Note: Descriptions are shown in the official language in which they were submitted.
2 ~
IA576
BICYCLIC ENDOT~IELIN ANALOGUES
This invention relates to polypeptide
endothelin (ET) analogues useful as ET receptor
antagonists, and in particular to bicyclic ET
analogues.
Compounds of formula I (see within) are
endothelin receptor agonists and antagonists
useful, inter alia, as hypotensive and hypertensive
agents. Throughout this specification, the symbols
in formula I are defined as follows:
R1 and R2 are each independently hydrogen
or lower alkyl, or R1 and R2 together with the
carbon atom to which they are attached are
cycloalkyl;
R3 and R4 are each independently hydrogen
or lower alkyl, or R3 and R~ together with the
carbon atom to which they are attached are
cycloalkyl;
R5 and R6 are each independently hydrogen
or lower alkyl, or R5 and R6 together with the
carbon atom to which they are attached are
cycloalkyl;
-2- HA576
R7 and R8 are each independently hydro~en
or lower alkyl, or R7 and R8 together with the
carbon atom to which they are attached are
cycloalkyl;
provided that at least one of R1 to R8 is
other than hydrogen;
A2, A4, A5, A6 and A7 are each independently
amino acid residues;
A8 is an aliphatic, aromatic, acidic, or
polar amino acid residue;
A9 is an amino acid residue;
A10 is an aliphatic, aromatic, D- or
L-acidic, or polar amino acid residue;
Al2 is an amino acid residue;
Al 3 iS an aliphatic, aromatic or polar
amino acid residue;
Al 4 iS an aromatic or aliphatic amino acid
residue;
A1 6 iS an amino acid residue;
Al7 is an aliphatic or polar amino acid
residue;
Al 8 iS an acidic, polar, aliphatic or
aromatic amino acid residue;
Al 9 and A20 are each independently aliphatic
amino acid residues; and
A21 is an aromatic amino acid residue;
The following moieties are preferred for
the foregoing symbols:
at least one of R1 and R2 is lower alkyl or
Rl and R2 together with the carbon atom to which
they are attached are cycloalkyl; R3 and R4 are
hydrogen; at least one of R5 and R6 is lower alkyl,
_~_ HA576 ~ ~ ~2~t-~
or R5 and R6 together with the carbon atom to
which they are attached are cycloalkyl; and R7 and
R8 are hydrogen; or
R1 and R2 are hydrogen; at least one of R3
and R4 is lower alkyl or R3 and R4 ~ogether with
the carbon atom to which they are attached are
cycloalkyl; R5 and R6 are hydrogen; and at least
one of R7 and R8 is lower alkyl or R7 and R8
together with the carbon atom to which they are
attached are cycloalkyl.
Methyl is the most preferred alkyl moiety
for Rl to R8 and cyclopropyl is the most preferred
cycloalkyl moiety.
Preferred is the compound of formula I
wherein:
A7 is norleucyl, methionyl, leucyl, lysyl,
seryl, or threonyli
A3 is aspartyl;
A10 is L-glutamyl, D-glutamyl, glutaminyl,
or alanyl;
Al 3 is tyrosyl, phenylalanyl, or
asparaginyl;
Al 4 iS tyrosyl or phenylalanyl;
`A17 is leucyl or glutaminyl;
A1 8 is aspartyl, asparaginyl, alanyl, or
leucyl;
A19 is isoleucyl or valyl;
A20 is isoleucyl;
A21 is tryptophyl or napthylalanyl; and
_4_ HA576 ~7~6~
A2 A4 A5, A6, A9, ~12 and A1 6 ar~
each independently amino acid residues.
Most preferred are compounds of formula I
wherein:
A2 is seryl or threonyl;
A~ is seryl, phenylalanyl, asparaginyl, or
lysyl;
A5 is seryl, threonyl, or aspartyl;
A6 is leucyl, methionyl, lysyl, seryl or
threonyl;
A9 is D- or L-lysyl, alanyl or glutamyl;
Al2 is valyl or leucyl; and
A1 6 iS alanyl, histidyl or phenylalanyl;
and the remaining residues are selected
from the preferred moieties listed above.
A novel process is also disclosed, in which
the preferred R1 to R8 moieties above are used to
form compounds of formula I in high yield.
Listed below are definitions of various
terms used to describe this invention. These
definitions apply to the terms as they are used
throughout this specification, unless otherwise
limited in specific instances, either individually
or as part of a larger group.
The term "amino acid residue" refers to
moieties of the formula
R' ~
-~ - C-~-
H ~
including natural, modified and synthetic moieties
in L-form unless otherwise specified, wherein:
~ ~ t~
_5_ ~576
R is hydrogen, alkyl, alkenyl, alkynyl,
aryl, aralkyl, cycloalkyl, cycloalkylalkyl,
cycloalkenyl, cycloalkenylalkyl, heteroaryl,
heteroarylalkyl, mercaptoalkyl, mercaptoalkenyl,
mercaptoaryl, alkylthioalkyl, hydroxyalkyl,
hydroxyaryl, hydroxyaralkyl, dihydroxyaryl,
dihydroxyaralkyl, aryloxyalkyl, aminoalkyl, amino-
guanidinoalkyl, guanidinoalkyl, carboxyalkyl,
carboalkoxyalkyl, carboxamidoalkyl, dicarboxyalkyl,
alkylcarbonyl, heterocyclo, heterocycloalkyl, -P04,
(P04 )-alkyl, (P04 )-aryl, (P04 )-aralkyl, (P04 )-
heterocyclo, or (P04 )-heterocycloalkyl; and
R' is hydrogen; or
R and R' together with the carbon and
nitrogen atoms to which they are attached complete
a 3- to 7-membered heterocyclo (e.g., pyrrolidinyl
for proline residues) group.
The terms "alkyl" and "alkoxy" refer to
both straight and branched chain groups having 1
to 10 carbon atoms. Those groups having one to
four carbon atoms are preferred. The terms "lower
alkyl" and "lower alkoxy" refer to groups of 1 to 4
carbon atoms.
The term "aryl" or "ar" refers to
monocyclic or bicyclic aromatic hydrocarbon groups
having 6 to 10 carbon atoms in the ring portion,
such as phenyl and naphthyl.
The terms "cycloalkyl" and "cycloalkenyl"
refer to cyclic hydrocarbon groups having 3 to 7
carbon atoms in the ring.
2~
HA576
--6--
The term "heterocyclo" refers to fully
saturated, partially saturated~ and unsaturated
mono- or bicyclic groups having 5 to 6 atoms in
each ring and one to four heteroatoms in at least
one ring, wherein the heteroatoms can comprise
one or two oxygen atoms, one or two sulfur atoms
and/or one to four nitrogen atoms, and wherein an
available carbon or nitrogen atom can be
substituted with hydroxyl, lower alkyl, lower
alkoxy, halo, mercapto, amino or car~oxyl.
Exemplary heterocyclo groups are pyrrolidinyl,
pyrazolidinyl, imidazolyl, imidazolidinyl,
thiazolidinyl, oxazolidinyl, indolyl, pyridyl and
the like.
The term "heteroaryl" refers to aromatic
heterocyclo groups, such as imidazolyl, indolyl,
oxazolyl, pyridyl, pyrrolyl and the like.
The term "acidic" as used with respect to
amino acid residues refers to residues that would
be negatively charged at about pH 6 to 7, such as
residues derived from aspartic acid, glu~amic acid,
and the like.
The term "polar" as used with respect to
amino acid residues refers to residues derived
from arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glutamine, histidine, lysine,
serine, threonine, tyrosine, ornithine and the
like.
The term "aromatic" as used with respect to
amino acid residues refers to amino acid residues
wherein R is aryl, aralkyl, heteroaryl, hetero-
arylalkyl, mercaptoaryl, hydroxyaryl, dihydroxy-
aryl, hydroxyaralkyl, dihydroxyaralkyl, aryloxy-
~ t~
_7_ HA576
alkyl, (P04 )~ aryl, (PO~)-aralkyl, (P04 )-heteroaryl,
or (P04 )-heteroarylalkyl; for example, moieties
derived from histidine, phenylalanine, tryptophan,
tyrosine and the like.
The term l'aliphatic" as used with respect
to amino acid residues refers to amino acid
residues having hydrocarbon sidechains that are not
aromatic, such as residues derived from alanine,
isoleucine, leucine, valine and the like (but not
proline). The term "aliphatic" thus includes
residues wherein R is alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkylalkyl, cycloalkenyl, and
cycloalkenylalkyl.
The term "natural amino acid residue" refers
to alanyl, argininyl, asparaginyl, aspartyl,
cysteinyl, glutamyl, glutaminyl, glycyl, histidyl,
isoleucyl, leucyl, lysyl, methionyl, phenylalanyl,
prolyl, seryl, threonyl, tryptophyl, tyrosyl, or
valyl.
The compounds of formula I are agonists
(e.g., Example 2 hereinafter), antagonists (e.g.,
Example 1 hereinafter), or partial agonists and
antagonists of ET-l, ET-2, and/or ET-3 and are
useful in treatment of all endothelin-dependent
disorders. They are especially useful as
antihypertensive agents. By the administration of
a composition having one (or a combination) of
the compounds of this invention, the blood pressure
of a hypertensive mammalian (e.g., human) host is
reduced.
~ ~ 7 ~ ci
HA576
--8--
The antagonists of the present invention are
also useful in the treatment of disorders related
to renal, glomerular, and mesangial cell function,
including chronic renal failure, glomerular injury,
renal damage secondary to old age, nephrosclerosis
(especially hypertensive n~phrosclerosis),
nephrotoxicity (including nephrotoxicity related
to imaging and contrast agents), and the like. The
compounds of this invention may also be useful in
the treatment of disorders related to paracrine and
endocrine function.
The antagonists of the present invention
are also useful as anti-ischemic agents for the
treatment of, for example, heart, renal and
cerebral ischemia and the like.
In addition, the antagonists of this
invention are also believed to be useful as:
anti-arrhythmic agents;
anti-anginal agents;
anti-fibrillatory agents;
anti-asthmatic agents;
agents to increase the ratio of HDL-
cholesterol to total serum cholesterol in the blood;
therapy for myocardial infarctioni
therapy for peripheral vascular disease
(e.g., Raynaud's disease);
anti-thrombotic agents;
anti-atherosclerotic agents;
treatment of cardiac hypertrophy (e.g.,0 hypertrophic cardiomyopathy);
treatment of pulmonary hypertension;
additives to cardioplegic solutions for
cardiopulmonary bypasses;
~23
_g_ ~A576
adjuncts to thrombolytic therapy;
treatment of central nervous system vascular
disorders; for example, as anti-stroke agents,
anti-migraine agents, and therapy for subarachnoid
hemorrhage;
treatment of central nervous system
behavorial disorders, including psychiatric
conditions such as depression, mania, anxiety and
schizophrenia;
anti-diarrheal agents;
therapy for dysmenorrhea;
therapy ~or tinnitus and other auditory
and vestibulatory disordersi
alleviation of the various forms of oedema;
reversal of adriamycin resistance;
regulation of cell growth;
treatment of glaucoma, hepatoxicity, sudden
death, drug-induced tardive dyskinesia, allergies,
electrolyte imbalance, muscular dystrophy and0 cancer.
The ET agonists of formula I are useful in
treating hypotension, congestive heart failure,
shock, endocrinological disorders and the like.
The compounds of ~his invention can also be
formulated in combination with endothelin
converting enzyme (ECE) inhibitors, such as
phosphoramidon and thiorphan; platelet activating
factor (PAF) antagonists; angiotensin II (AII)
receptor antagonists; renin inhibitors; angiotensin
converting enzyme (ACE) inhibitors such as
captopxil, zofenopril, fosinopril, ceranapril,
alacepril, enalapril, delapril, pentopril,
quinapril, ramipril, lisinopril, and salts of such
HA576 ~ ~
--10--
compounds; neukral endopeptidase ~NEP) inhibitors;
calcium channel blockers; potassium channel
activators; beta-adrenergic agents; antiarrhythmic
agents; diuretics, such as chlorothiazide, hydro-
chlorothia ide, flumethiazide, hydroflumethiazide,bendroflumethiazide, me~hylchlorothiazide, trichloro-
methiazide, polythiazide or benzothiazide as well
as ethacrynic acid, tricrynafen, chlorthalidone,
furosemide, musolimine, bumetanide, triamterene,
amiloride and spironolactone and salts of such
compounds; thrombolytic agents such as tissue
plasminogen activator (tPA), recombinant tPA,
streptokinase, urokinase, prourokinase, and
anisoylated plasminogen streptokinase activator
complex (APSAC, Eminase, Beecham Laboratories). If
formulated as a fixed dose, such combination
products employ the compounds of this invention
within the dosage range described below and the
other pharmaceutically active agent within its
approved dosage range. The compounds of this
invention may also be formulated with or useful in
conjunction with antifungal and immunosuppressive
agents such as amphotericin B, cyclosporins and the
like to counteract the glomerular contraction and
nephrotoxicity secondary to such compounds. The
compounds of this invention may also be used in
conjunction with hemodialysis.
The compounds of formula I can be fo~mulated
in compositions such as sterile solutions of
suspensions for parenteral administration. About
0.1 to 500 milligrams of a compound of formula I is
compounded with a physiologically acceptable
vehicle, carrier, excipient, binder, preservative,
C~7 23(~3
HA576
stabilizer, etc., in a unit dosage form as called
for by accepted pharmaceutical practice. The
amount of active substance in these compositions or
preparations is such that a suitable dosage in the
range indicated is obtained.
The compounds of the present invention may
be prepared as follows. This preparation may be
carried out on an automated peptide synthesizer
(e.g., Biosearch 9600) using standard software
protocols.
A compound of the formula
II
Prot-A21 -O-PAM RESIN
wherein Prot is an amino-protecting group (e.g.,
t-butoxycarbonyl) attached at the amino terminus
and A2l is A21 or a residue derived therefrom
having a sidechain-protecting group if A2l is an
amino acid residue having a reactive sidechain
functional group. Exemplary reactive sidechain
functional groups are hydroxyl, carboxyl, mercapto,
amino, guanidino, imidazolyl, indolyl and the like.
Compound II is treated with, in sequence:
(a) a deprotecting agent (e.g., trifluoro-
acetic acid) in an inert solvent (e.g., methylene
chloride) in the presence of one or more cation
scavengers (e.g., dimethylphosphite, anisole);
(b) a tertiary base (e.g., diisopropyl-
ethylamine); and
(c) an amino acid of the formula
III
Prot-A20 -OH
(wherein A20 is A20 or a residue derived therefrom
having a sidechain-protecting group if A20 is an
amino acid residue having a reactive sidechain
functional group) in an inert solvent (e.g.,
dimethylformamide) in ~he presence of a
, ~i t ~
~A576
coupling reagent (e.g., diisopropylcarbodiimide) in
an inert solvent ~e.g., methylene chloride);
to form an amino acid of the formula
IV
Prot~A2 -A2~ -O-PAN RESIN
Presence of an agent which suppresses racemization
or dehydration (e.g., hydroxybenzotriazole) is
optional. Step (c) above may be followed by
treatment with an amino acid acetylating agent
(e.g., acetylimidazole) which acetylates or "caps"
unreacted amino acids. When Prot is a base-labile
protecting group (e.g., fluorenylmethoxycarbonyl),
step (a) above is carried out with a base (e.g.,
piperidine, morpholine) in an inert solvent (e.g.,
dimethylformamide) and step (b) is unnecessary.
The foregoing process is repeated with the
resin-linked amino acid chain until the N-terminal
amino acid residue has been coupled to the
polypeptide. One may also use multiple peptide
synthesis technigues, which are generally known in
the art. See, e.g., Tjoeng et al., "Multiple
Peptide Synthesis Using A Single Support (MPS3)",
Int. J. Protein Peptide Res., 35 (1990), 141-146.
After the polypeptide chain is complete, it
may be cleaved from the resin with an acid, such
as hydrofluoric acid, trifluoromethanesulfonic
acid and the like. When Prot is a base-labile
protecting group, a mild acid such as trifluoro-
acetic acid and the like may be used.
As noted above, sidechain protecting groups
may be used in this process for sidechains having
reactive functionalities, such as hydroxyl,
carboxyl, amino, mercapto, guanidino, imidazolyl,
indolyl and the like. The particular protecting
groups used for any amino acid residues depend upon
the sidechains to be protected and are generally
known in the art. Exemplary sidechain protecting
~ ~ ?~
HA576
-13~
groups are benzyl, halocarbobenzoxy, and the like
for hydroxyl; cyclohexyl, benzyl and the like for
carboxyl; 4-methylbenæyl, 4 methoxybenzyl,
acetamidomethyl and the like for mercapto;
carbobenzoxy, halocarbobenzoxy and the
like for amino; 2,4-dinitrophenyl, benzylo~ymethyl
and the like for imidazolyli formyl and the like
for indolyl; and tosyl, nitro and the like for
guanidino.
Sidechain protecting groups may be rem~ved,
if desired, by treatment with one or more
deprotecting agents in an inert solvent or solvent
mixture (e.g., dimethylformamide, methylene
chloride). Suitable deprotecting agents are
generally known in the art. Exemplary deprotecting
agents are thiophenol, mercaptoethanol and the like
for removing 2,4-dinitrophenyl; trifluoroacetic
acid and the like for butoxycarbonyl; hydrofluoric
acid, trifluoromethanesulfonic acid and the like for
several different protecting groups.
The disulfide bonds in the compound of
formula I are formed by oxidation with, for
example, air/ammonium hydroxide, potassium
ferricyanide, air/urea/ammonium hydroxide,
water/methanol/iodine, and the like. The preferred
R1 to R8 moieties cause higher yields of the
desired 1 15 and 3-11 disulfide bonds upon
oxidation.
The invention will now be further described
by the following working examples, which are
preferred embodiments of the invention. These
examples are meant to be illustrative rather than
limiting. Unless otherwise indicated, all
reactions are conducted ~t about room temperature
using a Biosearch 9600 automated peptide
synthesizer.
~Q~6~
~o C~
C ,
~ Ll~ I =
. ~o~
N
C
~ ~1
I X
--
N
C . .
cn
.'~;~
~, Z
P~ N
~2~
HA576
-15-
Example 1
L-Penicillaminyl-L-seryl-L-cysteinyl-L-seryl-L-
seryl-L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-
glutamyl-L-penicillaminyl-L-valyl-L-tyrosyl-L-
phenylalanyl-L-cysteinyl-L-histidyl-L-leucyl-L-
aspartyl-L-isoleucyl-L-isoleucyl-L-tryptophan,
S,S-crosslinked(1,15:3,11) ____ _
Preparation began with 1.0 g of a resin
of the nitrogen-protected carboxyl terminal amino
acid, tert-butoxycarbonyl-tryptophan phenylacet-
amidomethyl resin (0.56 mg/g). The successive
amino acids were added according to Procedure A
(single-coupled) or Procedure B (double-coupled).
Procedure A
1. Wash once with dichloromethane (CH2Cl2).
2. Treat for 1 minute with CH2Cl2:trifluoro-
acetic acid (TFA):anisole:dimethyl-
phosphite (50.5:45:2.5:2 by volume).0 3. Treat for 20 minutes with
CH2Cl2:TFA:Anisole:Dimethylphosphite
(50.5:45:2.5:2.0 by volume).
4. Wash once with CH2C12.
5. Wash once with dimethylformamide (DMF).
6. Wash three times with CH2Cl~.
7. Treat three times for 50 seconds with 10%
(v/v) diisopropylethylamine (DIPEA) in
C~I2 Cl 2 -
8. Wash three times with CH2Cl2.
9. The suitably protected Boc-amino acid (7.1
equivalents) dissolved in dimethylformamide
(0.4 M) and diisopropylcarbodiimide
dissolved in C~2Cl2 (0.4 M) are combined
2~723~
HA576
-16-
to afford a 0.2 M solution of the amino
acid O-acylisourea in (1:1) DMF/CH2Cl2
which is added to the resin. The suspension
is mixed for 2 hours.
10. Wash three times with CH2Cl2.
11. Wash once with 10% DIPEA for 40 seconds.
12. Wash three times with CH2Cl2.
13. Treat for 30 minutes with acetylimidazole
in DMF (0.3 M).
14. Wash three times with DMF.
Procedure B
For amino acids which require double coupling,
after step 12 of Procedure A, steps 9-12 are
repeated.
Successive residues were coupled with the
resin using either Procedure A or B, as noted in
parenthesis hereafter.
The seguence of amino acids introduced was
Boc-I-OH-~2 H2O (B), Boc-I-OH-~ H2O (B),
Boc-D(chx)-OH (B), Boc-L-OH H20 (A), Boc-H(nNP~-
OH-isopropanol (A), Boc-C(MeBn)-OH (A), Boc-F-OH
(A), Boc-Y(2BrZ)-OH (A), Boc-V-OH (B), Boc-Pen-
(MeOBn)-OH (B), Boc-E(chx)-OH (B), Boc-K (2ClZ)-OH
(A), Boc-D(chx)-OH (A), Boc-Nle-OH (A), Boc-L-
OH-H2O (A), Boc-S(Bn)-OH (A), Boc-S(Bn)-OH, (A),
Boc-C(MeBn)-OH (A), Boc-S(Bn)-OH (A), Boc-Pen-
(MeOBn)-OH (B). The protected peptidyl resin was
treated with TFA:CH2Cl2:anisole: dimethylphosphite
(45:50.5:2.5:2 v/v/v~v) for 1 minute, then 20
minutes, washed three times with CH~Cl2, ~reated
with a mixture of 4 mL of dimethylformamide and
1 mL of thiophenol for 45 minutes, washed twice
with dimethylformamide, treated with a mixture of
-17- HA576 ~37~3~
4 mL of dimethylformamide and 1 mL of thiophenol
for 45 minutes, washed twice with dimethyl-
formamide, twice with wa~er, twice with ethanol and
three times with CH2Cl2 and dried under nitrogen to
give the TFA salt of the protected peptidyl resin
containing unprotected His.
The peptidyl resin was suspended in HF
containing 10% anisole for 1 hour at 0C. The HF
was removed by evaporation under vacuum. The
peptide/resin mixture was washed three times with
1:1 hexane:ether. The peptide was extracted with
50% aqueous acetic acid, with 50% aqueous TFA, and
with neat TFA. The combined filtrates were
evaporated to a gum, which was suspended in water.
The solution was lyophilized to afford 0.82 g
of the unoxidized sulfhydryl peptide as a brown
solid.
The crude peptide was dissolved in 1.5 L of
degassed 0.2% ammonium hydroxide and air was
bubbled through the solution for 4.5 hours. The
solution was stirred overnight and lyophilized to
afford the crude cyclized peptide as a tan fluffy
solid. The solid was dissolved in 75% agueous
CH3CN containing ammonium hydroxide and the
solution was chromatographed on Sephadex LH-60
using 75% agueous CH3CN containing 0.2% ammonium
hydroxide. Fractions were analyzed by analytical
reversed phase HPLC and those containing primarily
product were pooled and lyophilized to afford
114 mg of tan powder. The solid was dissolved in
75:25 water:C~3CN containing 0.1% TFA and the
solution was subjected to preparative ~PLC on an
octadecylsilane column using water:CH3CN gradients
HA576213~7239~j
-18~
containing 0.1% TFA. Fractions were analyzed by
analytical reversed phase HPLC and those containing
product with a minimum purity of 95% were pooled
and lyophilized to afford 12.1 mg of the
trifluoroacetate salt of Example 1 (0.8%) as a
white solid. MS (M+~) 2529Ø
Example 2
L-Cysteinyl L-seryl-L-penicillaminyl-L-seryl-L-
seryl-L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-
glutamyl-L-cysteinyl-L-valyl-L-tyrosyl-L-phenyl-
alanyl-L-penicillaminyl-L-histidyl-L-leucyl-L-
aspartyl-L-isoleucyl-L-isoleucyl-L-tryptophan,
S,S-crosslinked(1!15:3,11~ _ _ _ _ _
Example 2 was prepared following Procedures
A and B of Example 1, using 0.8 g of the same
starting resin. Amino acids were added sequentially,
as follows: Boc-I-OH-~ H2O ~B), Boc-I-OH-~ H2O (B),
Boc-D(chx)-OH (B), Boc-L-OH-H2O (B), Boc-H(DNP)-OH-
isopropanol (B), Boc-Pen(MeOBn)-OH (B), Boc-F-OH (A),
Boc-Y(2BrZ)-OH (A), Boc-V-OH (B), Boc-C(MeBn)-OH
(B), Boc-E(chx)-OH (B), Boc-K-(2ClZ)-OH (A),
Boc-D(chx)-OH (A), Boc-Nle-OH (A), Boc-L-OH H2O
(B), Boc-S(Bn)-OH (A), Boc-S(Bn)-OH (A),
Boc-Pen(MeOBn)-OH (B), Boc-S(Bn)-OH (A),
Boc-C(MeBn)-OH (B). The protected peptidyl resin
was treated with TFA:CH2Cl2:anisole:dimethyl-
phosphite (45:50.5:2.5:2 v/v/v/v) for 1 minute,
then 20 minutes, washed three times with CH2Cl2,
treated with a mixture of 8 mL of dimethylformamide
and 2 mL of thiophenol for 30 minutes, washed twice
with dimethylformamide, treated with a mixture of
2 ~ ?~ 5
HA576
-19-
8 mL of dimethylformamide and 2 mL of thiophenol
for 1 hour, washed twice with dimethylformamide,
twice with water, twice with ethanol and three
times with CH2C12 and dried under nitrogen to give
the TFA salt of the protected peptidyl resin
containing unprotected histidine.
The peptidyl resin was suspended in HF
containing 10% anisole for 1 hour at 0C. The HF
was removed by evaporation under vacuum. The
peptide/resin mixture was washed three times with
1:1 hexane:ether. The peptide was extracted with
50% aqueous acetic acid, with 50% aqueous TFA, and
with neat TFA. The combined filtrates were
evaporated to a gum, which was suspended in water.
The solution was lyophilized to afford 0.96 g
of the unoxidized sulfhydryl peptide as a brown
solid.
The crude peptide was dissolved in 1.3 L
of degassed 0.1% ammonium hydroxide and air was
bubbled through the solution for 3 hours. The
solution was stirred overnight and lyophilized to
afford the crude cyclized peptide as a tan fluffy
solid. The solid was dissolved in 70:30
water:CH3CN containing 0.1% trifluoroacetic acid
and the solution was centrifuged. The solution was
decanted and the solid was rinsed with additional
solvent. The combined supernatants were subjected
to preparative HPLC on an octadecylsilane column
using water:CH3CN gradients containing 0.1% TFA.
Fractions were analyzed by analytical reversed
phase EPLC and those containing product with a
minimum purity of 95% were pooled and lyophilized
2 ~ i b
HA576
-20-
to afford 45 mg of the trifluoroacetate salt of
Example 2 (3.5%) as a white solid.
MS (M+H) 2529Ø
ExamPle 3
L-Penicillaminyl-L-seryl-L-cysteinyl-L-seryl-L-seryl-
L-leucyl~L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-
L-penicillaminyl-L-valyl-L-tyrosyl-L-phenylalanyl-
L-cysteinyl-L-histidyl-L-leucyl-L-alanyl-L-iso-
leucyl-L-isoleucyl-L-tryptophan, S,S-crosslinked
(1,15:3,11) _
Example 4
L-Penicillaminyl-L-seryl-L-cysteinyl-L-seryl-L-seryl-
L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-
L-penicillaminyl-L-valyl-L-tyrosyl-L-phenylalanyl-
L-cysteinyl-L-histidyl-L-leucyl-L-asparaginyl-L-
isoleucyl-L-isoleucyl-L-tryptophan, S,S-cross-
linked_(l,l5:3,11) _ _
Example 5
L-Penicillaminyl-L-seryl-L-cysteinyl-L-seryl-L-seryl-
L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-
L-penicillaminyl-L-valyl-L-tyrosyl-L-phenylalanyl-
L-cysteinyl-L-histidyl-L-leucyl-L-leucyl-L-iso-
leucyl-L-isoleucyl-L-tryptophan, S,S-crosslinked
(1,15:3,11~
Examples 3, 4 and 5 were prepared
simultaneously using the technique of multiple
peptide synthesis using a single support (MPS3);
~ 3
HA576
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see F. S. Tjoeng et al., Int. J. Peptide Protei~
Res., 35, 141-146 (1990). The synthesis then
resulted in the simultaneous synth~sis of a mixture
of several peptides which contained identical amino
S acids at most of the positions and different amino
acids at one or more of the positions.
Purification of the mixture allowed the isolation
of each purified compound.
O.7 g of Boc-W-Pam-resin (O.S6 meq/g; Bachem
Inc., Torrance, CA) was subjected to either
Procedure A (single couple) or B (double couple),
as indicated in parentheses, with the following
sequence of amino acids introduced in order:
Boc-I-OH ~H20(B), Boc-I-OH-~H20(B), a 1:2:2
mixture of Boc-A-OH, Boc-N-OH and Boc-L-OH H2O (B),
Boc-L-OH-H2O (B), Boc-H(DNP)-OH-isopropanol
(B), Boc-C(MeBn)-OH (A), Boc-F-OH (A), Boc-Y
(2BrZ)-OH (A), Boc-V-OH (B), Boc-Pen(MeOBn)-OH (B),
Boc-E(chx)-OH (B), Boc-K(2ClZ)-OH (A),
Boc-D(chx)-OH (B), Boc-Nle-OH (B), Boc-L-OH H20
(B), Boc-S(Bn)-OH (A), Boc-S(Bn)-OH (A), Boc-C-
(MeBn)-OH (A), Boc-S(Bn)-OH (A), Boc-Pen(MeOBn)-OH
(B).
The protected peptidyl resin was, in
sequence:
treated with TFA:CH2Cl2:anisole (45:52.5:2.5
v/v/v) for 1 minute, and then for 20 minutes;
~ ~3 ~ ~ c~ -~,3
HA576
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washed three times with CH2Cl2;
treated with a mixture of 4 mL of dimethyl-
formamide and 1 mL of thiophenol for 30 minutes;
washed twice with dimethylformamide;
treated with a mixture of 4 mL of
dimethylformamide and 1 mL of thiophenol for 60
minutes;
washed twice with dimethylformamide, twice
with water, twice with ethanol and three times0 with CH2Cl2; and
dried under nitrogen to give the TFA salt of
the protected peptidyl resin containing unprotected
His.
The peptidyl resin was suspended in HF
containing 20% anisole for 1 hour at 0c. The HF
was removed by evaporation under vacuum. The
peptide/resin mixture was washed three times with
l:1 cyclohexane:ether. The peptide was extracted
with 50% aqueous acetic acid (3 x 25 mL) and with
neat TFA (3 x 25 mL). The combined filtrates were
evaporated to a gum, which was suspended in water.
The solution was lyophilized to afford 0.91 g
of the mi~ture of unoxidized sulfhydryl peptides as
a yellow solid.
The crude peptide was dissolved in 1.2 L of
degassed 0.5% ammonium hydroxide, and air was
bubbled through the solution for 3 hours. The
solution was stirred overnight and lyophilized to
afford the mixture of crude cyclized peptides as a
yellow solid. The solid was dissolved in 70:30
water:acetonitrile containing 0.1% TFA (55 mL), and
the solution was centrifuged. The solution was
decanted and subjected in aliquots to preparative
3 ~ ~
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HPLC on an octadecylsilane column using water:
acetonitrile gradients containing 0.1% TFA.
Fractions were analyzed by analytical reversed
phase HPLC and those containing the earliest
eluting peak with a minimum purity of 95% were
pooled and lyophilized to afford 7.5 mg of the
trifluoroacetate salt of Example 4 (0.6%) as a
white solid. MS (M+H) 2528.1. Fractions
containing the middle eluting peak with a minimum
purity of 95% were pooled and lyophilized to
afford 10.0 mg of the trifluoroacetate salt of
Example 3 (0.8%) as a white solid.
MS (M+~)+2485.1. Fractions containing the latest
eluting peak with a minimum purity of 95% were
pooled and lyophilized to afford 3 mg of the
trifluoroacetate salt of Example 5 (0.2%) as a
white solid. MS (M+H) 2527.7.
The abbreviations used throughout this
specification have the following meanings:
Bn Benzyl
BOC tert-butoxycarbonyl
2-BrZ 2-bromo-benzyloxycarbonyl
Chx cyclohexyl
2-ClZ 2-chlorobenzyloxycarbonyl
DIPEA diisopropylethylamine
DMF dimethylformamide
DNP 2,4-dinitrophenyl
MeBn 4-methylbenzyl
MeOBn 4-methoxybenzyl
Nle norleucyl
PAM phenylacetamidomethyl
Pen penicillaminyl
TFA trifluoroacetic acid
