Int. J. Peptide Protein Res.l3,1979,426-433 Published b y Munksgaard, Copenhagen, Denmark N o part may be reproduced by any process without written permission from the author(s)
S Y N T H E S I S A N D SOME P H A R M A C O L O G I C A L P R O P E R T I E S O F [ G l ~ ( 0 M e ])O~X Y T O C I N A N D [ M p r ' , G l ~ ( 0 M e 1)O~X Y T O C I N * IPHIGENIA PHOTAKI, CHRYSA TZOUGRAKI and CHRISTINE KOTSIRA-ENCONOPOULOS
Laboratory of Organic Chemistry, The University of Athens, Athens, Greece
Received 30 June, accepted for publication 1 December 1978 [Glu (OMe)4]oxytocin ( X V I ) and [Mpr', Glu (OMe)4]oxytocin (XVII) bearing a methyl ester group in place of the carboxamide group in position 4 of o x y tocin were synthesized by (3 -k 6 ) segment condensation using the S-trityl group f o r the protection of the cysteine side chains. Analogue X V I exhibited 10.5 Ulmg in vitro uterotonic, and 4 2 UJmg avian vasodepressor, activity, and analogue XVII 21.4 Ulmg and 8 2 UJmg of the respective activities. Both compounds showed no response in the rat pressor assay. Key words: decreased biological activities;Sdeprotection and disulphide formation by iodine; Na-o-nitrophenylsulphenylgroup; (3 tive group.
The position 4 of the cyclic octapeptide neurohypophyseal hormones is a site of natural variation, as can be seen in isotocin (Johl et al., 1963), glumitocin (Acher et al., 1965) and aspartocin (Acher et al., 1972). Studies of the structure-activity relationship performed on a number of synthetic oxytocin analogues have shown that the glutamine residue in position 4 of the molecule is not essential for exhibition of high biological activity (Rudinger, 1971; Manning et al., 1977). A striking example is the synthetic analogue [Cthreonine] oxytocin
* This paper is a part of the Special Anniversary issues (January and February, 1979) of the International Journal of Peptide and Protein Research, dedicated to the 25th anniversary of the synthesis of oxytocin. It was received too late to be includedin those two issues. Abbreviations used in this paper follow the rules of IUPAC-IUB Commi&on on Biochemical Nomen&Biochemistry 5 , 1445-1453, 2485-2489 mre, (1966) a d 6, 362-364 (1967). BOC,tert.-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide; X U , N,N'dicyclohexylurea; DMF, dimethylformamide; EtOAc, ethyl acetate;Et, N, triethylamine; IPflH, i~ProPano1; MPr, @-mercaPtoProPiofic acid; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
426
+ 6) segment condensation;S-trityl protec-
(Manning et al., 1970), which proved to be a highly potent and specific oxytocic agent. However, the deamido-analogue [4-glutamic acid] oxytocin (Photaki & du Vigneaud, 1965) has very low activity although the carboxyl group is nearly isosteric to the replaced carboxamide group, hydrophilic, and has also hydrogen bonding capacity. Therefore, it seemed interesting to investigate the influence of the uncharged methyl ester group which can be better compared to the carboxamide group as to reactivity and polarity. For this purpose the title analogues 'of oxytocin (XVI)-and (XVII) have been synthesized by replacing Ldutamine with y-methyl L-&tamate in POsition 4 of oxytoch and 1-deminoaxytocin ([Mpr' ] OXytOCin) respectively (for a short communication cf. Photaki et al., 1977). These the first oxytocinare, to Our analogues carrying a side-chain ester group. The synthesis of the new analogues has been accomplished via two protected nonapeptides (XIV), (XV) as shown in Scheme 1 for [Glu(OMer ] oxytocin. The trityl group was chosen as sulphydryl protective group because
0367-8377/79/040426-08 $02.00/0 0 1979 Munksgaard, Copenhagen
[Glu(OMe)' Z-Ty r ( Bzl )-lle-OMe
(VIII)
4
Boc-Cys (Tri)-Tyr-lle-OMe
I Boc-Cys (Tril-Tyr-Ile-N,
1 - A N D [ Mpr' ,G l ~ ( 0 M eJ)OXYTOCIN ~
H,
Tri-Cys (Tri )-Pro- Leu-Gly-N H, Nps-Asn-Cys (Tri j-Pro-Leu-Gl y-N H
(IX)
1 Nps-Glu (0Me)-Asn-Cys (Tri)-Pro-Leu-Gly-NH, I
(XI
I azide Tri
(11
1
I OMe
(Ill) ( V I1
0.24 N HCI in EtOAc-DMF Tri
1 I I BocCys-Tyr-l le-Glu-Am-Cys-Pro-Leu-Gly-NH 1 HCys-Tyr-lle-ijlu-Asn-Cys-Pro-Leu-GIy-NH,
( X IV )
(XVI)
SCHEME 1 Synthetic route to [ G l ~ ( o M e )oxytocin ~] (XVI).
of its ready introduction into cysteine and the availability of mild procedures for its selective cleavage (Photaki, 1973, 1976; Kamber & Rittel, 1968; Jones et al., 1973). As for the amino group protection, during the various steps of the synthesis, the trityl (Zervas & Theodoropoulos, 195.6), the o-nitrophenylsulphenyl (Zervas et al., 1963; Zervas & Hamalidis, 1965) and the tert.-butyloxycarbonyl (Anderson & McGregor, 1957; Schwyzer et al., 1959) groups were used. In addition, many of the known coupling procedures have been tried for lengthening the peptide chain and those which gave the best results, in yield of pure compound, are described. Both oxytocin analogues exhibited biological activity on the rat uterus in vitro (i.e. [ G l ~ ( o M e )oxytocin, ~] XVI: 10.5 U/mg, and [Mpr' ,G l ~ ( 0 M e ) oxytocin ~l XVII: 21.4 U/mg) and in vivo (PliSka et al., 1975; PliSka & Rudinger, 1976). The avian vasodepressor activity was determined in comparison with the I11 International standard, four-point assay (U.S.P. XIV, 1950) as 42U/mg for XVI, and 82U/mg for XVII. No response was obtained in the rat pressor assay (Dekanski, 1952;PUka eral., 1977) at dosages of 10,20, and lOOpg. It is interesting to note that replacement of the amide by the methyl ester grouping decreases the activity of oxytocin equally or even more drastically than complete omission of the carboxamide group (cf. the biological activities
of [ k u 4 ]-, "ie4] -, "a4] (Rudinger, 1971).
oxytocin, etc.)
EXPERIMENTAL SECTION
All amino acid derivatives were of the L configuration and were synthesized by standard methods. Anhydrous solvents were used for couplings and removal of N-protecting groups unless otherwise stated. Other chemicals and solvents used were of reagent grade. Melting points were determined in capillary tubes and are uncorrected. Optical rotations were measured in a l d m cell on a Perkin-Elmer Model 141 polarimeter. When necessary, solutions in organic solvents were dried over Na2SO4. Thin-layer chromatography was carried out on silica gel, type 60 (Fluka), plates with the following solvent systems (proportions by volume): (1) toluene-acetone (7:3), (2) toluene-acetone (1 :l), (3) toluene-acetone (4: l), (4) toluene-pyridine-AcOH (8 :2 :1), (5) toluenepyridine-AcOH (80:10:l), (6) EtOAc-EtOH (7:3), (7) EtOAc-EtOH (75:25), (8) tAcOHzFrOH-H20 (62:26:7), (9) CH3CN-H20 (3:1), (1 0) CH3 CN-HZ 0 (9: l), (1 1) ~BuOH-AcOHHzO (4:1:1), (12) ~BuOH-AcOH-H2O (10: 1:3), (1 3) nBuOH-AcOH-H20 (upper phase) (4: 1:5), (14) nBuOH-AcOH-H20 (upper phase) (5 :4: l), (1 5) nBuOH-AcOH-H20-pyridine (30: 6:24:20), (16) CHC13-CC4-MeOH(6:3:1),(17) CHC13-CC14-MeOH-AcOH(6:3: 1:1). Thin-layer chromatograms were run for 7-12cm and developed with ninhydrin sol421
1. PHOTAKI ET AL.
ution (0.5% in acetone-AcOH-HzO, 90:5:5 or 18:l:l) or with iodine vapors. For paper electrophoresis an LKB 3276 apparatus was used with Schleicher and Schdl2043-b paper. S,N-Ditrity 1-L -cysteiny 1-L -prolyl-L-1eucylglycinamide (I)
S,N-Ditrityl-Lcysteine (1 1.2 g, 16.5 mmol) was liberated from its diethylamine salt (Zervas & Photaki, 1962) by partitioning between ether and 0.5 N HzS04. To a stirred solution of the above residue in THF (70ml), N-methylmorpholine (1.2 ml, 17 mmol) and diphenylphosphoryl chloride (4.29 g, 16 mmol) dissolved in THF (20ml) were added at -loo, and after stirring for 15 min a freshly prepared, precooled (0") mixture of L-prolyl-L-leucylglycinamide hydrobromide (Zaoral & Rudinger, 1955) (5.77g, 15mmol) and Et3N (4.2m1, 30 mmol) in DMF-THF (40 ml, 1:1) was added. The pH of the solution was adjusted to 6-7 with Et3N and the mixture was stirred at 0" for 15 min, and at 25" for 1.5 h. The solvent was removed under reduced pressure and the residue was taken up in CHC13. The solution was washed with a dilute aqueous solution of Et3N, Hz 0, dried and evaporated. The residue was triturated with hot ether, filtered off and recrystallized from EtOAc; yield 8 S g , m.p. 205-207", [a]25 + 87.3" (c 2, DMF). The product was furtEer purified by passing through a Dowex 1/1 column (acetate form) using CHC13-DMF-MeOH(2 : 1 :7 v/v) as an eluant, to yield 7.2 g; a second crop (1.5 g) was obtained by passing through the same column the residue of the first recrystallization; (total yield 66%), m.p. 206-209", [at] 2: + 85.9" (c 2, DMF); t.1.c. in systems 4,12, 16. Anal. calc. for C&15,N@$ (872.16): C, 74.36; H, 6.58; N, 8.03; S,3.67. Found: C, 74.42; H, 6.67; N, 7.75; S,3.68. N-o-Nitrophenylsulphenyl-S-trityl-L -cysteinylL -prolyl-L -1eucylglycinamide ( l a )
a waterbath for 5 min. After 30 min at 25" the hydrochloride salt was precipitated with ether. It was recrystallized from CHC13ether; yield 6 g (90%), m.p. 150-153", [a]E+42.3" (c 2, EtOH); t.1.c. in systems 6, 12, 15. Anal. calc. for C ~ ~ H & S O $(663.30): ~ C, 63.09; H, 6.66; N, 10.51, C1, 5.32. Found: C, 62.68; H,6.69, N, 10.40; C1,5.40. B. Compound I1 was also obtained from Ia by a similar treatment (0.2 N HCI in MeOH).
N-o-Nitropheny lsulpheny 1-L -asparaginyl-Strityl-L-cysteinyl-L-prolyl-L-1eucylglycinamide
Vrll
N-o-Nitrophenylsulphenyl-L-asparagine(Zervas el al., 1963) (3.4 g, 12 mmol) was dissolved in THF (60 ml) by the addition of pyridine (1 .O ml, 12.5 mmol) and Nethylpiperidine (1.2 ml, 12 mmol). The solution was cooled to - lo", pivalic acid chloride (1.2 ml, 10 mmol) was added and the mixture was stirred for 5-10 min. A precooled mixture of I1 (6.5 g, 10 mmol) and E t a (1.4 ml, 10 mmol) in DMF (20 ml) was added. The mixture was stirred at 0" for 1 h and at 25" overnight. The solvent was evaporated off and the residue was taken up in CHC13. The solution was washed with a dilute aqueous E t a solution, HzO, 2 M AcOH, HzO, 58 NaHC03 and HzO,dried and evaporated to a small volume. The crude protected pentapeptide was precipitated with ether and recrystallized from MeOH; yield 5.8 g (65%), m.p. 128-1 30" (after softening at 123"), [ a ] g-62.2" (c 2, EtOH), [a] - 88.2" (c 2, CHC13); t1.c. in systems 6, 9,11. Anal calc. for C4sHs2NsO&S2, 0.5 CH30H (913.10): C, 59.85; H, 5.96; N, 12.27; S,7.02. Found: C,59.75;H,5.91;N1 12.10;S,7.24.
L-Asparaginyl-S-trityl-L-cysteinyl-L-prolyl-L-
leucylglycinamide hydrochloride (IV) To a cold solution of I11 (4.5 g, 5 mmol) in CHC13 (45 ml), mercaptoethanol (2.1 ml, 30 mmol) and 2.5 N HC1 in EtOAc (1.9 ml) were added. Upon cooling the hydrochloride salt separated out as a gel which was crystallized from MeOH-EtOAc; yield 3.5 g (90%), m.p. 145-147", [(u]~-3.9" (c 2, MeOH); t.1.c. in systems 6,9, 10,14. Anal. calc. for C3.&&0&l (780.40): C, S-Trityl-L -cysteiny1-L -prolyl-L-1eucylglycin60.03; H, 6.46; N, 12.56; S, 4.11; C1, 4.54. amide hydrochloride (II) A. Compound (I) (8.7 g, 10mmol) was dissolved Found: C,60.47;H,6.78;N, 12.52;Sl4.10;C1, in 0.2 N HCI in MeOH (50 ml) by warming it on 4.51.
Prepared by DCC coupling of N-o-nitrophenylsulphenyl-3-trityl-L-cysteine (Zervas et al., 1963) with L-P~O-L-L~U-G~Y-NH~ (Boissonnas et aL, 1955), m.p. 80" dec., [ a ] ~ - 5 8 . 7 " (c 2, MeOH); t.1.c. in systems 4, 15. Anal. calc. for C4&&0&, 0.5 EtOAc (827.05): C, 62.45; H, 6.09; N, 10.16;S, 7.75. Found: C, 62.89; H, 6.39; N, 10.02; S,7.88.
[Glu(OMe)']- AND [Mpr' ,G~u(OM~)~]OXYTOCIN
N-o-Nitrop heny lsulp heny l-L -glutam ic acid y methyl ester (V)
To a solution of L-glutamic acid y-methyl ester (Hanby et al., 1950) (1.6 g, 10 mmol) and KHC03* (1.8 g, 10 mmol) in HzO (20 ml) and dioxane (20 ml), a solution of o-nitrophenylsulphenyl chloride (2.4 g, 12.5 mmol) in dioxane (10 ml) and saturated KHC03* (10 ml) were added alternatively, in 10 equal portions, dropwise, during 1 h. Care was taken to keep the pH at 7-8. The solution was stirred for another 1 h, diluted with HzO (50ml), filtered, washed with ether, and acidified with 1 N HzS04 to a slightly Congo red reaction. The oily precipitate was taken up into ether and the organic layer was washed with HzO, dried and evaporated. The residue was dissolved in MeOH, cooled, some insoluble material was filtered off, and the solution was evaporated to dryness. Crystallization from EtOAc-hexane and recrystallization yielded 2.4 g (75%), m.p. 103-105', [ a ] ; - 62.9" (c 2, CHC13); t.1.c. in systems 4 , 14. Anal. calc. for ClZH1&0,$3 (314.33): C, 45.86; H, 4.49; N, 8.9 1; S, 10.20. Found: C, 45.38; H, 4.29; N, 8.69, S, 10.08. Compound V was also converted to its dicyclohexylammonium salt** (86%), m.p. ] ~51.9' (c 2, CHCI,); 180.5-182.S0 dec., [ o L t1.c. in systems 4,16.
Anal. calc. for C51H61N~O11SZ (1040.22): C, 58.89; H, 5.91; N, 12.12; S, 6.16. Found: C, 58.93; H, 6.29; N, 11.98; S, 5.90. y-0-Methy l-L -glutamyl-L-asparaginyl-S-trityI-L cysteinyl-L-prolyl-L-leucylglycinamide hydrochloride (VII)
To a cold (0") solution of VI (0.25 g, 0.25 mmol) in DMF-EtOAc (6 ml, 1 : 1 v/v), mercaptoethanol (0.42 ml, 6 mmol) and 2.4 N HCl in EtOAc (0.1 1 ml) were added. The hydrochloride salt was precipitated with EtOAc. Recrystallization from iPrOH yielded 0.23 g (92%), m.p. 144-146"dec., [a] - 18.5'(c 2, MeOH), t.1.c. in systems 6 , 9 , 12, 14; homogeneous on paper electrophoresis (270 V, 2 h) in AcOH-pyridineHzO (PH 4.9). Anal. calc. for C45HSPN80&Cl (923.51): C, 58.52; H, 6.44; N, 12.13; S, 3.47; C1, 3.84. Found: C,58.31;H,6.61;N, 12.13;S,3.64;Cl, 3.95. N-Benzyloxycarbonyl-0-benzyI-L -tyrosyl-L-
To a cold (0") solution of compound V, liberated from its dicyclohexylammonium salt (0.5 g, 1 mmol), in DMF (2 ml), a precooled (0') mixture of IV (0.7 g, 1 mmol) and Etfl(O.14 ml, 1 mmol) in DMF (2 ml) was added, followed by DCC (0.22 g, 1.1 mmol). The mixture was stirred overnight at 25", then cooled and the DCU removed by filtration. Workup as described for compound I11 and trituration with warm iPrOH yielded 0.55 g (53%), m.p. 198-200", [(~]E-39.6O (C 2, EtOH-DMF 1: 1 V/V) [a] - 29.7' ( c 2, DMF); t.1.c. in systems 6,9, 12,14.
isoleucine methyl ester (VIII) To a cold (0") solution of L-isoleucine methyl ester hydrochloride (Brenner & Huber, 1953) (1.63 g, 9 mmol) in DMF (14 ml), N-benzylonrcarbonyl-0-benzyl-L-tyrosine p-nitrophe.iy1 ester (Bodanszky & du Vigneaud, 1959) (4.7 g, 9.45 mmol) was added followed by Etfl (1.26 ml, 9 mmol) (dropwise). After 2 days at 25" the product was precipitated with H20 and extracted with EtOAc. The organic layer was washed with 0.5 N HCl, H20, aqueous Etfl solution, 1 M N H P H and HzO,dried and evaporated. Recrystallization from MeOH yielded 3.8 g, m.p. 113-1 13.5". A second crop (0.8 g, m.p. 110-1 12") was gained from the mother liquor; totalyield96%,m.p. 115-1 15.5" after recrystallization from CHC13-light petroleum; t.1.c. in system 1; [a] - 2.7" (c 2.5, MeOH), [a] + 15.0" (c 1.5, CHC13). Anal. calc. for C31H&JZO6(532.645): C, 69.90; H, 6.81; N, 5.26. Found: C, 70.28; H, 6.67; N, 5.38.
* Or equivalent amount of Et,N.
N-tert.-ButyloxycarbonyI-S-trityl-L-cysteinyl-L-. tyrosyl-L-isoleucinemethyl ester (IX)
N-o-Nitrophenylsulphenyl-y-0-methyl-L -
-
glutamy l-L -asparaginyl-S-trityl-L-cysteinyl-L
propyl-L -1eucylglycinamide (VI)
** Prepared originally in this laboratory by P. Mazarakis, unpublished.
A solution of VIII (1.06 g, 2 mmol) in glacial AcOH (2.7 ml) was treated with 5.5 N HBr in 429
I. PHOTAKI ET AL.
AcOH (2.3 ml). After 35 min at 25" the solution w a s poured into cold ether (200 ml). The precipitated salt was washed with ether several times, cooled, filtered off and dried over KOH and Pz0 5 ;yield 0.69 g (88%),(chromatographically pure in system 12). The hydrobromide salt (0.6 g, 1.54mmol) was coupled with N-terr.buty1oxycarbonyl-S-trityl-L-cysteine p-nitrophenyl ester (Jones er ul., 1973) (0.95 g, 1.62 mmol). Et3N was added to keep the pH of the solution to about 7. The product was isolated as described for VIII (acidic washing with 0.5 N H2SO4). Crystallization from EtOAc-hexane yielded 1.1 g(94%),m.p. 82" dec. Recrystallization from DMF-HzO, m.p. 87", [a] - 2.7" (c 1.5, CHC13); t.l.c. in systems 1,2,5. A d . C ~ C . for Cg3HsIN307S (753.95): C, 68.50; H, 6.82; N, 5.57. Found: C, 68.71; H, 7.12;N,5.11. Identical products were obtained by DCC or mixed anhydride coupling.
ml), p-nitrophenol (6.7 g, 48 mmol) and DCC (8.25 g, 40 mmol) were added. After stirring at 0" for 1 h and at 25" for 3 h, the precipitated DCU w a s filtered off. Water was added to the filtrate and after cooling the precipitate was collected. The crude p-nitrophenyl ester was recrystallized twice from EtOH; 14.5 g (77%), m.p. 125";tl.c.in systems 1 , 1 2 . Anal. calc. for CZsHz3NO4S(469.56): C, 71.62; H, 4.94; N, 2.98; S, 6.83. Found: C, 71.58, H, 4.96; N, 2.85; S, 6.84.
S-Trity l-&mercap topropiony I-l-tyrosy I-L -holeucine methyl ester (XI.)
Compound XI (1.59 g, 3.39 mmol) was added to a cold (0"), stirred solution of L-tyrosyl-L-isoleucine methyl ester hydrobromide (1.26 g, 3.23 mmol, prepared as described above) in DMF (15 ml), followed by Et3N (0.45 ml, 3.2 mmol) (pH cu. 7). After 4 days at 25", HzO was added and the mixture was extracted with N-tert.-Bufyloxycarbonyl-S-tn'tyl-L~ysteiny~-~EtOAc. The organic layer was washed with 0.5 tyrosyl-L+soleucine hydrazide (XI N HCl, HzO, an aqueous solution of Et3N, and Compound IX was dissolved in MeOH (5 ml) HzO, dried and evaporated. The product was and treated with HzNNHz*H20(0.6 ml) at 25" crystallized from EtOAc-light petroleum and for 36 h. The precipitated solid was filtered off, recrystallized from DMF-HzO and then from washed with EtOAc; yield 4 S g , (86%) m.p. MeOH-H20 to yield 1.75 g (85%), m.p. 84-86", with hot MeOH. Theproduct (1.05 g,m.p. 195- [a]g-6.3" (c 1.5, CHC13); t1.c. in systems 1 , 196") was recrystallized from DMF-H20 to 5. yield 1 g (88%),m.p: 197-198", [a] + 10.2" Anal. calc. for CUII€,zN30sS, 0.25 CH30H (c 2, DMF); t1.c. in systems 1,2. (646.83): C, 70.99; H, 6.64; N, 4.1 7. Found: C, A d . C ~ C . for C42HS1NS06S (753.96): C, 71.03; H, 6.70; N, 4.33. 66.91; H, 6.82; N, 9.29; S, 4.25. Found: C, 66.92; H, 6.80; N, 9.1 2; S, 4.3 1. S-Trityl-0-mercaptopropionic acid
S-TrityI- 0-mercap topropi0 ny I-L-tyrosY 1-L -is@ leucin e hy draz ide (XIII)
To a hot solution of triphenylcarbinol (4.3 g, 16.5 mmol) in EtOAc (35 ml) P-mercaptopropionic acid (1.6 g, 15 mmol) and conc. HzS04 (0.1 ml) were added and the mixture was warmed for 10 min at 60". The precipitated crystalline compound was filtered off after cooling and washed with EtOAc; yield 4Sg, (86%) m.p. 204-205" unchanged after recrystallization from EtOAc (lit. m.p. 208-209", Zee-Cheng & Cheng, 1970). T.1.c. in systems 5,12.
Prepared as described for X; yield 77%,m.p. 219.5-220" after repeated trituration with boiling MeOH. [a] + 2.7" (c 1.5, DMF); t.1.c. in systems 1,2. Anal. C ~ C . for C37bZN404S (638.83): C, 69.57; H, 6.63; N, 8.77. Found: C, 69.29; H, 6.59; N, 8.51.
S-Trity I-P-mercaptopropionic acid p-nitrophenyI
asparaginyl-S-trityl-L -cysteinyl-L -proly I-L -
N-text.-Butyloxycarbonyl-S-trityI-L-cysteinyl-L ty rosy I- L -is0le ucy 1-7-0-me thy l-L -glutamy I-L -
leucylglycinamide (XIV) ester (XI) To a cold solution (0")of S-trityl-o-mercapto- The tripeptide hydrazide X (452 mg, 0.6 mmol) propionic acid (13.9 g, 40 mmol) in DMF (200 in DMF (3.5 ml) was converted into the azide 430
[ G l ~ ( o M e])-~AND [ Mpr' ,G l ~ ( 0 M e])OXYTOCIN ~
(Honzl & Rudinger, 1961) by treatment with 2.5 N HCl in EtOAc (0.62 ml) and tert.-butylnitrite (0.084 ml) at - 15" for 15 min. A precooled mixture of VII (555 mg, 0.6 mmol) and Et3N (0.3 ml, 2.1 mmol) in DMF (3.5 ml) was added, followed by N-methylmorpholine (0.081 ml, 1.I 5 mmol). The mixture (pH ca. 7, maintained by adding some N-methylmorpholine from time to time) was left at - 12" for 1.5 h and at 25" for 5 days. After concentration to a small volume, the product was precipitated by adding 0.03 N HCl, cooled, filtered off and washed with HzO; yield 843 mg (87%), m.p. 143-147' unchanged after precipitation from EtOHether, [a]g-29.7" (c 1 , MeOH); t1.c. in systems 6 , 8 , 9 . Anal. calc. for C87H105N110~5Sz (1609.02): C, 64.94; H, 6.58; N, 9.58. Found: C, 64.43; H, 6.70;N, 9.53.
h the crude oxytocin salt was filtered off, washed with ether and dried in a desiccator over KOH and PzOs to provide 90 mg (95%). A sample (65 mg) was subjected to partition chromatography (Yamashiro, 1964) on a 1.1 X 96 cm column of Sephadex (3-25 with nBuOHtoluene-pyridine-O.l% AcOH (5 :3 :1 :9 v/v) as a solvent system. Lyophilization of the pooled fractions from the major, symmetrical peak (Rf 0.38, detection by Folin Lowry analysis) provided 23.5 mg of a material, [a] - 24.4' (C 0.5,1 M AcOH), Rf(12) 0.45, Rf(13) 0.5, Rf(15) 0.75. Paper electrophoresis (300 V, 2 h) gave a single band (bromophenol blue-HgClz detection) at pH 9.3 (glycine buffer). Amino acid analysis (6 N HCl-phenol, 110", 24 h), pr01,05 G1y1.03 , 1/2 ASPLOO G1U1.04, cystinel.87, Ileo.w, L e u l . ~ , Tyro.97 and NH3 1.74. Anal. calc. for C44H67N11013SZ,CH3COOH [4-L-Glutamic acid ymethyl ester] oxytocin (1082.26): C, 51.05; H, 6.61; N, 14.24;OCHB, 2.87. Found: C, 51.26; H, 6.53; N, 14.02; (X vrl A. Compound XIV (188 mg, 0.1 16 mmol) was OCH3, 2.80. dissolved in 90%MeOH (750 ml) and a solution of iodine (0.09 g, 0.34 mmol) in MeOH (1 20 ml) S-Trityl-Prnercaptopropionyl-L-tyrosyl-L-isowas added dropwise under vigorous stirring at leucy 1-y-0-methyl-L -glutarnyl-L-asparaginyl-Strityl-L -cysteinyI-L -proly1-L -1eucylglycinamide 25' during 1 h (Kamber & Rittel, 1968). After a further 5 h stirring, H 2 0 (150ml) was added (X V ) and excess iodine was removed (Marbach & The hydrazide XI11 (192 mg, 0.3 mmol) was conRudinger, 1974) by evaporation of the solution verted to the azide and coupled with VII (277 mg, 0.3 mmol) as described for XIV.The crude to cu. 70ml (pH ca. 4). It was extracted first with EtOAc (10ml x 2), to remove the tri- product was chromatographed on a silica gel column using the solvent system EtOH-EtOAc phenylcarbinol and then with n BuOH-benzene (3 :7 v/v). The pooled fractions containing XV, (1: 1 v/v) (4x), and with benzene. The comas detected by tl.c., were evaporated to a small bined nBuOH-benzene extracts were washed volume. Upon addition of EtOAc a microtwice with a few ml of H 2 0 to neutral pH, crystalline material was obtained, 0.2 g (45%), diluted with about half their volume with H2O m.p. 142-145", [a] g- 33" (c 1 , MeOH), Rf(6) and evaporated to a small volume. MeOH was 0.85, Rf(9) 0.84. Amino acid analysis (6 N HCladded and the mixture was reevaporated to a , 0.98, ~01.01, small volume. Upon addition of ether micro- phenol, llOo, 24 h) ~ P I . O OGlu crystalline N-tert.-butyloxy-carboonyl-oxytocinG~YLOI 1/2 cystinel.08, Ileo.94, precipitated, which after 12 h at 4 was filtered TYr0.97. (1493.85): c , off to yield 100mg \76%), m.p. 167" (dec., Anal. cdc. for C8ZH96N10013S2 after shrinking at 160 ). [ a ] g-55.5' (c 1.5, 65.93; H, 6.48; N, 9.38. Found: C, 65.41, H, 6.43, N, 9.09. EtOH); t.1.c. in systems 7, 1 1 , 12, 15. 9
3
f I -&Mercaptopropionic acid, 4-L -glutamic acid y-methyl ester] oxytocin (XVZZ) B . Removal of' the N-tert .-butyloxycarbonyl group. The above Boc-oxytocin (94 mg, 0.084 mmol) was treated with 90% TFA (0.8 ml) for 2.5 h at 25". Ether was added at 0" and after 3
Compound XV (104 mg, 70 pmol) was treated with iodine in MeOH as described for the preparation of XVI, A, except that the triphenylcarbinol was extracted with benzene. MeOH 43 1
I. PHOTAKI ET AL.
was added to the residue and after evaporation to a small volume EtOAc was added to precipitate a microcrystalline product 45 mg, (65%), m.p. 178-180" (softening at 170°), Rf(l 1) 0.6. A sample of 35 mg was purified by partition chromatography on a 1.1 X 96 cm column of Sephadex G-25 with the solvent-system nBuOH benzene-H20,containing 1.5% pyridine and 3% AcOH (1 :2 :3 v/v). The purification was repeated twice to obtain a symmetrical peak (Rf 0.58, Folin Lowry analysis). Pooling and lyophilization provided 27 mg of chromatographically pure material, [a]4:-35.lo, (c 2, MeOH), Rf(6) 0.35,Rf(9) 0.9, Rf(l1) 0.6, Rf(12) 0.55, Rf(7) 0.18. Amino acid analysis: (6 N HC1-phenol, 1 lo", 24 h) AsPi.00, G1ui.01, ~ 0 1 . 0 0 , G1~o.w~ Ileo.96, Leu0.w, 1/2 cystine&l, mixed disulphide of L-cysteine and 0-mercaptopropionic acid 0.41 * and NH3 1.94. Anal. calc. for C44H66N10013SZ (1007.224): C, 52.47; H, 6.60; N, 13.91; OCH3, 3.08. Found: C,52.45;H,6.56;N, 13.47;OCH,3.11. ACKNOWLEDGEMENTS The authors wish to thank Drs. A. Dirscherl and R. Studer, Hoffmann-La Roche Co., Basle for the elemental and the aminoacid analyses, and Dr. K. Plixka, Institut fur Molekularbiologie und Biophysik, E.T.H. Zurich, Dr. P. A. Jacquenoud, Sandoz Co., Basle, and Prof. R. Walter, Dept. of Physiology and Biophysics, University of Illinois Medical Center, for the bioassays.
REFERENCES Acher, R., Chauvet, J., Chauvet, M.T. & Crepy, D. (1965) Biochim. Biophys. Acta 107,393-396 Acher, R., Chauvet, J. & Chauvet, M.T. (1972) European J. Bwchem 29,12- 19 Anderson, G.W. & McGregor, A.C. (1957) J. Am. Chem. SOC. 79,6180-6183 Bodanszky, M. & du Vigneaud, V. (1959) J. Am. Chem. SOC.81,5688-5695
* These two sulphur compounds thus account for the half-cystine residue in this analogue (HopeetaZ., 1962). 432
Boissonnas, R.A., Guttmann, S., Jaquenoud, P.A. & Waller, J.P. (1955) Helv. a i m . Acta 38, 14911501 Brenner, M. & Huber, W. (1953) Helv.Chim. Acta 36, 1109-11 15 Dekanski, J . (1952) Brit. J. Pharmacol. 7,567-572 Hanby, W.E.,Wally,S.G. & Watson, J. (1950) J. Chem. SOC.,3239-3249 Honzl, J. & Rudinger, J. (1961) CON. Czech. Chem. Commun. 26,2333-2344 Hope, B.B., Murti, V.V.S. & du Vigneaud, V. (1962) J. Biol. Chem. 237,1563-1566 Johl, A., Hartmann, A. & Rink, H. (1963) Biochim. Biophys. Acta 69,193-195 Jones, D.A., Mikulec, R.A. & Mazur, R.H. (1973) J. Org. Chem. 38,2865-2869 Kamber, B. & Rittel, W. (1968) Helv. Chim. Acta 51, 2061 -2064 Manning, M., Coy, E. & Sawer,W. (1970) Biochemistry 9,3925-3930 Manning, M., Lowbridge, J., Haldar, I . & Sawyer, W . ( 1 977) Federation Proc. 36,1848-1 852 Marbach, P. & Rudinger, J . (1974) Helv. Chim. Acta 57,403-413 Photaki, I. (1973) in The Chemistry of Polypeptides, Essays in honor of Dr. Leonidas Zervas (Katsoyannis, P.G., ed.), pp. 59-85, Plenum Press, New York Photaki, I. (1976) in Topics on Sulfur Chemistry (Senning, A., ed.), pp. 111-183, Georg Thieme, Stuttgart Photaki, I. & du Vigneaud, V. (1965) J. Am. Chem. SOC.87,908-913 Photaki, I., Engonopoulos, C. & Tzougraki, C. (1977) H.B.B.S., News Letters 10,27 PliSka, V. & Rudinger, J. (1976) Clin. Endocrinol. 5, 735-84s PliXka, V., V a a , J., Niederer, E., Rudinger, J., KotsiraEngonopoulos, C. & Photaki, I. (1975) Endocrinology Abstr. (London), 4-5 Pligka, V.,Marbach,P.,VaSak, J. & Rudinger, J. (1977) Experientia 33,367-368 Rudinger, J. (1971) in Drug Design (Ariens, E.J., ed.), vol. 11, pp. 3 1 9 4 1 9 , Academic Press,New YorkLondon Schwyzer, R., Sieber, P. & Kappeler, H. (1959) Helv. Chim. Acta 42,2622-2624 Yamashiro, D. (1964) Nature 201,76-77 Zaoral, M. & Rudinger, J. (1955) CON.Czech. Chem. Commun. 20,1183-1188 ZeeCheng, K.-Y. &Cheng, C.C. (1970) J. Med. Chem. 13,4 14 -4 18 Zervas, L. & Hamalidis, C. (1965) J. Am. Chem SOC. 87,99-104 Zervas, L. & Photaki, 1. (1962) J. Am. Chem. SOC.84, 3887-3897
[ G l ~ ( o M e ) ~AND ] - [Mpr’ ,Glu(0Me)‘J OXYTOCIN Zervas, L. & Theodoropoulos, D. (1956) J. Am. Chem. SOC. 78,1359-1363 Zervas, L., Borovas, D. & Gazis, E. (1963) J. Am. Chem. SOC.85,3660-3666 United States Pharmacopeia XIV (1950), PP. 475477, Mack Printing Co., Easton, PA
Address: Dr. I. Photaki Laboratory of Organic Chemistry The University of Athens 13A Navarinou Str. Athens 144, Greece
ANNOUNCEMENT TWELFTH MILES INTERNATIONAL SYMPOSIUM “Polypeptide Hormones” Turner Auditorium, the Johns Hopkins Medical Institutions Baltimore, Maryland, USA: July 1 1-1 3,1979
This symposium will address itself to recent developments in research on mammalian polypeptides that exhibit hormonal properties. Highlights of current laboratory and clinical studies of their biological and chemical synthesis, transport, receptors, and cellular response will be presented. Several sessions devoted to hormones according to their specific site of secretion (e.g., hypothalamus, thymus) are also included. The topics to be discussed reflect the current high level of interest regarding polypeptide hormones and will appeal to many investigators involved in cell biology, endocrinology, and pharmacology. Symposium Chairman: Roland F. Beers, Jr., M.D., Ph.D.
Program Committee Christian B. Anfinsen National Institutes of Health
Andrew V. Schally VA Hospital, New Orleans
Karl A. Folkers University of Texas, Austin
Nathan Trainin Weizmann Institute of Science
Choh H. Li University of California San Francisco For further information contact: Edward G. Bassett, Ph.D., Symposium Coordinator Miles Laboratories, Inc. P.O.Box 40 Elkhart, IN, 46515, USA (219)264-8460
433
S Y N T H E S I S A N D SOME P H A R M A C O L O G I C A L P R O P E R T I E S O F [ G l ~ ( 0 M e ])O~X Y T O C I N A N D [ M p r ' , G l ~ ( 0 M e 1)O~X Y T O C I N * IPHIGENIA PHOTAKI, CHRYSA TZOUGRAKI and CHRISTINE KOTSIRA-ENCONOPOULOS
Laboratory of Organic Chemistry, The University of Athens, Athens, Greece
Received 30 June, accepted for publication 1 December 1978 [Glu (OMe)4]oxytocin ( X V I ) and [Mpr', Glu (OMe)4]oxytocin (XVII) bearing a methyl ester group in place of the carboxamide group in position 4 of o x y tocin were synthesized by (3 -k 6 ) segment condensation using the S-trityl group f o r the protection of the cysteine side chains. Analogue X V I exhibited 10.5 Ulmg in vitro uterotonic, and 4 2 UJmg avian vasodepressor, activity, and analogue XVII 21.4 Ulmg and 8 2 UJmg of the respective activities. Both compounds showed no response in the rat pressor assay. Key words: decreased biological activities;Sdeprotection and disulphide formation by iodine; Na-o-nitrophenylsulphenylgroup; (3 tive group.
The position 4 of the cyclic octapeptide neurohypophyseal hormones is a site of natural variation, as can be seen in isotocin (Johl et al., 1963), glumitocin (Acher et al., 1965) and aspartocin (Acher et al., 1972). Studies of the structure-activity relationship performed on a number of synthetic oxytocin analogues have shown that the glutamine residue in position 4 of the molecule is not essential for exhibition of high biological activity (Rudinger, 1971; Manning et al., 1977). A striking example is the synthetic analogue [Cthreonine] oxytocin
* This paper is a part of the Special Anniversary issues (January and February, 1979) of the International Journal of Peptide and Protein Research, dedicated to the 25th anniversary of the synthesis of oxytocin. It was received too late to be includedin those two issues. Abbreviations used in this paper follow the rules of IUPAC-IUB Commi&on on Biochemical Nomen&Biochemistry 5 , 1445-1453, 2485-2489 mre, (1966) a d 6, 362-364 (1967). BOC,tert.-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide; X U , N,N'dicyclohexylurea; DMF, dimethylformamide; EtOAc, ethyl acetate;Et, N, triethylamine; IPflH, i~ProPano1; MPr, @-mercaPtoProPiofic acid; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
426
+ 6) segment condensation;S-trityl protec-
(Manning et al., 1970), which proved to be a highly potent and specific oxytocic agent. However, the deamido-analogue [4-glutamic acid] oxytocin (Photaki & du Vigneaud, 1965) has very low activity although the carboxyl group is nearly isosteric to the replaced carboxamide group, hydrophilic, and has also hydrogen bonding capacity. Therefore, it seemed interesting to investigate the influence of the uncharged methyl ester group which can be better compared to the carboxamide group as to reactivity and polarity. For this purpose the title analogues 'of oxytocin (XVI)-and (XVII) have been synthesized by replacing Ldutamine with y-methyl L-&tamate in POsition 4 of oxytoch and 1-deminoaxytocin ([Mpr' ] OXytOCin) respectively (for a short communication cf. Photaki et al., 1977). These the first oxytocinare, to Our analogues carrying a side-chain ester group. The synthesis of the new analogues has been accomplished via two protected nonapeptides (XIV), (XV) as shown in Scheme 1 for [Glu(OMer ] oxytocin. The trityl group was chosen as sulphydryl protective group because
0367-8377/79/040426-08 $02.00/0 0 1979 Munksgaard, Copenhagen
[Glu(OMe)' Z-Ty r ( Bzl )-lle-OMe
(VIII)
4
Boc-Cys (Tri)-Tyr-lle-OMe
I Boc-Cys (Tril-Tyr-Ile-N,
1 - A N D [ Mpr' ,G l ~ ( 0 M eJ)OXYTOCIN ~
H,
Tri-Cys (Tri )-Pro- Leu-Gly-N H, Nps-Asn-Cys (Tri j-Pro-Leu-Gl y-N H
(IX)
1 Nps-Glu (0Me)-Asn-Cys (Tri)-Pro-Leu-Gly-NH, I
(XI
I azide Tri
(11
1
I OMe
(Ill) ( V I1
0.24 N HCI in EtOAc-DMF Tri
1 I I BocCys-Tyr-l le-Glu-Am-Cys-Pro-Leu-Gly-NH 1 HCys-Tyr-lle-ijlu-Asn-Cys-Pro-Leu-GIy-NH,
( X IV )
(XVI)
SCHEME 1 Synthetic route to [ G l ~ ( o M e )oxytocin ~] (XVI).
of its ready introduction into cysteine and the availability of mild procedures for its selective cleavage (Photaki, 1973, 1976; Kamber & Rittel, 1968; Jones et al., 1973). As for the amino group protection, during the various steps of the synthesis, the trityl (Zervas & Theodoropoulos, 195.6), the o-nitrophenylsulphenyl (Zervas et al., 1963; Zervas & Hamalidis, 1965) and the tert.-butyloxycarbonyl (Anderson & McGregor, 1957; Schwyzer et al., 1959) groups were used. In addition, many of the known coupling procedures have been tried for lengthening the peptide chain and those which gave the best results, in yield of pure compound, are described. Both oxytocin analogues exhibited biological activity on the rat uterus in vitro (i.e. [ G l ~ ( o M e )oxytocin, ~] XVI: 10.5 U/mg, and [Mpr' ,G l ~ ( 0 M e ) oxytocin ~l XVII: 21.4 U/mg) and in vivo (PliSka et al., 1975; PliSka & Rudinger, 1976). The avian vasodepressor activity was determined in comparison with the I11 International standard, four-point assay (U.S.P. XIV, 1950) as 42U/mg for XVI, and 82U/mg for XVII. No response was obtained in the rat pressor assay (Dekanski, 1952;PUka eral., 1977) at dosages of 10,20, and lOOpg. It is interesting to note that replacement of the amide by the methyl ester grouping decreases the activity of oxytocin equally or even more drastically than complete omission of the carboxamide group (cf. the biological activities
of [ k u 4 ]-, "ie4] -, "a4] (Rudinger, 1971).
oxytocin, etc.)
EXPERIMENTAL SECTION
All amino acid derivatives were of the L configuration and were synthesized by standard methods. Anhydrous solvents were used for couplings and removal of N-protecting groups unless otherwise stated. Other chemicals and solvents used were of reagent grade. Melting points were determined in capillary tubes and are uncorrected. Optical rotations were measured in a l d m cell on a Perkin-Elmer Model 141 polarimeter. When necessary, solutions in organic solvents were dried over Na2SO4. Thin-layer chromatography was carried out on silica gel, type 60 (Fluka), plates with the following solvent systems (proportions by volume): (1) toluene-acetone (7:3), (2) toluene-acetone (1 :l), (3) toluene-acetone (4: l), (4) toluene-pyridine-AcOH (8 :2 :1), (5) toluenepyridine-AcOH (80:10:l), (6) EtOAc-EtOH (7:3), (7) EtOAc-EtOH (75:25), (8) tAcOHzFrOH-H20 (62:26:7), (9) CH3CN-H20 (3:1), (1 0) CH3 CN-HZ 0 (9: l), (1 1) ~BuOH-AcOHHzO (4:1:1), (12) ~BuOH-AcOH-H2O (10: 1:3), (1 3) nBuOH-AcOH-H20 (upper phase) (4: 1:5), (14) nBuOH-AcOH-H20 (upper phase) (5 :4: l), (1 5) nBuOH-AcOH-H20-pyridine (30: 6:24:20), (16) CHC13-CC4-MeOH(6:3:1),(17) CHC13-CC14-MeOH-AcOH(6:3: 1:1). Thin-layer chromatograms were run for 7-12cm and developed with ninhydrin sol421
1. PHOTAKI ET AL.
ution (0.5% in acetone-AcOH-HzO, 90:5:5 or 18:l:l) or with iodine vapors. For paper electrophoresis an LKB 3276 apparatus was used with Schleicher and Schdl2043-b paper. S,N-Ditrity 1-L -cysteiny 1-L -prolyl-L-1eucylglycinamide (I)
S,N-Ditrityl-Lcysteine (1 1.2 g, 16.5 mmol) was liberated from its diethylamine salt (Zervas & Photaki, 1962) by partitioning between ether and 0.5 N HzS04. To a stirred solution of the above residue in THF (70ml), N-methylmorpholine (1.2 ml, 17 mmol) and diphenylphosphoryl chloride (4.29 g, 16 mmol) dissolved in THF (20ml) were added at -loo, and after stirring for 15 min a freshly prepared, precooled (0") mixture of L-prolyl-L-leucylglycinamide hydrobromide (Zaoral & Rudinger, 1955) (5.77g, 15mmol) and Et3N (4.2m1, 30 mmol) in DMF-THF (40 ml, 1:1) was added. The pH of the solution was adjusted to 6-7 with Et3N and the mixture was stirred at 0" for 15 min, and at 25" for 1.5 h. The solvent was removed under reduced pressure and the residue was taken up in CHC13. The solution was washed with a dilute aqueous solution of Et3N, Hz 0, dried and evaporated. The residue was triturated with hot ether, filtered off and recrystallized from EtOAc; yield 8 S g , m.p. 205-207", [a]25 + 87.3" (c 2, DMF). The product was furtEer purified by passing through a Dowex 1/1 column (acetate form) using CHC13-DMF-MeOH(2 : 1 :7 v/v) as an eluant, to yield 7.2 g; a second crop (1.5 g) was obtained by passing through the same column the residue of the first recrystallization; (total yield 66%), m.p. 206-209", [at] 2: + 85.9" (c 2, DMF); t.1.c. in systems 4,12, 16. Anal. calc. for C&15,N@$ (872.16): C, 74.36; H, 6.58; N, 8.03; S,3.67. Found: C, 74.42; H, 6.67; N, 7.75; S,3.68. N-o-Nitrophenylsulphenyl-S-trityl-L -cysteinylL -prolyl-L -1eucylglycinamide ( l a )
a waterbath for 5 min. After 30 min at 25" the hydrochloride salt was precipitated with ether. It was recrystallized from CHC13ether; yield 6 g (90%), m.p. 150-153", [a]E+42.3" (c 2, EtOH); t.1.c. in systems 6, 12, 15. Anal. calc. for C ~ ~ H & S O $(663.30): ~ C, 63.09; H, 6.66; N, 10.51, C1, 5.32. Found: C, 62.68; H,6.69, N, 10.40; C1,5.40. B. Compound I1 was also obtained from Ia by a similar treatment (0.2 N HCI in MeOH).
N-o-Nitropheny lsulpheny 1-L -asparaginyl-Strityl-L-cysteinyl-L-prolyl-L-1eucylglycinamide
Vrll
N-o-Nitrophenylsulphenyl-L-asparagine(Zervas el al., 1963) (3.4 g, 12 mmol) was dissolved in THF (60 ml) by the addition of pyridine (1 .O ml, 12.5 mmol) and Nethylpiperidine (1.2 ml, 12 mmol). The solution was cooled to - lo", pivalic acid chloride (1.2 ml, 10 mmol) was added and the mixture was stirred for 5-10 min. A precooled mixture of I1 (6.5 g, 10 mmol) and E t a (1.4 ml, 10 mmol) in DMF (20 ml) was added. The mixture was stirred at 0" for 1 h and at 25" overnight. The solvent was evaporated off and the residue was taken up in CHC13. The solution was washed with a dilute aqueous E t a solution, HzO, 2 M AcOH, HzO, 58 NaHC03 and HzO,dried and evaporated to a small volume. The crude protected pentapeptide was precipitated with ether and recrystallized from MeOH; yield 5.8 g (65%), m.p. 128-1 30" (after softening at 123"), [ a ] g-62.2" (c 2, EtOH), [a] - 88.2" (c 2, CHC13); t1.c. in systems 6, 9,11. Anal calc. for C4sHs2NsO&S2, 0.5 CH30H (913.10): C, 59.85; H, 5.96; N, 12.27; S,7.02. Found: C,59.75;H,5.91;N1 12.10;S,7.24.
L-Asparaginyl-S-trityl-L-cysteinyl-L-prolyl-L-
leucylglycinamide hydrochloride (IV) To a cold solution of I11 (4.5 g, 5 mmol) in CHC13 (45 ml), mercaptoethanol (2.1 ml, 30 mmol) and 2.5 N HC1 in EtOAc (1.9 ml) were added. Upon cooling the hydrochloride salt separated out as a gel which was crystallized from MeOH-EtOAc; yield 3.5 g (90%), m.p. 145-147", [(u]~-3.9" (c 2, MeOH); t.1.c. in systems 6,9, 10,14. Anal. calc. for C3.&&0&l (780.40): C, S-Trityl-L -cysteiny1-L -prolyl-L-1eucylglycin60.03; H, 6.46; N, 12.56; S, 4.11; C1, 4.54. amide hydrochloride (II) A. Compound (I) (8.7 g, 10mmol) was dissolved Found: C,60.47;H,6.78;N, 12.52;Sl4.10;C1, in 0.2 N HCI in MeOH (50 ml) by warming it on 4.51.
Prepared by DCC coupling of N-o-nitrophenylsulphenyl-3-trityl-L-cysteine (Zervas et al., 1963) with L-P~O-L-L~U-G~Y-NH~ (Boissonnas et aL, 1955), m.p. 80" dec., [ a ] ~ - 5 8 . 7 " (c 2, MeOH); t.1.c. in systems 4, 15. Anal. calc. for C4&&0&, 0.5 EtOAc (827.05): C, 62.45; H, 6.09; N, 10.16;S, 7.75. Found: C, 62.89; H, 6.39; N, 10.02; S,7.88.
[Glu(OMe)']- AND [Mpr' ,G~u(OM~)~]OXYTOCIN
N-o-Nitrop heny lsulp heny l-L -glutam ic acid y methyl ester (V)
To a solution of L-glutamic acid y-methyl ester (Hanby et al., 1950) (1.6 g, 10 mmol) and KHC03* (1.8 g, 10 mmol) in HzO (20 ml) and dioxane (20 ml), a solution of o-nitrophenylsulphenyl chloride (2.4 g, 12.5 mmol) in dioxane (10 ml) and saturated KHC03* (10 ml) were added alternatively, in 10 equal portions, dropwise, during 1 h. Care was taken to keep the pH at 7-8. The solution was stirred for another 1 h, diluted with HzO (50ml), filtered, washed with ether, and acidified with 1 N HzS04 to a slightly Congo red reaction. The oily precipitate was taken up into ether and the organic layer was washed with HzO, dried and evaporated. The residue was dissolved in MeOH, cooled, some insoluble material was filtered off, and the solution was evaporated to dryness. Crystallization from EtOAc-hexane and recrystallization yielded 2.4 g (75%), m.p. 103-105', [ a ] ; - 62.9" (c 2, CHC13); t.1.c. in systems 4 , 14. Anal. calc. for ClZH1&0,$3 (314.33): C, 45.86; H, 4.49; N, 8.9 1; S, 10.20. Found: C, 45.38; H, 4.29; N, 8.69, S, 10.08. Compound V was also converted to its dicyclohexylammonium salt** (86%), m.p. ] ~51.9' (c 2, CHCI,); 180.5-182.S0 dec., [ o L t1.c. in systems 4,16.
Anal. calc. for C51H61N~O11SZ (1040.22): C, 58.89; H, 5.91; N, 12.12; S, 6.16. Found: C, 58.93; H, 6.29; N, 11.98; S, 5.90. y-0-Methy l-L -glutamyl-L-asparaginyl-S-trityI-L cysteinyl-L-prolyl-L-leucylglycinamide hydrochloride (VII)
To a cold (0") solution of VI (0.25 g, 0.25 mmol) in DMF-EtOAc (6 ml, 1 : 1 v/v), mercaptoethanol (0.42 ml, 6 mmol) and 2.4 N HCl in EtOAc (0.1 1 ml) were added. The hydrochloride salt was precipitated with EtOAc. Recrystallization from iPrOH yielded 0.23 g (92%), m.p. 144-146"dec., [a] - 18.5'(c 2, MeOH), t.1.c. in systems 6 , 9 , 12, 14; homogeneous on paper electrophoresis (270 V, 2 h) in AcOH-pyridineHzO (PH 4.9). Anal. calc. for C45HSPN80&Cl (923.51): C, 58.52; H, 6.44; N, 12.13; S, 3.47; C1, 3.84. Found: C,58.31;H,6.61;N, 12.13;S,3.64;Cl, 3.95. N-Benzyloxycarbonyl-0-benzyI-L -tyrosyl-L-
To a cold (0") solution of compound V, liberated from its dicyclohexylammonium salt (0.5 g, 1 mmol), in DMF (2 ml), a precooled (0') mixture of IV (0.7 g, 1 mmol) and Etfl(O.14 ml, 1 mmol) in DMF (2 ml) was added, followed by DCC (0.22 g, 1.1 mmol). The mixture was stirred overnight at 25", then cooled and the DCU removed by filtration. Workup as described for compound I11 and trituration with warm iPrOH yielded 0.55 g (53%), m.p. 198-200", [(~]E-39.6O (C 2, EtOH-DMF 1: 1 V/V) [a] - 29.7' ( c 2, DMF); t.1.c. in systems 6,9, 12,14.
isoleucine methyl ester (VIII) To a cold (0") solution of L-isoleucine methyl ester hydrochloride (Brenner & Huber, 1953) (1.63 g, 9 mmol) in DMF (14 ml), N-benzylonrcarbonyl-0-benzyl-L-tyrosine p-nitrophe.iy1 ester (Bodanszky & du Vigneaud, 1959) (4.7 g, 9.45 mmol) was added followed by Etfl (1.26 ml, 9 mmol) (dropwise). After 2 days at 25" the product was precipitated with H20 and extracted with EtOAc. The organic layer was washed with 0.5 N HCl, H20, aqueous Etfl solution, 1 M N H P H and HzO,dried and evaporated. Recrystallization from MeOH yielded 3.8 g, m.p. 113-1 13.5". A second crop (0.8 g, m.p. 110-1 12") was gained from the mother liquor; totalyield96%,m.p. 115-1 15.5" after recrystallization from CHC13-light petroleum; t.1.c. in system 1; [a] - 2.7" (c 2.5, MeOH), [a] + 15.0" (c 1.5, CHC13). Anal. calc. for C31H&JZO6(532.645): C, 69.90; H, 6.81; N, 5.26. Found: C, 70.28; H, 6.67; N, 5.38.
* Or equivalent amount of Et,N.
N-tert.-ButyloxycarbonyI-S-trityl-L-cysteinyl-L-. tyrosyl-L-isoleucinemethyl ester (IX)
N-o-Nitrophenylsulphenyl-y-0-methyl-L -
-
glutamy l-L -asparaginyl-S-trityl-L-cysteinyl-L
propyl-L -1eucylglycinamide (VI)
** Prepared originally in this laboratory by P. Mazarakis, unpublished.
A solution of VIII (1.06 g, 2 mmol) in glacial AcOH (2.7 ml) was treated with 5.5 N HBr in 429
I. PHOTAKI ET AL.
AcOH (2.3 ml). After 35 min at 25" the solution w a s poured into cold ether (200 ml). The precipitated salt was washed with ether several times, cooled, filtered off and dried over KOH and Pz0 5 ;yield 0.69 g (88%),(chromatographically pure in system 12). The hydrobromide salt (0.6 g, 1.54mmol) was coupled with N-terr.buty1oxycarbonyl-S-trityl-L-cysteine p-nitrophenyl ester (Jones er ul., 1973) (0.95 g, 1.62 mmol). Et3N was added to keep the pH of the solution to about 7. The product was isolated as described for VIII (acidic washing with 0.5 N H2SO4). Crystallization from EtOAc-hexane yielded 1.1 g(94%),m.p. 82" dec. Recrystallization from DMF-HzO, m.p. 87", [a] - 2.7" (c 1.5, CHC13); t.l.c. in systems 1,2,5. A d . C ~ C . for Cg3HsIN307S (753.95): C, 68.50; H, 6.82; N, 5.57. Found: C, 68.71; H, 7.12;N,5.11. Identical products were obtained by DCC or mixed anhydride coupling.
ml), p-nitrophenol (6.7 g, 48 mmol) and DCC (8.25 g, 40 mmol) were added. After stirring at 0" for 1 h and at 25" for 3 h, the precipitated DCU w a s filtered off. Water was added to the filtrate and after cooling the precipitate was collected. The crude p-nitrophenyl ester was recrystallized twice from EtOH; 14.5 g (77%), m.p. 125";tl.c.in systems 1 , 1 2 . Anal. calc. for CZsHz3NO4S(469.56): C, 71.62; H, 4.94; N, 2.98; S, 6.83. Found: C, 71.58, H, 4.96; N, 2.85; S, 6.84.
S-Trity l-&mercap topropiony I-l-tyrosy I-L -holeucine methyl ester (XI.)
Compound XI (1.59 g, 3.39 mmol) was added to a cold (0"), stirred solution of L-tyrosyl-L-isoleucine methyl ester hydrobromide (1.26 g, 3.23 mmol, prepared as described above) in DMF (15 ml), followed by Et3N (0.45 ml, 3.2 mmol) (pH cu. 7). After 4 days at 25", HzO was added and the mixture was extracted with N-tert.-Bufyloxycarbonyl-S-tn'tyl-L~ysteiny~-~EtOAc. The organic layer was washed with 0.5 tyrosyl-L+soleucine hydrazide (XI N HCl, HzO, an aqueous solution of Et3N, and Compound IX was dissolved in MeOH (5 ml) HzO, dried and evaporated. The product was and treated with HzNNHz*H20(0.6 ml) at 25" crystallized from EtOAc-light petroleum and for 36 h. The precipitated solid was filtered off, recrystallized from DMF-HzO and then from washed with EtOAc; yield 4 S g , (86%) m.p. MeOH-H20 to yield 1.75 g (85%), m.p. 84-86", with hot MeOH. Theproduct (1.05 g,m.p. 195- [a]g-6.3" (c 1.5, CHC13); t1.c. in systems 1 , 196") was recrystallized from DMF-H20 to 5. yield 1 g (88%),m.p: 197-198", [a] + 10.2" Anal. calc. for CUII€,zN30sS, 0.25 CH30H (c 2, DMF); t1.c. in systems 1,2. (646.83): C, 70.99; H, 6.64; N, 4.1 7. Found: C, A d . C ~ C . for C42HS1NS06S (753.96): C, 71.03; H, 6.70; N, 4.33. 66.91; H, 6.82; N, 9.29; S, 4.25. Found: C, 66.92; H, 6.80; N, 9.1 2; S, 4.3 1. S-Trityl-0-mercaptopropionic acid
S-TrityI- 0-mercap topropi0 ny I-L-tyrosY 1-L -is@ leucin e hy draz ide (XIII)
To a hot solution of triphenylcarbinol (4.3 g, 16.5 mmol) in EtOAc (35 ml) P-mercaptopropionic acid (1.6 g, 15 mmol) and conc. HzS04 (0.1 ml) were added and the mixture was warmed for 10 min at 60". The precipitated crystalline compound was filtered off after cooling and washed with EtOAc; yield 4Sg, (86%) m.p. 204-205" unchanged after recrystallization from EtOAc (lit. m.p. 208-209", Zee-Cheng & Cheng, 1970). T.1.c. in systems 5,12.
Prepared as described for X; yield 77%,m.p. 219.5-220" after repeated trituration with boiling MeOH. [a] + 2.7" (c 1.5, DMF); t.1.c. in systems 1,2. Anal. C ~ C . for C37bZN404S (638.83): C, 69.57; H, 6.63; N, 8.77. Found: C, 69.29; H, 6.59; N, 8.51.
S-Trity I-P-mercaptopropionic acid p-nitrophenyI
asparaginyl-S-trityl-L -cysteinyl-L -proly I-L -
N-text.-Butyloxycarbonyl-S-trityI-L-cysteinyl-L ty rosy I- L -is0le ucy 1-7-0-me thy l-L -glutamy I-L -
leucylglycinamide (XIV) ester (XI) To a cold solution (0")of S-trityl-o-mercapto- The tripeptide hydrazide X (452 mg, 0.6 mmol) propionic acid (13.9 g, 40 mmol) in DMF (200 in DMF (3.5 ml) was converted into the azide 430
[ G l ~ ( o M e])-~AND [ Mpr' ,G l ~ ( 0 M e])OXYTOCIN ~
(Honzl & Rudinger, 1961) by treatment with 2.5 N HCl in EtOAc (0.62 ml) and tert.-butylnitrite (0.084 ml) at - 15" for 15 min. A precooled mixture of VII (555 mg, 0.6 mmol) and Et3N (0.3 ml, 2.1 mmol) in DMF (3.5 ml) was added, followed by N-methylmorpholine (0.081 ml, 1.I 5 mmol). The mixture (pH ca. 7, maintained by adding some N-methylmorpholine from time to time) was left at - 12" for 1.5 h and at 25" for 5 days. After concentration to a small volume, the product was precipitated by adding 0.03 N HCl, cooled, filtered off and washed with HzO; yield 843 mg (87%), m.p. 143-147' unchanged after precipitation from EtOHether, [a]g-29.7" (c 1 , MeOH); t1.c. in systems 6 , 8 , 9 . Anal. calc. for C87H105N110~5Sz (1609.02): C, 64.94; H, 6.58; N, 9.58. Found: C, 64.43; H, 6.70;N, 9.53.
h the crude oxytocin salt was filtered off, washed with ether and dried in a desiccator over KOH and PzOs to provide 90 mg (95%). A sample (65 mg) was subjected to partition chromatography (Yamashiro, 1964) on a 1.1 X 96 cm column of Sephadex (3-25 with nBuOHtoluene-pyridine-O.l% AcOH (5 :3 :1 :9 v/v) as a solvent system. Lyophilization of the pooled fractions from the major, symmetrical peak (Rf 0.38, detection by Folin Lowry analysis) provided 23.5 mg of a material, [a] - 24.4' (C 0.5,1 M AcOH), Rf(12) 0.45, Rf(13) 0.5, Rf(15) 0.75. Paper electrophoresis (300 V, 2 h) gave a single band (bromophenol blue-HgClz detection) at pH 9.3 (glycine buffer). Amino acid analysis (6 N HCl-phenol, 110", 24 h), pr01,05 G1y1.03 , 1/2 ASPLOO G1U1.04, cystinel.87, Ileo.w, L e u l . ~ , Tyro.97 and NH3 1.74. Anal. calc. for C44H67N11013SZ,CH3COOH [4-L-Glutamic acid ymethyl ester] oxytocin (1082.26): C, 51.05; H, 6.61; N, 14.24;OCHB, 2.87. Found: C, 51.26; H, 6.53; N, 14.02; (X vrl A. Compound XIV (188 mg, 0.1 16 mmol) was OCH3, 2.80. dissolved in 90%MeOH (750 ml) and a solution of iodine (0.09 g, 0.34 mmol) in MeOH (1 20 ml) S-Trityl-Prnercaptopropionyl-L-tyrosyl-L-isowas added dropwise under vigorous stirring at leucy 1-y-0-methyl-L -glutarnyl-L-asparaginyl-Strityl-L -cysteinyI-L -proly1-L -1eucylglycinamide 25' during 1 h (Kamber & Rittel, 1968). After a further 5 h stirring, H 2 0 (150ml) was added (X V ) and excess iodine was removed (Marbach & The hydrazide XI11 (192 mg, 0.3 mmol) was conRudinger, 1974) by evaporation of the solution verted to the azide and coupled with VII (277 mg, 0.3 mmol) as described for XIV.The crude to cu. 70ml (pH ca. 4). It was extracted first with EtOAc (10ml x 2), to remove the tri- product was chromatographed on a silica gel column using the solvent system EtOH-EtOAc phenylcarbinol and then with n BuOH-benzene (3 :7 v/v). The pooled fractions containing XV, (1: 1 v/v) (4x), and with benzene. The comas detected by tl.c., were evaporated to a small bined nBuOH-benzene extracts were washed volume. Upon addition of EtOAc a microtwice with a few ml of H 2 0 to neutral pH, crystalline material was obtained, 0.2 g (45%), diluted with about half their volume with H2O m.p. 142-145", [a] g- 33" (c 1 , MeOH), Rf(6) and evaporated to a small volume. MeOH was 0.85, Rf(9) 0.84. Amino acid analysis (6 N HCladded and the mixture was reevaporated to a , 0.98, ~01.01, small volume. Upon addition of ether micro- phenol, llOo, 24 h) ~ P I . O OGlu crystalline N-tert.-butyloxy-carboonyl-oxytocinG~YLOI 1/2 cystinel.08, Ileo.94, precipitated, which after 12 h at 4 was filtered TYr0.97. (1493.85): c , off to yield 100mg \76%), m.p. 167" (dec., Anal. cdc. for C8ZH96N10013S2 after shrinking at 160 ). [ a ] g-55.5' (c 1.5, 65.93; H, 6.48; N, 9.38. Found: C, 65.41, H, 6.43, N, 9.09. EtOH); t.1.c. in systems 7, 1 1 , 12, 15. 9
3
f I -&Mercaptopropionic acid, 4-L -glutamic acid y-methyl ester] oxytocin (XVZZ) B . Removal of' the N-tert .-butyloxycarbonyl group. The above Boc-oxytocin (94 mg, 0.084 mmol) was treated with 90% TFA (0.8 ml) for 2.5 h at 25". Ether was added at 0" and after 3
Compound XV (104 mg, 70 pmol) was treated with iodine in MeOH as described for the preparation of XVI, A, except that the triphenylcarbinol was extracted with benzene. MeOH 43 1
I. PHOTAKI ET AL.
was added to the residue and after evaporation to a small volume EtOAc was added to precipitate a microcrystalline product 45 mg, (65%), m.p. 178-180" (softening at 170°), Rf(l 1) 0.6. A sample of 35 mg was purified by partition chromatography on a 1.1 X 96 cm column of Sephadex G-25 with the solvent-system nBuOH benzene-H20,containing 1.5% pyridine and 3% AcOH (1 :2 :3 v/v). The purification was repeated twice to obtain a symmetrical peak (Rf 0.58, Folin Lowry analysis). Pooling and lyophilization provided 27 mg of chromatographically pure material, [a]4:-35.lo, (c 2, MeOH), Rf(6) 0.35,Rf(9) 0.9, Rf(l1) 0.6, Rf(12) 0.55, Rf(7) 0.18. Amino acid analysis: (6 N HC1-phenol, 1 lo", 24 h) AsPi.00, G1ui.01, ~ 0 1 . 0 0 , G1~o.w~ Ileo.96, Leu0.w, 1/2 cystine&l, mixed disulphide of L-cysteine and 0-mercaptopropionic acid 0.41 * and NH3 1.94. Anal. calc. for C44H66N10013SZ (1007.224): C, 52.47; H, 6.60; N, 13.91; OCH3, 3.08. Found: C,52.45;H,6.56;N, 13.47;OCH,3.11. ACKNOWLEDGEMENTS The authors wish to thank Drs. A. Dirscherl and R. Studer, Hoffmann-La Roche Co., Basle for the elemental and the aminoacid analyses, and Dr. K. Plixka, Institut fur Molekularbiologie und Biophysik, E.T.H. Zurich, Dr. P. A. Jacquenoud, Sandoz Co., Basle, and Prof. R. Walter, Dept. of Physiology and Biophysics, University of Illinois Medical Center, for the bioassays.
REFERENCES Acher, R., Chauvet, J., Chauvet, M.T. & Crepy, D. (1965) Biochim. Biophys. Acta 107,393-396 Acher, R., Chauvet, J. & Chauvet, M.T. (1972) European J. Bwchem 29,12- 19 Anderson, G.W. & McGregor, A.C. (1957) J. Am. Chem. SOC. 79,6180-6183 Bodanszky, M. & du Vigneaud, V. (1959) J. Am. Chem. SOC.81,5688-5695
* These two sulphur compounds thus account for the half-cystine residue in this analogue (HopeetaZ., 1962). 432
Boissonnas, R.A., Guttmann, S., Jaquenoud, P.A. & Waller, J.P. (1955) Helv. a i m . Acta 38, 14911501 Brenner, M. & Huber, W. (1953) Helv.Chim. Acta 36, 1109-11 15 Dekanski, J . (1952) Brit. J. Pharmacol. 7,567-572 Hanby, W.E.,Wally,S.G. & Watson, J. (1950) J. Chem. SOC.,3239-3249 Honzl, J. & Rudinger, J. (1961) CON. Czech. Chem. Commun. 26,2333-2344 Hope, B.B., Murti, V.V.S. & du Vigneaud, V. (1962) J. Biol. Chem. 237,1563-1566 Johl, A., Hartmann, A. & Rink, H. (1963) Biochim. Biophys. Acta 69,193-195 Jones, D.A., Mikulec, R.A. & Mazur, R.H. (1973) J. Org. Chem. 38,2865-2869 Kamber, B. & Rittel, W. (1968) Helv. Chim. Acta 51, 2061 -2064 Manning, M., Coy, E. & Sawer,W. (1970) Biochemistry 9,3925-3930 Manning, M., Lowbridge, J., Haldar, I . & Sawyer, W . ( 1 977) Federation Proc. 36,1848-1 852 Marbach, P. & Rudinger, J . (1974) Helv. Chim. Acta 57,403-413 Photaki, I. (1973) in The Chemistry of Polypeptides, Essays in honor of Dr. Leonidas Zervas (Katsoyannis, P.G., ed.), pp. 59-85, Plenum Press, New York Photaki, I. (1976) in Topics on Sulfur Chemistry (Senning, A., ed.), pp. 111-183, Georg Thieme, Stuttgart Photaki, I. & du Vigneaud, V. (1965) J. Am. Chem. SOC.87,908-913 Photaki, I., Engonopoulos, C. & Tzougraki, C. (1977) H.B.B.S., News Letters 10,27 PliSka, V. & Rudinger, J. (1976) Clin. Endocrinol. 5, 735-84s PliXka, V., V a a , J., Niederer, E., Rudinger, J., KotsiraEngonopoulos, C. & Photaki, I. (1975) Endocrinology Abstr. (London), 4-5 Pligka, V.,Marbach,P.,VaSak, J. & Rudinger, J. (1977) Experientia 33,367-368 Rudinger, J. (1971) in Drug Design (Ariens, E.J., ed.), vol. 11, pp. 3 1 9 4 1 9 , Academic Press,New YorkLondon Schwyzer, R., Sieber, P. & Kappeler, H. (1959) Helv. Chim. Acta 42,2622-2624 Yamashiro, D. (1964) Nature 201,76-77 Zaoral, M. & Rudinger, J. (1955) CON.Czech. Chem. Commun. 20,1183-1188 ZeeCheng, K.-Y. &Cheng, C.C. (1970) J. Med. Chem. 13,4 14 -4 18 Zervas, L. & Hamalidis, C. (1965) J. Am. Chem SOC. 87,99-104 Zervas, L. & Photaki, 1. (1962) J. Am. Chem. SOC.84, 3887-3897
[ G l ~ ( o M e ) ~AND ] - [Mpr’ ,Glu(0Me)‘J OXYTOCIN Zervas, L. & Theodoropoulos, D. (1956) J. Am. Chem. SOC. 78,1359-1363 Zervas, L., Borovas, D. & Gazis, E. (1963) J. Am. Chem. SOC.85,3660-3666 United States Pharmacopeia XIV (1950), PP. 475477, Mack Printing Co., Easton, PA
Address: Dr. I. Photaki Laboratory of Organic Chemistry The University of Athens 13A Navarinou Str. Athens 144, Greece
ANNOUNCEMENT TWELFTH MILES INTERNATIONAL SYMPOSIUM “Polypeptide Hormones” Turner Auditorium, the Johns Hopkins Medical Institutions Baltimore, Maryland, USA: July 1 1-1 3,1979
This symposium will address itself to recent developments in research on mammalian polypeptides that exhibit hormonal properties. Highlights of current laboratory and clinical studies of their biological and chemical synthesis, transport, receptors, and cellular response will be presented. Several sessions devoted to hormones according to their specific site of secretion (e.g., hypothalamus, thymus) are also included. The topics to be discussed reflect the current high level of interest regarding polypeptide hormones and will appeal to many investigators involved in cell biology, endocrinology, and pharmacology. Symposium Chairman: Roland F. Beers, Jr., M.D., Ph.D.
Program Committee Christian B. Anfinsen National Institutes of Health
Andrew V. Schally VA Hospital, New Orleans
Karl A. Folkers University of Texas, Austin
Nathan Trainin Weizmann Institute of Science
Choh H. Li University of California San Francisco For further information contact: Edward G. Bassett, Ph.D., Symposium Coordinator Miles Laboratories, Inc. P.O.Box 40 Elkhart, IN, 46515, USA (219)264-8460
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