SE448303B - OXYTOCIN ANALOGS WITH NATURURETIC EFFECT - Google Patents

Description

Dimethylamino or nitro.

- CH2CO ~ Act'7 X-Ile + Gln¿Asn-NH-èH-COfPro-Leu-Gly ~ NH2 CH 448 303 2 çHz-f Si, .CI-Izf-'rí CH2-CO ~ X-Ile-Gln The maple amino acids belong to the L series and X represents the amino acid phenylalanine or phenylalanine substituted in pairing with a The selected oxytocin analogs (I) can be conveniently prepared by cyclization between an amino group of an amino acid X and the carboxyl group of the S-carboxyethyl homocysteine residue on a V linear peptide of the general formula (II). ) :.

G 2 -C- S ---- CH 2 * -f * - "" ***** CH2 I (II) - wherein X represents the amino acid phenylalanine or phenylalanine substituent in pairing with a group selected from methyl, ethyl 1 '-Ethoxy, amino, dimethylamino or nitro and Act denote according to activator a carboxyl group as an active ester group.

The oxytocin analog of the following general formula (III): 1sl 'CH ICHI 2A 2_ 2 èH2-CO-NH-CH # CO-Ile-Gln-AsnfNH-èH-COfPro-Leu-Gly-NH2 (III)' n i.

.CH _ _. The Nnzn can be conveniently prepared as indicated by I, (a) reduction of β-nitrophenylalanyl-7-deamino-6-carba-oxytocin with sodium in tetrahedral ammonia or 7 (b) from β-Vpz-benzyloxy-diphenylamino -6- * -carba-okytocin Qenom cleavage of the protecting group with hydrogen bromine in family syrup. Some biological activities of the oxytocin analogues of the general formula I are given in Table I in comparison with oxytocin. 10 15 20 25 30 35 -448 303 3 Tabëi1'I 'An¿10g.IN Biological activity (in rats) XK, _' e "Uterus- Milk- Blood- Sodium- * f concomitant- drive- pressure- urinary tract the increasing levels of (a) (a) (a) (b) go-gytocin in 450 »1150 5.0 010.0 NH-OH (cH2f06H5) -co g ~ - 70 170 0.9 l fl š -NH + OH ( On2-conu-OH5) co a 0 M5 55 1 326 NH + cH (OH2-06H2 ~ c2H5) -co 27 1 '1, »(0.2 25H gNH-OH (cH2-cono-ocHH) - co <0.001 5.6 (0.2 31 _NH-c fi ccngfcö NH u-NH2) ~ 0o 15 7 40.2 - 87 NH-cH (cn2-cenu-N (0H3) 2) -co <0.1u ~ u, 5 (0.2 75 NHscH (cH2-c6HuéNH-co2cH2. 1 VI 0:07 (052 10 NH-cH <0H2-cööu-No¿> ~ co0 0 <0.001 1.1 a <0.2 66) al International Units_per The process for the preparation of the analogues of oxytocin is further described in the following working examples:> Example gel I This example describes the preparation of β-p-nitrophenylalanine-7-deamino-6-carbaoxytbcin} To initiate its preparation, an octapeptide of the formula II was prepared in the following manner: 0.29 g of 214,5-trichloropheno] or 0.33 g dicyclohexylcarbodiimide was added to a solution of ortho-nitrobenzenesulfenyl-p-nitro-phenylalanine (0.53 g) in a mixture of 15 ml of dichloromethane and 15 ml of dimethylformamide, cooled to -10 ° C. The resulting mixture was stirred for 1 hour at -10 ° C and then for 12 hours at room temperature; Thereafter, the mixture was concentrated in vacuo, which resulted in the formation of crystals, which were filtered through suction by suction. The filter cake thus prepared was then washed with dichloromethane, and the filtrate was evaporated to dryness (at 20 DEG C. bath temperature). The yellow residual oil was then triturated several times with petroleum ether and dissolved in 12 ml of dimethylformamide. Thereafter, the amide of the isoleucyl-glutaminyl-2 -nyl-asparaginyl-S (2-carbomethyl) -homocystinyl + propyl-leucyl--glycine (0.8 g) was suspended in this solution, and after stirring the resulting solution for 135 hours. hours at room temperature it was evaporated to dryness (bath temperature 35 ° C). The resulting oily product crystallized after decomposition with petroleum ether and was then successively washed on glass frit with water, 0.05 M sulfuric acid, water and ether to give 720 mg of an octapeptide having a melting point of 215-219 ° C, with the properties 'As shown in Table II below: 7 7 In Tables II and III below, S1 denotes 2-putanol-98% »formic acid-water 75í13.5: l1.57vo1 ~% S2 7.2-btanol-25% aqueous solution of ammonia-water 85 : 7.5: 7: 7.5 vol-% "S3 _ 1-butanol-acetic acid-water 4: 1: 1 vol-% -ZS4 _ 1-butanol-pyridineacetic acid-water 15: 10: 3: 6 vol- % Eglâ refers to the relative mobility of the compound in question in relation to the mobility of hiatidin in pyridine acetate buffer pH 7 ZVI 31% refers to the relative mobility of the compound in question in relation to the mobility of glycine in 1M acetic acid, pH 2.4 k 'means the liquid chromatography constant calculated according to the formula t _c k |: __' X 'tO "'. '* T 0 where tx is the elution time of the compound in question and to is the elution time of non-adsorbed compound, ^'.;. '. f »_ | o @ o ~ m @ mm @@ ~ N .Hm flflflfl w flflfi EOW nwg flfl wv fi ßf fi CQÜ MÜÜEWPWÖQ. .Åumovwsm møcm flfl mnwcc fl .Hmmws fi cwmmm .Hæm mmm Aumovmnm + .ÄB Gm fi ssm m s. 4. _ _ _ n: f. -. . 0 _. f u n za. _ _ »V L I if! wa oo m0.H n fl b fl Ho. fl wo. fl ßo.mH mß.wm @ .Hm .fmwfo fi fzßßzwmo @ o. @ fl. o ~ No, Nmxz »w; 1 MW« m. @ oo. ~ HQ1 ~ om. o fl m.mH om. @ w «, Nm Amm flfl v GN: ~ m @ .o. fl «. o H @ .o_ fl0. H mm.ø No. fl oo.H mo.« ~ _ @ ß. @ «m. ~ m .vww fi ow fl zmßïnmu o fi. o @«. o m0.o_ Hpmv fl ßm m @. @ mo.H _m0. ~ fl m.o @w. «H ßw.w _ m> .mm A fl w fifl w QNI @@. om« .o ww.o _ w @. ~ ßm.oo ~. fl vø. fl ßv . «H øm.m _« ß.Nm. ~ M «.oNH_ <ß; Hmu ß0.ø mm.o fl H.0 fl mxvwgm mw.o_ mw.Q; o fl.fl mw.0 mo. m @ .H mm.0 @ o. ~ ßo. fl _mw.n ~ @mw oo.mm .fi mm fl nv fl zøßïwmo m fl. o ß «.o flfl. o fi wmzvwfm mm. @ mm.ø No1 ~ _ _mw.o ~ m . <fi mm @@ mw.om _ ^ mmA ~ V_oN: m wm.nw «.0 _ mm.0_: _« QH fl o. fl fi ø. ~ mo. fl mH. mw.o @ m.o ßo. ~ ~ m @ o _mm.NH mm. @ _ ßw; ~ m __ ßomw fl v ON: m.A Nw.o w «.o wmfo w @ _. _ mo.H ß @. fl No. fl> oJ fl _oo. «fl mm. @ mo. ~ m _.« @ mf @ 0.z @ ß: ~ mu _ßo, o. @ n.n_ vd.o. . ^ »WøvwCm _Nm.O mm, ø ßo» fl ___ M @ .o ___ fl N.m fi_ m ~. @ O flflfl m ^ «ßHHv wm.om« »o _._ ßw.ow« ~ mh .__ _ ul al. ) \ l. 1.1. u n _ .auf nLÛ. ) .I a_ _n .. _ w._ .. ._ no. mn F vc _ mo _ fm m fl mo @ W rm_ »wLHovHzf> IomL_ ßo 0 nn 0« H o._ hwoyym fi m _ _ _ _ w _m__ ... _ WIUQU 0 _ Ü __ _ .w w_ m fl wm _ I _ _ J __ _ _ _ 1 .TNUV xfuï GX __ _ UAH 2% __ _ __ Tïm Ovm. _ .Apk fl b fl wmüåv Nw Hw_ nu Pi _ nam, _am_ <_ _. .Qmnszm _ __ _ amëhow _ ß._m_ _ _ __ __ _ _ _ _ _ _ pmcxmhmmf _ .Tnxm fl oå m. M fl Im x_ __ _ ~: z »> ~ @«: @ 410 @@ «oo« Iu-Izfcm < - = Ho | @ fl Hfxwwqz _. _. _ _ _ _ Thooowzum _.___ omm _._ u: w _ fifl wa fi ow mcsms flfl w: mv vms huø fi pawmwmxo men wmmmxwcwmm Mßmmz HH flfl wßma à .Ö ._ å »1 10 15¿f 20 '25 50 35; ....., ..-... ... a -__ __. bl -.- -of 20 MPa; The fraction for kf: 8.2 was concentrated in vacuo and the emulsion in 0.7 g of bis- (p-nitrophenyl) sulfite was then added to a solution of 200 mg of the octapeptide thus prepared in 7 ml of dimethylformamide and 7 ml of pyridine through which nitrogen had been bubbled. After stirring for 9 hours at room temperature from: *, an additional 0.7 g of sulfite was added and, after stirring for an additional 12 hours, an additional 0.35 g of sulfite was added.

After H hours, the reaction mixture was concentrated in vacuo; The product was precipitated with ether, filtered through suction and washed thoroughly with ether. The product was then dried, dissolved in 7 ml of dimethylformamide and 2,6-M hydrochloride in 0.52 ml of ether was added. The mixture thus obtained was allowed to stand for 7 minutes. and diluted with 100 ml of ether. The hydrochloride precipitate of the compound of formula II was then filtered by suction, washed with ether and dried in vacuo.

Cyclization to produce a peptide bond is carried out as follows: The hydrochloride, prepared by the above procedure, was dissolved in 7 ml of dimethylformamide and added at a rate of 2 ml / hour with a vigorously stirred mixture of 200 ml of pyridine and 50 microliters of N ethyl piperidine, through which nitrogen was bubbled, and which had been heated to 5000. After complete addition, the mixture was heated to 5000 for H hours and allowed to stand at room temperature for 12 hours. Then the solution was concentrated to a small volume (bath temperature 3000), and the product precipitated with ether. 170 mg of a microcrystalline material were obtained, 100 mg of the product was then dissolved in 3 ml of 3M acetic acid and applied to a column packed with polyacrylamide gel. Biofgel? P-U (100 x 1 cm) 7. Freeze-drying of the corresponding fractions gave 70 mg of a compound, which was redissolved in 3 ml of acetic acid and applied to a column packed as described above. Freeze-drying of the corresponding fractions gave H2 mg of a compound. 15 mg of this product were dissolved in 2 ml of a methanol-water mixture (?: 5), and the solution was applied to a column packed with modified silica gel (Setaron SI-01 ~; .15 x 0, 6 cm). Elution was then carried out with a methanol-water mixture (HH: 56) at a pressure I was lyophilized; to give 6.3 mg of the compound, the properties of which are shown in the following table llI.'¶ 10- 7l ~, in 443 so: jl Example 2 '' This example describes the preparation of ¿f2-p-ethoxyphenyl. alanine; 74deamino-6-carbaoxytocin; The starting compound of formula II was prepared as described in Example 1. 0.25 g of a suspension of the dicyclohexylammonium salt of the o-nitrobenzenesulfinyl-p-ethoxyphenylalanine in 50 ml of ethyl acetate därefter was then shaken with 0.05 M sulfuric acid, and the resulting The solution was dried over sodium sulfate and evaporated to dryness. The oil thus formed was then dissolved in dichloromethane, and the active acid was prepared in the manner described in Example 1, Condensation. With 0.25 g of heptapeptide was performed on In the same way, which gave 200 mg of a compound having a melting point of 20 DEG-218 DEG C. and exhibiting properties of the kind shown in Figs. 11 * 11; the cyclization described therein was carried out. in Example 1.

The yield, resulting from 200 mg of protected peptide, was 196 mg of the cyclization product. Part of the reaction mixture was purified by repeated gel filtration to give 8.3 mg of a pure compound having the properties shown in the following double phase.

IO å .. ha. , wm.o mo {H oo. ~; ~m.oHH@o.mH _mm.ß Hmowww H ~ .mmwvHowIHm.o _oß.o Hwwwo HH ß "mH«. «H _ H .H 1 ._ H HH HH Hß mv HHH.: U @ LmHH = @ row: om.v mm.o wo.H oo.H oo.H w <@mH m ~ .ß Nw.mm .mH 0 2 IU wH.o ß ~. o HHH ~ wvm; mH H om.o mo.H mo.H @mo mß. «H mH.ßH mm.Hw H <_ Nßm« .v H.. HHH HHH HH mm mw HH mnnnmwwaßfmomz mH Q mo H vm .o Ho.H mo.H m> .wH m <.ßH mß.Hm .m Ho z: .U wH.o ww.o HH @ z ~ w; u _ HH N Hd H 0o.H Nw »oHoo .HHNm.o_ om. «H Nm. @ Mm. Fl m _HHHwHHv ou: m ~ w.ø H ~ .ø _ HH ~" mHH ~. <. >. _ ;,"..HRS" .. . _ _. mH NH wß NmH. H HH; HH H: @ »Lm» +; D »: o @ z mm m. Hwo H mm.o.oo.H mo H Hmn. _ HH HH Ho.HHHHQ.H @nH ß @ HoH @ ~ HmHHHmm. @ O @ .wmH _HH ~ H «mmv UNI NM ~ wu HmH.0 N fl mHoN:»: uwzHoo.ß> mo wo »HHHo1H mm.oHHøm. mH Ho ~ .ß_HmH «mw H_.mHHoHHz ~ w: .. HH QQHH mw« o. «of fi ßm« oHHwß.mH HwN.ß _m ~ .mm HN ~ HHvwo «: ~ uHw Hmw.oH wN.oH _ HHN "n ~ .v H t. @ H ~>. H _. HHH H NH HH Hß mv H. H .HH _: Qp ~» »w; n |: o @ z ßw fl w .. oo.H« m. oH «o» H ßo.H wß.wH ° oH.ßH Hw.mm .mo 2 IHHQ ~ Hc »HwH.o ^ Hwo ~ @; u H Hmm.o Hmw.oH« o, HHmm.o ow.

H H. ..,. n H. H oH H. H .. Hc. s H n.H. o H HaH .H H0. .o u. HH H _ .H H ,. . H ul H. ....

H HHo ~: -: u@z.m~ w. Hmß.HH mu Q ^: oouv: NoVHHßH. HRS ; H Hp, _H »H HHH _H._w. rr. H. - HH H.x, HH _H> u: H. mxn H = mH H @HH; ma, Hmæ. uæH HHHH H H vw. HmmH _ HH .Ö H. EwumSmH ~. > cum o cum. 30. am <_ pwc fl smH H H H HÛBHHMÉUQEV Nm .Hm _ HHnu. n HH HH. _ H _ umcxmnmm. H fi mšnow fi ßxm fl oz H Hua _ HMWH ~ Iz |> Hw-: NH «0L1 | oo-Iu |: z | = m <| cHwfwHH |» Lou | N:% »wa H. HH Nmp H NH »H.> HH N. L IC 0 c al mšnom mcnmš fifl m Sleep WMS nw © f nQmmoHxæo_moQ hwmmxmcomw mnwwz HHH. Fl @ nwkH 448,303 .pmwmowas al ppmc: m f @. @ LW A1 ma wme cwppwwwø al _ _ mn al em fi MVM al mv> m mvmm fi Hwp_umE_mhämx f "" mModHw fl m @> m Cwws fi CWW fi mm fi ncwoonan fi amd äm_m ": mm øwë Suvmmmwsm _: n WLÜ .fifi UDm fl P ECM Gw fl ü fi fmnvwß flfl ëdw IN mm.o_ mß.o _N ~ .A__wo »~ oß.mH _mm_ @ om.om _ Amvo fl v o¶I _ <__ ~ mmo_ mu.o _ _ _ _ .w _ _ _ _ HH w fi wo ww _ _ _ __ N _ __ IQ »@w $» = @ | Iowz ~ ß.o_ ~ o1 ~ mm.o oH.H @mo mo «m ~ _ @m. m_ ßm.øm .mo z__: _ mo.o_ mo.o_ ^ Izvmæa f ___, _ ___ _ _Hw »o_ mw.o_ wo. fi __> @. o_ Hw.m ~ _ øwJ @ _ mw.ww ~ ^ ßm @Hv o ~ I_N H @ «o_ mo __ _ __ w” m__v1w _ __ _; __. _ _ .N. __ ___. »_ _ _ _ _Fl ~ NH Nß_wv _ _ _ ~ _ _ _ 1Q @ & mm» = ß »Io» z N @. ~. «O.H_ mo-H _wo. ~ __Mo. Fl _-. @ H _oo.ß w ~ .mm_ .wo 2: _0_; ~ mo _o fi1 o ß @ z2v»; @__ _ ^ .m @ »o ~ V_ .HHH H fl wnmæ ... h F .. a? lO 15 20 25 30 '. 35 '15 ml dimethylformamide. After stirring for 96 hours at room temperature. The active ester (0.8 g) was added to a suspension of 0.6 g of heptapeptide in 15 ml of dimethylformamide, and 448,305 in the pilot Example 5-7.

This example describes the preparation of 2-phenylalanine-7--deamino-ilecarba-oxytoein. The starting compound of formula II p was prepared in the manner shown in Example 1. Then the 2,4,5-trichlorophenyl ester of the o-nitrobenzenesulphenylphenylalanine was added to 0.8 g of a suspension of free heptapeptide at room temperature. * the reaction mixture as described in Example 1, 1 g (85%)] of a compound with a melting point of W223 ~ 225 ° C was obtained, the properties of which are shown in Table II. The cyclization was then carried out as described in Example 1. The product, which contained ninhydrin-positive material, dissolved 151 methanol-water mixture and was filtered through a 1 column of sulfonate cation exchanger (Dowex-Sd in H + form, 5 ml).

Concentration and lyophilization of 125 mg of a compound, which was then purified by gel filtration. 15 mg of the product thus obtained were then chromatographed on a column of modulated silica gel (sepafone s: C18; 1.5 x 0.6 cm) in a methanol-water mixture 33: 2. The concentration of k 2 = 7.0 fraction and lyophilization gave 2 mg of a compound, the properties of which are shown in Table 2. This Example describes the preparation of 1β2-benzyloxy-carbonylaminophenylalanine-74-deamino + 6-carbaoxytocin. The starting compound was prepared (formula II) in the same manner as described in Example 1. o-Nitrobenzenesulfonyl-p-benzyloxycarbonylaminophenylalanine was liberated from 1.05 g of its dicyclohexylammonium salt in the manner described in Example> 2, and it was transferred to the active ester in the manner described in Example 1. After trituration with petroleum ether, 1.0 g of a crystalline compound was obtained with a melting point of Û !! was processed as described in Example 1. 0.53 g (55%) of a compound melting at 220-222 ° C were obtained with the properties shown in Table II. = Å "iv I 101 15 20 25 50 s_f55 Mode - Example 5 111- 448 305 The cyclization was carried out as described in Example 1 starting from 200 mg, 155 mg of a product was obtained, and 30 mg of it was purified by repeated gel filtration. Further purification was performed by chromatography on a reversed phase column 18 (modified silica gel Separon SI C 15 x 0.6 cm) in a mixture of methanol with trifluoroacetate buffer (3: 2) with pH,, 22. The fraction containing the compound k '= 3.56, lyophilized, and 432_mg of a product were obtained, the properties of which are shown in Table IIlg I d I' This example describes the preparation of L_2-p-aminophenylalanine_7 + deamino-6-map-oxytocin. Sodium was added to 60 mg of a solution of .beta.-2-p-nitrophenylalanine-7-deamino-6-carbaoxytocin in liquid ammonia (5 ml), in which blue staining, which was stable for 50 seconds, appeared. Then the solution was decolorized by addition of acetic acid, and the residue, after evaporation of ammonia, was purified by gel filtration.

Example 6 IV »; . A solution of vatabramia 1 acetic acid (35%, 1 ml) was added to a suspension of 2-p-benzyloxycarbonylaminophenylalanine-7-o-deamino-6-carbaoxytocin (50 mg) in acetone (1 ml). ), and the resulting solution was allowed to stand for 1 hour at room temperature. After repeated evaporation from acetone and reprecipitation from methanol with ether, the product was dissolved in 3 M acetic acid (3 ml) and refined by gel filtration. Freeze-drying gave 8.7 mg of the compound which in its properties corresponds to the product of Example 5. Example 7H This example describes the preparation of Åf2-p-methylphenylalanine; 7-deamino-6-carbaoxytocin. The active ester of 10-nitrobenzenesulphenyl-p-methylphenylalanine was prepared as the corresponding dicyclohexylammonium salt (0.7 g) in the same manner as in Example 4. 0.58 g of a compound melting at 10 ° C. ànsjd ¿, ,,: _ hl? ~ 12? -135 ° C was obtained. This active ester was added to a suspension of hepcapepa fi ia * (0.6 g) in methylformamide (13 cm 3) and 0.85 g of a compound having a melting point of 220 DEG-226 DEG C. were obtained in the same manner as in Table II; NOT in Example 1. The properties found in Cyclization of 200 mg of peptide were performed in the same manner as in Example 1. The product was obtained in an amount of 180 mg, and a portion of it (50 mg) was purified by gel filtration.oeh column chromatography (Éeparon SI_C) while mixing methanol-water (3: 2) .Wi Fraction with k '# f5, D3 gave by lyophilization 13.6 mg of the compound, which is characterized in Table III. ¿F2-p-ethylphenyl- 'alanine7-deamino-64carbasoxytocin. The Protected Octapeptide This example describes the preparation of was prepared as described in Example 1 from ofnitrobenzenesulphenylphypethylphenylalanine (0.4 g) and the free heptapeptide (0.3 g). A compound having a melting point of 218 Å 22400 was obtained in an amount of 0.25 g, and its properties are given in Table II.

Cyclization and refining of the octapeptide were carried out in the same manner as in Example 7. 8.3 g of the compound [kf; 7, H Methanol-water (5: 2fl having the properties shown in Table III. .Z Example) This example describes the preparation of β-β-dimethylamino-1-phenylalanine-7-deamino [6-e] carboxy-oxytocin. The active ester of o-nitrobenzenesulfenylanylanylanylanyl was prepared "from the diocylohexylammonine salt (0.5 g) in the same manner as in Example 4. A compound having a melting point 113-f 11700 was obtained in an amount of mg05 mg; This active ester was added to a suspension of heptapentide_ (0; 3 .mu.l in dimethylformamide and 0.1 g of a compound, melting in the range 201 DEG-20 DEG C., were obtained by the same procedure as in Example 1. (Wash with dilute sulfuric acid was removed). in the same manner as in Example 1, gave 190 mg of a product which was refined by gel filtration, the product obtained by freeze-drying (100 mg) was dissolved in 20% of acetic acid and subjected to purification by flowing electrophoresis The compound, obtained in an amount of SO 1, I was further purified by gel filtration. The properties of the product (28 mg) are given in Table III.

Claims (1)

1.% 443 30¶3 Å Patent claims Oxyfocingnaioåér, _k "ä n ñ¶é tec kïn á, de av att har har ¶allmänna formeln.¶ 'fHz' sf E '' çHzi 2 _ ¶ _ _ __ ónz-co -x-Ile-çln-Asn-NH-ca-cofpro-Leu-sly-NH2 CH "wherein the excretedTamic acids belong to L-sepien and X denotes" phenylalanine "or phenylalanine subsite in pairing with a group selected from (ä) methyl group, (b) ethylene group, (Å) ethoxy group, (d) -amino group, (e) 1-dimethylamino group and (f) a nitro group- '' I 'í x hñz. . '* WA