US 2002O165132A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/0165132 A1 G00dman et al. (43) Pub. Date: Nov. 7, 2002

(54) LANTHIONINE BRIDGED tion of application No. 07/742,908, filed on Aug. 9, 1991, now abandoned. (76) Inventors: Murray Goodman, La Jolla, CA (US); George Osapay, La Jolla, CA (US) Publication Classification Correspondence Address: (51) Int. Cl...... C07K 7/52; A61K 38/12 Howard C. Lee (52) U.S. Cl...... 514/9; 530/317 Norris McLaughlin & Marcus, PA. 30th Floor (57) ABSTRACT 220 East 42nd Street New York, NY 10017 (US) Disclosed are lanthionine bridged peptides having the Struc ture (21) Appl. No.: 09/852,870 (22) Filed: May 10, 2001 O O H | Related U.S. Application Data R-N- th-c- -NH- ti- C-Rs (60) Division of application No. 09/384,061, filed on Aug. R- S -R 26, 1999, now Pat. No. 6,268,339, which is a con R7 R8 tinuation of application No. 08/467,472, filed on Jun. 6, 1995, now Pat. No. 6,028,168, which is a continu ation-in-part of application No. 08/021,606, filed on methods of their preparation and their use as pharmacologi Jan. 28, 1993, now abandoned, which is a continua cally active agents. Patent Application Publication Nov. 7, 2002 Sheet 1 of 4 US 2002/0165132 A1

BocCys(Fm)-MBHA Assembly of chain by Solid-phase CouplingS.

ZTyr(Bzi)SerGlyPheCys(Fm)-MBHA (1)

DSC

Y ZTyr(BzI)DhaGlyPheCys(Fm)-MBHA (2) piperidine Zyr(BZ)-NH-CO-Glyphecys-MBHAY CH2Y-2 -1 SH

ZTyr(Bzl)-D-Ala GlyPhe Ala-MBHA (3) -S- HF

H-Tyr-D-Ala GlyPhe Ala-NH2 (4) —S-

FIG. 1 Patent Application Publication US 2002/0165132 A1

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US 2002/0165132 A1 Nov. 7, 2002

LANTHONNE BRIDGED PEPTIDES BRIEF DESCRIPTION OF THE DRAWINGS 0001. This application is a Continuation of Ser. No. 0007. The invention will now be described in greater 09/384,601, filed on Aug. 26, 1999, now allowed, which in detail with reference to the drawings wherein: turn was a Continuation of Ser. No. 08/467,472 filed Jun. 6, 0008 FIGS. 1-4 describe different syntheses of lanthion 1995, now abandoned which in turn was a Continuation-in ine bridged compounds. Part of Ser. No. 08/021,606 filed Jan. 28, 1993, now aban doned, which in turn was a Continuation of Ser. No. 07/742, DETAILED DESCRIPTION OF THE 908 filed Aug. 9, 1991, now abandoned. INVENTION 0002. It is a basic goal in peptide chemistry to design 0009. It is an object of the present invention to provide molecules for medical or industrial application. Design analogs of peptide compounds having a monoSulfide bridge means that naturally occurring peptides which have a bio in the molecule and exhibiting an improved biological logical different activity are modified in order to obtain activity. The analogs of peptide compounds according to the molecules which have advantages over the naturally occur invention comprise analogs of compounds Such as: virus ring peptides in different respects. There are Several groups related peptides, Such as peptides of HIV, fragments of of peptides which act as hormones, as neurotoxins or as oncogenes, atrial natriuric peptides, epidermal growth fac plant regulating agents. These peptides are usually Small, tors, transforming growth factor and fragments thereof, flexible-molecules which may optionally have a disulfide Spanolysin, TRP, Xenopsin, PGL, PGA, custracean, bacte bridge. necin, relaxin, cyclotheonamides, casOXin D, mastoparan, azaline-B, GRF (growth hormaone releasing factor), leuci SUMMARY OF THE INVENTION nostatins, didemnins, naZumamides, theonellamides, glicen 0003. It is an object of the present invention to provide tin, aureobasidins, guanylin, NGF (nerve growth factor), peptides which comprise a monosulfide bridge. This thioet gluten exorphins, paradaxin P-1, neuromedin U (NMU), her bond is also designated as a lanthionine bridge and bactenecins, bacitracin, bombyxin, PTH, PTHrP (para-thy corresponds with the bridge with the exception that roid hormon-related protein), agelenin, insulin, glucagon, the disulfide bridge is replaced by a monoSulfide linkage. tyrocidins, polymyxins, valinomycin, conotoxins and Two residues having the general formula related neurotoxins, mast cell granulating peptides (MCD), HIV gp41 antigenic peptide 1 or peptide 4, TNF (tumor necrosis factor), RGD-peptides (peptides containing the -HN-CH-COO- -HN-CH-COO Sequence . . . -Arg-Gly-Asp- . . . ). RHC-S-CHR 0010. It is an especially preferred embodiment of the present invention the peptide or the fragment thereof having the thioether bond is derived from a molecule having the 0004 are designated to be joined into a lanthionine biological activity which is in the naturally occurring form bridge wherein the linkage of the two amino acids has the linear. meaning -RCH-S-HCR' , wherein R and R' respec tively represent -H, a lower (C1-C10) alkyl or aralkyl 0011. In a preferred embodiment of the present invention group. In a preferred embodiment RandR' are H. The amino the peptide has not more than two monoSulfide bridges and acid termini of the lanthionine Structure are designated as in an especially preferred embodiment the peptide has only Ala, if R and Rare H and Thr, when R or R are CH3. Other one monoSulfide bridge. B-Substituted lanthionine components are designated as Sub 0012. In a further preferred embodiment of the present Stituted Ala derivatives, e.g. fethylAla. invention the naturally occurring molecule has two or more cyclic Structures wherein at least one cycle is formed by a DESCRIPTION OF THE RELATED ART thioether bond. 0005. Thioether bonds of the lanthionine type are known 0013 The compounds of the present invention have a from Some fungal toxins and antibiotics, for example from higher biological activity than the corresponding naturally the , as , epidermin, dunamycin or merSaci occurring peptides. din. Naturally occurring compounds having the monoSulfide bridge always have more than two monoSulfide bridges in 0014. According to the present invention lanthionine the molecule. bridged peptides are disclosed having the general formula 0006 M. F. Bean et al. have reported in their article “Identification of a Thioether By-product in the Synthesis of (I) a Cyclic Disulfide Peptide by Tandem Mass Spectrometry” O O as published in the Proceedings of the 11th American H | Peptide Symposium, ESCOM, (Leiden 1990, p. 443) on a Somatostatin analog wherein the internal disulfide bond has R-n-i-o-o-ni- ti-c- been converted to a thioether link. The Somatostatin analog R----, with the putative amino acid sequence Phe-Ala-Phe-Trp R7 R8 Lys-Thr-Ala-Thr(oil), wherein the two Ala residues are linked via the thioether bridge, has been described as the by-product which was obtained by the Boc-TFA-preparation 0.015 wherein of Sandostatin analogs. The originally occurring Somatosta 0016 R is selected from the group consisting of tin derivative has a disulfide bridge. Sequences of 2 to 7 amino acids Selected from US 2002/0165132 A1 Nov. 7, 2002

naturally occurring amino acids and the D-enanti 0032. The radicals R, R or R can comprise peptido omers thereof and peptidomimetrics, mimetics. Non-natural amino acids which can be used in out (0017) R, is selected from the group consisting of invention are identified by the following: 0033 C.-methylated amino acids 0018 a) -H, C alkyl, C, aralkyl, -HCO, C, acyl or C2-1s aracyl, 0034 B-mono and dimethylated amino acids 0019 b) a naturally occurring amino acid or 0035 Tic T-hydroxy-1,2,3,4-tetrahydroisoquino Sequence of up to 25 amino acids where the -N line-3-carboxylic acid terminal -NH2 group is present or is replaced by 0036) Atc 2-aminotetralin-2-carboxylic acid Cs alkyl, C7 aralkyl, -HCO, or Cs acyl, -OH, -H or NHCOR, and 0037 Aic 2-amino-5-hydroxyimolinane-carboxy lic acid 0020 c) peptidomimetics; 0038 Hat 2-amino-6-hydroxytetralin-2-carboxylic 0021 R3. is selected from the group consisting9. of acid 0022) a) -OH, NH; 0039) Nal 2-(1-napththyl)alanine 0023 b) naturally occurring amino acid or 0040 APhe dehydro-phenylalanine Sequence of up to 25 amino acids where the -C terminal -COOH is present or is replaced by 0041) p-X-Phe p-halogeno-phenylalanine —CONH, or -CH-OH: 0.042 MePhe 2-dimethyl-f-methyl-phenylalanine 0024 c) peptidomimetics; and O 0043. Examples of dipeptide isosteres which may be 0025 d) CR may be replaced by CH-OH; with utilized are: the proviso that R is not Phe-Trp-Lys-Thr when R, is Phe and Rs is Throll; Benzodiazepine ring: 0026 R, Rs, R, and Rs are independently hydro CO gen, cyclohexyl or a Substituted or unsubstituted

C.1-10 alkyl, and 0027 R is an alkyl or aralkyl residue. 0028. In a preferred embodiments of the present inven tion R, Rs, R, and Rs are independently hydrogen or a methyl group. HydantoinC. ring: 0029. The amino acids can be selected from the group consisting of the naturally occurring amino acids including R i O R O the L-enantiomers and the D-enantiomers. The group of the naturally occurring amino acids comprises alanine, arginine, asparagine, aspartic acid, , cystine, glutamic acid, N- N glutamine, glycine, histidine, , isoleucine, 1. isk CO leucine, lysine, methionine, phenylalanine, proline, , O threonine, tryptophan, tyrosine, Valine, B-alanine, Y-ami R i nobutyric acid, betaine, carnitine, , creatine, orni thine, Saccharopine, 3,4-dihydroxyphenylalanine, 5-hydrox ytryptophan, thyroXine, , S-methylmethionine, 1. NN/ \, , pipecolic acid and nalidixic acid. Tetrazole ring: N 0.030. A regular peptide has the following structure: 1S \ n N

O R O R O -HN CO

-C-NH-CH-C-NH-CH-C- R R 3-oxoimidoliazidine: 0.031 whereas the retro-inverso structure has the follow O ing formula: N R

CO O R O R

-C-NH-CH-NH-C-CH-C- US 2002/0165132 A1 Nov. 7, 2002

0044) The terto-inverso structure is only one possible 0060 Among the especially preferred peptides of the modification to the regular peptide Structure. The regular present invention are: lanthionine-enkephalins SEQ ID Structure may be modified by the presence of a thioester NO:1) having the formula Structure according to the following formulae: (II) O R H-Tyr-D/L-Ala-Gly-Phe-D/L-Ala-R

1 S R O 0061 wherein R is OH or NH, lanthionine-Somatost O R atins SEQ ID NO: 2 to SEQ ID NO: 11) having the general formula

H ---R O (III) H-Xxx-Ala-Phe-D-Trp-Lys-Yyy-Ala-Zzz 0.045. In other embodiments, the regular peptide structure may be modified in accordance with the formula: 0062 wherein XXX=D-Phe, D-E-Nal or Phe; Yyy=Thr, R R O Val; ZZZ=TrpNH, ThrNH or Thr(oil) with the proviso that -NH-CH-X-CH-C- XXX is not Phe when ZZZ is Thr(oil) and Yyy is Thr. 0063 Lanthionine-vasopressin SEQ ID NO: 12 to SEQ 0046 where X is ID NO: 14 having the formula 0047 CH-S methylene thioester 0048 CH-S(O) methylene sulfoxide (IV) H-Ala-Tyr-Aaa-Gln-Asn-Ala-Pro-Bbb-Gly-NH2 0049 CH-O methylene ether 0050 C(S)-NH thioamide) 0051) CO-CH ketomethylene 0064 wherein Aaa=Phe and Bbb=Arg, Leu or Lys 0.052 CH-CH carba lanthionine-oxytocin SEQ ID NO: 13 to SEQ ID NO: 15 0053 NH-NHaza 0054 CH-NH methylene amino (V) 0055. In a preferred embodiment of the present invention H-Ala-Tyr-Aaa-Gln-Asn-Ala-Pro-Bbb-Gly-NH2 the moiety R of the peptides according to formula (I) comprises a Sequence of from 2 to 4 amino acids. 0056 Preferably the amino acids of the residues R and R are selected from the group of amino acids comprising 0065 wherein Aaa=lie and Bbb=Leu or Arg. D-Phe, D-B-Nal, Tyr, TrpNH2, ThrNH2, Thr(oil). Alterna tively the substituents R can be H, acyl or aracyl with 1 to 0066 Further preferred peptides of the present invention 18, preferably 2-12 carbon atoms and R can have the have the formula (VI) meaning of -OH or -NHand O-C-R3 can be replaced by CH-OH. 0057 Moreover R and R respectively can be a short (VI) amino acid Sequence of Pro-Arg-Gly or Pro-Leu-Gly. H-DPhe-Ala-Tyr-DTrp-Lys-Val-Ala-Trp-NH 0.058. In preferred embodiments of the present invention R is represented by a short amino acid Sequence Selected from the group consisting of Gly-Phe; Phe-D-Trp-Lys-Thr; Phe-D-Trp-Lys-Val; Tyr-Phe-Gln-ASn, Tyr-le-GIn-ASn, 0067 or the formula (VII) Tyr-D-Trp-Lys-Val, Gly-Asn-Leu-Ser-Thr, Ser-Asn-Leu Ser-Thr or Glu-Lys-Asp-Met-Leu-Ser-Ser. 0059 Although the naturally occurring biologically (VII) active molecules contain mainly the L-enantiomers of the H-D|Nal-Ala-Tyr-DTrp-Lys-Val-Ala-Thr-NH amino acids, in one preferred embodiment of the present invention at least one of the amino acid forming the lanthionine bridge is the D-enantiomer. US 2002/0165132 A1 Nov. 7, 2002

0068. Further preferred peptides SEQID NO: 16 to SEQ because the peptides will tend to be degraded by the ID NO: 21) have the general formula (VIII) enzymes of the alimentary tract. 0075. In respect to pharmaceutical compositions contain (VIII) ing the peptides disclosed herein, carrier and other ingredi ents should be Such that as not to diminish the therapeutic Rs-Ala-Ggg-Asn-Leu-Ser-Thr-Ala-Ro effects of the peptides. 0076. The pharmaceutical compositions of this invention thus will generally comprise an effective amount of the 0069 wherein R8 is H, acyl or aracyl, R9 is the fragment peptide in a Suitable pharmaceutically acceptable carrier 8-32 of human, Salmon or eel-calcitonin and Gigg is Gly or Such as water or alcohols which may contain fillers, Stabi Ser. lizers, Wetting agents, emulsifying agents and dispersing 0070. In another preferred embodiment of the present agents to name but a few conventional carriers, adjuvant and invention the peptide has the amino acid Sequence of endot excipients which may be employed. helin (Schematic Structure Asee below) wherein one or two of the naturally occurring disulfide bridges are replaced by 0077. The injectible compositions are formulated as a thioether bond. Therefore the compounds can be shown as shown in the art and may contain appropriate dispersing or described by the schematic structures BSEQ ID NO: 22), wetting agents and Suspending agents identical or similar to CSEQ ID NO: 23 and D SEQ ID NO: 24). those mentioned above. 0078 Depending on the nature of the biologically active A. peptide they can be used in injection Solutions, capsules, End o the lin tablets, ointments, creams, sprays and Suppositories. ls-s 0079 A representative example of the peptides of the S-S present invention can be produced according to the follow B ing procedure described for the enkephalin SEQ ID NO: 1 End o the lin -s- S-S C -s- End o the lin H-Tyr-D-Ala GlyPheAla-NH2

sS D 0080. The linear peptide chain was assembled on a meth End o the lin ylbenzhydrylamine resin using tert-butoxycarbonyl chemis try with the Symmetrical anhydride peptide coupling method. A Serine residue was preferably incorporated at L position 2 and later on converted to using disuccinimido carbonate. The S-protecting group (fluorenyl methyl) was selectively removed with piperidine. Schematic Structures 0081. A slightly basic milieu, preferably 5% piperidine/ 0071. The preferred peptides of the present invention will dimethylformamide, promoted the Michael addition of the have Sequentially overlapping thioether bonds as shown SH-group to the double bond. The amino acid analysis above, if the peptide has two thioether linkages. This means showed 48% conversion of Serine. A greater excess of the that in the amino acid Sequence one lanthionine-bridge is reagent disuccinimido carbonate would have resulted in an located between two Ala residues forming the Second increased yield for these two steps. “Low-high HF cleavage” lanthionine-bridge. was used to cleave the peptide from the resin and to remove the protecting groups. Purification of the resultant crude 0.072 The peptides of the present invention can be used product was achieved by preparative RP-HPLC using a as pharmaceutically active compounds. They can therefore gradient acetonitrile-water elution. The material obtained be used in pharmaceutical compositions comprising at least was further purified and desalted by gel filtration on a one of the peptides of the present invention. Sephadex G-15 column (20% acetic acid/water). 0.073 Pharmaceutical formulations of the peptides com 0082 The product was identified by amino acid analysis prising the peptides of this invention and a pharmaceutically and mass Spectrometry. Although the Michael addition is not acceptable carrier are part of this invention. AS used herein, stereoselective, this reaction resulted in only the (2D, 5L) the term “pharmaceutically acceptable' carrier encompasses diastereomer. The other diastereomer expected, (2L, 5L) any of the Standard pharmaceutical carriers, Such as phos could not be detected in the reaction mixture. Steric hin phate buffered Saline Solution, water, emulsions, Such as drance from the Solid Support near the SH group may be oil/water emulsions, and various types of wetting agents. responsible for the Stereoselectivity of the addition reaction. 0.074 The compounds may be administered intrave The Solid phase Synthetic approach allows a rapid assembly nously and parenterally using well known pharmaceutical of lanthionine-bridged cyclopeptides. FIG. 1 shows sche carriers or inert diluents. Oral administration is not preferred matically the process according to the invention. US 2002/0165132 A1 Nov. 7, 2002

Abbreviations Used in Peptide Synthesis Section Synthetic process. The Simultaneous application of the ben 0.083 Standard abbreviations for amino acids and pro Zyloxycarbonyl, t-butyloxycarbonyl and phenacyl (or tecting groups are followed according to the IUPAC-IUB methyl, trimethylsilylethyl, etc.) groups defines the Synthetic Joint Commission on Biochemical Nomenclature: J. Biol. Strategy of this invention to prepare Chem. 1971, 247,977. 0084 Abbreviations used: Acm, amidocarboxymethyl; Boc, ter-butoxycarbonyl; Bzl, benzyl; DCC, N,N'-dicyclo -s- hexylcarbodiimide; DCM, dichloromethane; Dha, dehy Z-Ala (Boc-Ala-OPac)-OH. droalanine; DMF, dimethylformamide; DIEA, N,N-diiso propylethylamine; DSC, disuccinimido carbonate; EtOAc, ethyl acetate; Fm, fluorenylmethyl; Fmoc, fluorenylmethy 0088 A new application of the PCOR method (Peptide loxycarbonyl; HOBt, 1-hydroxybenztriazole, Ala, Cyclization on an Oxime Resin) can provide the new cyclic lanthionine; MBHA, methylbenzhydrylamine resin; Pac, phenylacyl; TFA, trifluoroacetic acid; Tmse, trimethylsilyl Segment containing a defined lanthionine bridge (Scheme 2), ethyl; Z, benzyloxycarbonyl; NCA, N-carboxyanhydrid; Trt, where the chain can be elongated at both termini. The trityl. process without a lanthionine bridge has been described in more detail by Osapay et al. in J. Am. Chem. Soc. 1990, 112, Experimental Procedures p. 6046-6051 and Tet. Lett. 1990, 31, p. 6121-6124. The 0085 All amino acids were of the L-configuration except final deprotection Step followed by chromatographic purifi as indicated. Protected amino acids were purchased from cation yields the desired compounds.

Scheme 1. Synthesis of Protected Lanthionine from Dehydroalanine

1) CHSCOCHBr + TEA Z-Ser-OH --- Z-Dha-OPac 2) DSC+TEA 1) HOCH2CH2Si(CH3)3 + DCC Her Boc-Cys-OTmse 2) Hg(OAc)2; 3) H2S

Z-(DL)-Ala-OPac S Boc-Ala-OTmse

Bachem, Inc. ACS grade solvents (DCM, DMF, acetonitrile) were purchased from Fisher Scientific and purged with Scheme 2. Synthesis of Lanthionine-sandostatin Utilizing nitrogen, then Stored over molecular Sieves from Sigma. Cyclization on an Oxime Resin DIEA (Aldrich) was dried over KOH and distilled from Boc-Thr(BzI)-O-N=C-Resin ninhydrin. MBHA resin.HCl (Calbiochem) was swollen in DCM and washed with 5% DIEA/DCM followed by DCM 1) Chain elongation by BOP couplings | 2) TFA/DCM; 3) DIEA before use. TFA, piperidine, DSC (Aldrich) and DCC (Fluka) were used without further purification. Silica gel for zip-repre-scroll-o-Y= C-Resin flash chromatography was purchased from Baker. S 0.086 Peptides were analyzed on precoated silica gel H-Ala-OPac 60F-254 plates (Merck) using (A) chloroform:methanol:ace tic acid, 65:35:1; (B) butanol:acetic acid: water, 4:1:5-upper phase. Compounds were visualized by UV, ninhydrin, chlo Z-Ala-Phe-D-Trp.(For)-Lys(Tos)-Thr(Bzl)-Ala-OPac rine/o-tolidine and KMnO, solution. RP-HPLC analyses S were performed on a Waters (Model 510 and Waters 484 1) HBr/AcOH 3) Zn/AcOH detector) instrument with a C-18 analytical column. pages 2) Z-D-Phe-OH/HOBt + DCC 4) Threoninol/HOBt + DCC 11-17 are missing from the electronic version of the Speci fication 0087. There is, however, another synthetic method for the production of peptides. It is often desirable to have diaste Deprotection reomeric peptide analogs. The use of diastereomeric mixture D-Phe-Ala-Phe-D-Trp-Lys-Thr-Ala-Thr(oil) of lanthionine units can provide appropriate diastereomeric analogs, separable by chromatography (HPLC). By Such routes, two (or four) analogs can be prepared by a single US 2002/0165132 A1 Nov. 7, 2002

Scheme 2B. Synthesis of Lanthionine-SRIF(1-14 Boc-Ser(Bzl)-O-N=C < Oxime resin Peptide chain elongation by BOP couplings BocLys(Tos)AsnPhePheTrp(For)Lys(Tos)Thr(Bzl)PheThr(Bzl)Ser(Bz I -O-N=C < Oxime resin 2 1) TFA/DCM -NH- ti-cool Fmoc-NH- ti-cool 2) BOP +3 CH-S-CH 3 Facily (Tos)AsnPhePheTrp(For)Lys(Tos)Thr(Bzl)PheThr(Bzl)Ser(Bzl -O-NFC < Oxime resin 4 S 1) TFA/DCM 2) DIEA + AcOH BocAla OMe FmocAla Lys(Tos)AsnPhePheTrp(For)Lys(Tos)Thr(Bzl)PheThr(Bzl)Ser(Bzl-O-N=C < oxime resin

COOMe FmocAla Lys(Tos)AsnPhePheTrp(For)Lys(Tos)Thr(Bzl)PheThr(Bzl)Ser(Bzl)is Ser(Bzl)AlaLOMe 5

2) CbzAlaGlyOH (6) + EDC/HOBt CbzAlaGlyAla Lys(Tos)AsnPhePheTrp(For)Lys(Tos)Thr(Bzl)PheThr(Bzl)Ser(Bzl) Ser(Bzl)Ala-OMe 7 is 1) NaOH/HMPA HO

H-AlaGlyAlaLLysAsnPhePheTrpLysThrPheThrSerAlaL-OH 1.

0089 Another highly promising pathway involves the 0090 The B-lactone method is shown in FIG. 2 wherein Synthesis of two protected intermediates, followed by cou also the obtainable yields are given. pling of the two components in generating an optically pure 0091. Furthermore another route for the synthesis of the lanthionine. This is proceeded by the synthesis of both the protected lanthionine is disclosed where reactions proceed protected Serine ?-lactone (Arnold et al. J. Am. Chemn. Soc. with retention of configuration. This lanthionine derivative 1988, 110, p. 2237-2241) and the protected cysteine. The is prepared through the ring opening of an aziridine deriva latter compound acts as a nucleophile in opening the lactone tive (Wakamiya et al., Bull. Chem. Soc. Jpn., 1982, 55, ring at the Site of the methylene group (see Scheme 3). 3878-3881) by a nucleophile, namely cysteine or any appro priate SH-containing amino acid (Scheme 4).

Scheme 3. Synthesis of Protected Lanthionine from Serinelactone Scheme 4. Synthesis of Protected Lanthionine from AZiridine Derivatives DMAD Boc-Ser-OH 1. CHSCOCH2Br; 2. TFA; 3. TrtBr/TEA Z-Ser-OH - I 3 - C.H.CH-CO-NH- t to 4. Tosyl chloride/Pyridine; 5. TEA CH-O Trt-N-CH-COOPac Boc-Cys(Acm)-OH 1) CHSCOCHBr + TEA Boc-Cys-OPac V / 2) Hg(OAc); 3) H.S CH2 Z-CI Boc-Cys-OMe - NaHCO - Z-N-CH-COOPacV / --> Z-Ala-OPac CH2 S Z-Ala-OH S Boc-Ala-OMe Boc-Ala-OPac 0092. The Aziridine method is shown in FIG. 3 wherein also the yields obtainable by Said method are given. US 2002/0165132 A1 Nov. 7, 2002

0.093 Preparation of Lanthionine-opioids 0094. As will be shown later, all of the lanthionine -continued opioids Synthesized are Superactive both at the u- and SEQID NO: 16-21) Ö-receptor. To investigate Structural or pharmacochemical aspects of this new class of opioids, analogs can be Synthe AlaGlyAlaLLysAsnPhePheTrpLysThrPheThrSerAlaL sized in order to carry out bioassays and conformational analyses of the resulting molecules. Various peptidic or peptidomimetic units can be incorporated into cyclic Lanthionine-somatostatin enkephalin and dermorphin-deltorphin Structures including: —s— 0101 Lanthionine-calcitonins Tyr-D-Ala-Gly-Phe-Ala-X, 0102) It is possible to incorporate the lanthionine as the —s replacement of the cysteine-cysteine disulfide bridge in the Tyr-D-Ala. Phe-Ala-X and N-terminal loop. The loop can be prepared via the PCOR method (Scheme 5). Tyr-D-Ala-Phe-Asp-Ala-X (X = NH or OH). Ala-Aaa-Asn-Leu-Ser-Thr-Ala.

0.095 The incorporation of methyl group(s) at the B-car 0.103 Human and rat calcitonins: Aaa=Gly bon(s) and effects of chirality at the two main chain units of the lanthionine residue can also be included. 0104 Salmon and eel calcitonins: Aaa=Ser 0096. Thus the synthesis of B-methyl lanthionines and B.f3-dimethyl lanthionine results in modifications, which are Loops of Lanthionine-calcitonin expected to lead to Substantial differences in bioactivity profiles for closely related target molecules. Thus, critical 01.05 information about the “bioactive conformations” of the analogs can be obtained. In addition, Specific residues Such Scheme 5. Synthesis of Loops for Lanthionine-calcitonins as the Gly in Utilizing Cyclization on an Oxime Resin the Tyr-D-Ala-Gly-Phe-Ala-X Boc-Thr(BzI)-O-N=C-Resin —s— 1) Chain elongation by BOP spiel TFA/DCM; 3) DIEA O097 and the Asp of S H-Ala-OPac Cyclization Tyr-D-Ala-Phe-Asp-Ala-X

0.098 can be modified with natural and unnatural amino acids. This family of opioids are most promising for obtain ing novel and clinically useful opioid drugs. Deprotection 0099 Lanthionine-Somatostatins AlaL-Aaa-Asn-Leu-Ser-Thr-AlaL 0100 New lanthionine-Somatostatin derivatives can be —s— Synthesized. First, the cyclic Segment with a monoSulfide Human and rat calcitonins: Aaa = Gly bridge of Somatostatin or "key-hexapeptide' (Scheme 2) or Salmon and eel calcitonins: Aaa = Ser other analogs of Somatostatin according to the definition of R on a Kaiser-oxime resin has to be prepared. It will be elongated at both termini (D-Phe at the N-terminus and threoninol at the C-terminus) to obtain for example the 0106 The elongation at the C-terminus to get the final lanthionine analog of Sandostatin. The Same Synthetic Strat calcitonin-analog can be performed by normal classical egy can be used for the preparation of the lanthionine analog fragment condensations or by the Strategy shown later in the of the native Somatostatin tetradecapeptide. Potency and paragraph of lanthionine-oxytocin and -vasopressin Synthe receptor Selectivity of both target molecules are promising. sis (Scheme 6). 0107 Lanthionine-oxytocins and Lanthionine-vaso SEQ ID NO: 2) pressins D-Phe-Ala-Phe-D-Trp-Lys-Thr-Ala-Thr(oil) 0108. The incorporation of a lanthionine bridge to replace the existing disulfide bridge found in natural oxytocin (OT) and vasopressin (VP) is another example. This can be Lanthionine-sandostatin accomplished by the Synthesis of the lanthionine component prior to its incorporation in the peptide Sequence (Scheme 6). US 2002/0165132 A1 Nov. 7, 2002

SEQ ID NO: 15 1. 2 3 4 5 6 7 8 H-Ala-Tyr-Ile-Gln-Asn-Ala Pro-Leu-Gly-NH- lanthionine-oxytocin

SEQID NO: 12-14) 1. 2 3 4 5 6 7 8 H-Ala-Tyr-Phe-Gln-Asn-Ala-Pro-Lys-Gly-NH

lanthionine-vasopressin 1. 2 3 4 5 6 7 8 H-Ala-Tyr-Phe-Gln-Asn-Ala Pro-Arg-Gly-NH

0109 The following examples illustrate the present invention without limiting the Scope of the invention thereto. Scheme 6. Synthesis of Lanthionine-oxytocins/Lanthionine-Vasopressins

EXAMPLE 1. 0110) a) Preparation of S Cys(Fm)-MBHA (1) Boc-Ala-OMe 1) TFA | 2) Cyclization/PCOR methodology 0111) Methyl benzhydrylamine resin (3 g) was reacted with Boc-Cys(Fm)OH (10g, 2.5 mmol) and DCC (0.52g, 2.5 mmol) in DCM (30 mL) for 3 hr at room temperature in an SPPS vessel. The remaining amino groups were capped by acetylation. The resulting Boc-Cys(Fm)-MBHA resin (substitution level 0.36 mmol/g, based on picric acid titra his tion) was then deprotected with 30% TEA/DCM (v/v) and neutralized with 1% DIEA/DCM (v/v) solution. The peptide chain was then assembled by consecutive addition of the cool symmetrical anhydrides (2.5 equiv.) of BocFhe0H, BocG lyOH, BocSerOH, and ZTyr(Bzl)OH as well as deprotection Steps. The completeness of coupling was monitored by the HO-NEC-Resin Kaiser test. Coupling of ZTyr(Bzl)OH was repeated with 1 Peptide Chain Elongation molar equivalent reagent. Yield 1.06 mmol (84%) peptide |eason. DCC Boc-strategy based O Gly; amino acid analysis, Boc-Asn-O-NFC-Resin CyS(1)Gly1.ooPheososer 1.42Tyr 122. MBHA Resin 0112 b) Preparation of |colson. DCC Boc-Gly-MBHA-Resin (3) Peptide Chain Electics Z-Tyr(Bzl)-c(D-Ala-Gly-Phe-Ala)-MBHA Boc-Pro-Bbb-Gly-MBHA-Resin s— 1) TFA; 2) HOBI/DCC 0113. The protected peptidyl MBHA resin (1, 1.06 mmol) in the SPPS vessel was swollen and then suspended in DCM Z-cAlal-Tyr(Bzl)-Aaa-Gln-Asn-Ala-Pro-Bbb-Gly-MBHA-Resi (20 mL). A solution of DSC (387 mg, 1.51 mmol) in acetonitrile (10 mL) was added to the reaction mixture —s— followed by a 5% DIEA/DCM solution (5.22 mL, 1.5 mmol 1) t HPLC DIEA). The reaction was allowed to proceed for 4 hr, Shaking at room temperature in a nitrogen atmosphere. The cAlal-Tyr-Aaa-Gln-Asn-Ala-Pro-Bbb-Gly-NH2 reaction mixture was drained and the Solid phase was s— washed with DCM (4x). The product (2) was treated with a I Oxytocin: Aaa = Ile; Bbb = Leu solution of 20% piperidine/DMF solution (20 mL, V/V, 2x50 II Lysine-Vasopressin: Aaa = Phe; Bbb = Lys(2C-Z) min.) and shaken in a 5% piperidine/DMF-DCM solution III Arginine-Vasopressin: Aaa = Phe; Bbb = Arg(NO2) (40 mL, 1/1, V/v) overnight. The solution phase was drained and the resin was washed with DMF (1x), DCM (2x) and EtOH (2x) and dried. Yield 3.7 g. US 2002/0165132 A1 Nov. 7, 2002

0114 c) Preparation of 0118. The coupling was performed as follows: #1:3 eq. 20 min; #2:1 eq+DIEA 20 min (3) 0119 (a) Fmoc-Phe-NCA, H-Tyr-c(D-Ala-Gly-Phe-Ala)-NH2 0120) (b) Fmoc-Gly-NCA, 0121 (c) Fmoc-Ser-OH/DCC, 0115 The peptidyl resin (3, 1.0 g) was treated with 0122) (d) Fmoc-Tyr(Bzl)-NCA. anhydrous HF (20 mL) at 00C in the presence of anisole (1 0123. In the case of peptide chain elongation by the mL) for 1 hr in a teflon HF apparatus. After removal of Fmoc-strategy the -SH group of cystein was blocked with Volatile components the remaining material was washed a Trt group. with EtOAc (2x20 mL) and the product was extracted with acetic acid followed by 10% acetic acid/water solution. The EXAMPLE 4 combined extracts were freeze-dried (yield 200 mg). This 0.124. The preparation of lanthionine-bridged cyclic pep material was purified by preparative RP-HPLC on a Vydac tide fragments is demonstrated by the following prepara C-18 column (1.0x25 cm) eluted with 0.1% TFA in aceto tions: nitrile/water. A linear gradient from 15% to 22% acetonitrile over 12 min with a flow rate of 10 mL/min was employed. The appropriate fractions were lyophilized to give a Solid product (yield 87 mg). Finally, 30 mg of the product was a) Subjected to gel permeation chromatography (1.5x100 cm Z-D-Ala Phe-D-Ala-Opac Sephadex G-15 eluted with 20% acetic acid). The peptide fractions were pooled and lyophilized. Yield 16 mg (24% Z-D-Ala (Boc-D-Ala-Opac)-Phe-O-oxime calculated for compound 1). RF(A) 0.44; RF(B) 0.49. FAB MS m/e=557 (M+1). Amino acid analysis GlyoAla-S- Alali. Pheo.o.oTyroos.

EXAMPLE 2 0125 resin (10g, 187umolpeptide on resin) synthesized by regular Solid phase Synthetic method was Swollen in 0116 FIG. 4 shows schematically the synthesis of DCM (10 mL) in a solid-phase peptide synthesis vessel. The lanthionine-enkephalin in Solution. Other general methods Boc group was removed with 25% TFA/DCM, shaking the for chemical Synthesis can be followed using mixed anhy reaction vessel for 30 min. The peptidyl resin was then drides, carbodiimides, active esters and other coupling pro drained and washed (10 mL/wash) with DCM (2x), i-ProH cedures. Preferred solvents are CH2Cl and DMF. Cleavages (1x), DCM (2x), i-ProH (1x), and DCM (2x). The amino are following Standard Selective reactions. Purification fol group was neutralized by treating the peptidyl resin with 5% lows well-known extractions, precipitations and chromato DIEA in DCM (2x1 min.) and then washing with DCM (2x). graphic methods. The cyclization reaction was then carried out by Shaking the peptidyl resin in DCM/DMF (1:1, v/v, 10 mL) in the EXAMPLE 3 presence of 10 equiv. AcOH at RT for 72 h. The cyclic 0117 The lanthionine-enkephalin was also synthesized peptide product was collected from the reaction vessel by by the preferred Fmoc-NCA-Strategy by using the following draining and then washing the resin with DMF (3x). These Steps: Solutions were combined and evaporated to a reduced Vol ume, and then washed with water, 0.1 NHCl, 5% NaHCO, and brine. The Solvent was then evaporated and the crude product was purified by Silica gel flash chromatography (1) Deprotection (20% piperidine/DMF) 7 min 30 mL/min (2x20 cm, ethyl acetate-hexanes 1/1). The appropriate frac (2) Wash (DMF) 5 min 30 mL/min tions were pooled and the Solvent was evaporated. The pure (3) Coupling (see below) 20 min 30 mL/min (4) Wash (DMF) 5 min 30 mL/min solidified product was recrystallized from methanol/ether. (5) Repeat steps 1-4 Yield 40.5 mg (36.7%); mp 241-244 C. (decomp); R(E- tOAc/hexanes=2/1) 0.42; FAB-MS m/e=590 (MH"); theo retical 590.

b)

Z-L-Alai Phe-D-Trp.(For)

Z-P-Ala (Boc D-Ala Opac)-Phe-D-Trp(For) Lys(2C-Z)- US 2002/0165132 A1 Nov. 7, 2002

0126 Thr(Bzl)-O-oxime resin (100 mg., 6.0 umolpeptide wire entirely enclosed within the lumen. The other end on resin) Synthesized by regular Solid phase Synthetic of the preparation is tied over a piece of Stiff polyeth method was swollen in DCM (1.0 mL) in a solid-phase ylene tubing (4 cm, 2.5 mm O.D) which projects out of peptide Synthesis vessel. The Boc group was removed with the bath Solution and is tied to the Strain gauge. Another 25%TFA/DCM, shaking the reaction vessel for 30 min. The GRASSE 2B electrode is placed about 5 mm from the peptidyl resin was then drained and washed (1.0 mL/wash) intestine and parallel to it to achieve transmural Stimu with DCM (2x), i-ProH (1x), DCM (2x), i-ProH (1x), and lation. Single pulses of 4 msec during are delivered by DCM (2x). The amino group was neutralized by treating the a Harvard apparatus Stimulator at a frequency of 10 peptidyl resin with 2.5% DIEA in DCM (2x1 min.) and then min-1. Voltages in the range from 3 to 6 V are applied washing with DCM (2x). The cyclization reaction was then in order to obtain maximal response. Isometric con carried out by shaking the peptidyl resin in DCM/DMF (1:2, tractions of the ileum are recorded via a Harvard V/V, 1.0 mL) in the presence of 10 equiv. AcOH at RT for 72 isometric force transducer on a Harvard apparatus h. The cyclic peptide product was collected from the reac biograph which has been calibrated to produce a pen tion vessel by draining and then washing the resin with DMF displacement of 1 cm per tension change of 1 g. The (3x). These Solutions were combined and evaporated and the results are Standardized by expressing the reduction in crude product was purified by RP-HPLC on a Vydac C-18 tension obtained at each dose level as a percentage of column (1.0x25 cm) using 0.1% TFA in acetonitrile water. the mean tension produced by at least ten preceding A linear gradient from 50 to 80% acetonitrile over 15 min., control Stimulations. Semilogarithmic plots of percent with a flow rate of 4 mL/min., was employed. The product inhibition as a function of peptide concentration permit was eluted at 61% acetonitrile and lyophilized to give a solid the determination of IC50-values which are taken as the product. Yield 0.9 mg (24%); R(CHCl/MeOH/AcOH=18/ intercept of 50% inhibition. 1.5/1) 0.54; FAB-MS m/e=1,293 (MH"); theoretical 1,293. 0131 (2) The MVD (mouse vas deference) assay was EXAMPLE 5 performed essentially as described by Henderson. 0127 Comparative examples showing the Superior bio Briefly, adult, male albino mice (Swiss Webster 30-50 logical activity of compounds with the thioether bond com g) are killed by cervical dislocation and the vas defer pound compared with the compound having the disulfide entia are dissected out. After removal of extraneous fat bridge: and connective tissue, the Vas is Stripped of its associ ated blood vessel and the Somen is gently expressed 0128 Bioassays. Using Isolated Organ Preparations from the lumen. The vas is then mounted under 0.5 g. tension in a 5 ml organ bath containing warmed (37 0129. All of these assays represent standard procedures C.), oxygenated (95% O2, 5% CO2%)), Mg2+-free which have been well described in the literature. Krebs solutions of the following composition mM): 0130 (1) The GPI (guinea pig ileum) assay will be NaCl, 118; CaCl2, 2.54; KC1, 4.75; KH2PO4, 1.19; performed according to a modified version of a proce NaHCO3, 25; glucose, 11; L-tyrosine, 0.2. A modified dure first developed by Paton. Male guinea pigs (300 Harvard apparatus Stimulator is used to deliver repeti 450 g) are killed by a blow on the skull and exsan tive field Stimulation through platinum wire ring elec guinated. A 2-3 cm Segment of ileum not leSS than 10 trodes at the top and bottom of the bath, consisting of cm from the ileocecal junction is mounted in a 20 ml twin, rectangular pulses (80 V, 0.15 Hz, 10-ms delay, organ bath. The bath contains Krebs' solution of the 1.0-ms duration). Contractions of the muscle are following composition (in millimolar concentrations): recorded via a Hewlett-Packard Model FTA-I-I force NaCl, 150; KCl, 4.3; CaCl2, 1.25; MgCl2, 1.0; transducer connected to a Hewlett-Packard 7702B NaH2PO4.H2O, 1.7; NaHCO3, 25.0; glucose, 11.0. recorder. Determination of the reduction in the twitch The temperature is maintained at 370C and the solu height at various doses permits the construction of log tion is bubbled with 95% O2/% CO2. A GRASS E 2B dose-response curves and the determination of IC50 electrode is used as anode with the 1.5 cm platinum values.

TABLE 1. Inhibitory Potency and Selectivity of Lanthionine and Disulfide-Bridged Enkephalin Analogs in GPI (guinea pig ilium) and MVD (mouse was deferens) Bioassays Adequate to u and ö Receptor Binding Affinities, Respectively, and Rat Hot-plate in vivo Test GPI MVD GPI/MVD in vivo 8-Binding Structures ICs (nM) ICs (nM) ICs (nM) ICs (nM) ICs (nM) Tyr-cD-Ala-Gly-Phe-L-Ala-NH. O.62 O.54 1.13 O.11 7 Tyr-cD-Ala-Gly-Phe-D-Ala-NH. 1.67 1.67 1.O O.11 N/D Tyr-cD-Ala-Gly-Phe-L-Ala-OH O.45 O16 2.85 O.19 1. Tyr-cD-Cys-Gly-Phe-L-Cys-NH. 1.51 O.76 1.99 O.24 N/D Tyr-cD-Cys-Gly-Phe-D-Cys-NH. O.78 O.30 2.61 O.29 N/D Tyr-cD-Cys-Gly-Phe-L-Cys-OH 3.06 O.19 16.1 N/D N/D Tyr-cD-Val-Gly-Phe-L-Ala-OMe 88.1 3.65 24.1 N/D N/D Tyr-cD-Val-Gly-Phe-L-Ala-OH 39.0 0.27 143 N/D N/D Tyr-cD-Val-Gly-Phe-D-Ala-OH 535 1.51 354.3 N/D N/D Tyr-cID-Pen-Gly-Phe-D-Pen-OH 73OO 4.1 18OO 130 78 US 2002/0165132 A1 Nov. 7, 2002

TABLE 1-continued Inhibitory Potency and Selectivity of Lanthionine and Disulfide-Bridged Enkephalin Analogs in GPI (guinea pig ilium) and MVD (mouse was deferens) Bioassays Adequate to u and ö Receptor Binding Affinities, Respectively, and Rat Hot-plate in vivo Test

GPI MVD GPI/MVD in vivo 8-Binding Structures ICs (nM) ICs (nM) ICs (nM) ICs (nM) ICs (nM) Tyr-Gly-Gly-Phe-Leu 246 11.4 21.6 Morphine 58.6 644.O O.09 replacements of 3H-naltrindole; '40% of the maximum activity at 180 nmol dosage 0132) Table 1 shows that the compound according to the invention which has a thioether bond is in both methods, TABLE 3 particularly in the most relevant GPI test more potent than Potencies of Lanthionine and Disulfide-bridged Somatostatin Analogs the corresponding -S-S- compound. to Inhibit Radioligand Binding to Cloned somatostatin Receptors SSTR2 and SSTR4* EXAMPLE 6 Binding Constants 0133). Using protocols described by Schiller et al., Bio chem. Biophys. Res. Commun. 1983, 115, p. 864-870, we SSTR2 SSTR4 compared the half-lives of three compounds: Leu-enkepha Structures Receptor Receptor H-D-Phe-cAla Phe-D-Trp-Lys-Thr-Ala-OH O 500 lin. AS indicated in Table 2, the lanthionine-enkephalin is H-D-Phe-cAla Phe-D-Trp-Lys-Thr-Ala-Thr-ol S.OO 1.OO much more Stable than the other two compounds. H-D-Phe-cAla Phe-D-Trp-Lys-Thr-Ala-Thr- 1.O.O OSO NH Sandostatin TABLE 2 H-D-Phe-cAla Phe-D-Trp-Lys-Thr-Ala-Thr-ol 2.4 0.57 cAla 3, Ala 14-Somatostatin-14 O.3 Enzymatic Degradation of Enkephalin Analogs Somatostatin-14 O.28 O.86 *measured according to K. Raynor et al. (1993) Mol. Pharmacol. 43, Analog t12 (min) 838–844, and 44, 385-392 Lanthionine-enkephalin 1223 Disulfide-enkephalin 332 0.136 Although the lanthionine-enkephalins according to Leu-enkephalin 3O the invention possess Superactivity, they do not seem to have high receptor Selectivity. To imporoive thier Selectivity, it is possible to introduce one or more alkyl (methy) group(S) in f position(s) of the lanthionine segment. EXAMPLE 7 0137 In this case at least one of R, R., R., and Rs may be alkyl (methyl). 0134) The lanthionine opioid is highly active in the in 0.138. The two lanthionine octapeptides in Scheme 3 are vitro and in vivo tests (Table 3). In vivo bioactivity was highly potent on both SSTR2 and SSTR4 receptors. They determined using the rat hot plate test after intrathecal have similar affinities to SSTR4 as Sandostatin but their dosages. affinities for SSTR2 are slightly decreased. By comparing the ICso values for SSTR2/SSTR4 receptors, the disulfide and monoSulfide bridged octapeptide alcohols are practi cally the Same. On the contrary, the octapeptide amide has a four times higher selectivity for SSTR4 because its affinity Tyr-cID-Ala-Gly-Phe-Ala-NH2 to SSTR2 is decreased. The lanthionine analog of SRIF 14 shows the same binding affinity to SSTR2 as the natural disulfide bridged peptide. 0135 shows 37 times higher bioactivity than morphine 0139 Preparation A and twice the activity of DCLCE (Table 3). In the same tests, 0140 Preparation of Lanthionine Building Blocks for Leu-enkephalin shows only 40-50% of full agonistic Syntheses of Lanthionine Peptides activity after 100 pg dosage in the in vitro assays using GPI 0141 1) Z-Ala (Boc-Ala-OPac)-OH and MVD preparations. The lanthionine opioid exhibits 400 0142 Benzyloxycarbonyl-serine lactone (1.1 g, 5 mmol) times greater bioactivity at the GPI (u-receptor) and 20 times and t-butyloxycarbonyl-cysteine phenacyl ester (2.04 g, 6 greater bioactivity at the MVD (Ö-receptor) than Leu mmol) are dissolved in DMF (25 ml) and cesium carbonate (0.97g, 3 mmol) is added to the solution. The reaction enkephalin. These values are higher than those of its disul mixture is stirred at room temperature for one hour in fide bridged counterpart, DCDCE. The lanthionine analog nitrogen atmosphere, the Solvent is removed under reduced does not show a preference for the u- or the Ö-receptor. The pressure. The residue is dissolved in a mixture of 10% ICso ratio (MVD/GPI) is 0.9. NaHSO, and ethyl acetate. The organic phase is washed with US 2002/0165132 A1 Nov. 7, 2002

water, dried with sodium Sulfate and the solvent is removed 0° C. for 1 hr. The product was filtered and dried. Yield: 0.92 under reduced preSSure. The crude product is purified on g, R(EtOAc/BuOH/AcOH/HO)=0.93, Z-D-Phe(OH (3.2g, Silica gel column with a Solvent System of chloroform 3.29 mmol) and HOBt.HO (0.89 g, 6.58 mmol) were methanol (10/1, V/v). The fractions containing the product dissolved in DMF (5 mL), cooled to 0° C. and EDC 0.69 g, are combined and the Solvent removed. The product is a 3.62 mmol), DIEA (1.91 ml, 11.0 mmol) and the HBr salt of white foam. Yield: 1.46 g (52%); R(CHCl/MeOH-10/1)= the above peptide (0.91 g, 0.82 mmol) were given to the reaction mixture. After being stirred for 1 hr at 0° C. and at 0.20; FAB-MS (m/e)=561 (MH)". room temperature for overnight the product was precipitated 0143 2) Z-Ala (Tri-Ala-OMe)-OH by addition of water. The crude product was filtered, washed with 0.5 NHCl, 5% NaHCO and water, then recrystallized 0144. The compound can be prepared according to the from DMF-ether. Yield: 0.9 g (86%), RP-HPLC: R=15.3 literature: Photaki, I., Samouilidis, I., Caranikas, S. and min (same column and eluent written above; conditions: Zervas, L. J. Chem. Soc. Perkin I., (1979) 2599. linear gradient from 50 to 75% of acetonitrile during 20 0145 3) Z-Ala (BOC-Ala-OMe)-OPac min). FAB-MS: m/e 1,231 (M+H"). 0146 Benzyloxycarbonyl-aziridine phenacyl ester (111.7 0156) 3) Z-D-Phe-cAla Phe-D-TrpLys(Tos)ThrAla)- mg, 0.33 mmol) and t-butyloxycarbonyl-cysteine methyl ThrNH ester (106.1 mg, 0.45 mmol) are dissolved in DCM (2 ml). 0157 The protected heptapeptide (2,0.3.g., 0.24 mmol) in BF.EtO (2 drops) is added to the solution at room tem DMF (6 mL) was stirred with NHHO (0.6 mL) overnight perature, and the reaction Stirred under nitrogen. After five under nitrogen atmosphere. The Solvent was partly removed days, the Solvent is removed under reduced pressure. The under reduced pressure then water was added to the reaction residue is then dissolved in ethyl acetate, and washed mixture. The precipitated product was filtered and dried. sequentially with 10% NaHCO, water, and brine. Ethyl Yield: 220 mg, RP-HPLC: R=10.9 min (same conditions as acetate layer is dried over magnesium Sulfate and then described for compound 2). FAB-MS; m/e 1,204 (M+H"). removed under reduced pressure. The crude product is To a solution of this material (180 mg), 0.15 mmol) in DMF purified on a Silica gel column with a Solvent System of ethyl (2 mL) cooled to -15° C., 4N HCl/dioxane (0.225 mL) and acetate-hexane (1:2, V/v). Compound is a white powder from t-butyl nitrite (0.042 mL 0.35 mmol) were added. After ethyl acetate-hexane. Yield: 82 mg (43.2%); m.p. 93.5-94.5 being stirred for 25 min at the same temperature HC1.H- C; Rf(EtOAc/Hexane=1/2)=0.3; FAB-MS (m/e)= Thr-NH. (0.139 g, 0.9 mmol) and DIEA (0.195 mL, 1.25 575(MH)+. mmol) were added to the reaction mixture. After a further 1 0147 Found: C, 58.45; H, 5.99; N, 4.82; S, 5.46%. hr stirring at -15 C. and 48 hr at 4 C. the mixture was diluted with water. The precipitated product was filtered, 0148 Calculated: C, 58.51; H, 5.98; N, 4.88; S, 5.58%. washed with 1N HCl, 5% NaHCO and dried. The crude 0149 Preparation B material was recrystallized from DMF-ether. Yield: 192 mg, RP-HPLC: R=21.5 min (same column and eluent written 0150. Syntheses of Lanthionine-Somatostatin Analogs above; conditions: linear gradient from 50 to 100% of 0151. 1) Z-cAla Phe-D-Trp.(For)Lys(Tos)Thr(Bzl)A- acetonitrile during 25 min). FAB-MS; m/e 1,290 (M+H") la)-OMe and 1,312 (M+Na"). 0152 BocThr(Bzl)-oxime resin (5.4g, substitution level: 0158) 4) H-cAla Phe-D-TrpLysThrAla)-OH 0.26 mmol/gbased on picric acid titration) was deprotected 0159. The protected hexapeptide (1,58.6 mg, 0.05 mmol) with 25% TFA/DCM (v/v) and neutralized by 5% DIEA/ was dissolved in hexamethylphosphoramide (1.25 mL) DCM (v/v). The peptide chain was then assembled by cooled to 15° C., 0.5 N NaOH (0.5 mL) was added dropwise consecutive addition of 2.5 equiv. BOB ester of BocLys over 30 min. The reaction mixture was stirred for further 30 (ToS)OH, Boc-D-Trp.(For)OH, BocPheOH and ZAla (TrtA min at this temperature, then and diluted with water (15 mL) laOMe)OH as well as deprotection steps. After the Trt acidified to pH 2.5 with 2.5 NHCl at 0°C. After being kept group was removed with 25%TFA/DCM (V/V) the peptidyl for 1 hr at this temperature the product was filtered, dried resin was washed and neutralized according to the Standard and recrystallized from DMF-ether. Yield: 110 mg, RP Oxime resin protocol. The cyclization reaction was carried HPLC: R=13.7 min (same conditions as described for out by shaking the peptidyl resin in DMF-DCM (100 mL, compound 2). FAB-MS: m/e 1,131 (M+H"). This material 2/1 V/v) in the presence of 10 equiv. of acetic acid at room was deprotected with Sodium (23 mg) in liquid ammonia (40 temperature for 24 hr. The cyclic peptide product was mL). After removal of the ammonia the rest was dissolved obtained from the filtrate of the reaction mixture: The in water (2 mL) and the pH was adjusted to 5 with acetic Solvent was removed in reduced preSSure and the product acid. The Solution was Subjected to gel permeation chroma was precipitated from a mixture of DMF-water. tography (1.5x75 cm Sephadex G-10 eluted with 10% acetic 0153. Yield: 1.07 g (65%), R(CHCl/MeOH/AcOH)= acid) followed by RP-HPLC purification on a Vydac C-18 0.79, RP-HPLC: R=23.5 min (Vydac C-18 semipreparative Semipreparative column eluted with isocratic 22% acetoni column; eluent: acetonitrile-water mixture containing 0.1% trile/water containing 0.1% TFA. The pure peptide fractions TFA, conditions: linear gradient from 30 to 90% of aceto were pooled and lyophilized. nitrile during 30 min.). FAB-MS: m/e 1.173 (M+H"). 0160 Yield: 19 mg (51%), RP-HPLC: R=14.0 min 0154) 2) Z-D-Phe-cAla Phe-D-Trp.(For).Lys (conditions above). FAB-MS; m/e 753 (M+H"). (ToS)ThrAla)-OMe 0161 5) H-D-Phe-cAla Phe-D-TrpLysThrAla)- 0155 The protected hexapeptide (1, 1.0 g, 0.85 mmol) ThrNH, was partially deprotected with 33% HBr/AcOH (6 mL, V/v) 0162 The protected octapeptide (3,100 mg, 0.077 mmol) at 0°C. for 10 min and at room temperature for 50 min. Abs. was deprotected by Sodium (46 mg) in liquid ammonia (75 ether (50 mL) was given to the reaction mixture and kept at mL). After removal of the ammonia the residue was desalted US 2002/0165132 A1 Nov. 7, 2002 13 by gel permeation chromatography (1.5x75 cm Sephadex purification on a Vydac C-18 Semipreparative column. The G-10 eluted with 10% acetic acid) followed by RP-HPLC pure peptide fractions were pooled and lyophilized.

SEQUENCE LISTING

NUMBER OF SEQ ID NOS: 24 SEQ ID NO 1 LENGTH 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Cys-Ser lanthionine bridge <400 SEQUENCE: 1 Tyr Cys Gly Phe Ser 1 5

SEQ ID NO 2 LENGTH 8 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Cys-Ser lanthionine bridge <400 SEQUENCE: 2 Phe Cys Phe Trp Llys Thr Ser Thr 1 5

SEO ID NO 3 LENGTH 10 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Cys-Ser lanthionine bridge NAME/KEY: SITE LOCATION: (1) . . (8) OTHER INFORMATION: Xaa-D-Phe, D-beta-Nal, or Phe <400 SEQUENCE: 3 Xaa Ala Ala Cys Phe Trp Llys Thr Ser Thr 1 5 10

SEQ ID NO 4 LENGTH 10 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Cys-Ser lanthionine bridge NAME/KEY: SITE LOCATION: (1) . . (8) OTHER INFORMATION: Xaa- D-Phe, D-beta-Nal, or Phe <400 SEQUENCE: 4 Xaa Ala Ala Cys Phe Trp Llys Thr Ser Thr 1 5 10

SEQ ID NO 5 LENGTH 8 TYPE PRT ORGANISM: Artificial Sequence FEATURE OTHER INFORMATION: Cys-Ser lanthionine bridge

<400 SEQUENCE: 5 US 2002/0165132 A1 Nov. 7, 2002 14

-continued Phe Cys Phe Trp Lys Thr Ser Trp 1 5

<210> SEQ ID NO 6 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <221 NAME/KEY: SITE <222> LOCATION: (1) . . (8) <223> OTHER INFORMATION: Xaa- D-Phe, D-beta-Nal, or Phe <400 SEQUENCE: 6 Xaa Ala Ala Cys Phe Trp Lys Thr Ser Trp 1 5 10

<210 SEQ ID NO 7 &2 11s LENGTH 8 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <400 SEQUENCE: 7 Phe Cys Phe Trp Lys Val Ser Trp 1 5

<210 SEQ ID NO 8 &211's LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <221 NAME/KEY: SITE <222> LOCATION: (1) . . (8) <223> OTHER INFORMATION: Xaa-D-Phe, D-beta-Nal or Phe <400 SEQUENCE: 8 Xaa Ala Ala Cys Phe Trp Lys Val Ser Trp 1 5 10

<210 SEQ ID NO 9 &2 11s LENGTH 8 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <400 SEQUENCE: 9 Phe Cys Phe Trp Lys Val Ser Thr 1 5

<210> SEQ ID NO 10 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <221 NAME/KEY: SITE <222> LOCATION: (1) . . (8) <223> OTHER INFORMATION: Xaa- D-Phe, D-beta-Nal, or Phe <400 SEQUENCE: 10 Xaa Ala Ala Cys Phe Trp Lys Val Ser Thr 1 5 10 US 2002/0165132 A1 Nov. 7, 2002 15

-continued

<210> SEQ ID NO 11 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <221 NAME/KEY: SITE <222> LOCATION: (1) . . (8) <223> OTHER INFORMATION: Xaa- D-Phe, D-beta-Nal, or Phe <400 SEQUENCE: 11 Xaa Ala Ala Cys Phe Trp Lys Val Ser Thr 1 5 10

<210> SEQ ID NO 12 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <400 SEQUENCE: 12 Cys Tyr Phe Gly Asn Ser Pro Arg Gly 1 5

<210> SEQ ID NO 13 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <400 SEQUENCE: 13 Cys Tyr Ile Gly Asn. Ser Pro Arg Gly 1 5

<210> SEQ ID NO 14 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <400 SEQUENCE: 14 Cys Tyr Phe Gly Asn Ser Pro Leu Gly 1 5

<210 SEQ ID NO 15 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge <400 SEQUENCE: 15 Cys Tyr Ile Gly Asn. Ser Pro Leu Gly 1 5

<210> SEQ ID NO 16 &2 11s LENGTH 32 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge between Cys and US 2002/0165132 A1 Nov. 7, 2002 16

-continued

second Ser

<400 SEQUENCE: 16 Cys Gly Asn Lieu Ser Thr Ser Val Lieu Gly Lys Lieu Ser Glin Glu Lieu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asp Val Gly Ala Gly Thr Pro 2O 25 30

<210 SEQ ID NO 17 &2 11s LENGTH 32 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge between Cys and third Ser

<400 SEQUENCE: 17 Cys Ser Asn Lieu Ser Thr Ser Val Lieu Gly Lys Lieu Ser Glin Glu Lieu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asp Val Gly Ala Gly Thr Pro 2O 25 30

<210> SEQ ID NO 18 &2 11s LENGTH 32 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge between Cys and second Ser

<400 SEQUENCE: 18 Cys Gly Asn Leu Ser Thr Ser Met Leu Gly Thr Tyr Thr Glin Asp Phe 1 5 10 15 Asn Llys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro 2O 25 30

<210 SEQ ID NO 19 &2 11s LENGTH 32 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge between Cys and third Ser

<400 SEQUENCE: 19 Cys Ser Asn Leu Ser Thr Ser Met Leu Gly Thr Tyr Thr Glin Asp Phe 1 5 10 15 Asn Llys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro 2O 25 30

<210> SEQ ID NO 20 &2 11s LENGTH 32 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Cys-Ser lanthionine bridge between Cys and third Ser

<400 SEQUENCE: 20 Cys Ser Asn Lieu Ser Thr Ser Val Lieu Gly Lys Lieu Ser Glin Glu Lieu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro US 2002/0165132 A1 Nov. 7, 2002 17

-continued

2O 25 30

SEQ ID NO 21 LENGTH 32 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Cys-Ser lanthionine bridge between Cys and second Ser

<400 SEQUENCE: 21 Cys Gly Asn Lieu Ser Thr Ser Val Lieu Gly Lys Lieu Ser Glin Glu Lieu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro 2O 25 30

SEQ ID NO 22 LENGTH 21 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: -S- bridge between Cys and fourth Ser and -S-S- bridge between second and third Ser

<400 SEQUENCE: 22 Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val Thr Phe Ser His 1 5 10 15 Leu Asp Ile Ile Trp 2O

SEQ ID NO 23 LENGTH 21 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: -S- bridge between the second Cys and fourth Ser and -S-S- bridge is between first and third Cys

<400 SEQUENCE: 23 Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Ser Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp 2O

SEQ ID NO 24 LENGTH 21 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: -S- bridge between first Cys and fifth Ser and -S- bridge between second Cys and fourth Cys

<400 SEQUENCE: 24 Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Ser Val Tyr Phe Ser His 1 5 10 15

Leu Asp Ile Ile Trp US 2002/0165132 A1 Nov. 7, 2002

We claim: sequence of 3 amino acids where the -N terminal -NH 1. Biologically active peptides having improved biologi is replaced by -OH, -H or -NHCOR6. cal activity Selected from the group consisting of lanthion 6. The peptide of claim 1 wherein ine-bridged biologically active peptides of the formula R is Selected from the group consisting of D-Phe, D-B- Nal, Tyr, TrpNH, ThrNH, Thr(oil), Pro-Arg-Gly, Pro O O Leu-Gly, -H, Cs alkyl, C7-2 aralkyl, C-1s acyl and R-NH-CH-C-R-NH-CH-C-Rs C2-1s aracyl R-C-S C-Rs R is Selected from the group consisting of D-Phe, D-B- Nal, Tyr, TrpNH, ThrNH, Thr(oil), Pro-Arg-Gly or Pro-Leu-Gly, -OH, -NH2, and alkylated amino acids, and wherein R is selected from the group consisting of a sequence of 2 to 7 amino acids Selected from naturally occurring amino acids and the D-enantiomers thereof and CR peptidomimetrics, R is selected from the group consisting of -H, Cs may be replaced by CH-OH with the proviso that R is alkyl, C7-12 aralkyl, -HCO, C-1s acyl or C2-1s aracyl, not Phe-Trp-Lys-Thr when R is D-Phe and R is a naturally occurring amino acid or Sequence of up to 25 Thr(ol). amino acids where the -N terminal -NH group is 7. The peptide of claim 1 wherein R is selected from the present or is replaced by Cls alkyl, C7-2 aralkyl, group consisting of Gly-Phe, Phe-D-Trp-Lys-Val, Phe-D- -HCO, or Cs acyl-OH, -H or -NHCOR, and Trp-Lys-Thr, Thr-D-Trp-Lys-Val, Tyr-Phe-Gln-ASn, Tyr-Ile Gln-ASn, Gly-Asn-Leu-Ser-Thr, Ser-Asn-Leu-Ser-Thr and peptidomimetics, Glu-Lys-Asp-Met-Leu-Ser-Ser. R is Selected from the group consisting of -OH, NH2, 8. The peptide of claim 1 wherein R, Rs, R7, Rs are a naturally occurring amino acid or a Sequence of up to 25 individually selected from the group consisting of -H, amino acids where the -C terminal -COOH is -CH, and cyclohexyl. present or is replaced by -CONH or -CH-OH; and 9. The peptide of claim 1 wherein R and R are indi vidually selected from the group consisting of D-Phe, D-B- peptidomimetics, and Nal, Tyr, TrpNH, and Thr(ol). 10. Lanthionine bridged peptide analogs of naturally O occurring peptides of claim 1 having a biological activity greater than that of the naturally occurring peptide. CR3 may be replaced by CH2OH: 11. Lanthionine bridged enkephalin analog peptides of claim 1 having a biological activity greater than that of with the proviso that R is not Phe-Trp-Lys-Thr when R. enkephalin. 12. The peptides of claim 1 having no more than two is Phe and R is Throl; lanthionine bridges. R is hydrogen, cyclohexyl or a Substituted or unsubsti 13. The peptides of claim 1 having one lanthionine bridge. tuted Co alkyl, 14. Peptides of claim 1 having the general formula Rs is hydrogen, cyclohexyl or a Substituted or unsubsti tuted Co alkyl, H-Tyr-D/L-Ala-Gly-Phe-D/L-Ala-Rs. R is an alkyl or aralkyl residue, R7 is hydrogen, cyclohexyl or a Substituted or unsubsti tuted Co alkyl, and 15. Peptides of claim 1 having the general formula Rs is hydrogen, cyclohexyl or a Substituted or unsubsti tuted Co alkyl. 2. The peptide of claim 1 wherein R is selected from the H-Xxx-Ala-Phe-D-Trp-Lys-Yyy-Ala-Zzz group consisting of from 2 to 4 amino acids. 3. The peptide of claim 1 wherein R is a sequence of 3 amino acids, and R is a Sequence of 3 amino acids. 4. The peptide of claim 3 wherein R and R are indi wherein XXX is selected from the group consisting of D-Phe vidually Selected from the group consisting of Pro-Arg-Gly and D-B-Nal; Yyy is Selected from the group consisting of and Pro-Leu-Gly. Thr and Val, and ZZZ is Selected from the group consisting 5. The peptide of claim 1 where R and R are each of TrpNH, ThrNH and Thr(oil) with the proviso that XXX individually Selected from the group consisting of a is not D-Phe when ZZZ is Thr(oil). US 2002/0165132 A1 Nov. 7, 2002

16. Peptides of claim 1 having the general formula endothelin or endothelin analog has been replaced by a thioether bond and 2) the rings are sequentially overlapping. 21. A pharmaceutical composition comprising a peptide H-DPhe-Ala-Tyr-DTrp-Lys-Val-Ala-Trp-NH. of claim 1 in a physiologically acceptable carrier. 22. In a process for the preparation of a peptide according Of to claim 1 using an appropriate combination of Solid-phase H-DBNal-Ala-Tyr-DTrp-Val-Ala-Thr-NH2 peptide Synthesis and/or classical Synthesis methods, the improvement consisting of using at least one peptide frag ment containing a moiety which is cyclized either attached to the resin used or after cleavage from the resin to the 17. Peptides of claim 1 having the general formula desired lanthionine-bridged cyclic peptide fragment which can optionally be elongated at the -N and/or -C terminal to form the final peptide by fragment condensation or Step by H-Ala-Tyr-Aaa-Gln-Asn-Ala-Pro-Bbb-Gly-NH. Step Synthesis. 23. A process according to claim 22 for the preparation of a peptide according to claim 1 wherein the peptide frag ments containing the moiety to be cyclized are assembled on wherein Aaa is Selected from the group consisting of Phe and an appropriate resin using tert-butoxcarbonyl-chemistry Ile and Bbb is Selected from the group consisting of Arg and with any peptide coupling method, Serine is incorporated at Leu. the desired place, which is then converted to dehydroalanine 18. Peptides of claim 1 having the general formula using disuccinimido carbonate, the S-protecting group attached to the cystine coupled the desired place is Selec R2-AlaL-Gigg-Asn-Leu-Ser-Thr-AlaL-R tively removed, the Michael addition of the SH group to the double bond is promoted by a slightly basic milieu, and the peptide and the other protecting groups are cleaved from the resin by treatment with HF. wherein Gigg is Selected from the group consisting of Gly 24. The process according to claim 23 wherein the peptide and Ser, R is -H, C is acyl and aracyl, and R is selected chain is assembled at any appropriate resin using the Fmoc from the group consisting of fragment 8-32 of human, Strategy with any usable coupling agent, intermediately Salmon and eel-calcitonin. using cleavage of the Fmoc-protecting-group by the piperi 19. Naturally occurring peptides having a linear Structure dine-method, wherein the cleavage of the acid labile S-pro cyclized by a thioether bond to form a peptide of claim 1. tecting group is carried out by any appropriate acid or 20. Peptides having the amino acid Sequence of endothe reagent. lin or an endothelin analog modified to a peptide of claim 1 wherein 1) at least one of the disulfide bridges in the