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WO2000009544A1 - Analogues heptapeptidiques de lhrh - Google Patents

Analogues heptapeptidiques de lhrh Download PDF

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Publication number
WO2000009544A1
WO2000009544A1 PCT/US1999/017874 US9917874W WO0009544A1 WO 2000009544 A1 WO2000009544 A1 WO 2000009544A1 US 9917874 W US9917874 W US 9917874W WO 0009544 A1 WO0009544 A1 WO 0009544A1
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Prior art keywords
leu
ser
nmetyr
d4clphe
d3pal
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PCT/US1999/017874
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English (en)
Inventor
Fortuna Haviv
Wesley J. Dwight
Charles J. Nichols
Jonathan Greer
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Abbott Laboratories
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Abbott Laboratories
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Priority claimed from US09/232,425 external-priority patent/US6191115B1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel analogs of LHRH.
  • the novel analogs provide heptapeptides truncated from the C-terminus of LHRH antagonist peptides.
  • the invention also relates to processes for preparing the disclosed compounds, pharmaceutical compounds containing such compounds, and use of such compounds for modulating levels of sex hormones in male or female mammals.
  • Luteinizing hormone releasing hormone (LHRH) is released from the hypothalamus and binds to a receptor on the anterior pituitary gland causing the release of gonadotropin hormones.
  • Natural mammalian releasing hormone LHRH isolated and purified from porcine and human hypothalami has been characterized as having the sequence: ( Pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 as described in AN. Schally, Science, 202:6 (1978). Substitutions and derivatizations of amino acyl residues have been developed to achieve novel compounds useful in treating various disorders related to mammalian reproductive systems.
  • LHRH antagonists wherein the nitrogen atom of at least one of the amide bonds has been alkylated.
  • the decapeptide and undecapeptide analogs described in U.S. Pat. No. 5,502,035 have an acyl-substituted N-terminal nitrogen atom.
  • Truncated peptide compounds have been developed as series of smaller peptide analogs also exhibiting biological activity and having the added advantage of possibly improved oral bioavailability. These reduced-size peptides, described in U.S. Pat. No. 5,140,009, exhibit effective LHRH agonist or antagonist activity. They are "pseudo" hexapeptide, heptapeptide, octapeptide and nonapeptide analogs of LHRH, which have 1 to 3 amino acids eliminated from the N-terminus of a decapeptide antagonist sequence to achieve activity as LHRH antagonists. Distinctively, the peptides described in this invention have the 10 to 8 amino acids eliminated from the C-terminus. Copending U.S. Application Serial No. discloses and claims a class of pentapeptide LHRH analogs wherein the 1 to 3 amino acids are eliminated from the N-terminus and the 10 to 8 amino acids from the C-terminus of a decapeptide LHRH antagonist.
  • LHRH antagonists truncated from the C-terminus having biological activity provides novel compounds for treatment of hormone dependent diseases in male and female mammals.
  • Smaller synthetic peptide sequences provide significant advantages when compared to decapeptide LHRH analogs.
  • These LHRH antagonists are useful in the treatment of a variety of clinical condition in which the suppression of sex steroids plays a major therapeutic role, including delay of puberty, treatment of benign prostatic hyperplasia, palliative treatment or remission of hormonal- - dependent tumors of breast and ovaries, palliative treatment or remission of hormonal- dependent tumors of the prostate, the treatment of cryptorchidism. hirsutism in women, gastric motility disorders, dysmenorrhea, and endometriosis. Summarv of the Invention
  • the compounds of the invention comprise a peptide of the formula:
  • A is an amino acyl residue selected from the group consisting of: 3-(2-naphthyl)-D-alanyl; [3-(4-chloro)]-D-phenylalanyl; and sarcosyl;
  • B is an amino acyl residue selected from the group consisting of:
  • C is an amino acyl residue selected from the group consisting of: 3-(3-pyridyl)-D-alanyl; and 3-(l-naphthyl)-D-alanyl;
  • E is an acyl group selected from the group consisting of: arginyl;
  • F is an amino acyl residue selected from the group consisting of: D-arginyl;
  • G is an amino acyl residue selected from the group consisting of: cyclohexy lalany 1 ; leucyl; and
  • R 2 is of the formula -NR 4 R 5 ; wherein
  • R 4 is selected from the group consisting of: hydrogen; methyl; and ethyl
  • R 11 is selected from the group consisting of: lower alkyl; and lower alkyl-R 6 ;
  • R is selected from the group consisting of amino, guanidino, hydrogen, hydroxy, phenyl, morpholinyl, piperidinyl, pyrrolyl, pyridyl, pyrrolidinyl, pyrrolidinonyl, and quinuclidinyl; and wherein the piperidinyl, pyrrolyl, pyrrolidinyl, and pyrrolidinonyl groups are optionally substituted with a methyl group.
  • Another aspect of the invention relates to pharmaceutical formulations comprising the compounds of the invention or pharmaceutically acceptable salts, esters, or prodrugs thereof.
  • the invention relates to a method of modulating gonadotropin hormones in a mammal comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound as defined above.
  • Yet another aspect of the invention relates to a process for preparing compounds of the invention or pharmaceutically acceptable salts, esters, or prodrugs thereof.
  • the invention relates to novel LHRH analogs of seven amino acyl residues represented by the formula (I), wherein A, B, C, D, E, F, and G represent amino acyl residues as described above.
  • a lower alkyl group of one to ten carbons, represented by R 1 attaches via a carbonyl moiety to the N-terminal nitrogen atom of the representative amino acyl residue A.
  • An N-alkylated nitrogen atom forms an amide bond with the C-terminal carbon atom of the representative amino acyl residue G.
  • Compounds of the invention provide LHRH analogs wherein the 10 to 8 amino acids from the C-terminus carboxylic acid moiety of a decapeptide LHRH antagonist sequence is eliminated.
  • the compounds exhibit effective LHRH antagonist activity and are useful in treating disorders related to high levels of reproductive hormones.
  • the lower molecular weight of the present compounds allows for improved oral bioavailability in administering treatment for various hormone-dependent diseases.
  • lower alkyl refers to straight or branched chain alkyl radicals containing from 1 to 10 carbon atoms including, but not limited to methyl, ethyl, n-propyl, isopropyl (Isp), n-butyl (nBu), isobutyl, sec-butyl, t-butyl, diethyl, and the like.
  • D the stereochemistry of the alpha- carbon atom of the amino acids and aminoacyl residues in peptides described in this specification and the appended claims is the natural or "L" configuration.
  • the compounds of the present invention are useful in modulating levels of gonadotropin and androgen secretion in mammals.
  • the compounds are particularly useful for their activity as LHRH antagonists or agonists.
  • the term '"pharmaceutically acceptable salt” as used herein refers to acid addition salts of the compounds of formula (I) which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of mammals, including humans and lower animals, without undue toxicity, irritation, allergic response, and the like commensurate with a reasonable benefit/risk ratio, and which are effective for their intended use.
  • Pharmaceutically acceptable salts are well known in the art, and are summarized in S. M. Berge, et al, J. Pharmaceutical Sciences 66:1-19 (1977).
  • the salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid.
  • suitable organic acid examples include pharmaceutically acceptable, nontoxic acid addition salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene-sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate, per
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate. sulfate, phosphate, nitrate, loweralkylsulfonate and aryl sulfonate.
  • esters of the compounds of formula (I) refers to non-toxic esters of the compounds of formula (I) derived by the condensation of a compound of the invention with an alcohol.
  • examples of pharmaceutically acceptable, non-toxic esters of the compounds of the invention include Ci to C 6 alkanoyl esters wherein the alkanoyl group is a straight or branched chain, such as formate, acetate, propanoate, butyrate, isopropanoate. pentanoate. hexanoate, and the like.
  • Esters of the compounds of the present invention may be prepared according to conventional methods.
  • prodrug refers to biolabile compounds or derivatives which, upon delivery or administration to a treatment subject, are converted to in vivo parent compounds of the formula (I) above.
  • Prodrugs of compounds of the invention are suitable for use in contact with the tissues of mammals, including humans and lower animals, without undue toxicity, irritation, allergic response, and the like commensurate with a reasonable benefit/risk ratio as determined by one of ordinary skill in the medical arts within the scope of sound medical judgement, and which are effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
  • Prodrugs are well-known in the art, and generally refers to compounds that are rapidly transformed in vivo to yield the parent compound of formula (I), for example by hydrolysis in blood.
  • a summary of the art is described in T. Higuchi and V. Stella, "'Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
  • Such prodrugs are readily apparent to one of ordinary skill in the art and can be regarded as functional equivalents of the compounds of the invention.
  • prodrugs of derivatives of compounds of the present invention may be prepared by any suitable method.
  • the condensation of the amino group with amino acids and peptides may effected in accordance with conventional condensation methods such as the azide method, the mixed acid anhydride method, the DCC (dicyclohexylcarbodiimide) method, the active ester method (p-nitrophenyl ester method, N-hydroxysuccinic acid imide ester method, cyanomethyl ester method, and the like), the Woodward reagent K method, the DCC-HOBT ( 1 -hydroxybenzotriazole) method and the like.
  • Classical methods for amino acid condensation reactions are described in M.
  • Representative examples of compounds contemplated as within the scope of the present invention include, but are not limited to the following: NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-nBu;
  • NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH- (CH 2 ) 5 NH(C NH)NH 2 ; NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-(CH 2 ) 3 NH 2 ;
  • NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH- (CH 2 ) 2 NH(C NH)NH 2 ;
  • NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys( ic)-Leu-NH- (CH 2 ) 8 NH(C NH)NH 2 ; NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-(CH 2 ) ⁇ 0 NH- isopropyl:
  • NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH- (CH 2 ) 4 NH(C NH)NH 2 ;
  • NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH- (CH 2 ) 4 NH(C NH)NH 2 ; NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-CH 2 -(2-pyridine);
  • compositions comprising a compound of the present invention as the active ingredient and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers suitable for the pharmaceutical compositions of the invention comprise non-toxic compatible substances useful for preparing a composition for administering the compound to a mammal in need of treatment.
  • Suitable pharmaceutically acceptable carriers generally include, but are not limited to, non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • Exemplary material which can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc, excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl la
  • wetting agents such as sodium lauryl sulfate and magnesium stearate.
  • coloring agents such as sodium lauryl sulfate and magnesium stearate.
  • coloring agents such as sodium lauryl sulfate and magnesium stearate.
  • coloring agents such as sodium lauryl sulfate and magnesium stearate.
  • coloring agents such as sodium lauryl sulfate and magnesium stearate.
  • coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutically acceptable composition.
  • the invention in another aspect of the invention, relates to a method of modulating gonadotropic hormones in a mammal comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound as defined above.
  • Condition for which an amount of the compound may be effective can be described as excessive tissue swelling, precocious puberty, hormonal imbalance, and other related conditions.
  • Exemplary known symptoms and conditions for which the compounds are useful in treating include, but are not limited to, benign prostate hypertrophy, dysmenorrhea, endometriosis, precocious puberty, prostate cancer, uterine fibroids, prostate necrosis, and other sex hormone dependent diseases. These compounds provide novel peptides for treatment regimens useful in treating such conditions.
  • Compounds of the invention are administered to a mammal in need of such treatment by any of a variety of routes depending on the specific end use.
  • the means for administering the peptide to a mammal will be a method selected from treatments consisting of oral, parenteral, vaginal, rectal, buccal (including sublingual), transdermal, and intranasal administration.
  • Parental routes of administration include, but are not limited to subcutaneously, intramuscularly, and intravenously. The exact method and route of administration can be determined by one of ordinary skill in the medical arts having knowledge and the ability to develop a reasoned judgment as to the form of treatment administered to the mammal in need of treatment.
  • dose and regimen for administration may depend on a variety of any factors including, but not limited to, the need of the individual subject being treated, the type of treatment, the degree of affliction or need, and length and frequency of the treatment.
  • dosage for the treatment is between about 0.01 and 10 milligram of the active ingredient per kilogram body weight per day.
  • the dose administered is from about 0.1 to about 5.0 mg/kg body weight per day.
  • the administration may be accomplished in a single, daily administration or by distributing doses over several applications or by slow release in order to achieve the most effective results.
  • Peptides of the present invention may be prepared by any techniques that are known to those skilled in the art. Commonly employed methods known in the art of peptide synthesis generally referred to as “solid phase” peptide synthesis, wherein sequential coupling of amino acids is accomplished attached to an inert solid support, and “solution phase” synthesis, the technique wherein amino acids are coupled in solution. Solid phase methods of synthesis on a support resin are described in J.M. Steward and J. D. Young, Solid Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, 1963; and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, p. 46, Academic Press (New York), 1973. Summary of classical solution phase synthesis techniques is recited in G. Schroder and K. Lupk ⁇ , 77ze Peptides, vol. 1, .Academic Pressure (New York), 1965, and "The Practice of Peptide Synthesis" by M. Bodansky and Bodansky, A.
  • Starting materials used in these general methods of peptide synthesis comprise a suitable resin and one or more amino acids or derivatives thereof.
  • Naturally occurring and commonly protected amino acids are commercially available or, alternatively, can be prepared with readily available starting materials by methods commonly known in the art.
  • the peptide is synthesized in solution by methods known to those with skill in the art. The methods are described in "The Practice of Peptide Synthesis" by M. Bodansky and Bodansky, A. Briefly, an amino acid is N-protected with a protecting group and is coupled to the next N-free amino acid with a suitable coupling reagent (described below) at 0°C to ambient temperatures for about 1 to about 5 hours to afford a dipeptide fragment. The dipeptide fragment is deprotected to afford a free amine terminus and a subsequent protected amino acid is coupled to the fragment under the coupling conditions previously described.
  • Suitable protecting groups are selected from the group consisting of hydroxy protecting groups.
  • Exemplary suitable protecting groups include, but are not limited to, t-butyloxy-carbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (alpha.alpha)-dimethyl-3,5-dimethoxy- benzyloxycarbonyl, o-nitrophenylsulfenyl. 2-cyano-t-butyloxycarbonyl-9-fluoroenyl- methyloxycarbonyl (FMOC). and the like.
  • the preferred protecting group is t-butyloxy- carbonyl.
  • Coupling reagents suitable for the amino acid coupling are selected from the group consisting of N,N'-dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide (DIC) with or without 1 -hydroxybenzotriazole (HOBT), benzotriazol-l-yloxy-tris(dimethylamino)- phosphonium-hexafluorophosphate (BOP) and bis-(2-oxo-3-oxazolidinyl)phosphine chloride (BOPC1).
  • the preferred reagent is N,N'-dicyclohexylcarbodiimide.
  • Deprotecting reagents suitable for removing the protecting groups are anhydrous liquid hydrogen fluoride in the presence of anisole and dimethylphosphite or other carbonium ion scavenger, hydrogen fluoride/pyridine complex, tris(tri-fluoroacetyl)boron and trifluoroacetic acid, hydrogen and palladium on carbon on polyvinylpyrrolidone, sodium in liquid ammonia.
  • the deprotecting agent is liquid hydrogen fluoride in the presence of anisole and dimethylphosphite.
  • a solid support provides an inert surface to which an amino acid is attached.
  • the solid support materials typically resins, are inert to the reagents and reaction conditions of the peptide linkage formation as well as conditions for cleaving the final peptide from the solid support.
  • Suitable solid supports useful for the above synthesis are chloromethylpolystyrene-divinylbenzene polymer, hydroxymethylpolystyrene-divinylbenzene polymer, benzyldrylaminopolystyrene- divinylbenzene polymer, and the like.
  • the support is chloromethyl-polystyrene- 1% divinylbenzene polymer.
  • amino acid refers to amino acids, salts, esters and derivatives thereof suitable for sequencing in a peptide synthesis as determined by one of ordinary skill in the art.
  • the amino acid residues are attached to the resin or a formed polypeptide chain as a salt to synthesis the polypeptide chain having the desired sequence and having the desired length.
  • Suitable salts of the amino acid are cesium, tetramethylammonium, triethyl- ammonium, l,5-diazabicyclo-[5.4.0]undec-5-ene salts, or the like.
  • the amino acid is coupled with the solid support as a cesium salt.
  • Protecting groups preferred for preparing the peptides provide stable moieties for protecting the alpha-amino function of the amino acids.
  • the protecting groups used generally have properties of being stable to conditions of peptide linkage formation and can be readily removed without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein.
  • Suitable protecting groups are hydroxy protecting groups as described in the solution phase synthesis.
  • Coupling of the protected amino acid to the support is accomplished in an inert solvent.
  • Solvents suitable for the coupling reaction include, but are not limited to, ethanol, dichloromethane, methylene chloride, acetonitrile, N,N-dimethyl-formamide (DMF), and the like, or a mixture thereof.
  • the solvent is ethanol or dimethylformamide.
  • reaction is carried out between about 40°C and 60°C, from about 12 to about
  • Coupling of the attached amino acid and residue with additional amino acids involves reacting the attached amino acid with a suitable coupling reagent for about 1 to 24 hours.
  • the reaction is carried out for 12 hours at a temperature of between 10°C and 50°C in the inert solvent.
  • Each protected amino acid is introduced in 0.4 M concentration and approximately 3.5 molar excess.
  • the coupling reaction is carried out in a 1 :1 mixture of dichloromethane and DMF at ambient temperatures.
  • cleaving reagents are alkylamines or fluoroalkylamines in the presence or absence of boron tribromide. The most preferred is 1 -(2-aminoethyl)- pyrrolidine.
  • Deprotection is usually accomplished under anhydrous strong acidic conditions that remove the protecting groups without destroying the formed peptide chain or degrading the acid sensitive moieties present on the peptide chain.
  • Preferred temperatures for carrying out the deprotection reaction are from about -10°C to about +10°C. The most preferred reaction is carried out at 0°C for about 30 minutes with a deprotecting agent as described in the solution phase synthesis. Procedures of the invention can be better understood in accordance with the
  • reaction mixture was stirred at room temperature for one hr, then poured into a saturated solution of sodium bicarbonate and stirred overnight.
  • the solid precipitate was filtered, washed with water and dried in vacuo over P 2 O 5 .
  • the dried solid was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour.
  • the excess of reagent was removed in vacuo, the residue was washed with ether and then purified by HPLC using C-18 reverse phase column and running a gradient, over 30 minutes, of 25-50% acetonitrile/water containing 0.1% trifluoroacetic acid.
  • Example 5 The procedure described in Example 5 was used, but substituting 1 ,4-diamino- butane for 1 ,6-diaminohexane. After workup and drying of the product over P 2 O 5 overnight, the residue was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour. The excess of reagent was removed in vacuo. The residue was washed with ether and then purified by HPLC using C- 18 reverse phase column and running a gradient, over 30 minutes, of 25-50%) acetonitrile/water containing 0.1% trifluoroacetic acid.
  • the precipitate was filtered and then dried over P 2 O 5 overnight and then was treated with anhydrous anisole ( 1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour. The excess of reagent was removed in vacuo, the residue was washed with ether and then purified by HPLC using C-18 reverse phase column and running a gradient, over 30 minutes, of 25-50% of acetonitrile/water containing 0.1% trifluoroacetic acid.
  • Example 7 The procedure described in Example 7 was used, but substituting phenethylamine for ethanolamine. After workup and drying over P 2 O 5 overnight, the dry residue was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour. The excess of reagent was removed in vacuo. The residue was washed with ether and then purified by HPLC using C-18 reverse phase column and running a gradient of 25-50%, over 30 minutes, of acetonitrile/water containing 0.1 % trifluoroacetic acid.
  • NAcD2Nal-D4ClPhe-D3Pal-Ser(OBzl)-NMeTyr(OBzl)-DLys(Nic)-Leu-OBzl (0.2213 g) was treated at room temperature with 2-aminoethyl-N-isopropylamine (1.0 ml). The reaction mixture was stirred for two days, then was poured into 10%
  • Example 9 The procedure described in Example 9 was used, but substituting 1 ,7-diamino- heptane for 2-aminoethyl-N-isopropylamine (1.0 ml). The reaction mixture was stirred for two days, then was poured into 10% NaHCO 3 solution, the precipitate was filtered, dissolved in (1 : 1) acetonitrile/water and lyophilized. The dry residue was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour.
  • Example 7 The procedure described in Example 7 was used, but substituting 1 ,5-diamino- pentane for ethanolamine. The reaction mixture was stirred at room temperature overnight. then was poured into a 10% solution of NaHCO and stirred for 15 minutes. The precipitate was filtered, dissolved in (1 : 1) acetonitrile/water and lyophilized. The dry powder was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour. The excess of reagent was removed in vacuo. The residue was washed with ether and then purified by HPLC using C-18 reverse phase column and running a gradient, over
  • Example 11 The procedure described in Example 11 was used, but substituting NAcD2Nal- D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-(CH 2 ) 5 NH 2 for NAcD2Nal-D4ClPhe- D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-(CH2) 6 NH2.
  • the guanidino derivative was obtained, and purified by HPLC using C-18 reverse phase column and running a gradient, over 30 minutes, of 25-50% acetonitrile/water containing 0.1 % trifluoroacetic acid.
  • Example 10 The procedure described in Example 10 was used, but substituting 1,3-diamino- propane (1.0 ml) for 1 ,7-diaminoheptane. The reaction mixture was stirred for two days, then was poured into 10% NaHCO 3 solution. The precipitate was filtered, dissolved in (1 : 1) acetonitrile/water and lyophilized. The dry residue was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour. The excess of reagent was removed in vacuo.
  • the guanidino derivative was obtained, and purified by HPLC using C-18 reverse phase column and running a gradient, over 30 minutes, of 25-50% acetonitrile/water containing 0.1% trifluoroacetic acid.
  • Example 10 The procedure described in Example 10 was used, but substituting 1,10-diamino- decane (1.0 ml) for 1,7-diaminohexane. The reaction mixture was stirred for two days, then was poured into 10% NaHCO 3 solution, the precipitate was filtered, dissolved in (1 :1) acetonitrile/water and lyophilized. The dry residue was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour.
  • Example 10 The procedure described in Example 10 was used, but substituting 1,8-diamino- octane (1.0 ml) for 1,10-diaminodecane. The reaction mixture was stirred for two days, then was poured into 10% NaHCO 3 solution. The precipitate was filtered, dissolved in (1 :1) acetonitrile/water and lyophilized. The dry residue was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour.
  • Example 16 The procedure described in Example 16 was used, but substituting NAcD2Nal- D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-(CH 2 ) 8 NH 2 for NAcD2Nal-D4ClPhe- D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-(CH 2 ) 2 NH 2 .
  • the guanidino derivative was obtained, and purified by HPLC using C-18 reverse phase column and running a gradient, over 30 minutes, of 25-50% acetonitrile/water containing 0.1% trifluoroacetic acid.
  • the dry powder was treated with anhydrous anisole (1 ml) and anhydrous HF (5 ml) at 0 °C for 1 hour. The excess of reagent was removed in vacuo. The residue was washed with ether and then purified by HPLC using C-18 reverse phase column and running a gradient, over 30 minutes, of 25-50%) acetonitrile/water containing 0.1% trifluoroacetic acid. The desired product.
  • Example 14 The procedure described in Example 14 was used, but substituting NAcD2Nal- D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-(CH 2 ) 4 NH 2 forNAcD2Nal-D4ClPhe- D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-NH-(CH 2 ) 5 NH 2 .
  • the guanidino derivative was obtained, and purified by HPLC using C-18 reverse phase column and running a gradient, over 30 minutes, of 25-50% acetonitrile/water containing 0.1% trifluoroacetic acid.
  • Example 27 The procedure described in Example 27 was used but substituting amino- ethylpyridine for aminoethylmorpholine. After work up. the cleaving of the protecting groups and HPLC purification, the desired product, NAcD2Nal-D4ClPhe-D3Pal-Ser-
  • Example 29 The procedure described in Example 29 was used, but substituting 2-(2-amino- ethyl)- 1 -methyl-pyrrolidine for 2-(2-aminoethyl)- 1 -methyl-pyrrole.
  • the desired product NAcD2Nal- D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)- Leu-NH-CH 2 -CH 2 -[2-(l-methyl)-pyrrolidine]
  • R t 21.5 min
  • FAB Mass spec for C oH 87 N ⁇ 2 OnCl showed (M+1) @ 1306 m/z; Amino Acid Anal.: 0.43 Ser; 1.08 NMeTyr; 1.01 Leu;
  • Deblocking to remove the t-BOC group from the alpha-amino function of the peptide. was carried out using a solution of 45% trifluoroacetic acid (TFA). 2.5 % anisole. 2.0%> dimethyl phosphite, and 50.5% methylene chloride. The resin was prewashed with the deblocking solution for one minute and then the deblocking reaction was run for 20 minutes.
  • TFA trifluoroacetic acid
  • Base wash to remove and neutralize the TFA used for deprotection, was carried out using a solution of 10%> N,N'-diisopropylethylamine in methylene chloride. The resin was washed with base three times for one minute each time after a deblocking step.
  • Coupling reaction was carried out using a 3-fold molar excess of 0.3 M DMF solution of a t-BOC protected amino acid derivative along with a 3-fold molar excess of 0.3 M methylene chloride solution of diisopropylcarbodiimide as activator.
  • the activated amino acid was then coupled to the free alpha-amino grroup of the peptide-resin.
  • the reaction time was as described in the synthesis protocol described below.
  • amino acids were coupled to the resin according the following order, number and duration of couplings:
  • the peptide-resin was dried overnight over P 2 O 5 under vacuum and then was treated at room temperature with anhydrous methylene chloride (10 ml) with stirring under N 2 .
  • anhydrous methylene chloride (10 ml) with stirring under N 2 .
  • a 0.63 M solution of boron tribromide in methylene chloride (2 ml) and the mixture was stirred for one hr, then aminoethyl-N-pyrrolidine (0.3 ml) was added and stirring continued overnight.
  • Methanol (1 ml) was added and the mixture was stirred for 15 min and filtered. The resin was washed thoroughly with methanol three times and the filtrate and washes were combined and concentrated in vacuo.
  • Example 36 The procedure described in Example 36 is used, but substituting BOC- NMeTyr(OBzl) for BOC-Tyr(OBzl). After workup and HPLC purification the desired product, NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DCit-Leu-NH-(CH 2 ) 2 -(l-pyrrolidine), is obtained as trifluoroacetate salt.
  • Example 36 The procedure described in Example 36 is used, but substituting BOC-NMePhe for BOC-Tyr(OBzl). After workup and HPLC purification the desired product, NAcD2Nal- D4ClPhe-D3Pal-Ser-NMePhe-DLys(Nic)-Leu-NH-(CH 2 ) 2 -(l -pyrrolidine), is obtained as trifluoroacetate salt.
  • Example 36 The procedure described in Example 36 is used, but substituting BOC- NMeTyr(OBz) and BOC-DArg(Tos) for BOC-Tyr(OBzl) and BOC-DLys(Nic), respectively. After workup and HPLC purification the desired product, NAcD2Nal- D4ClPhe-D3Pal-Ser-NMeTyr-DArg-Leu-NH-(CH 2 ) 2 -(l-pyrrolidine), is obtained as trifluoroacetate salt.
  • Example 36 The procedure described in Example 36 is used, but substituting BOC-NMePhe- (4-NAc) and BOC-DPhe(4-NAc) for BOC-Tyr(OBzl) and BOC-DLys(Nic) respectively. After workup and HPLC purification the desired product, NAcD2Nal-D4ClPhe-D3Pal-Ser- NMePhe(4-NAc)-DPhe(4-NAc)-Leu-NH-(CH 2 ) 2 -(l -pyrrolidine), is obtained as trifluoroacetate salt.
  • Example 36 The procedure described in Example 36 was used, but substituting BOC- NMePhe(4-N-FMOC) and BOC-DPhe(4-N-FMOC) for BOC-Tyr(OBzl) and BOC- DLys(Nic), respectively.
  • the peptide-resin is treated with 30% piperidine/DMF for 2 to 24 hr, to cleave the FMOC protecting group from the 4-amino group on the phenyl rings of the side chains.
  • the peptide-resin is washed 3 times with methylene chloride, 3 times with DMF and reacted with 10- to 20-fold excess of diphenylcyanocarbodiimidate in DMF overnight, washed 3 times with methylene chloride, 3 times with DMF, and then reacted with 20- to 100-fold excess of hydrazine in DMF overnight.
  • the peptide-resin is again washed, as previously described, dried over P 2 O 5 overnight and then is treated at room temperature with anhydrous methylene chloride (10 ml) with stirring under N 2 .
  • Example 48 The procedure described in Example 48 is used, but substituting BOC-Phe- (4-N-FMOC) for BOC-NMePhe(4-N-FMOC). After workup and lyophilization the crude product is purified by HPLC to give the desired product NAcD2Nal-D4ClPhe-D3Pal-Ser- Phe(4-Atza)-DPhe(4-Atza)-Leu-NH-(CH 2 ) 2 -(l -pyrrolidine) as the trifluoroacetate salt.
  • Representative compounds of the present invention were evaluated in vitro for inhibition of LH release from rat pituitary cells (pA 2 ). Methods for the assay procedures are described by F. Haviv, et al. J. Med. Chem., 32:2340-2344 (1989). Values of pA 2 are the negative logarithms of the concentration of the particular antagonist test compound required to shift the response curve produced by the agonist leuprolide to two-fold higher concentration. Typically values of 7.0 or greater are indicative of good LHRH antagonist potency, with values of 8.0 or greater being preferred.
  • Leuprolide LHRH agonist disclosed and claimed in U.S. Pat. No. 4,005,063, has the structure 5-oxo-Pro'-His 2 -Trp 3 - Ser 4 -Tyr 5 -D-Leu 6 -Leu 7 -Arg 8 -Pro 9 -NHEt.

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Abstract

L'invention concerne une classe d'analogues heptapeptidiques de LHRH. Ces composés sont utiles pour traiter des états pathologiques induits par les hormones de reproduction notamment l'adénome prostatique, les tumeurs de la prostate, les tumeurs du sein et des ovaires, la cryptorchidie, l'hirsutisme, les troubles de la motilité gastrique, la dysménorrhée et l'endométriose.
PCT/US1999/017874 1998-08-12 1999-08-06 Analogues heptapeptidiques de lhrh Ceased WO2000009544A1 (fr)

Applications Claiming Priority (4)

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US13305598A 1998-08-12 1998-08-12
US09/133,055 1998-08-12
US09/232,425 US6191115B1 (en) 1998-08-12 1999-01-15 C-terminus modified heptapeptide LHRH analogs
US09/232,425 1999-01-15

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4087416A (en) * 1976-11-05 1978-05-02 Parke, Davis & Company New heptapeptides and methods for their production
EP0417454A2 (fr) * 1989-08-07 1991-03-20 Abbott Laboratories Analogues de LHRH à dimension réduite

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4087416A (en) * 1976-11-05 1978-05-02 Parke, Davis & Company New heptapeptides and methods for their production
EP0417454A2 (fr) * 1989-08-07 1991-03-20 Abbott Laboratories Analogues de LHRH à dimension réduite

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHANG E.A.: "Studies on analogs of the LHRH towards elucidation of the release mechanism", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 47, 1972, ORLANDO, FL US, pages 1256 - 1261, XP000857554 *
JANECKA A ET AL: "THE STRUCTURAL FEATURES OF EFFECTIVE ANTAGONISTS OF THE LUTEINIZINGHORMONE RELEASING HORMONE", AMINO ACIDS,AU,SPRINGER VERLAG, vol. 6, no. 2, pages 111-130, XP000566196, ISSN: 0939-4451 *
JANECKA E.A.: "Reduced-size antagonists of LHRH active in vitro", JOURNAL OF MEDICINAL CHEMISTRY, vol. 38, 1995, WASHINGTON US, pages 2922 - 2924, XP002125914 *

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