CA1179328A - Oxytocin analogs and method for preparation thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:
Analogs of oxytocin of general formula I

(I), where all amino acids are L-isomers and X is phenylalanine or phenylalanine substituted in the p-position by alkyl, ethoxy, amino, substituted amino, or nitro group, suitable for medical applications, and a method for their preparation consisting in the cyclization of linear octapeptides of general formula II

Description

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The invention relates to a method for the prepara-tion of oxytocin analogs. More specifically, the present in-vention relates to a method for the preparation of deamino-6-carba-oxytocin having a modified amino acid in position 2, and to the oxytocin analogs so produced.
It has been recognized that some of the numerous activities of the neurohypophysial hormone oxytocin may be enhanced or suppressed by chemical modification of the molecule, and the attainment of biological effects is a condition precedent for its successful clinical application. Thusj for example, studies have revealed that replacement of a tyrosine group in the 2 position of a deamino-6-carba-oxytocin molecule by a phenylalanine or a phenylalanine substituted in the para position yield compounds evidencing a high and specific na-triuretic effect which may be suitable ~or use in treating renal and heart insufficiency or in some cases hipertension.
Workers in the art (Fraser, A.M. J. Pharmacol.
Ex. Therapy 60, 89 ~1937))have long recognized that oxytocin evidences a natriuretic efect. Unfortunately, that effect is very weak and generally has superimposed thereon a strong antidiuretic activitv. Tlle deamino-6-carba-oxytocin evi-dences a substantially greater natriuretic effect and, even so, is combined with hiyh values of other biological acti-vlties, such as oxytocic, galactogogic and pressor activi-ties, which preclude use of the natriuretic effect o~ the compound in clinical applications. These analogs are interesting not only with respect to the absolute m~gnitude of natriuretic effect, but also for specificity of the effect of the ratio of different activities.
Moreover we cannot exclude apriori, that for different animals ~including man), the value as well as spe-- cificity o the natriuretic effect can be different.

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In accordance with the present invention, these prior art limitations are effectively obviated by means of novel analogs of oxytocin of the general formula fH2 ~ S - CH2 Cl 2 (I) CH2-CO-X-Ile-Gln-Asn-NH-CH-CO-Pro--Leu-Gly-NH2 wherein the amino acids are of the L-series and X represents the amino acid phenylalanine or a phenylalanine substituted in the para position with a group selected from among alkyl, (for example alkyl containing 1 to 4 carbon atoms), ethoxy, amino or substituted amino (for example, amino substituted either by benzyloxycarbonyl or by two methyl groups) r or nitro groups.
The compounds of the present invention can be administered by any suitable pharmaceutical means, e.g. by some fluent method (injection, infusion, nose drops etc.).
The compounds may be administered in association wi-th a suitable diluent e.g. in a saline solution.
The describe~ novel oxytocin analogs (I) may be conveniently prepared by cyclization between an amino group of an amino acid X and the carboxylic group of the S-carbo-xyethylhomocysteine residue of a linear peptide of the general Eormula, (II) :
CH S C~12 - - CH2 (II) CH2CO-ACt X-lle-Gln-Asn-NH-CH-CO-Pro-~eu-Gly-NH2 wherein X represents the amino acid phenylalanine or phenyl-alanine substituted in the para position by a group selected ~rom among alkyl (for example alkyl containing 1 to 4 carbon atoms), ethoxy, amino or substituted amine, (for example amino substituted by benzyloxycarbonyl or two methyl groups), or nitro groups and Act represents an activator for a carbo-xylic group such as an active ester group (i.e. Act is a - group activating the carboxylic group of S-carboxyethylhomo-3~8 cysteine residue).
In particular, the aACt group can be any arbitrary group activating a carboxyl group for nucleophilic reaction with the amino group e.g. p-nitrophenyl, 2,4,5-trichlorophenyl, pentachlorophenyl etc. Further details will respect to the activating group can be found, for example, by consulting E.Schroeder, X.Luebke: ~he peptides, Academic press, New York, 1966).
Various cyclization methods were c~xred and the method of active esters was selected as the best one ~CF. Jost K. :
collection of Czechoslovak Chemical Communications 36, 218-233 ~1971) - the Paper entitled: ~n Improved Synthesis of Deamino-carbal-oxytocin~ Comparison of various methods for peptide cyclisation).
The analog of oxytocin of the ~ollowing general formula (III) :
fH2 S CH2 ~~ H2 (III) CH2-CO-NH-fH-CO-Ile-Gln-Asn-NH-CH-CO-Pro-Leu-Gly-NE~2 [~

may conveniently be prepared either by ~a) reduction of L~p-nitrophenylalanlne7 deamino-6-carba-oxytocin with a suitable reducing agent e.g. wi-th sodium in liquid ammonia or (b) from ~-p-benzyloxycarbonyl-aminophenylalanineJ dea-mino-6 carba-oxytocin by cleavage of the protecting group with a suitable clea~ing agent, e.g. with hydrogen bromide in acetic acid.
Thus, in accordance with another aspect, the present invention provides a process for preparing an oxytocin analog of the general formula (I) L~

fH2 S CH2-- l H2 CH2-CO-X-Ile-Gln-Asn-~H-CH-CO-Pro-Leu-Gly-NH2 (I) wherein the designated amino acids are of the L-series and X represents phenylalanine or phenylalanine substituted in the para position with a member selected Erom the group consisting of (a) alkyl containing 1 to 4 carbon atoms, (b) ethoxy, (c) amino, (d) benzyloxycarbonylamino, (e) dime-. thylamino and (f) nitro, characterized in that (A) a linear peptide of the formula (II) lH2 - -- S - CH lH2 CH2-CO-Act X-Ile-Gln-Asn-NH-CH-CO-Pro-Leu-Gly-NH2 wherein X is as defined above and Act represents a suitable carboxyl group activatorl is cyc~ised, the cyclization being effected between the amino group of amino acid X and the carbonyl group of S-carboxy - ethylhomocysteine residue to obtain an oxytocin analog of the general formula ~I~ as ~efined above, or (b) ~2-p-n1trophenylalanirle7 deam.tno-5-carba-oxytocin is reduced with a suitable reducing agent or the protecting group is cleaved from r 2-p-benzyloxy-carbonylaminophenylalanine7 deamino-6-carba-oxytocin by the action of a suitable cleaving agent to obtain an oxytocin analog of the general formula (I) as defined above wherein X is phenylalanine substituted in the para position by amino.
Some biological activities of the analogs oE
oxytocin of general formula I are given in Table I in comparison to oxytocin.

- 3 a -:~t7932~3 TABLE I

ANALOGY I Biolo~ical Activity (in Rats) Ut t Galacto- Pressor Na-tri-X ero onlc gogiC uretic (a) (a) (a) ~b) _ .
oxytocin 450 450 3.0 I00 NH-CH(C~2-C6~l5)-co 70 170 0.9 143 NH-cH(cH2-c6H4-cH3)co 45 35 1 326 MH CH(CH2 C6H4 2 5) 27 1.4 <0.2 254 NH CH(C~2 C6 4 2 5) ~0.001 5.6 ~0.2 31 NH-CH(CH2-C6H4-NH2)-C ~ 15 7 <0.2 87 NH-CH(CH2-C6H4-N(CH3)2) ~0.14 4.5 <0.2 75 NH-cH(cH2-c6H4 NH CO2 2 0.07 <0.2 10 C6H5) CO
NH-CH(CH2-C6H4-NO2)-CO <0.001 1.4 <0.2 66 (~) Inter~ational units per ~g~ (bl percent of oxytocin activity.
The method Eor producing the analogs of oxytocin is Eurther illustrated in the following examples, the final products being obtained as lyofilisates; i.e. very light foams, which have no meltiny point.
EX~MPLE I
This example described the preparation of ~2-p-Nitrophenylalanine~deamino-6-carba-oxytocin. Prior to the initiating of the preparation thereof, an octapeptide of formula II was prepared, as follows :
0.29 grams 2,4,5-trichlorophenol and 0.33 grams of dicyclohexylcarbodiimide were added to a solution of orthonitrobenzenesulfenyl-para-nitrop~enylalanine (0.53 ~rams~

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in a mixture of 15 cm3 of dichloromethane and 15 cm3 of dime-thylformamide cooled to -10C. The resultant mixture was stirred for one hour at -10C and then for 12 hours at room temperature. Following, the mixture was concentrated in vacuum/ resulting in the formation of crystals which were filtered by suction. Th~ filter cake so produced was then washed with dichloromethane and the filtrate evaporated to dryness (at the 20C bath temperature). The yellow oil resulting was next triturated several times with petroleum ether and dissolved in 12 cm3 dimethylformamide. Following, the amide of isoleucyi-glutaminyl-asparaginyl~S-(2-carboxy-ethyl)homocysteinyl-prolyl-leucyl-glycine (0.8 g) was su-spended in this solution and after stirring the resultant ~olution for 135 hours at ambient temperature it was evaporated to dryness (bath temperature 35C). The resultant oily product crystallized on trituration with petroleum ether and was then successively washed on fritted glass with water, 0.05 M
sulfuric acid, water and ether, yielding 720 mq of an octapep-tide melting within 215-219C having the characteristics set forth in Table II, belo~

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In Table II Nps represents o-nitro~enzenesulphenyl protecting group; Hcy (C2H4COOH) stands for S-beta-carboxyethylhomo-cysteine; Phe (NO2) and t~e similar designations refer to phenylalanine substituted by the specified group in the para position; the first column after the definition for X
concerns electrophoretic mobilities related to mobilities of glycine or histidine; electrophoreis being carried out in moist chamber apparatus on a whatman 3 mm paper for 1 hour at a potential drop of 20V cm 1 in 1 M acetic acid (ph 2.4) or pyridine acetate buffer (ph 5.7). The next two columns denote RF values obtained by thin-layer chromatography (on silica gel-coated plates? in solvent systems :
Sl = 2 ~utanol - 98 0/0 Formic acid - water (75:13.5:11.5), S2 = 2-Butanol - 25 0/0 Aqueous Ammonia - water (85:7.5:7.5).
S3 = l-Butanol - Acetic acid - water (4:1:1) and S4 - l-Butanol - Pyridine - Acetic acid - water (15:10:3:6).
The last four columns give the results of amino acid analyses.
These values were obtained after acid hydrolysis of the compounds (conditions: 20 hours, 6M-HCl, 105 Grade C, Ampoules evacuated rro 150 PA) on the fully automatic amino acid analyser. The values give the amount in micromoles of the Lndividual amino acids which originates from one mi-micromole oE the title compound.
With respect to Table III,which follows, the first-two columns o figures in the table III have the same meaning as in the table II; the same is true for the columns entitled ASP, GLU, PRO ~ND GLY. The last column is the capacity factor from hight-performance liquid chromatography; follows the solvent mixture (EV. PH of a buffer, when it is used instead of water).
0.7 gram of bis-(p-nitrophenyl) sulfite was then added to a solution of 200 milligrams of khe octapeptide so produced in 7 cm' of dimethylformamide and 7 cm3 of pyridine ~7~Z~

through which nitrogen had been bubbled. After stirring for 9 hours at room temperature, an additional 0.7 gram of sul-fite was added and after stirring for another 12 hours an additional 0.35 gram of sulfite was added. A~ter 4 hours, the reaction mixture was concentra-ted in vacuum and the product precipitated by ether, filtered by suction and thoroughly washed with ether. The product was then dried, dissolved in 7 cm3 of dimethylformamide and 2.26 M hydrogen chloride in 0~52 c~3 of ether added thereto. The mixture so obtained was permitted to stand for 7 minutes and diluted with 100 cm3 of ether. The hydrochloride precipitate of the compound of formula II was then fil-tered by suction, washed with ether and dried in vacuum.
Cyclization to form a peptide bond was effected as follows: the hydrochloride prepared in the foregoing manner was dissolved in 7 cm3 of dimethylformamide and added at the rate of 2 cm3 per hour with a vigorously stirred mixture of 200 cm3 of pyridine and 50 microliters of N-ethylpiperidine which had nitrogen bubbled therethrouyh and which had been warmed to 50C. Following completion of the addition, the mixture was heated to 50C for 4 hours and permitted to stand at ambient temperature for 12 hours. Then, the solution was concentrated to a small volume (bath temperature 30C) and the product precipitated by ether. 170 my of a microcrystal-line material were obtained. 100 mg o~ the product were then dissolved in 4 cm3 of 3 M acetic acid and applied on a column packed with polyacrylamide gel ~Bio gel-P-4 (100 x 1 cm)~ . Freeze-drying of the corresponding fractions yielded 70 mg of a compound which was again dissolved in 3 cm3 of 3 M acetic acid and applied on a column packed as above described. Freeze-dryiny of the corresponding fractions yielded 42 mg of a compound. 15 mg of this product was ~7~Z~3 dissolved in 2 cm3 of a methanol-water (203) mixture, and the solution applied on a column packed with modified silica gel (Separon SI C , 15 x 0~6 cm). Elution was then performed with a methanol water mixture (44:56) at a a pressure or 20 MPa. The fraction of k~- 8.2 was concentrated in vacuum and freeze-dried, yielding 6.3 mg of the compound whose characteristics are set forth in Table III, on the following two pages.

This example describes the preparation of L~-para-ethoxyphenylalanine~ deamino-6-carba-oxytocin. The starting compound of formula II was prepared in the manner described in example 1. 0.23 gram of a suspension of the dicyclohexyl-ammonium salt of ortho-nitrobenzene-sulphenyl-p-ethoxyphenyl-alanine in 50 cm3 of ethyl acetate was then shaken with 0.05 M
sulfuric acid and the resulting solution dried over sodium sulfate and evaporated to dryness. The oil so formed was then dissol~ed in dichloromethane and the active ester prepared in the manner described in example 1. Condensation with 0.25 gram of heptapeptide was performed in the same manner, yielding 200 my of a compound having a melt:ing point of 215-218~C and evidencing characteristics of the type shown in ~able II.
Next, the cyc~ization described in example 1 was conducted. The yield resultlng from 200 mg oE protected peptide was 196 mg of cyclization. A part of the reaction mixture was purified by repeated gel Eil-tration, yielding 8.3 mg of a pure compound evidencing the characteristics set forth in Table III, Ef:

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31.~'~3Z~3 This example describes the preparation of ~2-phenyl-alanineJ deamino-6-carba-oxytocin. The starting compound of formula II was prepared in the manner set forth in example 1.
Then, 2,4,5-trichlorophenyl ester of o-nitrobenzenesulfenyl-phenylalanine was added to 0.8 gram of a suspension of free heptapeptide in 15 cm3 of dimethylformamide. After stirring for 96 hours at ambient temperature, the reaction mixture was treated as described in example 1. 1 gram (85~) of a compound having a melting point of 223-225C was obtained, the characteristics thereof being set forth in Table II.
Cyclization was then performed as described in example 1. The product, which contained ninhydrin-positive material was dissolved in a 1:1 methanol water mixture and filtered through a column of sulfonate cation exchanger ~Dowex 50 in the H+ cycle, 5 cm3 ) ~ Concentration and freeze-drying yielded 125 mg of a compound which was then purified by gel filtration. 15 mg of -the product so obtained was then chromatographed on a column with modified silica gel (Separon SI C , 15 x 0.6 cm) in a 3:2 methanol water mixture.
Concentration of the k'= 7.0 fraction and freeze-drying yielded 4.2 mg of a compound, the characteristics of which are set Eorth in Table III.
EXAM
This example describes the preparation of C2-para-benzyloxycarbonylaminophenylalanine7 deamino-6-carba-oxytocin.
The starting compound was prepared (formula II) in the manner set forth in example 1. Ortho-nitrobenzenesulfenyl-para-benzyloxycarbonylaminophenylalanine was liberated from 1.05 grams of its dicyclohexylammonium salt in the manner described in example 2 and it was transferred into the active ester in the manner set forth in example 1. On trituration with * Trademark petroleum ether, 1.0 gram of a crystalline compound was obtained having a melting point from 54 - 57C. The active ester (0.8 g) was added ~o a suspension of 0.6 gram of hep-tapeptide in 15 cm3 of dimethylforma~ide and processed as described in example 1. 0.53 gram (55~) of a compound melting at 220-222C was obtained having the characteristics set forth in Table II.
Cyclization was conducted as described in example 1, starting with 200 ~. 155 mg of a product was obtained and 30 mg thereof ~ere purified by repeated gel filtration.
Further refining was effected by chromatography on a column with reverse phase (modified silica gel Separon SI C 8 :
15 x 0.6 cm) in a mixture of methanol with trifluoroacetate bu~fer (3:2) of pH 4.4. The fraction containing the compound k'= 3.56 was freeze-dried and 4.2 mg of a product was obtained having the characteristics set forth in Table III.

This example describes the preparation of L~-para-aminophenylalanine~ deamino-6-carba-oxytocin.
Sodium was added to 3.6 mg of a solution of ~2-p-nitrophenylalenine7 deamino-6-carba-oxytocin in l:iquid am~onia (5 cm~) until a blue coloration which was stable for 30 seconds arose. Then, the solution was decolorized by addi.tion of acetic acid and the residue, after evaporation oE ammonia, was purified by cJel :Eiltration. The corresponding collected fractions gave by freeze-drying 2.1 mcJ of a compound which is characterized in Table III.

A solution of hydrogen bromide.~ acetic acid (35~, lcm3) was added to a suspension of r2-p-benzyloxycarbonylaminophe-nylalanine~ deamino-6-carba-oxytocin (30 mg) in acetone (1 cm3 ) andthe solution formed was allowed to stand for 1 hour at ambient temperature. After repeated eYaporation from acetone and reprecipitatio~ from methanol with ether, the product was dissolved in 3 M acetic acid (3 cm3 ~ and refined by gel filtration. Freeze-drying gave 8.7 mg of the compound which corresponded by its properties to the product according to Example 5.
_AMPLE 7 This example describes the preparation of r2-p-me-thylphenylalanine~ deamino-6-carba-oxytocin. The active ester of 0-nitrobenzenesulphenyl-p-methylphenylalanine was prepared from the corresponding dicyclohexylammnonium salt (0.7 g) in the same manner as in Example 4. 0.58 gram of a compound melting at 127-133C was obtained. This active ester ~as added to a suspension of hep~apeptide (0.65 g) in dimethylformamide (13 cm3 ~ and 0.83 g of a compound of m.p.
220-226C Was obtained in the same way as in example 1' the characteristics can be found in Table II.
Cyclization of 200 mg of peptide was carried out in the same way as in example 1. The product was obtained in the amount of 180 mg and a part of it (50 mg) was purified by gel filtration and a column chromatography tseParon SI
C18) in a mixture of methanol-water (3:2). The Erac:tion of k'= 5.03 yielded by freeze-drying 13.6 mg of the compound which is characterized in Table III.

This example desGribes the preparation of ~2-p-ethylphenylalaninel deamino~6-carba-oxytocin. The protected octapeptide was prepared as described in example 1 from o-nitrobenzenesulphenyl~p-ethylphenylalanine (0.4 g~ and the free heptapeptide (0.3 g). A compound melting 218-224C
was obtained in tha amount of 0.23 g and its properties are given in Table II.

~75~32~3 - Cyclization and refining of the octapeptide were effected in the same manner as in example 7. There was obtained 8.3 mg of the compound (k'= 7.4, methanol-water (3:2) which exhibited properties shown in Table III.

This example describes the preparation of ~2-p-di-methyl-aminophenylalanine7 deamino-6-carba-oxytocin. The active ester of o-nitrobenzenesulphenyl-p-dimethylaminophenyl-alanine was prepared from the dicyclohexylammonium salt (0.5 g) in the same manner as in example 4. A compound of m.p. 113-117C was obtained in the amount of 405 mg. This active ester was added to a suspension of heptapeptide ~0.3 g) in dimethylformamide and 0.43 g of a compound melting within the range of 201-204C was obtained by the same procedure as in example 1 (omitting washing with diluted sulphuric acid).
Its properties are shown in Table II.
Cyclization was carried out in the same way as in example 1 and yielded 190 mg of a product which was refined by gel filtration. The product obtained by freeze-drying (100 mg) was dissolved in 20~ acetic acid and subjected to purification by free-flow electrophoresis. The compound obtained in the amoun-t of 54 mg was further reElned by gel Eiltration. Propertles oE the product (28 mg) are ylven in Tabl~ III.

Claims (14)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing an oxytocin analog of the general formula (I) (I) wherein the designated amino acids are of the L-series and X represents phenylalanine or phenylalanine substituted in the para position with a member selected from the group consisting of (a) alkyl groups containing 1 to 4 carbon atoms, (b) ethoxy, (c) amino, (d) benzyloxycarbonylamino, (e) dimethylamino and (f) nitro, characterized in that a linear peptide of the formula (II) (II) wherein X is as defined above and Act represents a suitable carboxyl group activator, is cyclized, the cyclization being effected between the amino group of amino acid X and the carboxyl group of S-carboxy ethylhomocysteine residue.

2. An oxytocin analog of the general formula (I) (I) wherein the designated amino acids are of the L-series and X represents phenylalanine or phenylalanine substituted in the para position with a member selected from the group consisting of (a) alkyl groups containing 1 to 4 carbon atoms, (b) ethoxy, (c) amino, (d) benzyloxycarbonylamino, (e) dimethylamino and (f) nitro, whenever prepared by a process as claimed in claim 1 or an obvious chemical equivalent thereof.

3. A process as defined in claim 1, wherein said activator is a suitable ester.

4. An oxytocin analog of the general formula (I) as defined in claim 2, whenever obtained by the process as claimed in claim 3.

5. A process for preparing a compound of the formula wherein the designated amino acids are of the L-series, characterized by reducing [2-p-nitrophenylalanine] deamino-6-carba-oxytocin with a suitable reducing agent.

6. A compound of formula wherein the designated amino acids are of the L-series, whenever prepared by a process as claimed in claim 5 or an obvious chemical equivalent thereof.

7. A process as defined in claim 5 wherein [2-p-nitrophenylalanine] deamino-6-carba-oxytocin is reduced with sodium in liquid ammonia.

8. A compound of formula wherein the designated amino acids are of the L-series, whenever prepared by a process as claimed in claim 7 or an obvious chemical equivalent thereof.

9. A process for preparing a compound of the formula wherein the designated amino acids are of the L-series, characterized in that the protecting yroup is cleaved from [2-p-benzyloxycarbonylaminophenylalanine] deamino-6-carba-oxytocin by the action of a sultable cleaving agent.

10. A compound of formula wherein the designated amino acids are of the L-series, whenever prepared ny a process as claimed in claim 8 or an obvious chemical equivalent thereof.

11. A process for preparing a compound of the formula wherein the designated amino acids are of the L-series, characterized in that the protecting group is cleaved from [2-p-benzyloxycarbonylamine phenylalanine] deamino-6-carba-oxytocin by the action of hydrogen bromide in acetic acid.

12. A compound of formula wherein the designated amino acids are of the L-series, whenever prepared by a process as claimed in claim 11 or an obvious chemical equivalent thereof.

13. A process for preparing an oxytocin analog of the general formula (I) (I) wherein the designated amino acids are of the L-series and X represents phenylalanine or phenylalanine substituted in the para position with a member selected from the group consisting of (a) alkyl containing 1 to 4 carbon atoms, (b) ethoxy, (c) amino, (d) benzyloxycarbonylamino, (e) dime-thylamino and (f) nitro, characterized in that (A) a linear peptide of the formula (II) wherein X is as defined above and Act represents a suitable carboxyl group activator, is cyclized, the cyclization being effected between the amino group of amino acid X and the carboxyl group of S-carboxy ethyl-homocysteine residue to obtain an oxytocin analog of the general formula (I) as defined above, or (B) [2-p-nitrophenylalanine] deamino-6-carba-oxytocin is reduced with a suitable reducing agent or the protecting group is cleaved from [2-p-benzyloxycarbonylaminophenyl-alanine] deamino-6-carba-oxytocin by the action of a suitable cleaving agent to obtain an oxytoain analog of the general formula (I) as defined above wherein X
is phenylalanine substituted in the para position by amino.

14. An oxytocin analog of the general formula (I) (I) wherein the designated amino acids are of the L-series and X represents phenylalanine or phenylalanine substituted in the para position with a member selected from the group consisting of (a) alkyl groups containing 1 to 4 carbon atoms, (b) ethoxy, (c) amino, (d) benzyloxycarbonylamino, (e) dimethylamino and (f) nitro, whenever prepared by a process as claimed in claim 13 or an obvious chemical equivalent thereof.