferred compounds include:

[Pen , Tyr(OMe) , Thr , Orn ] oxytocin;

[Pen , D-Phe(4-Me) , Thr , Orn ] oxytocin;

[Pen , D-Phe(4-Et) , Thr , Orn ] oxytocin; and

[Pen , Phe(4-Me) , Thr , Orn ] oxytocin.

Specifically, the polypeptides of the present invention are potent antagonists of oxytocin which exhibit prolonged bio¬ logical activity both din vivo and in vitro. It is believed that the polypeptides are potent antagonists which exhibit prolonged biological activity due in part to the O-alkyl or 4-alkyl substi¬ tuted amino acid in position 2.

In a preferred group of compounds, the prolonged bio¬ logical activity is believed to be primarily attributable to the presence of a penicillamine amino acid derivative or a modified penicillamine amino acid derivative in position 1, a 4-sub- stituted alkyl or aryl L or D amino acid derivative at position 2, a carboxa ide or hydroxy-containing amino acid at position 4 and a basic amino acid at position 8. It is believed that the combination of the above factors accounts for the prolonged bio¬ logical activities of the compounds of the present invention.

The compounds of the present invention were tested for their duration of activity in vitro and their potency. The re¬ sults of these tests are summarized in Table I.

As shown in Table I, [Pen , D-Phe(4-Me) , Thr , Orn ] oxytocin is a potent antagonist having an antagonist potency (pA₂) greater than 7.3. pA« values represent the negative log to the base 10 of the average molar concentration of an antagonist which will reduce the response of the uterine horn of 2X units of pharmacologically active compound to X units of the agonist, in this case oxytocin. The pA„ values in Table I represent only the lower limit of antagonist activity and, thus, the actual antago¬ nist activity may be considerably higher due to the prolonged activity of the compound. An effective dosage of the compounds of the present invention can be determined by one of ordinary skill in the art without undue experimentation.

The t 1/2 values for several compounds of the invention are summarized in Table I. The t 1/2 value represents the abil¬ ity of an antagonist to stop the contraction of the uterine mus¬ cle over time. In the case of [Pen , D-Phe(4-Me) , Thr , Orn ] oxytocin the t 1/2 value was 2 hours and 28 minutes. The t 1/2 value of the aforementioned compound should be contrasted with the t 1/2 value for oxytocin and previously known antagonists wherein complete reversal is normally observed in vitro in sever¬ al minutes.

The prolonged biological activity coupled with the potent antagonist.activity of the compounds of the present inven¬ tion render them useful in the prevention of premature birth in mammals. The compounds of the claimed invention are also useful in blocking the release of prostaglandin and may also be useful in affecting the release of aldosterone. Additionally, it is be¬ lieved that the compounds of the present invention can be effec¬ tive in regulating the effects of oxytocin in the mammary glands and the central nervous system.

TABLE I Inhibitory Potencies and II Vivo Half-lives of the Inhibiting Re¬ sponse Recovery of Oxytocin Antagonist Analogs Analog pA2 t 1/2 for

Response

Recovery

[Pen , Tyr(OMe) , Thr , Orn ]oxytocin >7.3 130 min. [Pen , D-Phe(4-Me) , Thr , Orn ]oxytocin >7.3 148 min. [Pen , D-Phe(4-Et) , Thr , Orn ]oxytocin >7.3 155 min. [Pen , Phe(4-Me) , Thr , Orn ]oxytocin >7.3 160 min.

Preparation of compounds within the scope of the pres¬ ent invention appear in the following examples. Example I Preparation of N'¹ -benzyloxycarbonyl-S-benzyl-L- penicillaminyl-0-methyl-L-tyrosyl-L-isoleucyl-O-benzyl-L- threonylasparaginyl-S-benzy1-L-cysteinyl-L-prolyl-N -tosyl- I-ornithinylglycinamide.

Chloromethylated (1.02 mmol/g resin) polystyrene resin (1% crosslinked with divinylbenzene) was used for solid phase synthesis. The carboxyl terminal amino acid (N -Boc-Gly) was attached to the resin as an ester linkage to a substitution level of about' 0.45 mmols of the protected amino acid/g resin using the method of Gisen, B., Helv. Chim Acta, 56, 1476-1482 (1973) which is specifically incorporated by reference herein. The synthetic resin of the title compound was prepared, and the synthesis was carried out on approximately a 1.0 mmol scale (about 2.2-2.3 g of resin). 2.3 g of Boc-Gly-resin was placed into a solid phase peptide synthesis vessel and Boc-Orn(N -Tos), Boc-Pro, Boc-Cys (S-Bzl), Boc-Asn-ONp, Boc-Thr(O-Bzl) , Boc-Ile, Boc-Tyr(OMe) , and Z-Pen(S-Bzl) were then incorporated onto the growing peptide chain according to the protocol of Agenda A (set forth in the end of the example) and for Boc-Asn-ONp-the protocol of Agenda B (set forth in the end of the example) was followed to yield the resin compound Z-Pen(S-Bzl)-Tyr(OMe)-Ile-Thr(O-Bzl)-Asn-Cys-(S-Bzl)- Pro-0rn(N -To s)-Gly-0-Resin. Each coupling step was monitored for completeness using the Kaiser ninhydrin test. See Kaiser, E.T., Colescott, R.L., Cook, P.I. and Bossinger, C.S., Anal. Biochem. , 34, 595-598 (1970) which is specifically incorporated by reference herein.

Following the synthesis, the peptide resin was removed from the reaction vessel by washing the resin four times with 30 ml of N,N-dimethylformamide (DMF) into a sintered glass funnel, followed by washing the resin with four 30 ml portions of CH₂Cl₂. The resin was dried in vacuo for 3 hours and then weighed. A portion of the peptide was hydrolyzed in a sealed ampoule con¬ taining 2 ml of a 1:1 mixture of 12N HC1 and propionic acid for 22 hr. at 110°C. The sample was then cooled and the solvents evaporated off in vacuo. To obtain the carboxa ide peptide, the protected peptide resin was placed in a 250 ml round bottom flask contain¬ ing a mixture of 150 ml of freshly distilled anhydrous methanol (from Mg(OMe)₂) and anhydrous ammonia (freshly distilled from Na) at -5°C to saturation of the ammonia in methanol. The volume in the flask increased about 20 ml with the addition of the ammonia to the methanol. After addition of the resin the flask was wired shut and stirred in a dessicator containing KOH pellets for 4 days at 25°C. The solvents were removed, first, by aspiration, then by rotary evaporation in vacuo to give a dry solid mixture. The carboxamide terminal peptide was extracted from the resin by the addition of 125 ml of DMF and heating the resulting mixture at 62°C for 8 hours. The mixture was cooled, the resin filtered, and a fresh 125 ml portion of DMF added to the resin and the mix¬ ture heated at 85°C for 2 hours. The resin mixture was cooled, the resin filtered off, and the combined DMF solution evaporated down to 15 ml by rotary evaporatin in vacuo. Then, 125 ml of deionized water was slowly added and a white precipitate formed. The solution was cooled in an ice bath for 2 hours, and the pre¬ cipitate was collected by filtration. The* product was washed with 20 ml of water twice, then with 20 ml of ether twice, and then dried in vacuo. A second crop of the product generally could be obtained from the mother liquor; a sample of 0.99 g of the title compound was obtained having a melting point of 248-253°C (decomposition). Amino Acid Analysis: Asx, 1.06; Thr, 0.94; Pro, 0.98; Gly, 1.04; Cys(Bzl), 0.88; Pen(Bzl), 1.12; Orn(Tos), 0.13 (destroyed); lie, 0.98; Tyr(OMe), 0.26 (de¬ stroyed) .

Agenda A

1. Wash for 1 min. with CH₂C1₂ (repeat this step a total of 4 times) .

2. Treat with 50% trifluoroacetic acid (TFA) in CH„C1₂ containing 2% anisole (v/v/v) for 2 min.

3. Treat as in No. 2 for 20 min.

4. Wash for 1 min. with CH₂C1₂ (repeat this step 2 times) . 5. Treat with 10% diisopropylethylamine (DIEA) in CH₂C1₂ (v/v) for 2 minutes (repeat this step once).

6. Wash for 1 min. with CH₂C1₂ (repeat this step 3 times) .

7. Ninhydrin test —Kaiser et al., Anal. Biochem. , 34, 595 (1970), which is herein specifically incorporated by ref¬ erence. If positive, proceed to step 8; if negative, repeat steps 2-7.

8. Treat with 3 equivalents of the appropriate Boc-amino acid derivative dissolved in CH₂C1₂ (with some DMF as needed), 1-hydroxybenzotriazole (HOBt) (3 equivalents) in DMF, and 2.4 equivalents of dicyclohexylcarbodiimide (hereinafter DCC) in CH₂C1₂. Final volume 25 ml. Allow reaction to proceed for 45-90 minutes.

9. Wash for 1 min. with CH₂C1₂ (repeat this step 2 times. )

10. Wash with 100% EtOH for 1 min. (repeat this step 3 times) .

11. Wash for 1 min. with CH₂C1₂ (repeat this step 2 times) .

12. Ninhydrin test—If coupling incomplete, repeat steps 8-12; if complete, go on to the next amino acid residue.

Agenda B

1. Wash for 1 min. with CH₂C1₂ (repeat this step 3 times) .

2. Treat with 50% TFA in CH₂Cl₂ containing 2% anisole (v/v/v) for 2 min.

3. Treat as in No. 2 for 20 min.

4. Wash for 1 min. with CH₂C1₂ (repeat this step 2 times) .

5. Treat with 10% DIEA in CH₂C1₂ (v/v) for 2 minutes

(repeat this step once).

2

6. Wash for 1 min. with CH₂C1 (repeat this step 3 times) .

7. Wash for 1 min. with DMF (repeat this step 2 times) .

- 8. Ninhydrin test as in Agenda A, No. 7.

9. Couple with 4 equivalents of Boc-a ino acid p-nitrophenyl ester in DMF containing 4 equivalents of HOBt. Run reaction for 240-360 minutes.

10. Do the ninhydrin test and continue coupling if in¬ complete.

11. Wash with DMF for 1 min. (repeat this step 2 times) .

12. Wash for 1 min. with CH₂C1₂ (repeat this step 3 times:) ..

13. Washing for 1 min. with 100% EtOH (repeat this step 2 times) .

14. Do ninhydrin test.

15. If coupling still incomplete treat again with 2.5 equivalents of amino acid derivative and HOBt. If the reaction is complete, go on to next amino acid.

Example II Preparation of cyclic-L-penicillaminyl-L-tyrosine-(0- methyl-L-isoleucyl-L-threonyl-L-asparaginyl-L-cysteinyl-L- prolyl-L-ornithinylglycinamide cyclic disulfide.

A sample of 250 mg of the protected nonapeptide from Example I was dissolved in 200 ml of anhydrous ammonia (freshly distilled from sodium). The solution was warmed to the boiling point and treated with a sodium stick until a blue color persisted for 60 seconds. If the blue color persisted for longer than 60 sec, the addition of a few mg of NH.C1 crystals rapidly dissipated the color and prevented cleavage at the proline resi¬ due. The ammonia was evaporated off under nitrogen and the last 20 ml was evaporated by l.yophilization. The white powder was dissolved in 650 ml of deaerated 0.1% aqueous acetic acid under a nitrogen atmosphere. The pH of the solution was adjusted to 8.5 with 3N ammonium hydroxide. The solution was then oxidized with an excess of 0.01 N K_Fe(CN),. The resultant yellow solution was stirred for 30 min. while maintaining the pH at 8.5. The excess ferro- and ferri-cyanide ions were removed from the solution by first adjusting the pH to 5 with 20% acetic acid and then adding 5 ml of the anion exchange resin, Rexyn 203 (Cl cycle) or the anion exchange resin BioRad AG3X4A (Cl cycle). The mixture was stirred for 20 minutes. The resin was then filtered off and washed with 20% aqueous acetic acid 3 times using 20 ml portions. The combined aqueous solution was evaporated down to about 100 ml by rotary evaporation in vacuo at 20-30°C. The compound was then purified by partition chromatography and gel filtration after lyophilization of the 100 ml of solution to a dry powder, as sum¬ marized below.

The dry powder was then dissolved in the upper phase using the solvent system, 1-butanol-water (containing 3.5% acetic acid and 1.5% pyridine) (1:1), and placed on a column of Sephadex G-25 (block polymerizate) which had been equilibrated with the upper and lower phases according to Yamashiro, D., Nature, 201, 76-77 (1964) which is specifically incorporated by reference herein. Four ml fractions of each were collected and the eluent analyzed for peptide material using either UV measurement at 280 nm or Folin-Lowry analysis as described in Lowry, D.H. Rosebrough, N.J. Parr, A.L. and Randall, R.J., J. Biol. Che . , 193, 265-275 (1951) and Larson, B., Fox, B.L., Burke, M.F. and firuby, V.J., Int. J. Peptide Protein Res., 13, 12-21 (1979) which are herein specifically incorporated by reference. The fractions corresponding to the product (Rf 0.23) were collected in the usual manner and the product further purified by gel filtration on Sephadex G-25, using 0.2 N aqueous acetic acid as eluent sol¬ vent. There was obtained 47 mg of the title compound (31% yield), [< ] ^₇ + 95.1° (c=0.5 in HOAc). Amino Acid Analysis: Orn, 0.94; Asp, 1.04; Thr, 0.95; Pro, 1.03; Gly, 1.06; Cys-Pen, 1.62; lie, 0.84; Tyr(OMe), 0.16—destroyed by hydrolysis. Thin layer chromatography in three solvent system, and HPLC analysis according to Blevins, D.D., Burke, M.F. , and Hruby, V.J., Anal. Chem. 52, 420-424 (1980) which is herein specifically incorpo¬ rated by reference, showed the compound to be highly purified—98%.

Thin layer chromatography was performed on silica gel G plates using the following solvent systems: (a) 1-butanol-acetic acid-pyridine-water (15:3:10:12) (BAPW) ; (b) 1-butanol-acetic acid-water (4:1:5, upper phase only) (BAW-upper); and (c) 1-pentanol-pyridine-water (7:7:6) (PPW) . HPLC analysis was done using one of three aqueous solu¬ tions: 0.1% trifluoracetic acid, or 0.04 M ammonium acetate (pH 4), or 0.05 M triethylammonium acetate (pH 4). The organic modi¬ fier was acetonitrile. The analytic HPLC determinations were performed using a VYDAC 218 TP 4.6 15-16 CIS reverse phase col¬ umn 0.46 cm i.d. by 25 cπr. length (Separations Group, Inc., Hesperia, CA) . Semipreparative HPLC purifications were performed on a Waters C18 reverse phase column 0.8 cm i.d. by 10 cm length contained in a Radial Compression Module (RCM) . Flow rates were generally 1 ml/min in the analytical runs and 2-4 ml/min in the semipreparative runs.

Example III

Preparation of S-benzyl-L-penicillaminyl-D,L-4- methylphenylalanyl-L-isoleucyl-O-benzyl-L-threonyl-L- c asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-N -tosyl-L- ornithinylglycinamide.

The scheme outlined in Example I was followed except that N^-Boc-D,L-4-methylphenylalanine was used for coupling this amino acid to the two (2) position in the peptide resin. In this way both diastereoisomeric peptide analogs could be obtained.

Thus, Boc-Orn(N^-Tos) , Boc-Pro, Boc-Cys(S-Bzl) , Boc-Asn-ONp, Boc-

Thr(O-Bzl), Boc-Ile, Boc-D,L-Phe(4-Me) , and Boc-Pen(S-Bzl) were successively added to give the peptide resin Pen(S-Bzl)-D,L-

Phe(4-Me)-Ile-Thr(O-Bzl) -Asn-Cys(S-Bzl)-Pro-Orn(Tos)-Gly-O-

Resin. The peptide was cleaved from the resin as its carboxamide terminal peptide after removal first of the N^α-Boc group followed by neutralization (first 6 steps of Agenda A).

Methanolic ammonia was again used to cleave the peptide from the resin as its carboxamide. There was obtained 1.16 g of the title compound; m.p. 237-253°C. Amino Acid Analysis: Pen(S-Bzl),

1.10; Cys(S-Bzl), 0.90; Orn(Tos), 0.20; Asx, 1.01; Thr, 0.91;

Pro, 1.04; Gly, 1.14; lie, 0.90; Phe(4-Me), 0.48. NMR analysis showed the peptide to be that indicated. Example IV

Preparation of L-penicillaminyl-L-phenylalanyl(4- methyl)-L-isoleucyl-L-threonyl-L-asparaginyl-L-cysteinyl-L- prolyl-L-ornithinylglycinamide-cyclic disulfide.

The scheme outlined in Example II was used with a sam¬ ple of 250 mg of the peptide from Example III to obtain the title compound. Following treatment with sodium in liquid ammonia and K_Fe(CN), oxidation, purification was achieved using the solvent system 1-butanol-water (containing 3.5% acetic acid and 1.5% pyridine). The title compound eluted at Rf 0.19 and was further purified by gel filtration on Sephadex G-25 using 0.2 N aqueous acetic acid as eluent solvent to give 23 mg of the title com¬ pound, [«■] ^_? + 52.4° (c=0.5 2N HOAc).

TLC Analysis in all three solvent systems showed a sin¬ gle spot, and reverse phase HPLC also showed a purity of 98%. Amino Acid Analysis: Gly, 0.99; lie, 1.04; Phe(4-Me), 0.91; Pro, 1.06; Asp, 1.01; Orn, 1.01; Thr, 0.97; Pen-Cys, 0.86; Cys-Cys, 0.08.

Example V Preparation of L-penicillaminyl-D-phenylalanyl(4- methyl)-L-isoleucyl-L-threonyl-L-asparaginyl-L-cysteinyl- L-prolyl-L-ornithinylglycinamide cyclic disulfide.

The scheme outlined in Example IV was used since the title compound was present simultaneously with Examiner IV. On partition chromatography it eluted at Rf 0.37 and hence was read¬ ily separated from compound IV. Following gel filtration on

Sephadex G-25 using 0.2 N aqueous acetic acid as the eluent sol- vent, 25 mg of the title compound was obtained, [«£] 2 -5. -° + 42.5°

(c=0.5 IN HOAc) .

TLC analysis in all three .solvent systems showed a sin¬ gle spot, and reverse phase HPLC also showed a purity of 98%. Amino Acid Analysis: Gly, 1.00; lie, 1.01; Phe(4-Me), 0.91; Pro, 1.06; Asp, 1.05; Orn, 1.07; Thr, 0.96; Pen-Cys, 0.84; Cys-Cys, 0.10. Example VI Preparation of S-benzyl-L-penicillaminyl-D,I-4- ethylphenylalanyl-L-isoleucyl-O-benzyl-L-threonyl-L- asparaginyl-^-benzyl-L-cysteinyl-L-prolyl-N^-tosyl-L- ornithinylglycinamide.

The scheme outlined in Example I was followed except that N^-Boc-D,L-4-ethylphenylalanine was used for coupling this amino acid to the two (2) position in the peptide resin. The synthesis involved coupling the following amino acids to the Boc- glycine-resi r Boc-Orn(N^-Tos) , Boc-Pro, Boc-Cys(S-Bzl) , Boc- Asn-ONp, Boc-Thr(O-Bzl) , Boc-Ile, Boc-D,L-Phe(4-Et) , and Boc- Pen(S-Bzl) to give the peptide resin Pen(S-Bzl)-D,L-Phe(4-Et) -Ile-Thr(O-Bzl)-Asn-Cys(S-Bzl)-Pro-Orn(N^-Tos)Gly-Resin. The title compound was obtained in the usual manner: yield, 0.44 g; mp, 202-204°C; Amino Acid Analysis: Pen(Bzl), 1.09; Asx, 1.03; Cys(Bzl), 0.89; Thr, 0.84; Orn(Tos), 0.48; Pro, 1.13; Gly, 1.08; lie, 0.81; Phe(4-Et), 0.83.

Example VII Preparation of L-penicillaminyl-D-phenylalanyl(4- ethyl)-L-isoleucyl-L-threonyl-L-asparaginyl-L-cysteinyl- L-prolyl-L-ornithinylglycinamide cyclic disulfide.

The scheme outlined in Example III was used and all the L and D-containing diastereoisomeric peptides were separated by partition chromatography in the solvent system 1-butanol-water (containing 3.5% acetic acid and 1.5% pyridine). (1:1). The Rf of the title compound was 0.89. Final purification was by gel filtration in Sephadex G-25 using 20% aqueous acetic acid as the eluent solvent. The product was obtained as a white powder; 30 mg: [<*] ^_? + 60° (c=0.5 IN HOAc).

TLC analysis in all three solvent systems show a single spot, and reverse phase HPLC showed a purity of >98%. Amino Acid Analysis: Gly, 1.02; lie, 0.96; Phe(4-Et), 1.08; Pro, 1.02; Thr, 0.95; Asx, 1.03; Orn, 0.92; Cys-Pen, 0.26; Cys-Cys, 0.72.

Bioassay Methods

The in vitro oxytocic assays were performed on isolated uteri from rats in natural estrus as described by Chan et al. in Endocrinology, 81, 1267-1277 (1967), which is specifically incor- porated by reference herein, with the use of Mg +2-free van

Dyke-Hastings' solution as the bathing fluid.

Antioxytocic potencies were determined in the in vitro assay system using the pA2 method described by Schild, H.O., Brit J. Pharmacol. _r 2_ 189-206 (1947) which is specifically incorpo¬ rated by reference herein.

The in vitro experiments were conducted as follows: 21-22 days pregnant rats were given a standard dose of oxytocin intravenously at 30 minute intervals. At zero time a predeter¬ mined dose of the antagonist was injected into the rat. After 30 seconds a standard dose of oxytocin was injected. Regular oxytocin injections were then resumed at 30 minute intervals. The contractile activity during the 10 minutes following each oxytocin injection was determined by measuring the area under the contractile curve. The contractile activity was then converted to percent recovery of the inhibitory response. Zero recovery corresponds to the maximum inhibitory effect. The t 1/2's for the recovery of inhibitory response (50% recovery) was calculated and are listed in Table I.

It will be apparent to those skilled in the art that various modifications and variations can be made in the processes and products of the present invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide which is an analog of the hormone oxytocin and has the formula:

wherein

X is- hydrogen or -NH_ ;

Y. and Z are independently sulfur or CH„ ; R 1 and R2 are hydrogen, methyl, cyclopentamethylene or an alkyl group having 1 to 5 carbon atoms;

R is Phe, D-Phe, Tyr, D-Tyr, Tyr(OMe), D-Tyr(OMe),

Tyr(OEt), D-Tyr(OEt), Phe(4-Me), D-Phe(4-Me), Phe(4-Et) or

D-Phe(4-Et) ;

R ι - is Gin, Thr, Ser, Asn, or Val; and

.5 is Orn, Arg, Lys, Leu, or lie; provided, however, that when X is hydrogen and R is Tyr (OMe),

R .•_ may not be Thr or when X is hydrogen, R 3 is Phe and R4 is Asn, 5 may not be Orn.

2. A polypeptide according to claim 1 having the for¬ mula: H-Pen-Tyr(OMe)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH₂

3. A polypeptide according to claim 1 having the for- mula; H-Pen-D-Phe(4-Me)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH₂

4. A polypeptide according to claim 1 having the for- mula: H-Pen-D-Phe(4-Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH₂

5. A polypeptide according to claim 1 having the for¬ mula: H-Pen-Phe(4-M )-Ile-Thr-Asn-Cys-Pro-0rn-Gly-NH

6. A polypeptide according to claim 1 wherein R 1 and

2

R are both methyl and X is -NH₂.

7. A biologically, long-acting oxytocin antagonist compound comprising:

wherein X is hydrogen or - H₂; Y is sulfur or CH₂;

Z is sulfur or CH„ ; R 1 and R2 are hydrogen, methyl, ethyl, or cyclopentamethylene; 3 R is Phe, D-Phe, Tyr, D-Tyr, Tyr(OMe), D-Tyr(OMe),

Tyr(OEt), D-Tyr(OEt), Phe(4-Me), D-Phe(4-Me), Phe(4-Et), or

D-Phe(4-Et) ; R 4 is Gin, Thr, Ser, Asn, or Val; and

5 R is Orn, Arg, Lys, Leu, lie; provided, however, that when X is hydrogen and R 3 is Tyr(OMe) R4 may not be Thr or when X is hydrogen, R 3 is Phe and R4 is Asn, R5 may not be Orn.

8. A compound according to claim 7 having the formu-

9. A compound according to claim 7 having the formu¬ la: H-Pen-D-Phe(4-Me)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH₂

10. A compound according to claim 7 having the formu¬ la: H-Pen-D-Phe(4-Et)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH₂

11. A compound according to claim 7 having the formu- la: H-Pen-Phe(4-Me)-Ile-Thr-Asn-Cys-Pro-Orn-Gly-NH

12. A compound according to claim 7 wherein R 1 and R2 are both methyl and X is ~NH₂.

13. A method of inhibiting oxytocin induced contrac¬ tions in mammals by administering a safe and effective amount of the compound of claim 7.

14. A method of inhibiting oxytocin induced prostaglandin release in mammals comprising the step of adminis¬ tering to a mammal a safe and effective amount of the compound of claim 7.

15. A pharmaceutical composition comprising an oxytocin antagonist having prolonged biological activity of the formula:

wherein X is hydrogen or ~NH₂;

Y and Z are sulfur or CH ; R 1 and R2, which may be the same or different, are hy¬ drogen, methyl, cyclopentamethylene or lower alkyl having 1 to 5 carbon atoms; 3 R is Phe, D-Phe, Tyr, D-Tyr, Tyr(OMe), D-Tyr(OMe),

Tyr(OEt), D-Tyr(OEt), Phe(4-Me), D-Phe(4-Me), Phe(4-Et) or

D-Phe(4-Et);

4 R is Gin, Thr, Ser, Asn or Val; and

₅ R is Orn, Arg, Lys, Leu or lie, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carri¬ er.