CA2072395A1 - Bicyclic endothelin analogues - Google Patents
Bicyclic endothelin analoguesInfo
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- CA2072395A1 CA2072395A1 CA002072395A CA2072395A CA2072395A1 CA 2072395 A1 CA2072395 A1 CA 2072395A1 CA 002072395 A CA002072395 A CA 002072395A CA 2072395 A CA2072395 A CA 2072395A CA 2072395 A1 CA2072395 A1 CA 2072395A1
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- amino acid
- seryl
- leucyl
- acid residue
- cycloalkyl
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Abstract
Abstract BICYCLIC ENDOTHELIN ANALOGUES
Endothelin receptor antagonism is exhibited by novel compounds of the formula
Endothelin receptor antagonism is exhibited by novel compounds of the formula
Description
2 ~
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;
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~
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 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 and 0 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 ~ ~
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 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 ~ ~ ?~
-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~
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~
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 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 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 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 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 times 0 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 ~ ~
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
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;
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~
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 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 and 0 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 ~ ~
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 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 ~ ~ ?~
-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~
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~
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 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 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 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 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 times 0 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 ~ ~
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
Claims (13)
1. A compound of the formula wherein:
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 R4 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;
R7 and R8 are each independently hydrogen 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 residuei A10 is an aliphatic, aromatic, D- or L-acidic, or polar amino acid xesidue;
A12 is an amino acid residue;
A13 is an aliphatic, aromatic, or polar amino acid residue;
A14 is an aromatic or aliphatic amino acid residue;
A16 is an amino acid residue;
A17 is an aliphatic or polar amino acid residue;
A18 is an acidic, polar, aromatic or aliphatic amino acid residue;
A19 and A20 are each independently aliphatic amino acid residues; and A21 is an aromatic amino acid residue.
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 R4 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;
R7 and R8 are each independently hydrogen 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 residuei A10 is an aliphatic, aromatic, D- or L-acidic, or polar amino acid xesidue;
A12 is an amino acid residue;
A13 is an aliphatic, aromatic, or polar amino acid residue;
A14 is an aromatic or aliphatic amino acid residue;
A16 is an amino acid residue;
A17 is an aliphatic or polar amino acid residue;
A18 is an acidic, polar, aromatic or aliphatic amino acid residue;
A19 and A20 are each independently aliphatic amino acid residues; and A21 is an aromatic amino acid residue.
2. The compound of Claim 1, wherein:
at least one of R1 and R2 is lower alkyl or R1 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, or R5 and R6 together with the carbon atom to which they are attached are cycloalkyl; and R7 and R3 are hydrogen; or R1 and R2 are hydrogen; at least one of R3 and R4 is lower alkyl or R3 and R4 together 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.
at least one of R1 and R2 is lower alkyl or R1 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, or R5 and R6 together with the carbon atom to which they are attached are cycloalkyl; and R7 and R3 are hydrogen; or R1 and R2 are hydrogen; at least one of R3 and R4 is lower alkyl or R3 and R4 together 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.
3. The compound of Claim 2, wherein lower alkyl is methyl and cycloalkyl is cyclopropyl.
4. The compound of Claim 1, wherein:
A7 is norleucyl or methionyl;
A8 is aspartyl;
A10 is L-glutamyl, D-glutamyl, glutaminyl, or alanyl;
A13 is tyrosyl, phenylalanyl, or asparaginyl;
A14 is tyrosyl or phenylalanyl;
A17 is leucyl or glutaminyl;
A18 is aspartyl, asparaginyl, alanyl, or leucyl;
A19 is isoleucyl or valyl;
A20 is isoleucyl; and A21 is tryptophyl or napthylalanyl.
A7 is norleucyl or methionyl;
A8 is aspartyl;
A10 is L-glutamyl, D-glutamyl, glutaminyl, or alanyl;
A13 is tyrosyl, phenylalanyl, or asparaginyl;
A14 is tyrosyl or phenylalanyl;
A17 is leucyl or glutaminyl;
A18 is aspartyl, asparaginyl, alanyl, or leucyl;
A19 is isoleucyl or valyl;
A20 is isoleucyl; and A21 is tryptophyl or napthylalanyl.
5. The compound of Claim 2, wherein:
A7 is norleucyl or methionyl;
A8 is aspartyl;
A10 is L-glutamyl, D-glutamyl, glutaminyl, or alanyl;
A13 is tyrosyl, phenylalanyl, or asparaginyl;
A14 is tyrosyl or phenylalanyl;
A17 is leucyl or glutaminyl;
A18 is aspartyl, asparaginyl, alanyl, or leucyl;
A19 is isoleucyl or valyl;
A20 is isoleucyl; and A21 is tryptophyl or napthylalanyl.
A7 is norleucyl or methionyl;
A8 is aspartyl;
A10 is L-glutamyl, D-glutamyl, glutaminyl, or alanyl;
A13 is tyrosyl, phenylalanyl, or asparaginyl;
A14 is tyrosyl or phenylalanyl;
A17 is leucyl or glutaminyl;
A18 is aspartyl, asparaginyl, alanyl, or leucyl;
A19 is isoleucyl or valyl;
A20 is isoleucyl; and A21 is tryptophyl or napthylalanyl.
6. The compound of Claim 4, wherein:
A2 is seryl or threonyl;
A4 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;
A12 is valyl or leucyl; and A16 is alanyl, histidyl ox phenylalanyl.
A2 is seryl or threonyl;
A4 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;
A12 is valyl or leucyl; and A16 is alanyl, histidyl ox phenylalanyl.
7. The compound of Claim 5, wherein:
A2 is seryl or threonyl;
A4 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;
A12 is valyl or leucyl; and A16 is alanyl, histidyl or phenylalanyl.
A2 is seryl or threonyl;
A4 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;
A12 is valyl or leucyl; and A16 is alanyl, histidyl or phenylalanyl.
8. The compound of Claim 1, selected from the group consisting of:
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);
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-phenylalanyl-L-penicillaminyl-L-histidyl-L-leucyl-L-aspartyl-L-isoleucyl-L-isoleucyl-L-tryptophan, S,S-crosslinked(1,15:3,11);
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-isoleucyl-L-isoleucyl-L-tryptophan, S,S-crosslinked (1,15:3,11);
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-crosslinked (1,15:3,11); and 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-isoleucyl-L-isoleucyl-L-tryptophan, S,S-crosslinked (1,15:3,11).
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);
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-phenylalanyl-L-penicillaminyl-L-histidyl-L-leucyl-L-aspartyl-L-isoleucyl-L-isoleucyl-L-tryptophan, S,S-crosslinked(1,15:3,11);
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-isoleucyl-L-isoleucyl-L-tryptophan, S,S-crosslinked (1,15:3,11);
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-crosslinked (1,15:3,11); and 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-isoleucyl-L-isoleucyl-L-tryptophan, S,S-crosslinked (1,15:3,11).
9. A process for preparing a product of the formula (a) synthesizing an intermediate of the formula (b) oxidizing the intermediate to form the product;
wherein:
at least one of R1 and R2 is lower alkyl or R1 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, 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 together 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;
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;
A12 is an amino acid residue;
A13 is an aliphatic, aromatic, or polar amino acid residue;
A14 is an aromatic or aliphatic amino acid residue;
A16 is an amino acid residue;
A17 is an aliphatic or polar amino acid residue;
A18 is an acidic, polar, aromatic or aliphatic amino acid residue;
A19 and A20 are each independently aliphatic amino acid residues; and A21 is an aromatic amino acid residue.
wherein:
at least one of R1 and R2 is lower alkyl or R1 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, 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 together 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;
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;
A12 is an amino acid residue;
A13 is an aliphatic, aromatic, or polar amino acid residue;
A14 is an aromatic or aliphatic amino acid residue;
A16 is an amino acid residue;
A17 is an aliphatic or polar amino acid residue;
A18 is an acidic, polar, aromatic or aliphatic amino acid residue;
A19 and A20 are each independently aliphatic amino acid residues; and A21 is an aromatic amino acid residue.
10. The process of Claim 9, wherein lower alkyl is methyl and cycloalkyl is cyclopropyl.
11. The process of Claim 9, wherein:
each of A2 to A21 in the intermediate has a sidechain protecting group if it is a reactive amino acid residue;
the process further comprises treating the intermediate or the product with one or more deprotecting agents; and the term "reactive amino acid residue"
refers to amino acid residues having sidechains comprising hydroxyl, carboxyl, mercapto, amino, imidazolyl, indolyl or guanidino groups.
each of A2 to A21 in the intermediate has a sidechain protecting group if it is a reactive amino acid residue;
the process further comprises treating the intermediate or the product with one or more deprotecting agents; and the term "reactive amino acid residue"
refers to amino acid residues having sidechains comprising hydroxyl, carboxyl, mercapto, amino, imidazolyl, indolyl or guanidino groups.
12. A method of treating endothelin-related disorders in a mammal, which comprises administering an effective amount of an agonist or antagonist in accordance with Claim 1.
13. A method of treating renal, glomerular, or mesangial cell disorders, which comprises administering an effective amount of a compound of Claim 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US72399391A | 1991-07-01 | 1991-07-01 | |
| US723,993 | 1991-07-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2072395A1 true CA2072395A1 (en) | 1993-01-02 |
Family
ID=24908516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002072395A Abandoned CA2072395A1 (en) | 1991-07-01 | 1992-06-25 | Bicyclic endothelin analogues |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2072395A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6030975A (en) * | 1997-03-14 | 2000-02-29 | Basf Aktiengesellschaft | Carboxylic acid derivatives, their preparation and use in treating cancer |
| US6063911A (en) * | 1993-12-01 | 2000-05-16 | Marine Polymer Technologies, Inc. | Methods and compositions for treatment of cell proliferative disorders |
| US8858964B2 (en) | 2010-04-15 | 2014-10-14 | Marine Polymer Technologies, Inc. | Anti-bacterial applications of poly-N-acetylglucosamine nanofibers |
| US8871247B2 (en) | 2007-02-19 | 2014-10-28 | Marine Polymer Technologies, Inc. | Hemostatic compositions and therapeutic regimens |
| US10765698B2 (en) | 2011-04-15 | 2020-09-08 | Marine Polymer Technologies, Inc. | Treatment of disease with poly-N-acetylglucosamine nanofibers |
-
1992
- 1992-06-25 CA CA002072395A patent/CA2072395A1/en not_active Abandoned
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6063911A (en) * | 1993-12-01 | 2000-05-16 | Marine Polymer Technologies, Inc. | Methods and compositions for treatment of cell proliferative disorders |
| US6030975A (en) * | 1997-03-14 | 2000-02-29 | Basf Aktiengesellschaft | Carboxylic acid derivatives, their preparation and use in treating cancer |
| US8871247B2 (en) | 2007-02-19 | 2014-10-28 | Marine Polymer Technologies, Inc. | Hemostatic compositions and therapeutic regimens |
| US9139664B2 (en) | 2007-02-19 | 2015-09-22 | Marine Polymer Technologies, Inc. | Hemostatic compositions and therapeutic regimens |
| US9139663B2 (en) | 2007-02-19 | 2015-09-22 | Marine Polymer Technologies, Inc. | Hemostatic compositions and therapeutic regimens |
| US10383971B2 (en) | 2007-02-19 | 2019-08-20 | Marine Polymer Technologies, Inc. | Hemostatic compositions and therapeutic regimens |
| US8858964B2 (en) | 2010-04-15 | 2014-10-14 | Marine Polymer Technologies, Inc. | Anti-bacterial applications of poly-N-acetylglucosamine nanofibers |
| US9198928B2 (en) | 2010-04-15 | 2015-12-01 | Marine Polymer Technologies, Inc. | Anti-bacterial applications of poly-N-acetylglucosamine nanofibers |
| US9642871B2 (en) | 2010-04-15 | 2017-05-09 | Marine Polymer Technologies, Inc. | Anti-bacterial applications of poly-N-acetylglucosamine nanofibers |
| US10206938B2 (en) | 2010-04-15 | 2019-02-19 | Marine Polymer Technologies, Inc. | Anti-bacterial applications of poly-N-acetylglucosamine nanofibers |
| US10561677B2 (en) | 2010-04-15 | 2020-02-18 | Marine Polymer Technologies, Inc. | Anti-bacterial applications of poly-N-acetylglucosamine nanofibers |
| US10765698B2 (en) | 2011-04-15 | 2020-09-08 | Marine Polymer Technologies, Inc. | Treatment of disease with poly-N-acetylglucosamine nanofibers |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 19970625 |