OXYTOCIN ANALOGUES WITH INHIBITORY PROPERTIES,CONTAINING IN POSITION 2 A HYDROPHOBIC AMINO ACIDOF D-CONFIGURATION*

Michal LEBLj Tomislav BARTH, Linda SERvfTOVA, Jifina SLANINovAand Karel JOST

Institute ofOrganic Chemistry and Biochemistry,Czechoslovak Academy of Sciences, 16610 Prague 6

Received May 19th, 1984

Ten analogues derived from oxytocin, deamino-oxyt ocln and deamlno-carba-oxytocln wereprepared, which contained a n-amino acid in the position 2 of the parent system. The followingn-amino acids were introduced: tyrosine, phenylalanine, p-mcthylphcnylalanine, p-ethylphenyl­alanine and Ocethyltyrosine. Combination of two structural features which alone lead to stronginhibitors (a suitable c-amino acid in position 2 and a penicillamine moiety in position I) did notenhance the inhibitory effect. Compounds containing a-tyrosine are weak agonists in the utero­tonic assay; in case of Lcarba-analogues they can be converted into sulfoxides with low inhibitoryactivity. Analogues with n-phenylalanine substituted in the para-position arc the most potentantagonists of the uterotonic effect of oxytocin (pAz = 8'73) in vitro.

Recently, there is a considerable interest in the preparation and study of neuro­hypophyseal hormone analogues with inhibitory activity towards oxytocin and vaso­pressin in a great variely of biological tests' - 3. These compounds are interestingnot only because of their possible use in the study of mechanism of action or spatialarrangement but they may also find use in the clinical therapeutical practice. Thereare several structural modifications leading to oxytocin analogues with inhibitoryeffectsin the uterotonic test. We may mention the alkylation of the tyrosine" hydro­xyl'-7, particularly with simultaneous acylation of the primary o-amino groupof cysteine in position 1 (ref.8 - 11 ) or its replacement by a hydrogen atom!", Parti­cularly important is the attachment of two methyl groups'3.'4, two ethyl groups"or a cyclopentamethylene group'6 to the ~-carbon atom of cysteine in position1 which might be combined with deamination in this position. Various combinationsof this modification with substitution of the amino acids have been realized2 ,3, '4,' 7 -24(in positions 2, 4, 7 and 8) which gave very strong inhibitors.

* Part CXC in the series Amino Acids and Peptldes; Part CLXXXIX: This Journal 49, 2680(1984).** Unless stated otherwise, all chiral amino acids belong to the r-serles. The nomenclatureand symbols of theamino acids, their derivatives and peptides obey the published recommenda­tions4. Hey denotes homocysteine, Pen penicillamine and Mpa Il-rnerceptoproplonlc acid moie­tics.

Collection Czechoslovak Chern. Commun. [Vol. 501 [1985]

Amino Acids and.Peptidea 133

In the oxytocin series, replacement of tyrosine in position 2 by its n-enantiomerleads to analogues of very low biological activity",2., Introduction of an-aminoacid other than tyrosine (such as phenylalanine-Z, pentafiuorophenylalanine-Iv",3-nitrotyrosine29, leucine'· or tryptophan") givesanalogues with a certain inhibitoryactivity. Interestingly, the compound containingO-ethyl-D-tyrosine showed"an intrinsic activity in the in vitro uterotonic test which was .even higher than thatof the analogue with the t-enantiomer7 (which, contrariwise, in certain experimentalarrangement had an inhibitory effect).A similar modification (introduction ofO-alkyl­on-tyrosine) of [1-(p-mercapto-p,p-cyc1opentamethylenepropionic acid), 4-valine,8-arginine]vasopressin gave analogues with inhibitory activity in. the antidiureticas well as pressor tests; however, also the corresponding analogues with the L-aminoacid in position 2 showed inhibitory activity, similarly to the compound with un­substituted n-tyrosine32 ,33 . If, however, n-tyrosineis introduced into the vasopres­sin molecule as the only structural change, the resulting analogue ([2-D-tyrosine,8-arginine]vasopressin) exhibits relatively high vasopressin-like activities'". Thiscorresponds to the assumptions about the spatial arrangement of the tyrosine sidechain in the vasopressin molecule2.,'4. [1-(p-Mercapto-p,p-cyc1opentamethylene­propionic acid), 4~valine, 8R L- (or D-)arginine]vasopressin analogues containingn-phenylalanine or an aliphatic n-amino acid in position 2 showed inhibitory activityin the antidiuretic, pressor and uterotonic tests, the ratio of the inhibition constantsin various assays depending on the type of the aliphatic amino acid in position 2(rer.'3) or in position 4 (ref.'·).

A combination of deamino-penicillamine in position I with an alky!tyrosine in posi­tion 2 increases the inhibitory activity in the uterotonic test as compared with [Iede­amino-penicillamlne'[oxytocirr'". The same has been found" also with the corres­ponding amino derivatives: both [I-penicillamine, 2-0-methyltyrosine]oxytocin and[I-penicillamine, 2-0-methyltyrosine, 8-lysine]vasopressin are strong inhibitorsin the nterotonic and pressor tests, On the other hand, acetylation of the a-aminogroup of penicillamine in position I reduced the inhibitory activity", regardlesswhether the molecule contained a tyrosine or O-methyltyrosine in the position 2or not. Interestingly enough, the inhibitory activity increased upen introductionof leucine into the position 2 (ref.14) . The spatial arrangement of the obtained analo­gue was intensively studied by physical methods and its enhanced inhibitory activitywas explained" by a reduction of the sterie strain in the side chains of the aminoacids in positions I and 2. The sterie bulk of the substituent in position 2 can beinfluenced by change in configuration of this amino acid. The tyrosine side chaincan form hydrogen bonds and its hydroxyl is assumed'" to be a part of the so-calledactive site of the oxytocin molecule. On the other hand, hydrophobic interactionsof the side chains of amino acids in positions 3, 4, 7 and 8 are very important forbinding to the receptor". A change in configuration of the amino acid in position 2combined with replacement of the hydrophilic hydroxyl by a hydrophobic snbstitnent

Collection Czechoslovak Chern. Ocmrnun. IVo1. S0l [1985J

G "·mZRT

134

could strengthen the bond to the receptor with simnltaneons reduction or completeelimination of the intrinsic activity, i.e, the resulting analogne shonld be an inhibitor.

We prepared deamino-6-carba-oxytocin analognes containing D-tyrosine, n-phenyl­alanine, p-methyl-n-phenylalanine, p-ethyl-D-phenylalanine and O-ethyl-D-tyrosinein the position 2. Deamino-6-carba-oxytocinwas chosenbecause its high uterotonicactivity indicates a high affinity towards the uterotonic receptor and its possibleoxidation has a snbstantially smaller effecton the biological activities than oxidationof the l-carba-aualogue'Pr'". To checkthe suitability of the6-carbasubstitution com­bined with deamination for obtaining more potent inhibitors we synthesized oxy­tocin and deamino-oxytocln analogues with p-ethyl-n-phenylalanine in tbe position 2.We prepared also analogues with penicillamine in position 1 and p-ethylphenyl­alanine of D or L configuration in position 2.

The analognes were prepared by stepwise synthesis of the chain nsing activeesters. Acylations with phenylalanine derivatives (protected with o-nitrobenzene­sulfenyl group) were performed with the 2,4,5-trichlorophenyl ester or N-hydroxy­benzotriazolyl ester prepared ill situ'", Benzyloxycarbonyl-S-benzylpenicillaminewas attached as the p-nitrophenyl ester!", p-Ethyl-DL-phenylalanine was resolvedvia its phenylacetyl derivative using penicillin amidohydrolase (E.C.3.5.1.11)(ref.4 2

." ) . n-Tyrosine was alkylated with diethyl snlfate as o-nitrobenzenesulfenylderivative in which the acylation of the amino gronp as well as the alkylation at theoxygen conld be curried out withont isolation of the N-acyl intermediate. This alkyla­tion reqnired a substantially higher pH of the reaction mixture and a longer reactiontime than the analogous alkylation with dimethyl sulfate and was followed by li­quid chromatography.

The carba-analognes were cyclized via N-hydroxybenzotriazolyl ester preparedfrom hydrochloride of the free octapeptide'". Disulfide-containing analognes wereobtained by removal of the protecting gronps with sodium in liquid ammonia and oxi­dation with potassium ferricyanide. The prodncts were desalted either on AmberliteIRC-50 or using Sep-Pak CiS cartridges'", All the obtained analognes were purifiedby reversed phase liqnid chromatography.

In syntheses of some analogues we made use of the fact that diastereoisomericpeptides can be easily separated by reversed phase chromatography'vr'". We there­fore performed the synthesis with racemic para-substituted phenylalanine derivativesand the formed diastereoisomers were separated only after cyelisation. Their identitywas determined by incubation of the hydrolysate with L-amino acid oxidase.

In the diastereoisomeric analognes Ie and If, hydrolysis of the peptide bondD-Phe(Et)-Ile is markedly slower than the L-Phe(Et)-Ile bond, as seen from Table 1.

Whereas in ali the studied cases the analogue with an aromatic D-amino acid iseluted in the reversed phase chromatography later than the corresponding t-dia­stereoisomer, the compound containing n-tyrosine differs from its t-analogue onlyvery little (IX = 1'05) and is eluted first. It was particularly important to exclude

Collection Czec.!l0slovak Chern, commun. Ivol. 501 [1985J

Amino Acids and.Peptides 135

contamination of the analogue Ib with the t-tyrosine analogue (which is very active)and it was therefore purified via its sulfoxide (preparedhy periodate oxidatlon't'')whose capacity factor differs substantially from that of the sulfoxide of the t-tyrosinecompound (a = 1'47). The sulfoxide was then reduced with hydrogen bromide inacetone'" ,48 and the obtained analogue purified hy liquid chromatography.

R'I

R2_C R4 CH21 I 5 . I

R -CH-CO-R -I1e-Gln-Asn-HN-CH-CO-Pro-Leu-Gly-NHz

R 1 R 2 R' R4 R S

Ia NH2 H H S-S TyrIb I-I H H S-CH2 DRTyrIe H H H S-CH2 n-PheId H H H S-CHz D-Phe(Me)Ie H I-I I-I S-CI-Iz Phe(Et)If I-I I-I H S-CI-Iz D-Phe(EI)Ig H H H SO-CH2 D-Phe(EI)Iii H H H S-CH2 D-Tyr(EI)II NH2 H H S-S D-Phe(EI)If H H H S-S D-Phe(EI)II< NH 2 CH, CH, S-S Phe(EI)II NH2 CH, CH, S-S D-Phe(Et)

The biological activities of the synthesized analogues are given in Table II. A com­parison with uterotonic activities of analogues containing t-amino acids in position2 can be found in our preliminary communication'P. As seen, both in the in vitro

TABLE I

Dependence of amount of liberated amino acids on the time in acid hydrolysis of compounds Ieand /f(related to glycine)

Hydrolysis time Phe(Et) lie Giu

hIfIf Ie If Ie Ie

5 0'57 0·72 0'39 0·44 0·72 0'7610 0'69 0·79 0'66 0·74 0·90 0'9420 0'87 0'97 0'87 0'90 1'02 1'0040 0'97 0'99 0'98 0·99 0·99 1'00

Collection Czechoslovak Chern. commun. [Vol. 501 [1985}

and in vivo assays on uterus the highest inhibitory activity was found with the analo­gue If containing p-ethyl-n-phenylalanine and carba-substitution in the disulfidebridge. Compounds with non-modified bridge show a slightly lower inhibitory activity,confirming the assumption of higher affinityofcarba-compounds towards the utero­tonic receptor. The amino compound is. slightly more potent inbibitor than theanalogue Ij.<A similar situation was observed also with other analogues containingpenicillamine or deaminopeniciliamine22 • The combination of substitution at theJl-carbon atom of cysteine in position 1 with introduction of a n-amino acid intoposition 2 is not of additive character; on the contrary, it reduces slightly the inhibi­tory activity of the analogue. A comparison of properties of compounds Ii, 1/, and 11shows that a configurational inversion at the ex-carbon of the amino acid in position 2alone is more important than the introduction of penicillamine into the position I.

However, a mere configurational inversion is not sufficient; very important isremoval of hydroxyl from the tyrosine moiety because compounds containing n-tyro­sine have their own activity. Alkylation of n-tyrosine preserves a polar oxygenatom in the molecule which, as it seems, is unfavourable for the inhibitory activity.The activity of [2-0-ethyl-o-tyrosine]oxytocin31 (as compared with the inhibitoryactivity of compound 1II) again shows the suitability of the carba-modificationfor obtaining an inhibitor.

136 Lebl, Barth, Servltovd, Slaninovd, Jost :

TABLE II

Some biological activities ofthe prepared compounds (I.U.fmg or pAz values)

Uterotonic Antidi-Compound" Galactogogic Pressor uretic

ill vitro ill vivo

Ib [n-Tyr2IdC 6OXT 28 59 40'3 4 0·12Ie [n-Phe2IdC 6OXT pA2 ~ 8'06 pA2 ~ 7·15 1'25 0'04Id [n-Phe(Me)2IdC6OXT pA2 = 8·73 pA2 ~ 7·51 10'3 0·17If [n.Phe(EtJ'ldc"OXT pAz = 8·73 pA2 = 7-82 pA2 ~ 5'82 pA2 = 6'32 0'08Ig [n-Phe(Et)2jdC6OXT.

-sulfoxide pA2 = 7'91 pA2 = 7·5 pA, = 6·8 pA2 = 5·75 0'002

III [n-Tyr(Et)2 jdC·OXT pA2 = 7'45 0 0Ii [n.Phe(Et)210XT pA2 ~ 8·15 pA2 = 7·70 1·22 1·5Ij [Mpa', n-Phe(El)21-

OXT pAz = 8·06 pAz = 7·12 0'3 pA2 ~ 6·82lk [Pen', Phe(Et)210 XT pA, ~ 7·78 pA2 ~ 5'92 0-45 b pA, ~ 7'8 0'025II [Pen', n-Phe(Etj21-

OXT pA, = 8'09 pAz = 7·25 pA2 = 7·7 pA2 = 7'4 0·0007

a dC60XT means deamino-d-cnrba-oxytocin; b in some cases inhibition was observed.

Collection Czechoslovak Chern. Commun. [Vor. 501 [1985]

Amino Acids and Peptides

R-lie-Gln-Asn-Hcy(CzH.COOH)-Pro-Leu-Gly-NHz

lIa, R =HlIb, R = Nps-D-Tyrttc, R = Nps-n-Phetu. R = NPS-DL-Phe(Me)lIe, R = Nps-D-Phe(Et)Ill. R = Nps-D-Tyr(Et)

R-lie-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NHzlIla, R = NpsIIIb, R = NPS-DL-Phe(Et)Illc, R = Z-Cys(Bzl)-DL-Phe(Et)IIId, R = Mpa(Bzl)-DL-Phe(Et)IIIe, R = Z-Pen(Bzl)-DL-Phe(Et)

137

Oxidation of carba-analogues of neurohypophyseal hormones affords sulfoxidesexhibiting mostly lower activities than the corresponding sulfides'9,.0 (for exceptionssee ref!0,.9), the l-carba-analogues being substantially less active than the 6-carba­-analogues, We prepared the sulfoxide of compound If which showed about sixtimes lower inhibitory activity than the starting sulfide.

The inhibitory activities found in the in vivo test on the rat uterus parallel theactivities determined in vitro. The galactogogic activity was inhibited only in thecase of compound If and, surprisingly, its sulfoxide Ig was even more potent in­hibitor. On the contrary, compound Ij which does not contain a carba-bridge, showsno inhibition. Except compound I k, the prepared analogues are not effective inhibi­tors of vasopressin pressor activity. This indicates that for this inhibitor type (con­trary to the uterotonic inhibitor) alkylation of the p-carbon atom of cysteine in posi­tion 1 represents a much more effective structural feature than configurationalinversion in position 2 (compound Ib retains to a high degree its own pressor activity).Except compound Ii, all the prepared analogues exhibit a very low antidiureticactivity.

EXPEillMENTAL

The analytical samples were dried ill vacuo (150 Pa) over phosphorus pentcxide at room tempera­ture. Melting points were determined on a Kofler block and are uncorrected. Thin-layer chromato­graphy was carried out on silica gel plates (Silufol, Kavalier, Czec~oslovakia) in the systems2-butanol-98% formic acid-water (75: 13·5: 11,5) (SI), 2-butanol-25% aqueous ammonia-water(85: 7·5: 7·5)-{S2). l-butanol-acetic acid-water (4: 1 : 1) (S3). and Icbutanob-pyridine-aceticacid-water (15: 10 : 3 : 6) (S4). Electrophoresis was performed on a Whatman 3MM paperin moist chamber (20V/cm) for 1 h in 1 moll- 1 acetic acid (pH 2,4) and a pyridine-acetatebuffer (pH 5·7). The compounds were detected by ninhydrin or by the chlorination method. Sol­vents were evaporated on a rotatory evaporator at bath temperature 30°C; dimethylformamidewas evaporated at the same temperature at 150 Pa. Samples for amino acid analyses were hydro-

Collection Czechoslovak Chern. Commun. IVol. 50} [1985}

7

Iyzed with 6 mol 1-1 He! at 105°C for 20 h and analyzed on an automatic analyzer, type 6020(Development Workshops of Czechoslovak Academy of Sciences). The high performance liquidchromatography (lIPLC) was carried out on an SP~8700 instrument equipped with an SP-8400detector and an SP-4100 integrator (all from Spectra-Physics, Santa Clara, USA). L-Amino acidoxidase (0' 3 lJlrog) was a Serva product.

138 Lebl, Barth, Servttova.Blanlnova.Jost :

Dicyclohcxylammonium Salt of o-NitrobenzencsulfenyI-O-ethyl-n-tyrosine

c-Nitrobenzenosulfenyl chloride (5-78g) was added to a solution of n-tyroslne (2-83 g) in 4 mol.. 1-1 NaOH (9-8 ml) and dioxane (16 ml), the pH of the mixture being kept at 10 and the tempera­ture below Soc. After stirring for 30 min, diethyl slllfate(6'9 g)and 4 mol I-I NaOH were addedso as to keep the pH at 10'7. The alkylation course was followed by reversed-phase HPLC (70%of methanol and 30% of 0'05% aqueous trifluoroacetic acid; kNp5Tyr = 2'21, kNp5Tyr(Et) = 9'56).After disappearance of the starting compound (80 min), the mixture was filtered, extracted withether and ethyl acetate and the aqueous layer was acidified at O°C with 0·5 molI- 1 H 2S04to pH 3 and extracted with ethyl acetate. The organic layer was washed with water, dried oversodium sulfate, concentrated and poured into an ethereal solution of dicyclohexylamine (3'44 mlin 200 ml). After standing overnight the crystals were collected on filter and dried, affording 6·5 g(76%) of the product, m.p. 174-178°C, [lXlo -49" (c 0'19; methanol). For C29H.uN30SS(543,7) calculated: 64'06% C, 7'60%H, 7'73% N; found: 64·53% C, 7·55% H, 7'86% N.

General Method of Preparation of Octapeptides IIb- II!

The dicyclohexylammonium salt of the amino acid derivative (0'8 mmol) was suspended in ethylacetate (30 ml) atO°C and the amino acid derivative was liberated by addition of 0'05 moll- 1

H 2S04 , The ethyl acetate solution was dried over sodium sulfate and taken down. The residuewas dissolved in dimcthylformamide (4 ml), Nehydroxybenzotriazole (122 mg) was added,and, after cooling to OaC, dicyclohexylcarbodiimide (182 mg). After stirring for 1 h at DoC and1 h at room temperature, the mixture was filtered into a flask containing the heptapeptide IIa(350 mg). The solid cake was washed with dimethylformamide (3 ml) and the obtained solutionwas stirred for 12 h at room temperature. After filtration and evaporation of solvent, the resi­due was triturated with ether and water, the product was dried and an analytical sample wasprecipitated with ether from dimethylformamide; its properties are given in Table III.

o~Nitrobenzenesulfenyl-p~ethyl-DL-phenylalanyl-isoleucyl-glutaminyl-asparaginyl­

-Swbenzylcysteinyl-prolyl~leucyl~glycineAmide (II/b)

A solution of the heptapeptide II/a (2·5 g) in dimethylformamide (100 ml) was mixed with3·4 moll- 1 Hel in ether (1'7 ml). After standing for 5 min at room temperature, the heptapeptidehydrochloride was precipitated with ether, filtered, dried ill vacuo and dissolved in dimethyl­formamide (100 ml). o-Nitrobenzenesulfenyl-DL-p~ethylphenylalanine 2,4,5-trichlorophenyl ester(1,8 g) was added, the mixture was adjusted to pI-I 8 (moist indicator paper) and stirred at roomtemperature for 130 h. The solution was concentrated to a small volume, the product was pre­cipitated with ether, filtered, washed with ether and water and dried ill vacuo; yield 3·1 g (89%)of IIIb (for data see Table ill).

Benzyloxycarbonyl-S~benzylcysteinylkp·ethyJ-DL-phenylalanylwisoleucylwglutaminylwasparagi­

nyI-S-benzylcysteinyl~prolyl-Jeucyl-glycineAmide (llIe)

The octapeptide IIIb (0·5 g) was dissolved in dimethylformamide (25 ml) and the solution

Collection Czechoslovak Chern. Commun. [Vol. 501 [19851

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140 Lebl, Barth, Servltova, Slanlnova, Jost:

was mixed with 3'45 moll- t HCI in ether. After standing for 5 min the product was precipitatedwith ether, filtered, dried in vacuo and dissolved in dimethylfcrmamide (23 rot). p-Nitrophenylester of benzyloxycarbonyl-S-benzylcysteine (0'8 g) was added, the mixture was adjusted to pH 8with Nrethylpiperidine, stirred at room temperature for 125 h, and concentrated to a smallvolume. Theproduct was precipitated with ether, filtered, washed consecutively with ether,1 moll- t act, water, 0·5 mol 1-1 NaHC03• water, and dried; yield 0-42 g (73%). For data seeTable III.

Benzyloxyearbonyl-S-benzylpenicillaminyI-p-ethyl-DL;.phenylalanyl-isoleucyl-glutaminyl­-asparaginyl-S~benzylcystcinyl-prolyl-Ieucyl-glycineAmide (IIIe).

Bcnzyloxycarbonyl-S-benzylcysteine p-nitrophenyl ester (0'9 g) was added to a solution of theoctapeptide hydrochloride prepared in the same manner as in the case of II/c. The condensationand work up were carried out as described for compound I1Ie, yielding 0·46 g of IIIe (Table III).

S-BenzylmercaptopropionyI-p-ethyl-DL~phenylalanyl-isoleucyl~gtutaminyl-asparaginyI­

-S~benzylcysteinyl-prolyl-Ieucyl-glycine Amide (IIId)

A solution of the octapeptide hydrochloride, prepared as described for IIIe, was adjusted to pH 8with Neethylpiperidine. S-Benzylmercaptopropionic acid (95 mg), followed by N-hydroxybenzo­triazole (62 mg), was added and the mixture was cooled to -20GC and treated with dicyclohexyl­carbodiimide (104 mg). After stirring at -20"C for 1 h, at DoC for 30 h, and at room tempera­ture for 2 h, the mixture was filtered and taken down. The residue was suspended in water,filtered, washed successively with. I mol 1- 1 Hel, water and ether and dried, affording 380 rngof the product whose properties are given in Table III.

[2-o-Phenylalanine]deamino-6-carba-oxytocin (Ie)

A solution of the octapeptide IIe (150 mg) in dimethylformamide (4 ml) was mixed with 3·2 mol .. 1- 1 HCI in ether (0,4 ml). After standing for 6 min at room temperature, the octapeptidehydrochloride was precipitated with ether. The hydrochloride was reprecipitated from dimethyl­formamide with ether, filtered, dried in vacuo and dissolved in dimethylformamide (4 mI).N­-Hydroxybenzotriazole (l81 mg) was added, the mixture was cooled to DoC and treated withdicyc1ohexy1carbodiimide (200 rng). After stirring at DoC for 1 h and at room temperature for1·5 h, the separated crystals of dicyetohexylurea were filtered off and the filtrate was introducedinto warm (45°C) methanol (150 ml). The mixture was adjusted to pH 8·5 with N-ethylpipcridinc,kept at 45°C for 2 h and concentrated to a small volume. The product was precipitated withether, filtered and dried in vacuo; yield 140 mg. A part (30 mg) was dissolved in aqueous methanol(1 : 1, 10 ml), applied on a column of PartisH ODS~2 (50 X 0·9 cm) and eluted with a mixtureof 55% methanol and 0'05% aqueous trifluoroacetic acid. The product-containing fraction(k' = 7'3) was taken down ill llaeuo and freeze-dried, affording 6·5 mg of the product Ic, pureaccording to HPLC and TLC. Its properties are given in Table IV.

[2-0-Tyrosine[deamino-fi-carba-oxytocln (Ib)

The octapeptide IIb. (ISO mg) was cycllzed in the same manner as the analogue Ic. The crudeproduct was purified on the same column using a mobile phase containing 40% of methanol.Freeze-drying of the product fractions afforded 22 mg (16%) of the product which, accordingto HPLC of sulfoxides prepared by periodate oxidation, contained 0'8% of the i.-tyrosinc analo­gue (compared with the authentic sultoxide-"). Therefore, a part of the product (10 mg) was

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oxidized with periodate and purified again on the above-mentioned column (elution with 35~"~

methanol). A part of the sulfoxide, obtained by freeze-drying of the corresponding fractions,was used for.biological assays, another part (7 rng) was suspended in acetone (200 Ill), treatedwith 35% HBr in acetic acid (200 Ill) at room temperature for 5 min and again purified on a rever­sed phase column (40% methanol) to give 4'6 mg of the compound.

[2-p-Methyl-D-phenylalanine]deamino-6-carba_oxytocin (Id)

The octapeptide IId (280 rug) was cyclized as described for the analogue Ie. A part of the crudeproduct (lOOmg) was dissolved in a mixture of methanol (4 ml) and water (6 ml) and appliedon a column of Partisil ODS (50 X 0'9 em). Elution with a mixture of methanol (55%) and 0'05~~

aqueous trifluoroacetic acid afforded fractions of k' = 5·7 (12'6 mg) and k' = 7'6 (13'7 mg), theformer containing a compound identical with the already described [2-p-methyl-L-phenylalanine]­deamino-ti-carba-oxytocin. The acid hydrolysate of both fractions was incubated with L-arninoacid oxidase which removed p-methylphenylalanine from the former but not from the lattermaterial. Properties of the analogue Id are given in Table IV.

[2-p-Ethyl-D-phenylalanine]deamino-6-carba-oxytocin (If)

Cyclisation of the octapeptide lIe (300 mg) was carried out as described for the analogue Ieand afforded 249 mg of the crude product. This material was dissolved in a mixture of methanol(to ml) and water (18 ml) and the filtered solution was purified in three portions on a columnof Partisil ODS (50 X 0·9 em) using methanol-0'05% trifiuoroacetic acid (56 : 44) as the mobilephase. Concentration and freeze-drying of fractions of k' = 9·4 gave 68 mg of the productwhose properties are given in Table IV.

[2-p-Ethyl-D-phenylalanine]deamino-6-carba-oxytocin Sulfoxide (/g)

Sodium periodate was added to a solution of the analogue 11(4 mg) in aqueous methanol (l : 1;400 Ill). After standing for 2 h at room temperature, the mixture was applied on a columnof Partisil ODS (50 X 0·9 em) and eluted with a methanol-water (3 : 2) mixture. Freeze-dryingof the product fractions afforded 2·8 rng (64%) of the product, RF 0'14 (51), 0'05 (52), 0·16 (53),0'62 (54). For C46H 71 N l1 °125.4 H20 (I 074) calculated: 51'43% C, 7'41% H, 14'34%N; found:51'15% C, 6'85% H, 14'26% N. Amino acid analysis: Asp 1'00, Glu 1'00, Pro 1'03, Gly 1'02,lie 0'93, Leu 1'01, Phe(Et) 1,01, Hcy(C3H,02) 0·53.

[2-0-Ethyl-D-tyrosine]dearnino-S-carba-oxytocin (Ih)

The octapeptide III (130 rug) was cyclized in the same way as described for the analogue Ic,affording 135 mg of the crude product. A part of this material (30 mg) was dissolved in a metha­nol-water mixture (4: 6), applied on a column of Partlsil OD8-2 (50 X 0·9 ern) and elutedwith a mixture of methanol and 0'05% trifluoroacetic acid (1 : 1). The fraction of k' = 26'3was concentrated and freeze-dried, affording 6·2 mg of the product, pure according to HPLCand TLC. Its properties are given in Table IV.

[2-p-Ethyl-D-phenylalanine]deamino-oxytocin (If)

The peptide IIId (50 mg) was dissolved in liquid ammonia and reduced with a sodium rod untilthe blue colouration persisted for I min. The excess sodium was destroyed with a drop of aceticacid and the mixture was freeze-dried. The residue was dissolved in 0·1 mol 1- 1 Hel (8 ml},

Collection Czechoslovak Chern, Commun. [Vol. 501 [12B5)

Amino Acids and Peptides 143

the solution was diluted to 100 ml and adjusted to pI-1 7 with 0:1 moll- 1 NaOH; A solutionof K3Fe(CN)6 (25 mg in 5 ml of water) was added during Zu rnin the pl-l value being kept at 7.After stirring for 40 min at room temperature, the mixture was adjusted to pH 4 by additionof acetic acid and applied on a column of Scpharon SIwC-18 (25 X 0·4 em} by. means of a highpressure pump. The column was washed with water and the peptides were eluted with a mixtureof SO% methanol and 0·05% aqueous trifiuoroacetic acid. The eluate was concentrated ill vacuo,freeze-dried, the residue was again dissolved in aqueous methanol (1 :1; SmI) and applied on a co­lumn of Partisil ODS (50 X O·gem). Elution with a mixture of methanol and O'05/~ aqueoustrifiuoroacetic acid (65 : 35) afforded two fractions of k' = 10'2 (8'1 mg: 3S%) and k' = 15'9(7'6 mg; 36%). The former product contained p-ethyl-L-phenylalaninewhereas the latter con­tained p-ethyl-c-phcnylalantne (according to incubation of the hydrolysate with r.-amino acidoxidase). The properties of the latter compound are given in Table IV.

[2-p-Ethyl-n-phenylalanine[oxytocin (II)

The nonapeptide IlIc (200 mg) was. reduced and oxidized as described for the compound I],The cyclization solution was applied on a column of Amberlite IRC-50 (30 ml), the columnwas washed with 0'25% acetic acid and the peptides were eluted with 50% acetic acid; The productobtained by freeze-drying was dissolved in 50% aqueous methanol (8 ml), applied on a columnof Partlsil ODS (50 X 0·9 em) and eluted with a mixture of methanol (60%) with 0·1 moll- 1

ammonium acetate (40%), pH 7. Concentration and freeze-drying of the product fraction af­forded 16·4 mg (21%) of compound, k' = 11'6. containing p-ethyl-t-phenylalauine, and 15·8 mg(20%) of compound, k' = 23·3, containing p-ethyl-n-phenylalanine (incubation with L-aminoacid oxidase). The former product was identical with the sample prepared 16 years ago 7

, storedin the form of lyophilisate at room temperature. For properties of the latter compound seeTable IV. As side product we obtained compounds which (according to amino acid analysis) didnot contain the terminal tripeptide (k' = 7·3 and 14'4; 5·2 and 3'8 rng, respectively).

[l-Penicillamine, 2-p-ethyl-D- and t-phenylalaninejoxytocln (Lk and Ie)

The nona peptide IIIe (200 mg) was reduced and oxidized as described for the analogue Ii. Thecyclization mixture was processed and purified in the same manner as in the case of 11' exceptthat the elution was carried out with a mixture containing 65% of methanol. Concentrationand freeze-drying of the corresponding fractions atTorded17·4 mg (22%) of product containingp-ethyl-t-phenylalanine (Ie' = S·05) and 14·6 mg (l9;C;;) of compound containing p-ethyl-n-phenyl­alanine (k' = 13·S); for their properties see Table IV.

Pharmacological Methods

Uterotonic activity was determined on an isolated strip of rat uterus 51,52. the activity ill vivowas estimated according to PIiska53. Galactogogic activity was determined on ethanol-anestheti­zed rats 54

•55, pressor activity on despinalized rats 56 and antidiuretic y.otency on anesthetizedrats 57,58. Inhibitory properties were expressed by the pA., value; for the test in vitro (rat uterus)the recornmendations-" were obeyed, for the assay in vivo;e followed the described method1 5

,6 0 .

~Ve are indebted to Mrs H. KovtiroL'ti and J. Kellerova for the technical assistance in carrying autthe biological tests, to Mrs H. Farkafot"(l for carryillg out the am/no add analyses and to Mrs Z.Ledninoixi for optical rotation measurements. Tlte elemental analyses were performed ill lite Analy­tical Laboratory of this Institute (Dr J. Horacek, Head).

Collection Czechoslovak Chern. Commun. [Vol. 501 119851

144

REFERENCES

Labl , Barth, Servitova, Slaninova, Jost ;

1. Rudinger J., Krejci 1. in the book: Handbook ofExperimental Pharmacology (B. Berde, Bd.},Vol. 23;'p. 748. Springer, Berlin 1968.

2. Suwycr.w. H., Grzonka Z., Manning M.: Mol. Cell. Endocrinol. 22, 117 (1981),3. Hruby.V. J., Mosberg I-I, L: Hormone Antaqonists (M. K. Agarwal, Ed.}, p. 433. de Gruyter,

Berlin 1982.4. Biochemical Nomenclature and Related DOCIl11leJl!s. International Union of Biochemistry,

London 1978.5. Law H. D., du Vigneaud Y.: J. Amer. Chern. Soc. 82, 4579 (1960).6. J05t K" Rudinger J., Sorm F.: This Journa128, 1706 (1963).7. Zhuze A. L., Jost K., Kasnffrek E" Rudinger J.: This Journal 29, 2648 (1964).8. Chimiak A., Eisler K., Jost K., Rudinger J.: This Journal 33, 2918 (1968).9. Jost K., Sorm F.: This Journal 36, 297 (1971).

10. Krojidlo M., Barth T., Scrvitova L., Dobrovsky K., Just K., Sorm F.: This Journal40, 2708(1975).

1i. Krojidlo M., Barth T., Blaha K., Jest K.: This Journal 41, 1954 (1976).12. Kasaffrek E., Eisler K., Rudinger J.: This Journal 34, 2848 (1969).13. Schulz H., du Vigneaud Y.: J. Med. Chern. 9, 647 (1966).14. Hruby V. J., Deb K. K., Yamamoto D. M., Hadley M. E., Chan W. Y.: J. Med. Chern. 22,

7 (1979).15. Vavrek R. J., Ferger M. F., Allen G. A., Rich D. R., Blomquist A. T., du Vigneaud V.:

J. Med. Chern. 15, 12J (1972).16. Nestor J. J., Ferger M. F., du Vigneaud V.: J. Med. Chern. 18, 284 (1975).17. Lowbridge J., Manning M., Seto J., Halder J., Sawyer W. H.: J. Med. Chern. 22, 565 (1979).18. Sawyer W. H., Haldar J.,Gazis D., Seto J., Bankowski K., Lcwbridge J., Turan A., Manning

M.: Endocrinology 106, 81 (1980).19. Ferger M. F.: J. Med. Chern. 18, 1020 (1975).20. Dyckes D. E, Nestor J. J., Ferger M. E, du Yigneaud V., Chan W. Y.: J. Med. Chern. 17,

969 (1974).21. Manning M., Lowbridge J., Seto J., Haldar J., Sawyer W. H.: J. Med. Chern. 21, 179 (1978).22. Hruby V., Mosberg H. J., Hadley M. E., Chan W. Y., Powell A. M.: Int. J. Peptide Protein

Res. 16, 372 (1980).23. Grzonka Z., Knsprzykowski E, Larnmek B., Gazis D., Schwartz I. L.: Peptides 1982. Proc.

17th Bur. Pept, Syrup, (K. Blaha, P. Malon, Eds) , p. 445. de Gruyter, Berlin 1983.24. Hruby V. J., Knittel J. J., Mosberg H. I., Rcckway T. W., Wilkes B. C.: Pepttdes 1982. Proc.

17th Eur. Pept. Symp, (K. Blaha, P. Malon, Eds), p. 19. de Gruyter, Berlin 1983.25. Drabarek S., du Vigneaud V.: J. Arner. Chern. Soc. 87, 3974(1965).26. Hruby V. J., Upson D. A., Yamamoto D. M., Smith C. W., WaIter R.: J. Amer. Chern. Soc.

101,2717 (1979).27. Kaurov O. A., Martynov Y. F., Smirnova M. P.: Zh. Obshch. Khim. 43, 217 (1973).28. Kaurov O. A., Martynov V. F., Smimova M. P.: Khim. Prir. Soedin.1977, 392.29. Auna Z. Pc.Kaurov O. A., Martynov V. F., Morozov V. B.:2h. Obshch. Khim. 41, 674(1971).30. Kaurov O. A., Mikhailov Yu. D.,Srnirnova M.P.: Bioorg. Khirn. 4, 619 (1978).31. Kaurov O. ~., Martynov V. F., Mikhailov Yu. D., Auna Z. P.: Zh. Obshch. Khim.42,1654

(1972).32. Manning M., Olma A., Klis W. A., Kolodziejczyk A. M., Seto J., Sawyer W. H.: J. Med.

Chern. 25, 45 (1982).33. Manning M., Klis W. A., Olma A., Seto J., Sawyer W. H.: J. Med. Chern. 25, 414 (1982).34. Fric I., Kodicek M., Flegel M., Zaoral M.: Eur. J. Biochem. 56, 493 (1975).

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35. Simek P., Barth T., Bttnik E, Slanlnova J., Jest K.: Peptides 1982. Proc. 17tft Elll'. Pept.Symp. (K. Blaha, P. Malon, Eds), p. 461. de Gruyter, Berlin 1983.

36. Manning M., Olma A., Klis W. A., Seto J., Sawyer W. H.: J. Med. Chern. 28, 1507 (983).37. Hruby V. J., Deb K. K., Fox J., Bjarnason J., Tu A. T.: J. BioI. Chern. 253, 6060 (1978).38. Walter R.: Fed. Proc., Fed. Amcr. Soc. Exp. BioI. 36, 1872 (1977).39. Lebl M., Barth T., JoSt K.: This Journal 43, 1538 (1978).40. Lebl M., BarthT., rest K.: Peptides 1980, Proc. 16th Eur, Pept, Symp. (K. Brunfeldt, Ed).,

p. 719. Scriptor, Copenhagen 1981.41. Konig W., Geiger R.: Chern. BeLl03, 788 (1970).42. Simek P., Barth T., Barta M., Vojtfsek V., Jest K.: This Journal 46, 2263 (1981).43. Lebl M., Hrbas P., Skopkovd L, Slaninova J., Machova A.,Barth T., rest K.:This Journal 47,

2540 (1982).44. Krojidlo M., Flegel M., Lebl M.: Peptides 1982. Proc. 17th. EliI'. Pept. Symp, (K. Bhiha,

P. Malon, Eds}, p. 199. de Gruyter, Berlin 1983.45. Bohlen P., Castillo F., Ling N., Guillemin R.: Jnt. J. Peptide Protein Res. 16, 306 (1980).46. Larsen B., Fox B. L., Burke M. E, Hruby V. J.: Int. J. Peptide Protein Res. 13, 12 (1979).47. Lebi M.: J. Chromatogr. 264, 459 (1983).48. Iselin B.: Helv. Chim. Acta 44, 61 (1961).49. Lebl M., Barth T., Jost K.: This Journal45, 2855 (1980).50. Lebl M., Barth T., Servitovd L., Slaninova J., Jost K.: Peptides 1982. Proc. 17th Bur. Pept.

Syrup, (K. Blaha, P. Malon, Eds), p. 456. de Gruyter, Berlin 1983.51. Holton P.: Brit. J. Pharmacal. 3, 328 (1948).52. Munsick R. A.: Endocrinology 66, 451 (1960).53. Pliska V.: Eur. J. Pharmacal. 5,253 (1969).54. Bisset G. W., Clark B. J., Haldar J., Harris M., Lewis G. P., Rocha e Silva M.: Brit. J. Phar-

macal. Chemotherap. 31, 537 (1957).55. Barth T., Jost K., Rychlik 1.: Endocrincl. Exper. 9, 35 (1974).56. Krejci I., Kupkova B., Vavra 1.: Brit. J. Pharmacol. Chemotherap. 30,497 (1967).57. Jeffers W. A., Livezey M. M., Austin J. H.: Proc. Soc. Exp. BioI. Med. 50,184(1942).58. Pliska V., Rychlik I.: Acta Endocrinol. 54,129 (1967).59. Eggena P., Schwartz I. L., Walter R.: J. Gen. Physfol. 56, 250 (1970).60. Schild I-I. 0.: Brit. J. Pharmacal. 2,189 (1947).

Translated by M. Tichy.

Collection Czechoslovak Chern. Commun. [Vol. 491 [19841