Baláspiri, Lajos

Kovács, Kálmán

Papp, György

Sirokmán, Ferenc

Tóth, M.

English

Baláspiri Lajos

Papp György

Tóth M.

Sirokmán Ferenc

Kovács Kálmán

University of Szeged

SYNTHESIS OF BRADYKININ ANALOGUES CONTAINING OPTICALLY-ACTIVE PIPECOLJC ACID By L. BALASPIRI, GY. PAPP, M. TOTH, F. SIROKMAN* and K. KOVACS Institute of Medical Chemistry, University of Medicine, Szeged (Received 25th October, 1979) Seven structural analogues of bradykinin, containing optically-active L- and D-pipecolic acid**, were prepared and purified, and their physical constants were determined. Slight changes in the structures of known peptide hormones by substituting amino acids of similar character for constituent amino acids are very important for investigation of the relationship between chemical structure and biological activity [1—3]. The amino acids substituted may be similar or not; they can be proteinogenic or nonproteinogenic. The known tissue hormone bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg can be detected in the brain. Since this hormone contains three proline residues, it is easy to get more information by synthesis and biological study of bradykinin analogues containing one or more pipecolic acid residues in place of proline. Up to now, numerous papers on the substitution of proline by optically-active pipecolic acid can be found in the literature of peptide chemistry. This synthesis may be achieved both classical and solid phase methods [4]. Among others, peptide analogues reported containing optically-active pipecolic acids include collagen models [5], oxytocin [6], angiotensin II [7], thyreotropin releasing hormone [8, 9], sequence-polypeptide [10] and model peptides [11]. Some others now under pub-lication [12]. In order to fill gaps in the literature, special attention has been paid to the peptide chemical application of optically-active pipecolic acids [13]. Subsequently, the preparation of different protected and active derivatives of both L- and D-pipe-colic acid was reported [14, 15] in detail. First, 3-L-pipecolic acid bradykinin appeared in the literature [16]; later, our rdsearch group reported 2-L- and 3-D-pipecolic acid * Biological Center of the Hungarian Academy of Sciences, Szeged ** Nomenclature and'abbreviations are those accepted by IUPAC—IUB for peptide chemistry: pip = pipecolic acid; Z = benzyloxycarbonyl; BOC = r-butyloxycarbonyl; OMe = methylester; ONb = =p-nitro-benzylester. Except for glycine, all proteinogenic amino acids are in the £.-form. 97 180 L. BALASPIRI ct.al. Physical properties and analytical P e p t i d e Method1 Puri-fication3 Yield % Z-Arg(N02)-L-Pip-Pr0-Gly-Phe-Ser-L-Pip-Phe-Arg(N02)-0Me (1) Arg-L-Pip-Pro-Gly-Phe-Ser-L-Pip-Phe-Arg (2) A A Aa Bd 55 44 Z-Arg(N0,i)-Pr0-L-Pip-Gly-Phe-Ser-L-Pip-Phe-Arg(N02)-0Me (3) Arg-Pro-L-Pip-Gly-Phe-Ser-L-Pip-Phe-Arg (4) A A Aa Bd 52 48 Z-Arg(N02)-D-Pip-Pro-Gly-Phe-Ser-Pro-Phe-Arg(N02)-C>Nb (5) Arg-D-Pip-Pro-Gly-Phe-Ser-Pro-Phe-Arg(NO2)-0Nb (6) A A Ac Bd 46 70 Z-Arg(N02)-Pr0-Pr0-Gly-Phe-Ser-D-Pip-Phe-Arg(N02)-0Nb (7) Arg-Pro-Pro-Gly-Phe-Ser-D-Pip-Phe-Arg (8) A A Ac Bd 50 76 Z-Arg(N02)-Pro-Pro-Gly-Phe-Ser-L-Pip-Phe-Arg(N02)-ONb (9) Arg-Pro-Pro-Gly-Phe-Ser-L-Pip-Phe-Arg (10) B B Ac Bd 50 84 Z-Arg(N02)-L-Pip-L-Pip-Gly-Phe-Ser-Pr0-Phe-Arg-(N02)-0Me (11) Arg-L-Pip-L-Pip-Gly-Phe-Ser-Pro-Phe-Arg (12) B B Aa Bd 52 39 Z-Arg (N02)-L-Pip-L-Pip-G!y-Phe-Ser-L-Pip-Phe-Arg(N02)-0Nb (13) Arg-L-Pip-L-Pip-Gly-Phe-Ser-L-Pip-Phe-Arg (14) B B Ac Bd 52 79 1 all analogous are tabled 2 A=stepwise condensation, B=fragment condensation 3 A = s i l i c a gel column, B = Whatman CM 32 CMC-column, a=sys tem 2, b=system 3, c = EtOAc-MeOH system, d=0.01—0.5 M ammonium acetate 4 A=ace t ic acid, D = dimethylformamide bradykinin [17] and some fragments of further analogues [18, 19]. In this paper we describe the preparation of the following protected and free bradykinins: 2,7-L-pipecolic acid (1—2), 3,7-L-pipecolic acid (3—4), 2-D-pipecolic acid (5—6), 7-D-pipecolic acid (7—8), 7-L-pipecolic acid (9—10), 2,3-L-pipecolic acid (11—12) and 2,3,7-L-pipecolic acid bradykinins (13—14). The first four analogues were synthesized in a stepwise manner, the others by a fragment condensation method. The biological data of these analogues will be published elsewhere [20]. SYNTHESIS OF BRADYKININ ANALOGUES 181 Table I data of the synthesized analogues1 M. p. °c M D C = 1 T = 24 °CJ F o r m u l a (Mol. weight) Elemental analysis (N%) Calc. Found Amino acid analysis -128—137 171—184 - 4 7 ° D - 8 0 ° A CßI H 8 30 1 7 N17 (1326.5) C 5 8 H „ O U N 1 5 X 3 CH3COOH (1268.3) 17.9 17.7 Arg 1.82 Pro 1.02 Pip 1.9 Gly 1.0 Pre 1.97 Ser 0.88 132—140 176—186 - 5 0 ° D - 8 0 ° A C6IH8 301 7N1 7 (1326.5) C 5 8 H 7 7 O N N 1 5 X 3 CH 3 COOH (1268.3) 17.9 17.7 Arg 1.8 Pro 1.02 Pip 1.8 Gly 1.0 Phe 1.97 Ser 0.83 138—140 190—198 - 3 4 ° D - 5 8 ° A C6 6H8 1 OIGN18 (1433.5) c „ H75 o „ N15 X 3 CH3 COOH (1254.3) 17.6 17.4 Arg 1.9 Pro 2.0 Pip 0.9 . Gly 1.0 Phe 2.1 Ser 0.9 134—138 190—200 - 3 4 ° D - 5 8 ° A C6(;H84 0 1 9 N 1 8 (1433.5) c 5 7 H75 o „ N15 X 3 CH3 COOH (1254.3) 17.6 17.4 Arg 1.84 Pro 1.86 Pip 0.8 Gly 1.0 Phe 1.88 Ser 0.91 140—150 168—178 - 4 7 ° D - 8 0 ° A CßßHgj O19 Njg (1433.5) C S 7 H 7 5 0 „ N 1 5 X 3 CH 3 COOH (1254.3) 17.6 17.5 Arg 1.81 Pro 1.86 Pip 0.9 Gly 1.0 Phe 1.96 Ser 0.8 125—136 165—175 - 4 8 ° D - 7 6 ° A C 6 1H 8 3O 1 7N 7 (1326.5) C 5 8 H 7 7 O N N 1 5 X 3 CH 3 COOH (1268.3) 17.9 17.6 Arg 1.82 Pro 0.9 Pip 1.8 Gly 1.0 Phe 1.98 Ser 0.88 117—124 191—200 - 4 0 ° D - 8 2 ° A C„8H8 8O1 9N1 8 (1461.6) C 5 9 H 7 9 O N N 1 5 x 3 CH 3 COOH (1282.4) 17.2 17.1 Arg 1.87 Pip 2.79 Gly 1.0 Phe 2.04 Ser 0.81 Experimental Melting points were determined with a Kofler-block and optical rotations with a Zeiss-polarimeter. Amino acid analysis was performed on a Czechoslovakian HD-1200 E analyser after hydrolysis in 6 N HC1. IR spectra were taken in KBr on a Unicam SP 200 instrument. TLC on Kiesel G plates was used for purity control and identification in the following systems: 1. n-butanol—acetic acid—water 4:1:1 7* 7182 L. BALASPIRt et.al. 2. ethyl acetate—pyridine—acetic acid—water 60:20:6:11 3. ethyl acetate—pyridine—acetic acid—water 80:20:6:11 4. ethyl acetate—pyridine—acetic acid—water 90:20:6:11 5. chloroform—methanol 8:2 6. chloroform—methanol—acetic acid 85:10:5. The protected analogues were purified on a Merck Kieselgel 60 (0.063—0.2) •column, eluted with system 2 or 3 or with ethyl acetate—methanol. The free peptides were purified by ion-exchange chromatography on a Whatman CM 32 carboxy-methyl-cellulose column, eluted with a 0.01—0.5 M ammonium acetate gradient. Development of the TLC was carried out with ninhydrin, chlorine/o-toluidine and iSakaguchi reagent. All data of described analogues can be found in Table I. Methyl benzyloxycarbonvl-arginyl(nitro)-L-pipecolyl-prolyl-glycyl-phenylalanyl-seryl-L-pipecolyl-phenylalanyl-nitroargininate (1) 1.37 g (1.5 mmole) protected octapeptide ester [19] was treated with excess of •trifluoroacetic acid at room temperature for 1 hour. The acid was evaporated in vacuum and the remaining trifluoroacetate cf the octapeptide ester precipitated with ether, filtered, and dissolved in 8 ml dimethylformamide, cooled to — 5 °C, :and neutralized with triethylamine. 389 mg (1.65 mmole) benzyloxycarbonyl-(nitro)ar-ginine was added, followed by 190 mg (1.65 mmole) N-hydroxysuccinimide (HOSu) and 310 mg (1.5 mmole) dicyclohexylcarbodiimide (DCCI). The reaction mixture was stirred at 0°C for 4 hr, and then at room temperature for 24 hr. After filtering off the dicyclohexylurea, the filtrate was evaporated in vacuum, and the residue was triturated with ether, washed in turn with water, acetone and ether, and dis-solved in methanol for column chromatography. The fractions were evaporated nn vacuum, triturated in ether and dried. Arginyl-L-pipecolyl-prolyl-glycyl-phenylalanyl-seryl-L-pipecolyl-phenylalanyl-arginine triacetate (2) 620 mg (1) was dissolved in dioxan—methanol 5:1, and 0.5 ml 2 N NaOH was added. After a 4-hr stirring at room temperature, 0.5 ml 2 N H2S04 was added, and the mixture was evaporated in vacuum till half volume, poured into ice-water, filtered and washed with ether. The residual powder was dissolved in acetic acid— methanol 3:1, and the solution was hydrogenated in the presence of 10% Pd-C and Pd-black catalysts in a bubbling-apparatus at room temperature. The catalysts were filtered off and the filtrate was evaporated in vacuum. The residue was dissolved in start buffer (pH 5) and chromatographed. The main peak was collected and Jiophilized. The latter process was repealed Hvice from dilute acetic acid and water. SYNTHESIS OF BRADYKININ ANALOGUES 101 Methyl benzyloxycarbonyl-arginyl(nitro) -prolyl-L-pipecolyl-glycyl-phenylalanyl-seryl-L-pipeco/yl-phenyialanyl nitroargininate (3) 1.0 g (1.0 mmole) protected octapeptide ester [19] was treated with trifluoro-acetic acid, and the trifluoroacetate of the octapeptide ester, coupled with benzyl-oxycarbonyl(nitro)arginine in the presence of HOSu and DCCI, was worked up-and chromatographed as described for (1). A rginyl-prolyl-L-pipecolyl-glycyl-phenylalanyl-seryl-L-pipecolyl-phenylalanyl-arginine triacetate (4) 150 mg (3) was hydrolyzed, hydrogenated, chromatographed and worked up-as described for (2). p-Nitrobenzyl benzyloxycarbonyl-arginyl(nitro)-D-pipecolylproIyl-glycyl-phenylalanyl-seryl-prolyl-phenylalanyl-nitro-nitroargininate (5) 1.2 g (1.0 mmole) protected octapeptide ester [19] was treated with trifluoro-aeetic acid, and the trifluoroacetate of the octapeptide ester, coupled with benzyl-oxycarbonyl-(nitro)arginine in the presence of HOSu and DCCI, was worked up as described for (1), chromatographed in ethyl acetate—methanol and worked up similarly. Arginyl-D-pipecolyl-prolyl-glycyl-phenylalanyl-seryl-prolyl-phenylalanyl-arginine triacetate (6) 143 mg (5) was hydrogenated as described for (2) (but only in the presence-of Pd-black catalyst) and the crude product was chromatographed and worked up-similarly. p-Nitrobenzyl benzyloxycarbonyl-arginyl(nitro) -prolyl-prolyl-glycyl-phenyl-alanyl-seryl-D-pipecolyl-phenylalanyl-nitroargininate (7) 1.2 g (1.0 mmole) protected octapeptide ester [19] was treated with trifluoro-acetic acid, and the trifluoroacetate of the octapeptide ester, coupled with benzyl-' oxycarbonyl-(nitro)arginine in the presence of HOSu and DCCI, was worked up-as described for (1), with chromatography in ethyl acetate—methanol as for (5).. Arginyl-prolyl-prolyl-glycyl-phenylalanyl-seryl-p-pipecolyl-phenylalanyl-arginine triacetate (8) 380 mg (7) was hydrogenated, chromatographed, and worked up as described< for (6). 184 L. BALASPIRI et.ai. p-Nitrobenzyl benzyloxycarbonyl-arginyl(nitro) -prolyl-glycyl-phenylalanyl-seryl-L-pipecolyl-phenylalanyl-nitroargininate (9) 1.54 g (1.5 mmole) protected hexapeptide ester [19] was dissolved in acetic acid — HBr. After 1 hr the solution was evaporated in vacuum, solidified with ether, washed with ether and dried. The crude product hydrobromide of the hexa-peptide ester was dissolved in dimethylformamide, cooled to —15 °C and neutralized with N-methyl morpholine. From 840 mg benzyloxycarbonyl-arginyl(nitro)-prolyl-proline the azide was prepared in situ at — 15°C. The two cooled solutions were poured together and stirred for 48 hr at 0°C, and then filtered, evaporated in vacuum, dissolved for chromatography, and worked up as described for (5). Arginyl-prolyl-prolyl-glycyl-phenylalanyi-seryl-L-pipecolyl-phenylalanyl-arginine triacetate (10) 800 mg (9) was hydrogenated, chromatographed, and worked up as described for (6). Methyl benzyloxycarbonyl-arginyl(nitro) -L-pipecolyl-L-pipecolyl-glycyl-phenylalanyl-seryl-prolyl-phenylalanyl-nitroargininate (11) 1.35 g (1.5 mmole) protected hexapeptide ester [19] was treated with acetic acid—HBr, and the hydrobromide of the hexapeptide ester, coupled with 864 mg benzyloxycarbonyl-arginyl(nitro)-L-pipecolyl-L-pipecolic acid (converted in situ to the azide), and worked up and chromatographed as described for (9). Arginyl-L-pipecolyl-L-pipecolyl-glycyl-phenylalanyl-seryl-propyl-phenylalanyl-arginine triacetate (12) 540 mg (11) was hydrogenated, chromatographed, and worked up as described Tor (2). p-Nitrobenzyl benzyloxycarbonyl-arginyl(nitro)-L-pipecolyl-L-pipecolyl-glycyl-phenylalanyl-seryl-L-pipecolyl-phenylalanyl-nitroargininate 13 1.56 g (1.5 mmole) protected hexapeptide ester [19] was treated with acetic acid — HBr, and the hydrobromide of the hexapeptide ester, coupled with 864 mg benzyloxycarbonyl-arginyl(nitro)-L-pipecolyl-L-pipecolic acid (converted in situ to the azide), and worked up and chromatographed as described for (9). Arginyl-L-pipecolyl-L-pipecolyl-glycyl-phenyla!anyl-seryl-L-pipecoIyl-phenylalanyl-arginine triacetate 14 700 mg (13) was hydrogenated, chromatographed, and worked up as described for (6). * * * SYNTHESIS OF BRADYKININ ANALOGUES 185 The authors are indebted to thanks to the microanalytical and infrared laboratory of the Organic Chemistry Institute of Attila József University, Szeged, for elemental and infrared analyses, to Mr. R. Ferenczi for amino acid analysis, and to Miss I. Bagi and J. Fülöp for technical assistance. . References [1] Schröder. £., K. Lübke: "The Peptides", Vol. 2. Acad. Press, New York—London, 1966. [2] Rudinger, J.: "Drug Design", Vol. 1. Acad. Press, New York—London, 1971. [3] Sheppard, B. (senior reporter): "Amino-acids, Peptides, and Proteins." A Special Periodical Report. The Chemical Society, London. Vol. 1—9. (1969—1977). [4] Merrifield. R. B.: J. Amer. Chem. Soc. 85, 2149 (1963). Merrifield, R. B.: Advances in Enzymology 32, 221 (1969). [5] Katchalski, E., A. Berger, J. Kurtz: Internat. Symposium on Protein Structure and Crystallo-graphy, Madras, 1963. [6] Bespalova, Z. D., O. A. Kairov, U. F. Martinov, V. U. Natosky, M. I. Titov, E. I. Sachamtova: Vest. Leningrad. Univ. Ser. Fiz. Khim. 21, 157 (1966). [7] Chaturvedi. N. C., W. K. Park, R. R. Smeby. F. M. Bumpus: J. Med. Chem. 13, 177 (1970). [8] Bankowski, K., S. Drabarek: Acta Polon. Pharm. 33, 81 (1976). [9] Baláspiri, L., Zs. Csapó, M. Fekete, G. Tetegdy, K. Kovács: unpublished results. [10] Fairweather, R., J. H. Jones: J. C. S. Perkin I. 1972, 2475. [11] Vicar, J., M. Budésinsky, K. Blaha: Coll. Czechosl. Chem. Commun. 38, 1940 (1973). Vicar, J., J. Smolikova, K. Blaha: Coll. Czechosl. Chem. Commun. 38, 1957 (1973). [12] Baláspiri, L., A. Török, Zs. Csapó, I. Nógrádi, K. Kovács: unpublished results. [13] Kovács, K., B. Penke, J. Czombos, J. Petres, L. Baláspiri: Acta Phys. et Chem. Szeged 17, 61 (1971). [14] Baláspiri, L., B. Penke, J. Petres, K. Kovács: Monatshefte 101, 1177 (1970). [15] Baláspiri, L„ Gy. Papp, K. Kovács: Monatshefte 103, 581 (1972). [16] Nicolaides. E. D., H. A. DeWald, M. K. Craft: Ann. N. Y. Acad. Sei. 104, 14 (1963). [17] Neubert, K., L. Baláspiri and G. Losse: Monatshefte 103, 1575 (1972). [18] Baláspiri, L„ Gy. Papp, P. Pallai and K. Kovács: Acta Phys. et Chem. Szeged 20, 105 (1974). [19] Baláspiri, L., B. Penke, Gy. Papp and K. Kovács: Acta Phys. et Chem. Szeged 20, 465 (1974). [20] Baláspiri, L., J. Lonovics, K. Kovács: unpublished results. СИНТЕЗ АНАЛОГОВ БРАДИКИНИНА, СОДЕРЖАЩИХ ПИПЕКОЛИНОВУЮ КИСЛОТУ Я. Балашпири, Дь. Папп, М. Тот, Ф. Широкман и К. Ковач Описан синтез, очистка и физические характеристики аналогов брадикинина, содержащих оптически активную — L- и о-пипеколиновую кислоту. 

Synthesis of bradykinin analogues containing optically-active pipecolic acid

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Synthesis of bradykinin analogues containing optically-active pipecolic acid

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