AU604785B2 - Peptides with sulfate ester groups - Google Patents

Peptides with sulfate ester groups Download PDF

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Publication number
AU604785B2
AU604785B2 AU81335/87A AU8133587A AU604785B2 AU 604785 B2 AU604785 B2 AU 604785B2 AU 81335/87 A AU81335/87 A AU 81335/87A AU 8133587 A AU8133587 A AU 8133587A AU 604785 B2 AU604785 B2 AU 604785B2
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Prior art keywords
asp
fmoc
met
gly
trp
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AU8133587A (en
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James D. Rosamond
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Fisons Corp
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Fisons Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/595—Gastrins; Cholecystokinins [CCK]
    • C07K14/5955—Gastrins; Cholecystokinins [CCK] at least 1 amino acid in D-form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Description

I
1 AUSTRALIA TllS^UmCTT~f^^ AUSTRALIA -Tis document contains the Patents Act amendments made under Patents Act Section 49 and i correct for printing.
COMPLETE SPECIFICATION
(ORIGINAL)
Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: I 'her F :i /o ~e~ a-.
APPLICANT'S REFERENCE: 905-203(CIP)2AU Name(s) of Applicant(s): -Pennwalt Corporation Address(es) of Applicant(s): G ti~;.
C -Three Parkway, -K -Philadelph i a, N)oc,.str f Pennsylvania 19102-, UNITED STATES OF AMERICGA 1 K i Address for Service is: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled: PEPTIDES WITH SULFATE ESTER GROUPS Our Ref 75258 POF Code: 1012/1444 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 6003q/l 1 BACKGROUND OF THE INVENTION This invention concerns sulfate ester containing peptides possessing feeding inhibition properties and capable of stimulating the contraction of the gallbladder.
These peptides have 6 to 9 amino acids. They all differ structurally, however, from two similarly sized peptides known to have feeding inhibition properties: CCK-8, which has the structure, Asp-Tyr(S03H)-Met-Gly-Trp-Met- 9* 9* -1A- Asp-Phe-NH- 2 and ceruletide, which has the structure, Glp-Gln-Asp-Tyr(SO 3 H) -Thr-Gly-Trp-Met-Asp-Phe-NH 2 The peptides of this invention are not found in nature but, I L rather, must be synthesized.
SUMMARY OF THE INVENTION A The compounds of the invention are peptides of the formula (1) OSO H 3 CH 2 CH 2 H Q YCHC- tt W -X -j -F CHCO- Z 4* 9 wherein 10 Q is H, H-Asp, H-PAsp, H-DAsp, H-MeAsp, For, Ac, Suc, desQ, or R'R 2
CHOGO,
is H, (R)-R 3 N, or (S)-R 3
N,
.4M is Met, DMet, MeMet, MetO, Ahx, DAhx, MeAhx, Leu, MeLeu, Pro, Ile, MeIle, or Lys, Gis Gly, laProrS, W is Trp, MeTrp or Nal, X is Met, MeMet, MetO, Ahx, MeAhx, Leu, MeLeu, Ile, Metle, Pro, or Lys, I- 3- J is Asp, DAsp, MeAsp, or Asn,
F
1 is (S)-R 4 N, or (R)-R 4
N,
F
2 is H, C1, I, Br, F, NO 2
NH
2
R
5 or OR 6 Z is "H 2
NHR
7 or NR 7
R
8
R
1 and R 2 are independently H or lower alkyl,
R
3
R
4 and R 5 are lower alkyl,
R
6 is H or lower alkyl, and
R
7 and R 8 are lower alkyl, ij i and pharmaceutically acceptable salts thereof, provided that i Q is desQ when Y is H, S. F 2 is not H if, in the same peptide, Q is H-Asp or Ac, Y is M is either Met, MetO, Ahx or Leu, X is either Met, MetO, Ahx or Leu, G is Gly, DAla or Pro, .*15 W is Trp, J is Asp, F 1 is and Z is NH 2
F
2 is not H if, in the same peptide, Q is H, Hi Asp or For, Y is M is Met, Ahx or Leu, G is Gly, W is Trp, X is Met, Ahx or Leu, J is Asp, F 1 is (S)-NH, I and Z is NH 2
F
2 is not H if, in the same peptide, Y is H, M Sis Met, X is Met, G is Gly, W is Trp, J is Asp, F' is SNH, and Z is NH 2 and
F
2 is not H if, in the same peptide, Q is Suc, Y is M is Met, X is Met, G is Gly or DAla, W is Trp, J is Asp, F 1 is (S)-NH or (S)-R 4 N, and Z is NH 2 4- The invention is also a process of making the peptides of the invention.
The invention is also methods of treating obesity and preventing obesity, respectively; each such method comprising the administration, by either an intraperitoneal, intravenous, intramuscular, subcutaneous or intranasal route, to a mammal in need of such treatment, of a peptide of the formula (1) wherein Q is H, H-Asp, H-PAsp, H-DAsp, H-MeAsp, For, Ac, Suc, 0-.10 desQ, H-Arg-Asp, Glp-Asp, Glp-Glu, Glp-Gln, Suc-Asp, Glt-Asp, Pht-Asp, R 9 CO-Asp, Boc-Asp, Cbz-Asp, H-Abu, H-Ala, Boc, Cbz, or R'R 2
CHOCO,
Y is H,
(R)-R
3 N, or (S)-R 3
N,
M is Met, DMet, MeMet, MetO, Aknx, DAhx, MeAhx, Leu, H .15MeLeu, Pro, Ile, Metle, Lys, Thr, Abu, Val, Mox, Gly, Phe, Tyr, or Trp, G is Gly, DAla, Sar, DTrp, Pro, or pAla, W is Trp, MeTrp, Nal, DTrp, Trp(Me), or Trp(6-F), X is Met. McMet, MetO, Ahx, MeAhx, Leu, MeLeu, Ile, Meile, Pro, Lys, DMet, Abu. or Mox, J is Asp, DAsp, MeAsp, PAsp, or Asn, F1 is
(S)-R
4 N, or (R)-R 4
N,
F
2 is H, Cl, 1, Br, F, NO 2
NH
2
R
5 or OR', Z is NH 2 NRR 7 or NR 7R 8 j -5- 5
R
1 and R 2 are independently H or lower alkyl,
R
3
R
4 and R 5 are lower alkyl,
R
6 is H or lower alkyl, and
R
7
R
8 and R 9 are lower alkyl, and pharmaceutically acceptable salts thereof, provided that Q is desQ when Y is H, and I F 2 is not H if, in the same peptide, Q is H-Asp, S Y is M is Met, X is Met, G is Gly or Pro, i ".10 W is Trp, S J is Asp, F 1 is and Z is NH 2 In its first subgeneric aspect, the invention is defined Sas compounds of the invention with the exception that it is further limited so that Q is H, H-Asp, H-pAsp, H-DAsp, For, Ac, Suc, desQ, or I R
I
R
2
CHOCO;
S..Y is H, or (S)-R 3
N;
I M is Met, MeMet, Ahx, MeAhx, Leu, MeLeu, Ile, MeIle, or Pro; '20 G is Gly or DAla; W is Trp; X is Met, MeMet, Ahx, MeAhx, Leu, MeLeu, Ile, MeIle, or Pro; J is Asp;
F
1 is (S)-NH or (S)-R 4
N;
I
5
S
S S S.5 5* S S S
F
2 is H, Cl, NO 2
NI-
2
R
5 or OR 6 and Z is NH 2 In a second subgeneric aspect, the invention is defined as in its first subgeneric aspect with the exception that it is further limited so that M is neither MeMet, MeAhx, MeLeu nor MeIle, and X is neither MeMet, MeAhx, MeLeu, nor MeIle.
Compounds with increased feeding inhibition activity over a 3-hour feeding period as compared to CCK-8 are: A peptide of the formula CH 2 CH 2 1 I1 Q -YCHCO- M a W X 3 -F CHCO- z wherein Q is H-,PAsp, For, Suq desQ, or R'R 2
CHOCO,
Y is H or (S)-NH, M is Met, Ahx, Leu, or Ile, G is Gly, W is Trp, X is Met, Ahx, Leu, or Ile, J is Asp, F1 is (S)-NH or (S)-R 4
N,
F
2 is H, NO 2
R
5 or OR" 6 Z is NH 2 R1 and R 2 are independently H or lower alkyl, *S 10 15 7
R
3
R
4
R
5 and R 6 are lower alkyl, and pharmaceutically acceptable salts thereof, provided that Q is desQ when Y is H,
F
2 is not H if, in the same peptide, Q is H-pAsp For,--e-As, Y is M is either Met, Ahx, or Leu, X is either Met, Ahx, or Leu, and F 1 is (S)-NH,
F
2 is not H if, in the same peptide, Y is H, M is Met, X is Met, and F 1 is (S)-NH, 10 F 2 is not H if, in the same peptide, Q is Sue, Y QS) Nk o"is M is Met, X is Met, and F 1 is(S)-R4N.
In additional subgeneric aspects of the invention, the method of treating obesity or the method of preventing obesity is further limited to the administration of either the compounds of the invention or the compounds identified in the first or second generic aspects of the invention.
In a closely related invention, the compounds of the invention have Y as (R)-NH with the additional proviso that
F
2 is not H if, in the same peptide, Q is H-Asp, M and X S 20 are Ile, G is Gly, J is Asp, F 1 is and Z is NH 2 so that compounds of this related invention are peptides of the formula wherein Q is H, H-Asp, H-pAsp, H-DAsp, H-MeAsp, For, Ac, Suc, desQ, or R1R 2
CHOCO,
,/3 f/I -8 Y is (R)-NH, M is Met, DMet, MeMet, MetO, Ahx, DAhx, MeAhx, Leu, MeLeu, Pro, Ile, MeIle, or Lys, G is Gly, DAla, Pro or Sar, W is Trp, MeTrp or Nal, X is Met, MeMet, MetO, Ah--x, MeAhx, Leu, MeLeu, Ile, Melle, Pro, or Lys, J is Asp, DAsp, MeAsp, or Asn,
F
1 is or (R)-R 4
N,
F
2 is H, Cl, I, Br, F, NO 2
NI-
2
R
5 or OR 6 *Z is NH 2
NHR
7 or NR 7
R
8 RI and R 2 are independently H or lower alkyl,
R
3
R
4 and R 5 are lower alkyl,
R
6 is H or lower alkyl, and -1 RI and R 8 are lower alkyl, 08 and pharmaceutically acceptable salts thereof, provided that
F
2 is not H if, in the same peptide, Q is H-Asp, M \Qr and X are Ile, G is Gly, J is Asp, F1 is and Z is NH 2 DETAILED DESCRIPTION Definitions "lower alkyl"' contains 1 to 6 carbon atoms.
and refer to the absolute configurations, about the adjacent methine carbon. When Y is then 9- 9so 3 9 .9 9 9 99* 9 9t *9 9 9 *9 9 9 999999 9 *9 9 *99 *9 a ad 9 *9999* a 999b9 90 9 9 99 999999 is of the L-configuration and when Y is then oso 3 2 is of the D-configuration. Similarly, when F' is (S)-NH or then is of the L or D-configuration, respectively.
All optically active amino acids are of the L-configuration unless otherwise indicated.
ii i Li 10 The H in H-Asp, H-pAsp, H-DAsp, H-MeAsp, H-Arg, H-Abu, and H-Ala stands for hydrogen.
DesQ, which arises when Y is H, means that there is no Q.
RIR
2 CHOCO is the formula R H 0 C-O-C
R
2 ae M* a S" Hpp(SO 3 H) is the formula 3 1
CH
Each claim to a compound includes its pharmaceutically acceptable base addition salts. Base addition salts include those derived from both organic and inorganic bases, such as, 10 for example, ammonia, sodium hydroxide, calcium hydroxide, a 0. barium hydroxide, tetraethylammonium hydroxide, ethylamine, diethylamine, triethylamine, and the like.
In an alternative representation to formula used for purposes of brevity, peptides are also represented in accordance with conventional representation, for example, H-Asp-DTyr(SOsH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 which stands for the compound of formula in which Q is I
M
11 H-Asp, Y is M is Met, G is Gly, W is Trp, X is Met, J is Asp, F' is F 2 is H, and Z is NH 2 When amino acids, peptides, protecting groups, active groups, etc. are represented by syznbols in this specification and appended claims, usual symbols as defined by IUPAC and TUB or as used in the art ar e employed.
Examples of symbols are given below.
S S a *5 ,a a, A a a a aa~&OS a
S
a~ a..
,~l5 a a
S.
a h S e S.
2-aminobutyric acid acetyl 2-aminohexanoic acid 2-aminoisobutyric acid alanine arginine asparagine aspartic acid beta-aspartic acid tert-butyloxycarbonyl BrCH 2 -Pam 4-(bromomethyl)pheny'LacetamidomethyI carbobenzoxy S-methylcysteine D-alanine D,2-aminohexanoic acid D-aspartic acid D-methionine D-phenylalanine DPhe-NH 2 D-phenylalanine amide 12 9 5 4) 8* 4s 4. 9
S
10 4 Q 0 a 50.05.
a 9. 5 59 0 54* 15 0 0 me
S
4 006 @6 a 0 500S0*
S
5 54 S 4 9* a JO 009 a 20 DTyr EtOCO Etphe EtPhe-NH 2 Emoc For Gin Gip Glu Git Gly His Hpp Hpp(S0 3 H) iBuOCO Ile Leu Lys MeAhx MeAsp MeLeu Mele MeMet MeOGO D-tryptophan D-tyrosine ethyloxycarbonyl N-ethylphenylalanine N-ethyiphenylalanine amide 9- fluorenylmethyloxycarbonyl formyl.
glutamine pyrogiutamyl glutamic acid
HOOC(CH
2 3 C0glycine histidine (4-hydroxyphenyl)propionyi 3-(O-sulfo-4-oxyphenyl)propionyl isobutyloxycarbonyl.
isoleucine leucine lysine N-methyi-2-aminohexanoic acid N-methylaspartic acid N-methylleucine N-methylisoleucine N-methylmethionine methyloxycarbonyl, N-me thylphenylalanine -13- MePhe-NH 2 N-methylphenylalanine amide methionine methionine sulfoxide N-u-methyltryptophai N-methyltyrosine N,O-dimethyltyrosizie MeTyr(Me)-NH 2 N,O-dimethyltyrosine amide methoxinine 3-(2-naphthyl)alanine 1-benzotriazolyl ester OCH 4-(oxymethlphenyl)acetamidomethyl succinimidyloxy ester tert-butyl ester phenylalanine Phe-NH 2 phenylalanine amide l ln ne e h lm d phenylalanine methylamide 0VS.SS heyaain ithlmd Phe-N(-Me.. phenylalanine dmethylamide Phe-N(Et.. .phenylalanine acdieh md Phe(4-C1) 3-(4-cehlphenyl)alanine pPhe(4-G1)-NH 2 3-(4-cnehlphenyl)alanine amide 3-(4-methylphenyl)alanine Phe(4-NMe)-NH 2 3-(4-rnethyphenyl)alanine amide 14 Phe(4-NH 2 Phe(4-NH 2 )-N11 2 3- (4-aminophenyl)alanine.
3-(4-aminophenyl)alanine amide
.COON
Pht co- '9 5 9 9 999 9 9. C.
4 *9 .i 9 9 9 9*9999 9 .9 9.
9.
999 99 *9 9999 *9 15 9 .9 9 9 49
C
Pro PrOCO resin Sar Ser Suc tBu Thr Trp Trp(6-F) Trp(Me) Tyr Tyr-NH 2 Tyr(Me) Tyr(Me)-NH 2 Tyr(SO 3 H) Tyr(SO 3
H)-NH
2 Val praline n-propyloxycarbonyl polystyrene sarcosine serine
HOOC(CH
2 2
GO-
tert-butyl threonine tryptophan 5- fluorotryptophan 6-fluorotryptophan 1-methyltryptophan tyrosine tyrosine amide 0-methyltyrosine 0-methyltyrosine amide 0-sulfotyrosine 0-sulfotyrosine amide valine Preferred Compounds The preferred compounds of the invention are those that during the 0.5 hour feeding period inhibited feeding by 48- 100% when administered at 30 pg/kg in the test described under "Utility" below. (See Table 2 below.) The most preferred compound, at the time of filing this continuation-in-part application, from the point of view of gallbladder contraction, is the compound Hpp(SOaH)-Met-Gly-Trp-Met-Asp-MrePhe-NH 2 preferred compoun-ds, at the time of filing this continuation-in-part application, from the point of feeding inhibition, were those that during the 0.5 hour V. feeding period inhibited feeding by 20-72% when administered at 0.3 pg/kg in the test described under "Utility"' below.
They are: H T r S H -ealy T p M t As h H H-DOsp-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 i-uOCO-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MPheNH 2 Suc-Tyr(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-MPheNH 2 H-uAsp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-Dpp (SO 3 H)-Met-Gly-TrtAp-MeApheHe.
ErC-Tyr(SO 3 H-Met-Gly-Trp-MetAsp-Phe-NH 2 OCO-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 HppO 3 H) ,(S-Met-Gly-Trp-Met-Asp-Phe-NH HPOCO-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 -16i Suc-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Hpp(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Asp-Tyr(So 3 H)-Leu-Gly-Trp-Leu-Asp-Phe-NH 2 4For-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Suc-Tyr(S0 3 H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 iBuOGO-Tyr(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 Hpp(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 iBOCO-Tyr(S3H)-Ahx-Gly-Trp-Ahx-Asp-Phe i OHpp(ST 3 H)-Ahx-Gly-Trp-Ahx-Asp-Phe-NH 2 i~ 10l Suc-Tyr( SO3H) -Met-Gly.-Trp-Met-Asp-MePhe-NH2 SH-DAsp-Tyr(SO3H)-Ile-Gly-Trp-Ile-Asp-MePh-H For-Tyr(S03H)-Ile-Gly-Trp-Ile-Asp-MePhe-N S1 Suc-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 Ij .iBuOCO-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 H-Asp-DTyr(S 3 H)-le-Gly-Trp-le-Asp-MePhe-NH 2 Hpp(S0 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 i The compound, EtOCO-Tyr(S3H)-Met-Gly-Trp-Met-Asp- Phe-NH2, was the one that inhibited feeding by 72% at 0.3 pg/kg.
The most preferred compounds, at the time of filing this .1 continuation-in-part application, from the point of view of feeding inhibition, were those that during the 3 hour feed period inhibited feeding by 50-90% when administered at 3 g/kg in the test described under "Utility" below. They ari iBuOCO-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 -17- Suc-Tyr(SO 3 H) -Ahx-Gly-Trp-Ahx-Asp-Phe-NH 2 For-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 iBuOCO-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 HpHOH-e-l-r-e-s-eh-H HPpOsTr(H)-Met-Gly-Trp-Met-Asp-he-N2 POG-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Suc-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MPhe-NH 2 fi SuOc-Tyr(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 iBuOCO-Tyr(SO, 3 H)-Ahx-Gly-Trp-Ahx-Asp-MPhe-NH 2 Hpp(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-Phe-NH 2 iBuOGO-Tyr(SO 3 H)-Ahx--Gly-Trp-Ahx-Asp-MPhe-NH 2 SucT(SO 3 H)-e-Gly- TrpMe-Asp-MePhe-NH 2 ThFomndru-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhemehoswelknw to the(Sart,-IforGexamppleetheyMemae-be Thpaedb comun iucdyividua-le-minorideonaspolide 2 p hwas he one atinhibted feding by by cobnn duigth ous fedin perids nasldhs ei
AI
I -Y 18
M
*9
S.
*9 to yield the desired peptidyl-resin intermediate. Such additions, as is known, are accomplished by protecting the amino group of the amino acid or group of amino acids by converting it to, for example, its tert-butyloxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) derivative, and then activating the carboxylic group of such amino acid or group of amino acids by converting it, for example, to its l-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (HOSu) ester derivative. Such a protected-activated inter- 10 mediate is then allowed to react with an amino acid-resin or peptidyl-resin with a free amino group, thus extending the peptide chain to provide the peptidyl-resin of formula 2, wherein Pg is a suitable protecting group, for example, Boc or Fmoc.
-II
15 C (2) 2 C2 Pg Q -YCHCO- M X -alCCO- resin The phenolic OH group within formula is converted to a sulfate ester by the use of a usual sulfating agent, such as sulfur trioxide pyridine complex. More specifically, the reaction is conducted, for example, by suspending a peptidyl-resin of formula 2 in dimethylformamide (DMF), pyridine or like solvent, and adding sulfur trioxide pyridine -19 complex in about 10-40 molar excess to provide the sulfated peptidyl-resin of the formula 3.
oso H 72 O (3) CH2
CH
pg Q -YCHCO- M G W X J -CHCO- resin Since the sulfate ester containing peptide end-products S 5 of this invention are C-terminal amides, the chemical link S which cor sects the peptide chain to the resin must be such i 4 that its cleavage with suitable reagents readily provides amides. Due to the lability of the sulfate ester group to strong acids (for example, liquid hydrogen fluoride), the peptidyl-resin linkage may be cleavable with either weaker acids (for example, brief treatment with trifluoroacetic S' acid, TFA) and/or nucleophiles (for example, ammonia, amines, S hydroxide, and alkoxides). Among the suitable resin derivai tives may be mentioned oxymethyl-polystyrene, 4-(oxymethylj 15 phenyl)(CH 2 )nCO-aminomethyl-polystyrene (n 0-3) and 4-(oxymethylphenyl)oxymethyl-polystyrene. Similarly substituted polyacrylamide resins are equally well suited as the above polystyrene based resins. For the purposes of this invention the 4-(oxymethylphenyl)CH 2 CO-aminomethyl-polystyrene [herein referred to as 4 -(oxymethylphenyl)acetamidomethylpolystyrene or OCH 2 -Pam-resin] is best suited for the generation of L_ peptide amides. Thus, this invention describes a process for the synthesis of sulfated peptidyl-OCH 2 -Pam-resins of formula wherein the resin is polystyrene (the term i i In forming peptide sequences of this invention, the amino functions may be protected by commonly used amino protecting groups such as Boc, Fmoc, (4-methoxybenzyl)oxycarbonyl, 2-nitrophenylsulfenyl, and so forth. The Boc and Fmoc i t Sprotecting groups are preferred. The carboxyl and hydroxyl protecting group may be methyl, tert-butyl (tBu), benzyl, i4-methoxybenzyl and so forth. The tBu group is preferred.
The amino acid defined by the F' and F 2 groups of formula 4 may be attached to the OCH- Pam-resin in several ways. (a) For example, Boc proLected-phenylalanine, wherein F' is SIH and F 2 is H) may be reacted with a suitable 4-(bromomethyl)phenylacetate ester (for example, phenacyl ester) and processed further to provide Boc-Phe-(4-oxymethylphenyl)acetic acid which may be coupled to aminomethyl-polystyrene to provide Boc-Phe- 4 -oxymethylphenyl)acetamidomethylpolystyrene (Boc-Phe-OCH 0 1 I~ i 21 Pam-resin). Alternatively, 4-(bromomethyl)phenylacetic acid may be coupled to aminomethylpolystyrene to provide 4- (bromomethyl)phenylacetamidomethylpolystyrene (BrCH -Pamresin) which may be reacted with the cesium salt of Boc-Phe- OH to provide Boc-Phe-OCH -Pam-resin.
Among the suitable activating groups may be mentioned any combination of groups which causes the acid function of the amino acid to become more reactive, such as acid chlorides, mixed and symmetrical anhydrides, reaction product with carbodiimide (for example, dicyclohexylcarbodiimide, DCC), and active esters (for example, esters derived from HOBt, HOSu, 2- or 4-nitrophenol, and 2,4,5-trichlorophenol). The use of DCC and esters of HOBt and HOSu is particularly preferred from the standpoint of yield, lack of by-products, and consequent ease of purification.
The protecting groups are removed by known reactions such as treatment with dilute TFA (50% in dichloromethane, DCM) for Boc and/or tBu removal and treatment with dilute S piperidine (20% in DMF) for Fmoc removal, to name a few, to provide the sulfated peptidyl-resin of the formula C2 2 Q -y3CC- M W X J C o -P -rsi L 22 The sulfate ester containing peptides of formula 1 may be obtained by cleavage of the peptidyl-OCH -Pam-resin linkage of with the appropriate reagent. The C-terminal sulfated peptide amides are derived, for example, by treatment of the sulfated peptidyl-resin of formula with methanolic solutions of ammonia, alkylamines and dialkylamines.
An automatic peptide synthesizer was used for the solid S phase synthesis of the sulfated peptide amides of this invention. The protocol of coupling onto aminomethyl-resin S or peptidyl-OCH, Pam-resin (1 mmole of available nitrogen), deprotection, sulfation, cleavage, and product purification is set forth in Table 1.
i Table 1. Protocol for solid phase synthesis of sulfated peptide amides (1 mmole scale). Each step volume is 50 ml unless otherwise indicated. All wash steps are repeated three times. Abbreviations: DCC, dicyclohexylcarbodiimide; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine, DMF, Sdimethylformamide; HOBt, l-hydroxybenzotriazole;
TFA,
I trifluoroacetic acid.
J
I
23 Step Reagent or Solvent Purpose Mix Time 1 2 3 4
DCM
Go to Step 3, 5, or 8 Add filtered, pre-activated 1 hr) mixture of protected amino acid (or protected dipeptide, 3 mmole), HOBt mmole), and DCC (3 mmole) in 1:4 DMF/DCM Go to Step 10, 16, 21, or 26 Add protected amino acid (or protected dipeptide, 3 mmole) and HOBt (4.5 mmole) in 30 ml 1:2 DMF/DCM then DCC (3 mmole) in ml DCM 2-Propanol Go to Step 4 Add active ester or anhydride (3 mmole) in DCM, DMF, or mixture thereof Go to Step 4
S..
***15 *9 S. 9 Wash Pre-activated DCC/ HOBt coupling In situ activated DCC/HOBt coupling Wash Non DCC/HOBt activated coupling Wash Boc and tBu remova Wash Neutralize Wash Wash Fmoc removal Fmoc removal Wash 2-15 hr 1 min 2-15 hr 1 min 2-15 hr 9
S.
*S
S
S
9 20 10 11 12 13 14 25 15 16 17 18 19
DCM
Treat
DCM
Treat
DCM
Go to
DMF
Treat Treat
DMF
with 49:1:50 TFA/anisole/DCM with 1:19 DIEA/DCM Step 1, 16, 21, or 26 with 1:4 piperidine/DMF with 1:4 piperidine/DMF 20 Go to Step 21 DMF 22 1:2 pyridine/DMF 23 Add sulfur trioxide pyridine complex mmole) in 60 ml 1:2 pyridine/DMF 24 DMF 25 Go to Step 4 26 Methanol 27 Ammonia saturated (-20 0 C) methanol or 20% methanolic amine (250 ml) 28 Methanol 29 Combine, concentrate filtrates from Steps 27-28 Chromatograph residue on column(s) of Amberlite XAD-2 (Rohm and Haas, 2.5 x cm, methanol gradient 0.1 M in ammonia), Trisacryl M DEAE (LKB Inc., 2.5 x 47 cm, ammonium bicarbonate gradient), and/or ODS-3 (Whatman, 4.8 x 50 cm, methanol gradient 0.2% in ammonium acetate) Wash Wash Sulfation Wash Wash Resin cleavage Wash Isolation Purification 1 min 1 30 min 1 min 1 min 1 min 1 min 3 min 7 min 1 min 1 min 1 min 20-24 hr 1 min 1 min 2-5 day 1 min L -24 The sulfate ester containing peptides of formula (1) thus prepared may be desalted and purified by the usual methods. For example, the product may be purified by ion-exchange chromatography with use of Trisacryl M DEAE, DEAE-cellulose or the like, partition chromatography with use of Sephadex LH-20, Sephadex G-25 or the like, reverse phase chromatography with use of Amberlite XAD-2, ODSsilica gel or the like, normal phase chromatography with use of silica gel or the like, or high-performance liquid 0O chromatography (HPLC).
Analogous procedures, wherein the reactions are carried out without the solid phase component (resin), are well known in the art and well suited to large scale production. [See, U.S. Patent 3,892,726.] Utility The peptides of this invention have the ability to inhibit feeding activity in mammals. As a result they have utility in the prevention and treatment of obesity. Feeding inhibition activity can be demonstrated in rats as follows: Male Sprague-Dawley rats (weighing 300-350 g) are individually caged and maintained on a 12 hr light, dark cycle and trained for at least 14 days to feed during a three hr period of the dark cycle but not the 21 hours preceding that three hr period. The day of the study, rats are dosed intraperitoneally with saline (controls) or test compound (dissolved in saline; usually at a concentration of 0.3 to Ohl-e 300 micrograms of test compound per kg of rat weight).
Food is introduced 10 minutes after administration of saline or test compound. Test compounds are deemed to be active if the test group consumes significantly less food than the saline controls during the feeding period, which ends either or 3 hr after presentation of food. The feeding inhibition for the 1/2 hr and 3 hr feeding periods obtained by i administering a dose of 30 pg/kg of test compound is given in Table 2 for CCK-8 and various compounds of the invention.
0 For example, the feeding inhibition obtained with CCK-8 i (first line of Table 2) was 70% for the 1/2 hr feeding period iI and 25% for the 3-hour period.
i i *5555 Ii ji _r 26 TABLE 2 4 9bc a. 4 4 a 4 a a.
4.4 *4 a I sate's
S
*4**tq
S
a a a.
S
STRUCTURE IN H-sp-yr(S 3 H)Met-ly-rp-Mt-Ap-Ph-0.
3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 i-uOGO-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 H-As-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-N1 2 H-Asp-Tyr(SOH)-Met-Gly-Trp-IMet-Asp-Phe-NH 2 10 H-Asp-Tyr(SO 3 H)-D~hx-Gly-Trp-Met-Asp-Phe-NH 2 10 H-Asp-Tyr(SO 3 H) -Metx-Sa-Trp-Met-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H)-Met-Gly-MTrp-Met-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H) -Met-Gly-Nal~r-Met-Asp-Phe-NH 2 H-Asp-Tyr(SO3H) -Met-Gly-Trp-Met-Asp-Phe-N- 2 3 H) -Met-Gly-Trp-Met-As-Phe-NH 2 15H-Asp-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-MPhe-NH 2 H-Asp-Tyr(SO 3 H)-Met-Gly-.Trp-Met-Asp-Me~y(e-NH 2 H-Asp-Tyr(S03H)--Met-Gly-Trp-Met-Asp-Mhe(yr(Me)-NH 2 H-Asp-Tyr(S03H)-Met-Gly-Trp-Met-Asp-Phe(4-NC)-NH 2 20 H-Asp-Tyr(SO3H).-Met-Gly-Trp-Met-Asp-Phe(4-NH 2
)-NH
2 20H-Asp-Tyr(SO 3 H) -Met-Gly-.Trp-Met-Asp-Tyr(Me)-NH 2
-H
H-Asp-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NMe-H H-Asp-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH~e H-Asp-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-N(ME) H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-N(e) 2 H-Asp-Tyr(S 3 H)-Met-Gly-Trp-Met-Asp-Phe-NEt 2 H-DAsp-DTyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 Hspc-DTr(S 3 H) -Met-Gly-Trp-Met -Asp-Phe-NH 2
FEEDING
HIBITION
hr-3 hr 70-25 99-40 100- 94 74-24 17- IA 14-17* 55*-12*,' 31-11 52*-20* 32- 43*-17* 70-24 65-17 43-17 54- 28* 27-IA 72*-10*,' 13*-IA 16-IA 64-27 18*-IA 47- 33 53-15 82-60 27 9 0** *9 9* 99 999**, 9.
9., 9 *99*91 999 994 9.
9 .9 H-Asp-Tyr(SO 3 H) -Ile-Gly-Trp-Ile-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H)-Lys-Gly-Trp-Lys-Asp-Phe-NH 2 Hpp(SO 3 H) -Met-DAla-Trp-Met-Asp-Phe-NH 2 Suc-Tyr(SO 3 H) -Ahx-Gly-Trp-Ahx-Asp-Phe-NH 2 H-PAsp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-DAsp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-M(!Phe-NH 2 For-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-MePhe-NH 2 iBuOCO-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-Asp-DTyr(SO 3 H) -Met-Gly-Trp-Met-Asp-MePhe-NH 2 .10 Hpp(SO 3 H)-Met-Gly-Trp-Met-Asp-t~1ePhe-NH 2
L~
Hpp(SO 3 H) -Met-DAla-Trp--Met-Asp-MePhe-NH 2 PrifCO-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 EtOCO-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 MeOCO-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 H-rAsp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe -NH 2 H-T.,,r(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Suc-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 Hpp(SO 3 H)-Met-Gly-Trp-Met-A5p-Phe-NH 2 H-Asp-Tyr(SO 3 H)-Ahx-Gly-Trp-Met-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H)-Met-Giy-DTrp-Met-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H) -Met-Gly-Trp-Ahx-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-DPhe-NH 2 H-Asp-Tyr(SO 3 H) -MetO-Gly-Trp-MetO-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H) -Ahx-Gly-Trp-Ahx-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H)-Leu-Gly-Trp-Leu-Asp-Phe-NH 2 For-Tyr(SO 3 H) -Met-Gly-Trp-Met-Asp-Phe-NH 2 85-20 59-16 100- 97 100 -78 100-72 100-96 100- 99 85-35 100- 100 69-27 90-74 89-63 100-91 91-21 50- 75-55 99-75
NT-NT
42*-IA 82-23 46-14* 82-27 89-24 100-66 28 H-Asp-Tyr(SO 3 H)-MetO-Gly-Trp-Met-Asp-Phe-NH 2 H-Asp-Tyr(S03H)-Met-DAla-Trp-Met-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-DMet-Asp-Phe-NH 2 H-Asp,-Tyr(SO 3 H)-Met-Gly-Trp-MetO-Asp-Phe-NH 2 H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Ap-Phe(4-Me)-NH 2 H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Tyr-H 2 Hpp(SO 3 H)-Met-Gly-Trp-Pro-Asp-Phe-NH 2 Hpp(SO 3 H)-Pro-Gly-Trp-Pro-Asp-Phe-NH 2 Suc-Tyr(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 9 '0 iBuOCO-Tyr(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 Hpp(SO 3 H)7Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 iBuOCO-Tyr(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-PheNH 2 Hpp(S0 3 H)-Ahx-Gly-Trp-Ahx-Asp-Phe-NE 2 Suc-Tyr(S0 3 H)-Met-DAla-Trp-Met-Asp-Phe-NH 2 9 a* 15 For-Tyr(SO 3 H)-Ie-Gly-Trp-Ile-Asp-Phe-NH.
2 Suc-Tyr(SQ 3 H)-Ile-Gly-Trp-le-Asp-Phe-NH 2 iBuOCO-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-Phe-NH 2 Hpp(SO 3 H)-Ile-Gly-Trp-Ile-Asp-Phe-NH 2 Suc-Tyr(S03H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-DAsp-Tyr(S03H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 For-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-MH 2 Suc-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 iBuOGO-Tyr(SO3H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 H-Asp-DTyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 Hpp(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 89-25 16-18* 45-21* 57-18 23*-IA 48-IA 100-98 100-100 94-96 100-100 100-99 75-30
NT-NT
73-54 62-58 87-85 100-95 95-75 100-85 100-100 96-92 100-97 i 29 IA means 'linactive'f.
inhibition at 30 pg/kg; number shown represents% inhibition at dose of 300 pg/kg.
NT means "not tested" (END OF TABLE 2) 4
I
4J 4,
S
V
*V 64 V
V
V *4 49S C S
S
54 Cl 64 .4.1 0~* V S .4
S
S
4 a.
V
AS
S
bASS
S
I ~I 30 An appropriate procedure for administering a peptide designated here as suitable for intraperitoneal, intravenous, intramuscular, subcutaneous, or intranasal administration, to a mammal in need of either treatment for obesity or prevention of obesity, is at a dose of about 0.3 micrograms (pg) to 3 mg per kg of body weight per day, either as single dose or divided among two to four doses. The dosage may be varied, depending upon the requirements of the patient and I the compound being employed.
10 The peptides of this invention have the ability to stimulate gallbladder contraction in mammals. Thus, they also find utility as diagnostic aids in X-ray examination of t the gallbladder. The use of gallbladder contracting agents as diagnostic aids is a well established medical procedure.
15 EXAMPLES i The invention may be further illustrated by the following examples. Examples 1-18 illustrate the synthesis of intermediates. Examples 19-93 illustrate the synthesis of compounds that have been designated above as either a compound of the invention or a compound useful in the treatment and prevention of obesity. The examples are intended to illustrate the invention, not to limit it in any manner.
Peptide syntheses, unless otherwise stated, were initiated with 1 milliequivalent of aminomethyl resin, where the resin was 99:1 by weight styrene:divinylbenzene copolymer.
31 Reactions were performed at room temperature unless otherwise stated.
Washing steps were performed three times with 50 ml of the specified solvent unless otherwise stated.
EXAMPLE 1 Isobutyl Succinimidyl Carbonate (iBuOCO-OSu) To a solution of isobutyl chloroformate (26 ml, 200 mmole) in 600 ml of chloroform was added in portions the dicyclohexylamine (DCHA) salt of N-hydroxysuccinimide S'.0 (HOSu, 49.28 g, 200 mmole). After stirring the resulting S suspension overnight, the precipitated DCHA hydrochloride was filtered off and washed with chloroform. The concentrated filtrate (ca. 50 ml) and washings were diluted with 400 ml of ethyl acetate (EtOAc) and washed with citric acid (4 x 100 ml), brine (2 x 100 ml), 10% sodium bicarbonate (3 x 100 ml), and brine (4 x 100 ml) and then dried (magnesium sulfate), filtered, and concentrated to ca. 100 ml. On diluting with ether and precipitating with hexane, 26.6 g (62% yield) of iBuOCO-OSu was obtained, mp 33-35 0
C.
EXAMPLE 2 Fmoc-DTyr-OH D-Tyrosine (1.81 g) was dissolved in 20 ml of water and ml of tetrahydrofuran (THF) with 10 ml of N sodium hydroxide. Solid 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu, 3.37 g) was added with rapid stirring. The 32 suspension was adjusted to pH 7 with N sodium hydroxide and stirred overnight. Solid citric acid (3 g) was added followed by 60 ml of EtOAc. The EtOAc layer was collected, washed with 10% citric acid, brine, and dried (magnesium sulfate). Evaporation of the EtOAc solution gave a light i tan syrup which was crystallized from dichloromethane (DCM) to give 3.9 g of Fmoc-DTyr-OH, mp 178-181 0
C.
EXAMPLE 3 I Fmoc-Tyr-OH S *10 Following the procedure of Example 2 but substituting l L-tyrosine (9.06 g) for D-tyrosine, 18.4 g of Fmoc-Tyr-OH was obtained, mp 172-177 0
C.
EXAMPLE 4 Fmoc-MeTyr-OH Following the procedure of Example 2 but substituting N-methyltyrosine (1.95 g) for D-tyrosine, 1.22 g of S Fmoc-MeTyr-OH was obtained, mp 152-158 0
C.
EXAMPLE Boc-MePhe- (4-oxymethylphenyl)acetic Acid S 20 To a solution of Boc-MePhe-OH (27.93 g) and 4-(bromomethyl) phenylacetic acid phenacyl ester (33.32 g) in 1000 ml of Sacetonitrile was added potassium fluoride dihydrate (18.28 The suspension was stirred overnight, filtered and the filtrate evaporated to dryness. The residue, Boc-MePhe-(4oxymethylphenyl)acetic acid phenacyl ester, was dissolved in 85% acetic acid (1200 ml), treated with zinc dust (128 g), 33 and stirred for 2-4 hrs. Concentration of the filtered reaction mixture to ca. 400 ml and dilution with ca. 3200 ml of water gave an oil which was dissolved in EtOAc and treated with DCHA to give 41.31 g of the DCHA salt of title compound, mp 120-122 0
C.
0 0 0 15
.S
EXAMPLE 6 Boc-EtPhe-(4-oxymethylphenyl)acetic Acid Following the procedure of Example 5 but substituting Boc-EtPhe-OH (7.33 g) for Boc-MePhe-OH, 5.69 g of the DCHA salt of Boc-EtPhe-(4-oxymethylphenyl)acetic acid was obtained, mp 137-141 0
C.
EXAMPLE 7 Boc-Phe(4-Cl)-(4-oxymethylphenyl)acetic Acid Following the procedure of Example 5 but substituting Boc-Phe(4-Cl)-OH (2.5 g) for Boc-MePhe-OH, 3.44 g of the free base of Boc-Phe(4-Cl)-(4-oxymethylphenyl)acetic acid was obtained.
EXAMPLE 8 Boc-Tyr(Me)-(4-oxymethylphenyl)acetic Acid Following the procedure of Example 5 but substituting Boc-Tyr(Me)-OH (2.5 g) for Boc-MePhe-OH, 1.83 g of the free base of Boc-Tyr(Me)-(4-oxymethylphenyl)acetic acid was obtained, mp 64-67 0
C.
EXAMPLE 9 Fmoc-Tyr(tBu)-(4-oxymethylphenyl)acetic Acid Following the procedure of Example 5 but substituting
L_
i r r--rus~~rrrru*=~~;ucl;~i -ayZ~- Lls**II~ 34 Fmoc-Tyr(tBu)-OH (6.86 g) for Boc-MePhe-OH, 4.88 g of the free base of Fmoc-Tyr(tBu)-(4-oxymethylphenyl)acetic acid was obtained, mp 192-195 0
C.
EXAMPLE I 5 Fmoc-Met-Asp(OtBu)-OH Fmoc-Met-OSu was prepared, in situ, by the reaction of Fmoc-Met-OH (14.87 HOSu (5.52 and dicyclohexylcarbodiimide (DCC, 8.26 g) in THF (200 ml) at 0OC for 3.5 hrs. Precipitated dicyclohexylurea (DCU) was i removed by filtration and the THF filtrate was added to a cold solution of H-Asp (OtBu)-OH in 220 ml of 10:1 water/THF to which had been added 40 ml of N sodium hydroxide. After stirring the reaction mixture at room temperature overnight, solid citric acid (20 g) was added along with EtOAc (600 15 ml). The EtOAc layer was separated, washed with 10% citric acid and brine, and dried (magnesium sulfate). Evaporation of the EtOAc solution gave a residue which was dissolved in 200 ml of EtOAc and treated with DCHA (7.84 ml) to precipitate 17.93 g of the DCHA salt of the desired product, mp 159-162 0
C.
EXAMPLE 11 H-Phe-OCH2-Pam-resin Boc-Phe-(4-oxymethylphenyl)acetic acid (0.83 g, 2 mmole), l-hydroxybenzotriazole (HOBt, 0.46 g, 3 mmole) and DCC (0.41 g, 2 mmole) were dissolved in 50 ml of 4:1 DCM/DMF and stirred at 0°C for 1 hr. Aminomethyl-resin (1.34 g, 1 mmole l 'li-rL; 3r 1 Lt~L IC~-P C~--LIIC~ available nitrogen (was suspended in the filtered reaction mixture (precipitated DCU removed) and shaken for 2 to hours. The product, Boc-Phe-OCH 2 -Pam-resin, was isolated by filtration and treated according to Table 1 (Steps 10-14) to give the desired free base, H-Phe-OCHz-Pam-resin.
EXAMPLE 12 H-MePhe-OCH 2 -Pam-resin Boc-MePhe-(4-oxymethylphenyl)acetic acid (from 1.82 g, 3 mmole, of its DCHA salt, Example 5) and HOBt (0.69 g, 10 mmole) in 40 ml of 1:3 DMF/DCM followed by DCC (0.62 g, 3 mmole) in 20 ml of DCM were added to aminomethyl-resin (1.34 g, 1 mmole available nitrogen) to give a suspension Swhich was shaken for 2 to 15 hours. The desired product, Boc-MePhe-OCH 2 -Pam-resin, was isolated by filtration, .15 washed with 2-propanol and DCM, and treated according to Table 1 (Steps 10-14) to give the desired free base, H-MePhe-OCH 2 -Pam-resin.
EXAMPLE 13 I H-EtPhe-OCH 2 -Pam-resin Following the procedure of Example 12 but substituting Boc- EtPhe-(4-oxymethylphenyl)acetic acid (from 1.87 g, 3 mmole, of its DCHA salt, Example 6) for Boc-MePhe-(4-oxymethylphenyl)acetic acid, H-EtPhe-OCH 2 -Pam-resin was obtained.
EXAMPLE 14 H-Phe(4-Cl)-OCH 2 -Pam-resin Following the procedure of Example 11 but substituting Boc- L 36 Phe(4-C1)-(4-oxymethylphenyl)acetic acid (0.90 g, 2 mmole, Example 7) for Boc-Phe-(4-oxymethylphenyl)acetic acid, H- Phe(4-C1)-OCH 2 -Pam-resin was obtained.
EXAMPLE H-Phe(4-N0 2
)-OCH
2 -Pam-resin Boc-Phe(4-NO2)-OH (1.39 g) was dissolved in 100 ml of methanol (MeOH) and adjusted to pH1 7 with the addition of N 4: cesium bicarbonate. The solution wa3 evaporated to dryness S with the residue being evaporated three more times with added DMF. The resultant dried cesium salt of Boc-Phe(4- ~N0 2 )-OH was dissolved in 60 ml of DMF and shaken with BrCH 2 Pam-resin (1 meq o4 Br) overnight. The desired product, 2
)-OCH
2 -Pam-resin, was isolated by filtration, washed with DCM, and treated according to Table 1 (Steps 15 10-14) to give the desired free base, H-Phe(4-N0 2
)-OCH
2 S Pam-resin.
EXAMPLE 16 H-Tyr(Me)-OCH 2 -Pam-resin Following the procedure of Example 11 but substituting Boc- Tyr(Me)-(4-oxymethylphenyl)acetic acid (0.87 g, 2 mmole, Example 8) for Boc-Phe-(4-oxymethylphenyl)acetic acid, H- Tyr(Me)-OCH 2 -Pam-resin was obtained.
EXAMPLE 17 H-Tyr(tBu)-OCH 2 -Pam-resin Fmoc-Tyr(tBu)-(4-oxymethylphenyl)acetic acid (1.82 g, 3 mmole, Example 1-hydroxybenzotriazole (HOBt, 0.69 g, 37 mmole), and DCC (0.62 g, 3 mmole) were dissolved in ml of 4:1 DCM/DMF and stirred at 0°C for 1 hr. Aminomethylresin (1.34 g, 1 mmole available nitrogen) was suspended in the filtered reaction mixture (precipitated DCU removed) and shaken for 2 to 15 hours. The product, Fmoc-Tyr(tBu)-OCH 2 -Pam-resin, was isolated by filtration I and treated according to Table 1 (Steps 16-20) to give Sthe desired free base, H-Tyr(tBu)-OCH 2 -Pam-resin.
EXAMPLE 18 .10 H-MeTyr(Me)-OCH 2 -Pam-resin H 4 Following the procedure of Example 15 but substituting Boc-MeTyr(Me)-OH (from 1.47 g of its DCHA salt) for Boc- Phe(N 2 H-MeTyr(Me)-OCH 2 -Pam-resin was obtained.
V* EXAMPLE 19 5 H-DAsp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled I* with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- SOH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Fmoc-DAsp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by Fmoc '20 removal Steps 16-20) to provide Fmoc-DAsp(OtBu)-Tyr-Met-Gly- I Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, J deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 198 mg of the ammonium salt of the title compound. Amino IL i 38 acid analysis following acid decomposition gave Asp 2.11 Tyr 1.04 Met 2.07 Gly 1.08 and Phe 1.04 J Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.35 (TLC herein refers to chromatography of the title ;,pound on Merck silica gel thin layer plates in the sol,. nt system chloroform-methanol-acetic acid-water, 6:3:1:1.
EXAMPLE iBuOCO-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled S' with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- SOH, Fmoc-Met-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to give H-Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin S. which was coupled with iBuOCO-OSu (Example 1) according to Table 1 (Steps 8-9) to give iBuOCO-Tyr(tBu)-Met-Gly-Trp-Met- S Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, i Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 206 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.06 Tyr 1.04 Met 2.04 Gly 1.06 and Phe 1.04 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid .~e 39 ester at 1050 cm- 1 TLC Rf 0.56.
EXAMPLE 21 H-Asp-DTyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-Oh, Fmoc-DTyr-OH (Example and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to give Boc-Asp(OtBu)-DTyr-Met-Gly-Trp- Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, depro- .10 tected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step to give 241 mg of the ammonium salt of the title compound.
15 Amino acid analysis following acid decomposition gave Asp 2.14 Tyr 1.06 Met 2.12 Gly 0.95 Phe 0.98 S and NH 3 1.30 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.32.
f EXAMPLE 22 H-Asp-Tyr(SO 3 H)-DMet-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-DMet-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-DMet-Glyi -i _e Trp-Met-Asp(OtBu)-Phe-OCH2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step to give 219 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 2.07 Tyr 1.02 Met 2.11 Gly 0.95 Phe 1.11 and NH 3 1.31 Infrared absorption spectrum showed 410 a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.37.
EXAMPLE 23 H-Asp-Tyr(SO 3 H)-DAhx-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled ".15 with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-DAhx-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-DAhx-Gly- Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step to give 248 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 2.11 Tyr 1.03 Ahx 0.97 Gly 0.99 Met 1.07 i
-I
___CI
41 and Phe 1.07 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.34.
EXAMPLE 24 H-Asp-Tyr(S03H)-Met-Sar-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -FPam-icsin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Sar- OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by 10 Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-Met- Sar-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step to give 240 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 1.93 Tyr 0.97 Met 2.00 Sar 1.03 Phe 1.02 and NH 3 1.54 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.32.
EXAMPLE H-Asp-Tyr(S0 3 H)-Met-Gly-MeTrp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-MeTrp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH aca a.
a a lL _I 42 cording to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)-Met- Gly-MeTrp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE I. according to Table 1 (Step 30) to give 310 mg of the ammonium salt of the title compound. Amino acid analysis following 10 acid decomposition gave Asp 2.03 Tyr 1.02 Met 1.95 Gly 0.93 and Phe 1.01 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.35.
EXAMPLE 26 15 H-Asp-Tyr(S0 3 H)-Met-Gly-Nal-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Nal-OH, Fmoc- Gly-OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc-Asp- (OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr- Met-Gly-Nal-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was Ssulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 260 mg of the ammonium salt of the title
I
43 a.
4 a compound. Amino acid analysis following acid decomposition gave Asp 1.97 Tyr 0.91 Met 2.13 Gly 1.09 Nal 0.76 and Phe 1.14 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.38.
EXAMPLE 27 H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-Met-DAsp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-DAsp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-Met-Gly- Trp-Met-DAsp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 15 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 121 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.03 Tyr 0.98 Met 2.05 Gly 1.07 and Phe 1.08 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.43.
EXAMPLE 28 H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asn-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled
L_
:i II )1 1 4.
r as *r at a t 4.
44 with Fmoc-Asn-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)- Met-Gly-Trp-Met-Asn-Phe-OCH2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16- 20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on LO Trisacryl M DEAE .'nd P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 30 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.02 Tyr 1.03 Met 1.93 Gly 1.01 and Phe 1.01 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.43.
EXAMPLE 29 H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (free base of Example Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)- OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-MePhe-
OCH
2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15,
S.,
a a.
04*sc L Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 243 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.14 Tyr 1.02 Met 2.04 Gly 1.07 and NH 3 1.87 Infra- .red absorption spectrum showed a strong peak typical of S a sulfuric acid ester at 1050 cm 1 TLC Rf 0.37.
EXAMPLE H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-MeTyr(Me)-N 2 H-MeTyr(Me)-OCH 2 -Pam-resin (Example 18) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (free base of Example Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp (OtBu)-Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-MeTyr(Me)-OCH 2 Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically P purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 100 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.06 Tyr 1.06 Met 1.98 and Gly 1.05 Infrared absorption spectrum showed 46 i a strong peak typical of a sulfuric acid ester at 1050 cm 1 Vi TLC Rf 0.45.
EXAMPLE 31 i H-Asp-Tyr(S03H)-Met-Gly-Trp-Met-Asp-Phe(4-NO2)-NH2 H-Phe(4-N0 2
)-OCH
2 -Pam-resin (Example 15) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc- Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-.
**10 Asp(OtBu)-Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe( 4 -NO2)-
OCH
2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically 15 purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 128 mg of the ammonium salt of the title compound. Amino acid analysis following Sacid decomposition gave Asp 1.98 Tyr 1.05 Met 1.92 S. and Gly 1.05(1). Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.37.
EXAMPLE 32 H-Asp-Tyr(S03H)-Met-Gly-Trp-Met-Asp-Phe( 4 -Cl)-NH 2 H-Phe(4-Cl)-OCH 2 -Pam-resin (Example 14) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc- L 47 Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)- Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe(4-Cl)-OCH2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 299 mg of the ammonium salt of the 0* 9 title compound. Amino acid analysis following acid decompo- S sition gave Asp 1.85 Tyr 1.02 Met 1.78 and Gly 0.92 Infrared absorption spectrum showed a strong peak 4.
typical of a sulfuric acid ester at 1050 cm t TLC Rf 0.36.
EXAMPLE 33 H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe(4-NH 2
)-NH
2 H-Asp-Tyr(S03H)-Met-Gly-Trp-Met-Asp-Phe(4-NO 2 )-NH2 (64 mg, S* Example 31) was dissolved in 85% acetic acid (20 ml) and treated with zinc dust (69 mg) with stirring. After 30 min, the filtered reaction mixture was evaporated to dryness and the residue was chromatographically purified on P-40 ODS-3 according to Table 1 (Step 30) to give 30 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.92 Tyr 0.98 Met 1.87 and Gly 1.00 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.35.
48 EXAMPLE 34 H-Asp-Tyr (SO 3 i,)Met-Gly-Trp-Met-Asp-Tyr(Me)-a{2 H-Tyr(Me)-OCH- 2 -Pam-resil (Example 16) was sequentially coupled with Fmoc-Asp(OtBu)-Oi, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc- Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc- Asp(OtBu)-Tyr(tBu)-Met-Gly-Trp-MetAsp(OtBu)-Tyr(Me)- OCH,1-Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia). to give the title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, :9 sequentially, according to Table 1 (Step 30) to give 313 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.05 Tyr 1.98 Met 1.88 and Gly 1.09 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm TLC R f0.38.
*go EXAMPLE H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-Phe-NHMe H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Emoc- Gly-OH, Fmoc'-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)-Met- 49 Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with methylamine) to give the title compound which was chromatographically purified on Trisacryl M DEAE a-Tr--l- D-E and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 196 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.99 Tyr 1.00 Met 1.98 Gly, 1.00 and Phe 1.01 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.47.
EXAMPLE 36 H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-Phe-NHEt H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled S with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 S followed by Fmoc removal Steps 16-20) to provide Boc- Asp(OtBu)-Tyr-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ethylamine) to give the title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step to give 180 mg of the ammonium salt of the title compound.
50 Amino acid analysis following acid decomposition gave Asp 1.99 Tyr 0.84 Met 2.03 Gly 1.01 Phe 0.97 and NH3 1.14 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.49.
EXAMPLE 37 H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-N(Me) 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)- Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with dimethylamine) to give the title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 100 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.02 Tyr 1.01 Met 1.97 Gly 0.99 and Phe 1.01 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.35.
-^ewtf -51 EXAMPLE 38 H-Asp-Tyr(S03H)-Met-Gly-Trp-Met-Asp-Phe-N(Et) 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)- Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the 10 resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with diethylamine) to give the title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 102 mg of the 15 ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.16 Tyr 1.03 Met 1.92 Gly 1.00 and Phe 1.09 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm TLC Rf 0.38.
EXAMPLE 39 H-pAsp-DTyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, Fmoc-DTyr-OH (Example 2) and Boc-pAsp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to give Boc-pAsp(OtBu)-DTyr-Met-Gly-Trp- 52 Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected according to Table 1 (Steps 10-15) and coupled with Fmoc-OSu (1.1 g) according to Table 1 (Steps 8-9) to give Fmoc-pAsp-DTyr-Met- Gly-Trp-Met-Asp-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to gave 121 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.94 Tyr 1.02 Met 1.91 Gly 1.11 and Phe 1.01 Infrared absorption spectrum showed a strong peak typical of *a sulfuric acid ester at 1050 cm 1 TLC Rf 0.37.
15 EXAMPLE H-DAsp-DTyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH2-Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc- Gly-OH, Fmoc-Met-OH, Fmoc-DTyr-OH (Example 2) and Fmoc- DAsp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to give Fmoc- DAsp(OtBu)-DTyr-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pamresin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically .4
CI;_
53 purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, j according to Table 1 (Step 30) to give 100 mg of the ammonium salt of the citle compound. Amino acid analysis following Sacid decomposition gave Asp 2.08 Tyr 1.06 Met 1.83 Gly 1.05 nd Phe Infrared absorption spectrum showed a st.rqg .eak .tgpal of a sulfuric acid ester at 1050 cm- 1 TLC R.f 0:24. EXAMPLE 41 Suc-DTyr(SOsH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, and Fmoc-DTyr-OH (Example 2) according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to give H-DTyr-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 15 Pam-resin which was coupled with succinic anhydride (0.6 g, 6 mmole, in DMF) according to Table 1 (Steps 8-9) to give 4 Suc-DTyr-Met-Gly-Trp-Met-Asp(OtBu)-Phe-O 2-Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step to give 290 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.08 Tyr 1.04 Met 1.70 Gly 1.14 and Phe 1.03 Infrared absorption spectrum showed a
L-
54 :i strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.51.
EXAMPLE 42 H-Asp-Tyr(SO3H)-Ile-Gly-Trp-lle-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to give Fmoc-Asp (OtBu)-Tyr(tBu)-Ile- Gly-Trp-Ile-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin i according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 370 mg of the ammonium salt .of the title compound. Amino acid analysis following acid decomposition gave Asp 2.05 Tyr 1.00 lie 1.95 Gly 1.00 and Phe 1.00 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.33.
EXAMPLE 43 H-Asp-Tyr(S0 3 H)-Lys-Gly-Trp-Lys-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to give Fmoc-Asp(OtBu)- Tyr(tBu)-Lys(Boc)-Gly-Trp-Lys(Boc)-Asp(OtBu)-Phe-OCH2-Pamresin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and sea 6 ODS-3, sequentially, according to Table 1 (Step 30) to give 61 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.98 Tyr 1.01 Lys 2.00 Gly 0.99 and Phe 1.02 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC R 0.10.
"15 EXAMPLE 44 Hpp(S0 3 H)-Met-DAla-Trp-Met-Asp-Phe-NH 2 H1-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc- DAla-OH, and Fmoc-Met-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to give H-Met-DAla-Trp-Met-Asp(OtBu)-Phe-OCH2 -Pam-resin which was coupled with Hpp-OSu according to Table 1 (Steps 8-9) to give Hpp-Met-DAla-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was purified_ onAbrieXD2 rsarlMDAadP4
-II
56 chromatographically purified on Trisacryl M DEAE and ODS-3, sequentially, according to Table 1 (Step 30) to give 140 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.02(1), Met 1.97 Ala 0.98 and Phe 1.03 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm-1. TLC Rf 0.52.
EXAMPLE Suc-Tyr(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-Phe-NH 2 **10 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled S with Fmoc-Asp(OtBu)-OH, Fmoc-Ahx-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Ahx-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to give H-Tyr(tBu)-Ahx-Gly-Trp-Ahx-Asp(OtBu)-Phe-OCH 2 -Pam-
A.
resin which was coupled with succinic anhydride (0.6 g, 6 mmole, in DMF) according to Table 1 (Steps 8-9) to give Suc-Tyr(tBu)-Ahx-Gly-Trp-Ahx-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin
A.
according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 240 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.01 Tyr 0.95 Ahx 2.10 Gly 1.06 and Phe 0.88 Infrared absorption L
-L-LYI
57 spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.36.
EXAMPLE 46 H-pAsp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (Example 10), Fmoc-Trp-OH, Fmoc- Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Boc-pAsp(OtBu)- OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to give Boc-pAsp(OtBu)-Tyr(tBu)-Met-Gly- Trp-Met-Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was deprotected according to Table 1 (Steps 10-15) and coupled with Fmoc-OSu (1.1 g) according to Table 1 (Steps 8-9) to give Fmoc-pAsp- Tyr-Met-Gly-Trp-Met-Asp-MePhe-OCH2-Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Am e XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequential According to Table 1 (Step to give 72 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 2.03 Tyr 1.05 Met 1.85 and Gly 1.10 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.33.
EXAMPLE 47 H-DAsp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled i- 58 with Fmoc-Met-Asp(OtBu)-OH (Example 10), Fmoc-Trp-OH, Fmoc- I Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-DAsp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to give Fmoc-DAsp(OtBu)-Tyr(tBu)-Met- Gly-Trp-Met-Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl 1 M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 110 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.01 Tyr 1.01 Met 1.91 and Gly 1.07 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.35.
EXAMPLE 48 For-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (Example 10), Fmoc-Trp-OH, Fmoc- Gly-OH, and Fmoc-Met-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to give Fmoc- Met-Gly-Trp-Met-Asp(OtBu)-MePhe-OCH2-Pam-resin which was deprotected according to Table 1 (Steps 10-20) and coupled with For-Tyr-OH according to Table 1 (Steps 5-7) to give For-Tyr-Met-Gly-Trp-Met-Asp-MePhe-OCH 2 -Pam-resin which was sulfated and cleaved from the resin according to Table 1 i i ir and Gly 1.05 Infrared absorption spectrum showed LP~iii*iT;~ 59 (Steps 21-25 and then Steps 26-29 with ammonia) to give the title compound which was chronatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step 30') to give 78 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.02 Tyr 1.01 Met 1.95 and Gly 1.02 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm-1. TLC Rf 0.45.
.*10 EXAMPLE 49 iBuOCO-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (Example 10), Fmoc-Trp-OH, Fmoc- Gly-OH, Fmoc-Met-OH, and Fmoc-Tyr(tBu)-OH according to **15 Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to give U-Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-MePhe-
OCH
2 -Pam-resin which was coupled with iBuOCO-OSu (Example 1) according to Table 1 (Steps 8-9) to give iBuOCO-Tyr(tBu)- Met-Gly-Trp-Met-Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step to give 302 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp II I 60 9 1.03 Tyr 1.02 Met 1.91 and Gly 1.04 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.67.
EXAMPLE H-Asp-DTyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (Example 10), Fmoc-Trp-OH, Fmoc- Gly-OH, Fmoc-Met-OH, Fmoc-DTyr(tBu)-OH, and Fmoc-Asp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to give Fmoc-Asp(OtBu)-DTyr(tBu)-Met- Gly-Trp-Met-Asp(OtBu)-MePhe-OCH 2 -Pam which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE and P- 40 ODS-3, sequentially, according to Table 1 (Step 30) to give 29 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.02 Tyr 0.97 Met 1.93 and Gly 1.07 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.28.
15 baa..
a a EXAMPLE 51 Hpp(S0 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (Example 10), Fmoc-Trp-OH, Fmoc- Gly-OH, and Fmoc-Met-OH according to Table 1 (coupling I s 61 Steps 3-4 followed by Fmoc removal Steps 16-20) to give H-Met-Gly-Trp-Met-Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was coupled with 3-(4-hydroxyphenyl)propionic acid Nhydroxysuccinimide ester (Hpp-OSu) according to Table 1 (Steps 8-9) to give Hpp-Met-Gly-Trp-Met-Asp(OtBu)-MePhe-
OCH
2 -Pam-resin which was deprotected, sulfated, and cleaved i from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title S compound which was chromatographically purified on Trisacryl *10 M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 170 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.02 Met 1.97 and Gly 1.01 Infrared absorption spectrum showed a strong peak typical of a sulfu- I* '15 ric acid ester at 1050 cm- 1 TLC Rf 0.58.
I EXAMPLE 52 Hpp(SO 3 H)-Met-DAla-Trp-Met-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (Example 10), Fmoc-Trp-OH, Fmoc- S 20 DAla-OH, and Fmoc-Met-OH according to Table 1 (coupling j Steps 5-7 followed by Fmoc removal Steps 16-20) to give u H-Met-DAla-Trp-Met-Asp(OtBu)-MePhe-OCH2-Pam-resin which was coupled with Hpp-OSu according to Table 1 (Steps 8-9) to give Hpp-Met-DAla-Trp-Met-Asp(OtBu)-MePhe-OCH2-Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 k I 62 i i with ammonia) to give the title compound which was chromatographically purified on Amberlite ,XAD-2, Trisacryl M DEAE, Vi and P-40 ODS-3, sequentially, according to Table 1 (Step to give 84 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 0.99 Met 1.94 and Ala 1.08 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.49.
f EXAMPLE 53 i PrOCO-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled 1 with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 i '(coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to give H-Tyr-(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pami resin which was coupled with PrOCO-OSu (mp 31-34°C, prepared according to the procedure of Example 1 except that propyl Schloroformate was substituted for isobutyl chloroformate) according to Table 1 (Steps 8-9) to give PrOCO-Tyr(tBu)-Met- S 20 Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deproteci ted, sulfated, and cleaved from the resin according to Table S1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 270 mg of the ammonium salt of the title compound. Amino acid analysis 4 -r 63 following acid decomposition gave Asp 1.01 Tyr 1.03 Met 1.87 Gly 1.02 and Phe 1.06 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.45.
EXAMPLE 54 SEtOCO-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 S* 10 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) i to give H-Tyr-(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pamresin which was coupled with EtOCO-OSu (mp 52-54.50C, prepared according to the procedure of Example 1 except that ethyl chloroformate was substituted for isobutyl "15 chloroformate) according to Table 1 (Steps 8-9) to give EtOCO-Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pamresin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 J (Step 30) to give 300 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.02 Tyr 1.01 Met 1.91 Gly 1.02 and Phe 1.04 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 L .1 64 cm- 1 TLC Rf 0.44.
EXAMPLE MeOCO-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to give H-Tyr-(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe--OCH 2 -Pamresin which was coupled with MeOCO-OSu (mp 87-89 0 C, prepared according to the procedure of Example 1 except that methyl chloroformate was substituted for isobutyl chloroformate) according to Table 1 (Steps 8-9) to give MeOCO-Tyr(tBu)- Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and ODS-3, sequentially, according to Table 1 (Step 30) to give 270 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.04 Tyr 1.05 Met 1.82 Gly 1.03 and Phe 1.06 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm i. TLC Rf 0.44.
EXAMPLE 56 H-pAsp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2 The title compound has previously been prepared (Digestive 65 Diseases, 15, 149-156 (1970)). H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc- STyr-OH (Example and Boc-pAsp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) Sto provide Boc-pAsp(OtBu)-Tyr-Met-Gly-Trp-Met-Asp(OtBu)-Phe-
OCH
2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound '10 which was chromatographically purified on Trisacryl M DEAE i" according to Table 1 (Step 30) to give 283 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.03 Tyr 0.94 Met 2.08 Gly 0.99 and Phe 0.96 Infrared absorption 15 spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm-. TLC R 0.52.
*f EXAMPLE 57 H-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 .I The title compound has previously been prepared Patents 3,839,315 and 3,705,140). H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, and Boc-Tyr-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Tyr-Met-Gly-Trp-Met-Asp(OtBu)- Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, L i i i 4 4* 4* 9 4 66 Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 240 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.05 Tyr 1.04 Met 2.10 Gly 1.07 and Phe 1.08 Infrared absorption spectrum showed a strong S peak typical of a sulfuric acid ester at 1050 cm-1. TLC S Rf 0.55.
10 EXAMPLE 58 Suc-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 The title compound has previously been prepared (European Patent Application 0107860). H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc- Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, and Fmoc- Tyr-OH (Example 3) according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide H-Tyr-Met- Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was coupled with succinic anhydride in DMF according to Table 1 (Steps 8-9) to give Suc-Tyr-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 246 mg of the ammonium salt of the title compound. Amino acid I ,e -Wxrs i~w~- 67r analysis following acid decomposition gave Asp 1.03 Tyr 0.95 Met 2.08 Gly 0.98 and Phe 0.96 i Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm TLC Rf 0.54.
EXAMPLE 59 Hpp(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 The title compound has previously been prepared (Int. J.
Peptide Protein Res., 16, 402-411 (1980)). H-Phe-OCH 2 -Pami resin (Example 11) was sequentially coupled with Fmoc-Asp :i *10 (OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, and Fmoc- Met-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide H-Met-Gly-Trp-Met-Asp (OtBu)-Phe-OCH 2 -Pam-resin which was coupled with Hpp-OSu in i* DMF according to Table 1 (Step 8-9) to give Hpp-Met-Gly-Trp- S 15 Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give mg of the ammonium salt of the title compound. Amino acid i analysis following acid decomposition gave Asp 1.04 Met y 2.04 Gly 0.95 and Phe 1.00 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.47.
EXAMPLE H-Asp-Tyr(SO 3 H)-Ahx-Gly-Trp-Met-Asp-Phe-NH 2 _1 68 The title compound has previously been prepared Patent 3,892,726). H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Finoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc- Gly-OH, Fmoc-Ahx-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)-Ahx- Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and S .'o40 then Steps 26-29 with ammonia) to give the title compound *i which was chromatographically purified on Trisacryl M DEAE i and P-40 ODS-3, sequentially, according to Table 1 (Step S to give 188 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp I .'15 1.97 Tyr 0.99 Ahx 1.06 Gly 1.07 Met 0.93 and Phe 0.98 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm TLC Rf 0.57.
EXAMPLE 61 H-Asp-Tyr(S0 3 H)-Met-Gly-DTrp-Met-Asp-Phe-NH 2 1 The title compound has previously been prepared Patent 3,892,726). H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-DTrp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc- Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-Met- 69 Gly-DTrp-Met-Asp(OtBu)-Phe-OCH2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step to give 314 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 2.07 Tyr 1.01 Met 2.01 Gly 1.06 and Phe 0.98 Infrared absorption spectrum showed a strong peak 10 typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.30.
EXAMPLE 62 H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-Ahx-Asp-Phe-NH 2 The title compound has previously been prepared Patent 3,892,726). H-Phe-OCH 2 -Pam-resin (Example 11) was sequen- **0"15 tially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Ahx-OH, Fmoc-Trp- OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp S (OtBu)-Tyr-Met-Gly-Trp-Ahx-Asp(OtBu)-Phe-OCH2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26- 29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 87 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.11 Tyr 1.00 Met 1.11 Gly iwsawi 70 1.02 Ahx 1.04 and Phe 0.99 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.23.
EXAMPLE 63 H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-DPhe-NH 2 The title compound has previously been prepared Patent 3,892,726). H-DPhe-OCH 2 -Pam-resin {prepared in an analogous fashion as H-Phe-OCH 2 -Pam-resin [Example 11, Boc-Phe-(4-oxymethylphenyl)acetic acid replaced with Boc-DPhe-(4-oxymethyl- *410 phenyl)acetic acid] from Boc-DPhe-(4-oxymethylphenyl)acetic S" acid [Example 5, Boc-MePhe-OH replaced with Boc-DPhe-OH]} was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example a: and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp (OtBu)-Tyr-Met-Gly-Trp-Met-Asp(OtBu)-DPhe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26a.
29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 141 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.97 Tyr 0.98 Met 2.03 Gly 1.09 and Phe 1.05 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm TLC Rf 0.32.
i I i 1 -rr- l- r 71 94 4 4 4 .9 4 EXAMPLE 64 H-Asp-Tyr(SO 3 H)-MetO-Gly-Trp-MetO-Asp-Phe-NH 2 The title compound has previously been prepared (Ncbel Symp., 16, (Front. Gastrointest. Horm. Res.), 41-56 (1973)). H- Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-MetO-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-MetO-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-MetO-Gly- 10 Trp-MetO-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step to give 162 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 2.09 Tyr 1.00 MetO 1.83 Gly 1.08 and Phe 1.08 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1
TLC
Rf 0.14.
EXAMPLE H-Asp-Tyr(S0 3 H)-Ahx-Gly-Trp-Ahx-Asp-Phe-NH 2 The title compound has previously been prepared Patent 3,892,726). H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Ahx-OH, Fmoc-Trp- OH, Fmoc-Gly-OH, Fmoc-Ahx-OH, Fmoc-Tyr-OH (Example and *r 9 4 4 4 4. O 4 9 4 4tJ~
L
_1 ii~3~1-Y~.- 72 Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp (OtBu)-Tyr-Ahx-Gly-Trp-Ahx-Asp(OtBu)-Phe-OCH2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26- 29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 94 mg of the ammonium salt of the r i'O* title compound. Amino acid analysis following acid decompo- 10 sition gave Asp 2.18 Tyr 1.06 Ahx 2.01 Gly 1 1.10 and Phe 0.94 Infrared absorption spectrum
C.
showed a strong peak typical of a sulfuric acid ester at 1050 cm-. TLC Rf 0.32.
i EXAMPLE 66 S" 15 H-Asp-Tyr(SO3H)-Leu-Gly-Trp-Leu-Asp-Phe-NH 2 The title compound has previously been prepared (Digestive S. Diseases, 15, 149-156 (1970)). H-Phe-OCH 2 -Pam-resin (Example S 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, FmocaI Leu-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)- OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)-Leu-Gly-Trp-Leu-Asp(OtBu)-Phe-OCH 2 Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically 73 purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 150 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.02 Tyr 0.99 Leu 2.02 Gly 0.98 and Phe 0.98 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.35.
EXAMPLE 67 For-Tyr(SOsH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 The title compound has previously been prepared Patent i 3,705,140). H-Phe-OCHe-Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, and Fmoc-Met-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide :15 H-Met-Gly-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected according to Table 1 (Steps 10-15) to provide H-Met-Gly-Trp-Met-Asp-Phe-OCH 2 -Pam-resin which was coupled with For-Tyr-OH according to Table 1 (Step 3-4) to give For- Tyr-Met-Gly-Trp-Met-Asp-Phe-OCH 2 -Pam-resin which was sulfated and cleaved from the resin according to Table 1 (Steps 21-25 and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step to give 30 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 1.00 Met 1.98 Gly 1.07 and Phe 1.00 74 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm-1. TLC Rf 0.37.
EXAMPLE 68 H-Asp-Tyr(SO 3 H)-MetO-Gly-Trp-Met-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly- S OH, Fmoc-MetO-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-MetO-Gly- 0 Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically i "'purified on Trisacryl M DEAE according to Table 1 (Step 15 to give 230 mg of the ammonium salt of.the title compound.
Infrared absorption spectrum showed a strong peak typical S. of a sulfuric acid ester at 1050 cm 1 TLC R 0.26.
S.9 EXAMPLE 69 H-Asp-Tyr(S0 3 H)-Met-DAla-Trp-Met-Asp-Phe-NH 2 The title compound has previously been prepared (Peptides 1984, 383-385 (1984)). H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-DAla-OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide Boc-Asp (OtBu)-Tyr-Met-DAla-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin L .i I- 75 which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE and Amberlite XAD-2, sequentially, according to Table 1 (Step to give 140 mg of the ammonium salt of the title compound.
I Amino acid analysis following acid decomposition gave Asp 1.93 Tyr 0.94 Met 1.81 Ala 0.97 and Phe 0.95 i Infrared absorption spectrum showed a strong peak typical of 10 a sulfuric acid ester at 1050 cm- 1 TLC R 0.34.
EXAMPLE H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-DMet-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-DMet-OH, Fmoc-Trp-OH, Fmoc-Gly- :15 OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by Fmoc S* removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-Met-Gly- Trp-DMet-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 (Step 30) to give 368 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.90 Tyr 0.99 Met 1.97 Gly 0.92 and Phe 0.98 Infrared absorption spectrum showed a _e l ll I -76strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.31.
EXAMPLE 71 H-Asp-Tyr(SO 3 H)-Met-Gly-Trp-MetO-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-MetO-OH, Fmoc-Trp-OH, Fmoc-Gly- OH, Fmoc-Met-OH, Fmoc-Tyr-OH (Example and Boc-Asp(OtBu)- OH according to Table 1 (coupling Steps 3-4 followed by Fmoc I removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-Met-Gly- Trp-MetO-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Trisacryl M DEAE according to Table 1 15 (Step 30) to give 126 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.03 Tyr 0.96 Met 0.98 Gly 1.10 S MetO 0.97 and Phe 0.96 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm-. TLC R 0.29.
EXAMPLE 72 H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-Phe(4-Me)-NH 2 H-Phe(4-Me)-OCH 2 -Pam-resin {prepared in an analogous fashion as H-Phe-OCH 2 -Pam-resin [Example 11, Boc-Phe-(4-oxymethylphenyl)-acetic acid replaced with Boc-Phe(4-Me)-(4-oxymethylphenyl)acetic acid] from Boc-Phe(4-Me)-(4-oxymethylphenyl) 77 acetic acid [Example 5, Boc-MePhe-OH replaced with Boc-Phe(4- Me)-OH]} was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc- Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH. according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide Fmoc-Asp(OtBu)-Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)- Phe(4-Me)-OCH 2 -Pam-resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 :10 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step to give 420 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Asp 2.02 Tyr 1.00 Met 1.91 Gly 1.06 and Phe S (4-Me) 1.12 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.44.
EXAMPLE 73 H-Asp-Tyr(S0 3 H)-Met-Gly-Trp-Met-Asp-Tyr-NH 2 H-Tyr(tBu)-OCH 2 -Pam-resin {prepared in an analogous fashion j as H-Phe-OCH 2 -Pam-resin [Example 11, Boc-Phe-(4-oxymethylphenyl)-acetic acid replaced with Fmoc-Tyr(tBu)-(4-oxymethyloc phenyl)-acetic acid] from eB-Tyr(tBu)-(4-oxymethylphenyl) acetic acid [Example 5, Boc-MePhe-OH replaced with Fmoc-Tyr (tBu)-OH]} was sequentially coupled with Fmoc-Asp(OtBu)-OH, N L I I_ 78 a 5* *r S.
Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc- Tyr-OH (Example and Boc-Asp(OtBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide Boc-Asp(OtBu)-Tyr-Met-Gly-Trp-Met-Asp(OtBu)-Tyr (tBu)-OCH 2 -Pam-resin which was sulfated, deprotected, and cleaved from the resin according to Table 1 (Steps 21-25, Steps 10-15, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 42 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 2.09 Tyr 2.16 Met 1.66 and Gly 1.09 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid 15 ester at 1050 cm- 1 TLC Rf 0.29.
EXAMPLE 74 Hpp(S0 3 H)-Met-Gly-Trp-Pro-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, and Fmoc-Met-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Met-Gly- Trp-Pro-Asp-(OtBu)-Phe-OCH 2 -Pam-resin which was coupled with Hpp-OSu in DMF according to Table 1 (Steps 8-9) to give Hpp- Met-Gly-Trp-Pro-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 t* _I r 79 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step to give 70 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Met 1.01 Gly 1.04 Pro 0.97 Asp (1.04) S: .o and Phe 0.94 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 rLC Rf 0.51.
i 10 EXAMPLE *ii Hpp(S03H)-Pro-Gly-Trp-Pro-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, and Fmoc-Pro-OH according to Table 1 (coupling Steps 5-7 S ,15 followed by Fmoc removal Steps 16-20) to provide H-Pro-Gly- Trp-Pro-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was coupled with i Hpp-OSu in DMF according to Table 1 (Steps 8-9) to give Hpp- Pro-Gly-Trp-Pro-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and ODS-3, sequentially, according to Table 1 (Step to give 480 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Pro 2.02 Gly 1.05 Asp 1.00 and Phe 0.93 i -I i jl 80 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm-1. TLC Rf 0.44.
EXAMPLE 76 Suc-Tyr(S03H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Ahx-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced with Fmoc-Ahx-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ahx-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Tyr (tBu)-Ahx-Gly-Trp-Ahx-Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was coupled with succinic anhydride in DMF according to 1 Table 1 (Steps 8-9) to give Suc-Tyr(tBu)-Ahx-Gly-Trp-Ahx- Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was deprotected, sul- 99 I fated, and cleaved from the resin according to Table 1 :15 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographi- I cally purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to Sgive 100 mg of the ammonium salt of the title compound.
Amino acid analysis following acid decomposition gave Tyr S0.97 Gly 1.00 and Asp 1.00 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.46.
EXAMPLE 77 iBuOCO-Tyr(S0 3 H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled i i i ii -I-C-UI~I-il-- 1- 1111- 111 S81 with Fmoc-Ahx-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced with Fmoc-Ahx-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ahx-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Tyr (tBu)-Ahx-Gly-Trp-Ahx-Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was coupled with iBuOCO-OSu (Example 1) in DMF according to Table 1 (Steps 8-9) to give iBuOCO-Tyr(tBu)-Ahx-Gly-Trp-Ahx- Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified or Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 290 mg of the ammonium salt of the title compound. Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.54.
EXAMPLE 78 Hpp(SO 3 H)-Ahx-Gly-Trp-Ahx-Asp-MePhe-NH 2 S H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Ahx-Asp(OtBu)-OH (Example 10, Fmoc-Me- H replaced with Fmoc-Ahx-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, and Fmoc-Ahx-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Ahx-Gly-Trp-Ahx-Asp(OtBu)- MePhe-OCH 2 -Pam-resin which was coupled with Hpp-OSu in DMF according to Table 1 (Steps 8-9) to give Hpp-Ahx-Gly-Trp-Ahx- Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was deprotected, sulfated, 82 and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2, Trisacryl M DEAE, and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 91 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Ahx 1.92 Gly 1.03 and Asp i. 1.05 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.40.
EXAMPLE 79 iBuOCO-Tyr(S0 3 H)-Ahx-Gly-Trp-Ahx-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Asp(OtBu)-OH, Fmoc-Ahx-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ahx-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Tyr(tBu)-Ahx-Gly-Trp-Ahx-Asp(OtBu)-Phe-OCH 2 -Pamresin which was coupled with iBuOCO-OSu (Example 1) in DMF 0 according to Table 1 (Steps 8-9) to give iBuOCO-Tyr(tBu)-Ahx- Gly-Trp-Ahx-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 800 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Tyr 0.90 Ahx 1.84 Gly
L__
83 1.12(1), Asp 1.12 and Phe 0.92 Infrared absorp- I tion spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.58.
EXAMPLE Hpp(Sa3H)-Ahx-Gly-Trp-Ahx-Asp-Phe-NH2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled :i with Fmoc-Asp(OtBu)-OH, Fmoc-Ahx-OH, Fmoc-Trp-OH, Fmoc-Glyi t OH, and Fmoc-Ahx-OH according to Table 1 (coupling Steps 5-7 4.i followed by Fmoc removal Steps 16-20) to provide H-Ahx-Gly- :10 Trp-Ahx-Asp-(OtBu)-Phe-OCH 2 -Pam-resin which was coupled with Hpp-OSu in DMF according to Table 1 (Step 8-9) to give Hpp-Ahx-Gly-Trp-Ahx-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then S 15 Steps 26-29 with ammonia) to give the title compound which .*Sag.
as chromatographically purified on Amberlite XAD-2 and ODS-3, sequentially, according to Table 1 (Step 30) to give 450 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Ahx 1.92 Gly 1.01(1), Asp 1.10(1), and Phe 0.97(1). Infrared j absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.47.
EXAMPLE 81 Suc-Tyr(SO3H)-Met-DAla-Trp-Met-Asp-Phe-NH 2 The title compound has previously been prepared (Peptides 1984, 373-378 (1984)). H-Phe-OCH 2 -Pam-resin (Example 11) IL _I I Irr~marrrzruuu~a~ 84 was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (Example 10), Fmoc-Trp-OH, Fmoc-DAla-OH, Fmoc-Met-OH, and Fmoc-Tyr(tBu)-OH according to Table 3 (coupling Steps 3-4 followed by Fmoc removal Steps 16-20) to provide H-Tyr (tBu)-Met-DAla-Trp-Met-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was coupled with succinic anhydride in DMF according to Table 1 (Steps 8-9) to give Suc-Tyr(tBu)-Met-DAla-Trp-Met- S Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, 10 Steps 21-25, and then Steps 26-29 with ammonia) to give the b* a title compound which was chromatographically purified on Trisacryl M DEAE and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 110 mg of the ammonium salt of the title compound. Amino acid analysis following acid decompo- 15 sition gave Tyr 1.02 Met 1.93 Ala 1.00 Asp a 1.05 and Phe 1.01 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at S 1050 cm 1 TLC Rf 0.38.
EXAMPLE 82 For-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Ile-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, and Fmoc-Ile-OH according to Table 1 (coupling Steps 5-7 followed by Fmc removal Steps 16-20) to provide H-Ile-Gly-Trp-Ile-Asp(OtBu)- Phe-OCH 2 -Pam-resin which was deprotected according to Table 85 1 (Steps 10-15) to provide H-Ile-Gly-Trp-Ile-Asp-Phe-OCH 2 Pam-resin which was coupled with For-Tyr-OH according to Table 1 (Steps 3-4) to give For-Tyr-Ile-Gly-Trp-Ile-Asp-Phe-OCH 2 Pam-resin which was sulfated and cleaved from the resin according to Table 1 (Steps 21-25 and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 200 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Tyr 0.98 Ile 1.94 Gly 1.00 Asp 1.09 and Phe 0.98 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.59.
EXAMPLE 83 ;15 Suc-Tyr(SOaH)-Ile-Gly-Trp-Ile-Asp-Phe-NH 2 H-Phe-OCH,-Pam-resin (Example 11) was sequentially coupled with Fmoc-Ile-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced 6* with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Tyr(tBu)-Ile- Gly-Trp-Ile-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was coupled with succinic anhydride in DMF according to Table 1 (Steps 8-9) to give Suc-Tyr(tBu)-Ile-Gly-Trp-Ile-Asp(OtBu)-Phe-OCH 2 -Pamresin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was 86 chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 300 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Tyr 1.00 Ile 2.07 Gly 0.97 Asp 0.97 and Phe 0.99 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.42.
EXAMPLE 84 S* iBuOCO-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-Phe-NH 2 '10 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled Swith Fmoc-Ile-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced Swith Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Tyr(tBu)- Ile-Gly-Trp-I.e-Asp(OtBu)-Phe-OCH 2 -Pam-resin which was coupled with iBuOCO-OSu (Example 1) in DMF according to Table 1 (Steps S 8-9) to give iBuOCO-Tyr(tBu)-Ile-Gly-Trp-Ile-Asp(OtBu)-Phe-
OCH
2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and ODS-3, sequentially, according to Table 1 (Step 30) to give 390 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Tyr 1.07 Ile 2.21 Gly 1.04 Asp 1.00 and Phe 1.03 Infrared absorption spectrum showed a strong peak typical of sulfuric acid ester at 1050 cm TLC R 0.61.
sulfuric acid ester at 1050 cm TLC Rf 0.61.
S.
o 0 0**041* 0* *0 0 S 4 0 S 00 87 EXAMPLE 8 Hpp(SO 3 H) -Ile-Gly-Trp-Ile-Asp-Phe-NH 2 H-Phe-OCH 2 -Pam-resin (Example 11) was sequentially coupled with Fmoc-Ile-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, and Fmoc-lle-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc 9 000 ft.
0 0*0 *00 0S 5 *4~ [1I 9 9 9 9 *r 9 *1 9 9* r ii oooo9 .69* o* :i 9 88 removal Steps 16-20) to provide H-Ile-Gly-Trp-Ile-Asp(OtBu)- Phe-OCH 2 -Pam-resin which was coupled with Hpp-OSu in DMF according to Table 1 (Step 8-9) to give Hpp-Ile-Gly-Trp-Ile- Asp(OtBu)-Phe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 280 mg of the ammonium salt of the title 10 compound. Amino acid analysis following acid decomposition gave Ile 2.04 Gly 0.99 Asp 0.98 and Phe 0.99 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.52.
EXAMPLE 86 15 Suc-Tyr(SO 3 H)-Met-Gly-Trp-Met-Asp-MePhe-NH 2 The title compound has previously been prepared Yanaihara, et al., in Peptides 1984: Proceedings of the 18th European Peptide Symposium, U. Ragnarsson, Ed., Almqvist and Wiksell International, Publisher, Stockholm, Sweden, 1985, pp 373-378). H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Met-Asp(OtBu)-OH (Example Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Tyr(tBu)-Met-Gly-Trp-Met- Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was coupled with succinic anhydride in DMF according to Table 1 (Steps 8-9) _r -89to give Suc-Tyr(tBu)-Met-Gly-Trp-Met-Asp(OtBu)-MePhe-OCH 2 -Pamresin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 170 mg of -he ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Tyr 1.02 Met 1.94 Gly 1.09 Asp 1.00 and MePhe 1.02 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.58.
i EXAMPLE 87 S* H-DAsp-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH S2 i;f H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled I with Fmoc-Ile-Asp(OtBu)-Oh (Example 10, Fmoc-Met-OH replaced Vi with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc- Tyr(tBu)-OH, and Fmoc-DAsp(OtBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide Fmoc-DAsp(OtBu)-Tyr(tBu)-Ile-Gly-Trp-Ile-Asp(OtBu)- T MePhe-OCH 2 -Pam-resin which was deprotected, sulfated, I deprotected, and cleaved from the resin according to Table 1 0 0 j (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromatoii graphically purified on Amberlite XAD-2 and P-40 ODS-3, Ssequentially, according to Table 1 (Step 30) to give 30 mg of Ithe ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Asp 1.96 Tyr j 0.97 Ile 1.92 Gly 0.98 and MePhe 1.18 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 ctzf 1 TLC Rf 0.36.
EXAMPLE 88 For-Tyr(S0 3 H)-Ile-Gly-Trp-le-Asp-MePhe-'H 2 H-tMePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Ile-Asp(OtBu)-OH (Example 10, Fmoc-Met--OH replaced with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH and Fmoc-Ile-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Ile-Gly-Trp.-Ile-Asp(OtBu)a MePhe-OCH 2 -Pam-resin which was deprotected according to Table 1 (Steps 10-15) to provide H-Ile-Gly-Trp-Ile-Asp-MePhe-OCH 2 Pam-resin which was coupled with For-Tyr-O- according to Table 1 (coupling Steps 3-4) to give For-Tyr-Ile-Gly-Trp-Ile-Asp- MePhe-0CH- 2 -Pam-resin which was sulfated and cleaved from the resin according to Table 1 (Steps 21-25 and then Steps 26-29 *.with ammonia) to give'the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS'-3, sequentially, according to Table 1 (Step 30) to give 90 mg cf the ammnonium salt of the title compound. Amino acid analysis following acid decomposition gave Tyr 1.04 Ile 1.95 Gly 0.97 Asp 1.01 and MePhe 1.03 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 TLC R f 0.60.
-91 EXAMPLE 89 Suc-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Ile-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provide H-Tyr(tBu)- Ile-Gly-Trp-Ile-Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was coupled with succinic anhydride in DMF according to Table 1 (Steps 8-9) to give Suc-Tyr(tBu)-Ile-Gly-Trp-Ile-Asp(OtBu)- MePhe-OC.H 2 -Pam-resin which was deprotected, sulfated, and S.q. cleaved -from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) to give the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 130 mg of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Tyr 0.94 Ile 1.92 Gly 1.04 Asp 1.09 and MePhe 1.00 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm-1. TLC R 0.37.
f EXAMPLE iBuOCO-Tyr(SOH)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Ile-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and i -e -92- A, Fmoc-Tyr(tBu)-OH according to Table 1 (coupling Steps follwedby mocremoval Steps 16-20) to provide H-Tyr(tBu)- Ile-Gly-Trp-Ile-Asp(OtBu)-MePhe-OCH2 -Pain-resin which was coupled with iBuOCO-OSu (Example 1) in DMF according to Table 1~ (Steps 8-9) to give iBuOCO-Tyr(tBu)-Ile-Gly-Trp-Ile-Asp- (OtBu)-MePhe-OCH 2 -Pam-resin which was deprotected, sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with amnmonia) to give the title compound which was chromatographically purified on Amberlite XA.D-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 50 mng of the ammonium salt of the title compound. Amino acid analysis following acid decomposition gave Tyr 1.00 Ile 1,92 Gly 1.02 Asp 1.03 and MePhe 1.03 Infrared absorption spectrum -15 showed~a strong peak typical of a sulfuric acid ester at 1050 *cm-1. TLC R f 0.74.
EXAMPLE 91 H-Asp-DTyr(S0 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-N-H 2 H-MePhe-OCH- 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Ile-Asp(OtBia)-OH (Example 10, Fmoc-Met-OH replaced with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-DTyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH according to Table 1 (coupling Steps 5-7 followed by Fmoc removal Steps 16-20) to provid e Fmoc-Asp(otBu)-DTyr(tBu)-Ile-Gly-Trp-le-Asp(otBu).
MePhe-OCB 2 Pam-Resin which was deprotected, sulfated, deprotected, and cleaved from the resin according to Table 1 93 (Steps 10-15, Steps 21-25, Steps 16-20, and then Steps 26-29 with ammonia) to give the title compound which was chromato- I graphically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 200 mg of the ammonium salt of the title compound, Amino acid analysis following acid decomposition gave Asp 2.08 Tyr 1.01 Ile 1.94 Gly 0.98 and MePhe 0.99 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm 1 TLC Rf 0.38.
a 10 EXAMPLE 9 2 Hpp(SO 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe-NH 2 S H-MePhe-OCH 2 -Pam-resin (Example 12) was sequentially coupled with Fmoc-Ile-Asp(OtBu)-OH (Example 10, Fmoc-Met-OH replaced with Fmoc-Ile-OH), Fmoc-Trp-OH, Fmoc-Gly-OH and Fmoc-Ile-OH .15 according to Table 1 (coupling Steps 5-7 followed by Fmoc S removal Steps 16-20) to provide H-Ile-Gly-Trp-Ile-Asp(OtBu)- MePhe-OCH 2 -Pam-resin which was coupled with Hpp-OSu according I to Table 1 (coupling Steps 8-9) to give Hpp-Ile-Gly-Trp-Ile- Asp(OtBu)-MePhe-OCH 2 -Pam-resin which was deprotected, S 20 sulfated, and cleaved from the resin according to Table 1 (Steps 10-15, Steps 21-25, and then Steps 26-29 with ammonia) i to give the title compound which was chromatographically purified on Amberlite XAD-2 and P-40 ODS-3, sequentially, according to Table 1 (Step 30) to give 70 mg of the amnonium salt of the title compound. Amino acid analysis following acid decomposition gave lie 1.94 Gly 1.01 Asp 1,02 94 and MePhe 1.04 Infrared absorption spectrum showed a strong peak typical of a sulfuric acid ester at 1050 cm- 1 TLC Rf 0.63.
~f 4t 1: 0 0

Claims (7)

1. The MePhe-NH 2
2. The NH 2
3. The NH 2
4. The MePhe-NH 2
5. The NH 2
6. The MePhe-NH 2 defining the invention are as follows: peptide H-DAsp-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp- peptide For-Tyr(SO 3 H)-Ile-Gly-Trp-I'ie-Asp-MePhe- peptide Suc-Tyr(S0 3 H)-Ile-Gly-Trp-Ile-Asp-MePhe- peptide iBuOCO-Tyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp- peptide Hpp(SO0 3 H) -Ile-Gly-Trp-Ile-Asp-MePhe- peptide H-Asp-DTyr(SO 3 H)-Ile-Gly-Trp-Ile-Asp- 0 *0 00 00.0.0 0 0 *090 0000 0 0 00 0
7. A method of preventing or treating obesity comprising the administration by either an intraperitoneal, intravenous, intramuscular, subcutaneous or intranasal route, to a mammal in need of such treatment, an effective amount of the peptide of any of claims 1 to 6. DATED: 10 September 1990 PHILLIPS ORMONDE FITZPATRICK Attorneys For: FISONS CORPORATION A 0000 0 0 0 0 L0 0000KC
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Publication number Priority date Publication date Assignee Title
AU596388B2 (en) * 1985-12-19 1990-05-03 Fisons Corporation Peptides with sulfate ester groups

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* Cited by examiner, † Cited by third party
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