The present invention is directed to peptide analogues of glucagon-like peptide-1, the pharmaceutically-acceptable salts thereof, to methods of using such analogues to treat mammals and to pharmaceutical compositions useful therefor comprising said analogues.
Glucagon-like peptide-1 (7-36) amide (GLP-1) is synthesized in the intestinal L-cells by tissue-specific post-translational processing of the glucagon precursor pre-proglucagon (
In 1994, the therapeutic potential of GLP-1 was suggested following the observation that a single subcutaneous (s/c) dose of GLP-1 could completely normalize postprandial glucose levels in patients with non-insulin-dependent diabetes mellitus (NIDDM) (
This combination of actions gives GLP-1 unique potential therapeutic advantages over other agents currently used to treat NIDDM.
Numerous studies have shown that when given to healthy subjects, GLP-1 potently influences glycemic levels as well as insulin and glucagon concentrations (
GLP-1 is, however, metabolically unstable, having a plasma half-life (t1/2) of only 1-2 min in vivo. Exogonously administered GLP-1 is also rapidly degraded (
In one aspect, the present invention is directed to a compound of formula (I),
A7 -A8 -A9 -A10 -A11 -A12 -A13 -A14 -A15 -A16 -A17 -A18 -A19 -A20 -A21 -A22 -A23 -A24 -A25 -A26 -A27 -A28 -A29 -A30 -A31 -A32 -A33 -A34 -A35 -A36 -A37-R1, (I)
wherein
A7 is, Ura, Paa, Pta or Hppa,
A8 is Ala, D-Ala, Aib, Acc, N-Me-Ala, N-Me-D-Ala, Arg or N-Me-Gly;
A9 is Glu, N-Me-Glu, N-Me-Asp or Asp;
A10 is Gly, Acc, Ala, D-Ala, Phe or Aib;
A11 is Thr or Ser;
A12 is Phe, Acc, Aic, Aib, 3-Pal, 4-Pal, β-Nal, Cha, Trp or X1-Phe;
A13 is Thr or Ser;
A14 is Ser, Thr, Ala or Aib;
A15 is Asp, Ala, D-Asp or Glu;
A16 is Val, D-Val, Acc, Aib, Leu, Ile, Tle, Nle, Abu, Ala, D-Ala, Tba or Cha;
A17 is Ser, Ala, D-Ala, Aib, Acc or Thr;
A18 is Ser, Ala, D-Ala, Aib, Acc or Thr;
A19 is Tyr, D-Tyr, Cha, Phe, 3-Pal, 4-Pal, Acc, β-Nal, Amp or X1-Phe;
A20 is Leu, Ala, Acc, Aib, Nle, lie, Cha, Tle, Val, Phe or X1-Phe;
A21 is Glu, Ala or Asp;
A22 is Gly, Acc, Ala, D-Ala, β-Ala or Aib;
A23 is Gln, Asp, Ala, D-Ala, Aib, Acc, Asn or Glu;
A24 is Ala, Aib, Val, Abu, Tle or Acc;
A25 is Ala, Aib, Val, Abu, Tle, Acc, Lys, Arg, hArg, Orn, HN-CH((CH2)m-NR10R11)-C(O) or HN-CH((CH2)e-X3)C(O);
A26 is Lys, Ala, 3-Pal, 4-Pal, Arg, hArg, Orn, Amp, HN-CH((CH2)n-NR10R11)-C(O) or HN-CH((CH2)e-X3)-C(O);
A27 is Glu, Ala, D-Ala or Asp;
A28 is Phe, Ala, Pal, β-Nal, X1-Phe, Aic, Acc, Aib, Cha or Trp;
A28 is lie, Acc, Aib, Leu, Nle, Cha, Tle, Val, Abu, Ala, Tba or Phe;
A30 is Ala, Aib, Acc or deleted;
A31 is Trp, Ala, β-Nal, 3-Pal, 4-Pal, Phe, Acc, Aib, Cha, Amp or deleted;
A32 is Leu, Ala, Acc, Aib, Nle, lie, Cha, Tle, Phe, X1-Phe, Ala or deleted;
A33 is Val, Acc, Aib, Leu, lie, Tle, Nle, Cha, Ala, Phe, Abu, X1-Phe, Tba, Gaba or deleted;
A34 is Lys, Arg, hArg, Orn, Amp, Gaba, HN-CH((CH2)m-NR10R11)-C(O), HN-CH((CH2)e-X3)-C(O) or deleted;
A35 is Gly or deleted;
A36 is L- or D-Arg, D- or L-Lys, D- or L-hArg, D- or L-Orn, Amp, HN-CH((CH2)n-NR10R11)-C(O), HN-CH((CH2)-X3)-C(O) or deleted;
A37 is Gly or deleted;
- X1 for each occurrence is independently selected from the group consisting of (C1-C6)alkyl, OH and halo;
- R1 is OH, NH2, (C1-C12)alkoxy, or NH-X2-CH2-Z0, wherein X2 is a (C1-C12)hydrocarbon moiety, and Z0 is H, OH, CO2H or CONH2;
- X3 is
- e for each occurrence is independently an integer from 1 to 4;
- n for each occurrence is independently an integer from 1-5; and
- R10 and R11 for each occurrence is each independently H, (C1-C30)alkyl, (C1-C30)acyl, (C1-C30)alkylsulfonyl, -C((NH)(NH2)) or
- R12 is (C1-C30)alkyl;
A preferred compound of the immediately foregoing compound of formula (I) is where A11 is Thr; A13 is Thr; A14 is Ser, Aib or Ala; A17 is Ser, Ala, Aib or D-Ala; A18 is Ser, Ala, Aib or D-Ala; A21 is Glu or Ala; A23 is Gln, Glu, or Ala; and A27 is Glu or Ala; or a pharmaceutically acceptable salt thereof.
A preferred compound of the immediately foregoing compound of formula (I) is where A9 is Glu, N-Me-Glu or N-Me-Asp; A12 is Phe, Acc or Aic; A16 is Val, D-Val, Acc, Aib, Ala, Tle or D-Ala; A19 is Tyr, 3-Pal, 4-Pal or D-Tyr; A20 is Leu, Acc, Cha, Ala or Tle; A24 is Ala, Aib or Acc; A25 is Ala, Aib, Acc, Lys, Arg, hArg, Orn, HN-CH(CH2)n-NH-R10)-C(O); A28 is Phe or Ala; A29 is lie, Acc or Tle; A30 is Ala, Aib or deleted; A31 is Trp, Ala, 3-Pal, 4-Pal or deleted; A32 is Leu, Acc, Cha, Ala or deleted; A33 is Val, Acc, Ala, Gaba, Tle or deleted; or a pharmaceutically acceptable salt thereof.
A preferred compound of the immediately foregoing compound of formula (I) is where A8 is Ala, D-Ala, Aib, A6c, A5c, N-Me-Ala, N-Me-D-Ala or N-Me-Gly; A10 is Gly, Ala, D-Ala or Phe; A12 is Phe, A6c or A5c; A16 is Val, Ala, Tle, A6c, A5c or D-Val; A20 is Leu, A6c, A5c, Cha, Ala or Tle; A22 is Gly, Aib, β-Ala, L-Ala or D-Ala; A24 is Ala or Aib; A29 is Ile, A6c, A5c or Tle; A32 is Leu, A6c, A5c, Cha, Ala or deleted; A33 is Val, A6c, A5c, Ala, Gaba, Tle or deleted; or a pharmaceutically acceptable salt thereof.
A preferred compound of the immediately foregoing compound of formula (I) is where R1 is OH or NH2 or a pharmaceutically acceptable salt thereof.
A most preferred compound of formula (I) is where said compound is [Hppa7]hGLP-1(7-36)-NH2 or a pharmaceutically acceptable salt thereof.
Another most preferred compound of formula (I) is wherein said compound is [Ura7]hGLP-1(7-36)-NH2; [Paa7]hGLP-1(7-36)-NH2; [Pta7]hGLP-1(7-36)NH2; or a pharmaceutically acceptable salt thereof.
In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) as defined hereinabove or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.
In still another aspect, the present invention provides a use of compound of formula (I) as defined hereinabove or a pharmaceutically acceptable salt thereof in the preparation of a medicament for eliciting an agonist effect from a GLP-1 receptor in a subject in need thereof.
In yet a further aspect, this invention provides a use of a compound of formula (I) as defined hereinabove or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease selected from the group consisting of Type I diabetes, Type II diabetes, obesity, glucagonomas, secretory disorders of the airway, metabolic disorder, arthritis, osteoporosis, central nervous system disease, restenosis and neurodegenerative disease, renal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pulmonary edema, and hypertension, in a subject in need thereof which comprises administering to said subject an effective amount. Preferred of the foregoing uses is where the disease is Type I diabetes or Type II diabetes.
With the exception of the N-terminal amino acid, all abbreviations (e.g. Ala) of amino acids in this disclosure stand for the structure of -NH-CH(R)-CO-, wherein R is the side chain of an amino acid (e.g., CH3 for Ala). For the N-terminal amino acid, the abbreviation stands for the structure of (R2R3)-N-CH(R)-CO-, wherein R is a side chain of an amino acid and R2 and R3 are as defined above except in the case where A7 is Ura, Paa, Pta or Hppa in which case R2 and R3 are not present since Ura, Paa, Pta and Hppa are considered here as des-amino amino acids. The abbreviations: β-Nal, Nle, Cha, Amp, 3-Pal, 4-Pal and Aib stand for the following α-amino acids: β-(2-naphthyl)alanine, norleucine, cyclohexylalanine, 4-amino-phenylalanine, β-(3-pyridinyl)alanine, β-(4-pyridinyl)alanine and α-aminoisobutyric acid, respectively. Other amino acid definitions are: Ura is urocanic acid; Pta is (4-pyridylthio) acetic acid; Paa is trans-3-(3-pyridyl) acrylic acid; Tma-His is N,N-tetramethylamidino-histidine; N-Me-Ala is N-methyl-alanine; N-Me-Gly is N-methyl-glycine; N-Me-Glu is N-methyl-glutamic acid; Tle is tert-butylglycine; Abu is α-aminobutyric acid; Tba is tert-butylalanine; Orn is ornithine; Aib is α-aminoisobutyric acid; β-Ala is β-alanine; Gaba is γ-aminobutyric acid; Ava is 5-aminovaleric acid; and Aic is 2-aminoindane-2-carboxylic acid.
What is meant by Acc is an amino acid selected from the group of 1-amino-1-cyclopropanecarboxylic acid (A3c); 1-amino-1-cyclobutanecarboxylic acid (A4c); 1-amino-1-cyclopentanecarboxylic acid (A5c); 1-amino-1-cyclohexanecarboxylic acid (A6c); 1-amino-1-cycloheptanecarboxylic acid (A7c); 1-amino-1-cyclooctanecarboxylic acid (A8c); and 1-amino-1-cyclononanecarboxylic acid (A9c). In the above formula, hydroxyalkyl, hydroxyphenylalkyl, and hydroxynaphthylalkyl may contain 1-4 hydroxy substituents. COX5 stands for -C=O·X5. Examples of -C=O·X5 include, but are not limited to, acetyl and phenylpropionyl.
What is meant by Lys(Nε-alkanoyl) is represented by the following structure:
The full names for other abbreviations used herein are as follows: Boc for t-butyloxycarbonyl, HF for hydrogen fluoride, Fm for formyl, Xan for xanthyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMF for dimethylformamide, DCM for dichloromethane, HBTU for 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, DIEA for diisopropylethylamine, HOAc for acetic acid, TFA for trifluoroacetic acid, 2CIZ for 2-chlorobenzyloxycarbonyl and OcHex for O-cyclohexyl.
A peptide of this invention is also denoted herein by another format, e.g., [A5c8]hGLP-1 (7-36)NH2, with the substituted amino acids from the natural sequence placed between the set of brackets (e.g., A5c8 for Ala8 in hGLP-1). The abbreviation GLP-1 means glucagon-like peptide-1, and hGLP-1 means human glucagon-like peptide-1. The numbers between the parentheses refer to the number of amino acids present in the peptide (e.g., hGLP-1 (7-36) is amino acids 7 through 36 of the peptide sequence for human GLP-1). The sequence for hGLP-1 (7-37) is listed in
The peptides of this invention can be prepared by standard solid phase peptide synthesis. See, e.g.,
When R1 is NH-X2-CH2- CONH2 (i.e., Z0=CONH2), the synthesis of the peptide starts with BocHN-X2-CH2COOH which is coupled to the MBHA resin. If R1 is NH-X2-CH2-COOH (i.e., Z0=COOH) the synthesis of the peptide starts with Boc-HN-X2-CH2-COOH which is coupled to PAM resin.
The following describes a synthetic method for making a peptide of this invention, which method is well-known to those skilled in the art. Other methods are also known to those skilled in the art.
Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc., Louisville, KY) (0.9 g, 0.3 mmole) in the chloride ion form is placed in a reaction vessel of an Advanced ChemTech Peptide Synthesizer Model 200 programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 15 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f) 10% diisopropylethylamine in methylene chloride.
The neutralized resin is stirred with Boc-protected amino acid which is to be the C-terminal amino acid of the desired peptide to be synthesized and diisopropylcarbodiimide (3 mmole each) in methylene chloride for 1 hour and the resulting amino acid resin is then cycled through steps (a) through (f) in the above wash program. The other amino acids (3 mmol) of the desired peptide are then coupled successively by the same procedure. The finished peptide is cleaved from the resin by mixing it with anisole (5 ml), dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at about 0 °C and stirring it for about 45 min. Excess hydrogen fluoride is evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide is then dissolved in a minimum volume of dilute acetic acid and eluted on a column (2.5 x 25 cm) of VYDAC® octadecylsilane silica (10 mM) and eluted with a linear gradient of 20-60% acetonitrile over about 1 h in 0.1% trifluoroacetic acid in water. Fractions are examined by thin layer chromatography and analytical high performance liquid chromatography (40-70% B at 1%/min, solution B is 80% acetonitrile/water containing 0.1% TFA) and pooled to give maximum purity rather than yield. Repeated lyophilization of the solution from water gives the product as a white, fluffy powder.
The product peptide is analyzed by HPLC. Amino acid analysis of an acid hydrolysate of the product peptide can confirm the composition of the peptide. Laser desorption MS is used to determine the molecular weight of the peptide.
The protected amino acid 1-[N-tert-butoxycarbonyl-amino]-1-cyclohexanecarboxylic acid (Boc-A6c-OH) was synthesized as follows. 19.1 g (0.133 mol) of 1-amino-1-cyclohexanecarboxylic acid (Acros Organics, Fisher Scientific, Pittsburgh, PA) was dissolved in 200 ml of dioxane and 100 ml of water. To it was added 67 ml of 2N NaOH. The solution was cooled in an ice-water bath. 32.0 g (0.147 mol) of di-tert-butyldicarbonate was added to this solution. The reaction mixture was stirred overnight at room temperature. Dioxane was then removed under reduced pressure. 200 ml of ethyl acetate was added to the remaining aqueous solution. The mixture was cooled in an ice-water bath. The pH of the aqueous layer was adjusted to about 3 by adding 4N HCl. The organic layer was separated. The aqueous layer was extracted with ethyl acetate (1 x 100 ml). The two organic layers were combined and washed with water (2 x 150 ml), dried over anhydrous MugSO4, filtered, and concentrated to dryness under reduced pressure. The residue was recrystallized in ethyl acetate/hexanes. 9.2 g of the pure product was obtained. 29% yield.
Boc-A5c-OH was synthesized in an analogous manner to that of Boc-A6c-OH. Other protected Acc amino acids can be prepared in an analogous manner by a person of ordinary skill in the art as enabled by the teachings herein.
In the synthesis of a peptide of this invention containing A5c, A6c and/or Aib, the coupling time is about 2 hrs. for these residues and the residue immediately following them. For the synthesis of [Tma-His7]hGLP-1(7-36)NH2, HBTU (2 mmol) and DIEA (1.0 ml) in 4 ml DMF were used to react with the N-terminal free amine of the peptide-resin in the last coupling reaction; the coupling time is about 2 hours.
The full names for the abbreviations used above are as follows: Boc for t-butyloxycarbonyl, HF for hydrogen fluoride, Fm for formyl, Xan for xanthyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMF for dimethylformamide, DCM for dichloromethane, HBTU for 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, DIEA for diisopropylethylamine, HOAc for acetic acid, TFA for trifluoroacetic acid, 2CIZ for 2-chlorobenzyloxycarbonyl, 2BrZ for 2-bromobenzyloxycarbonyl and OcHex for O-cyclohexyl.
A compound of the present invention can be tested for activity as a GLP-1 binding compound according to the following procedure.
RIN 5F rat insulinoma cells (ATCC-# CRL-2058, American Type Culture Collection, Manassas, VA), expressing the GLP-1 receptor, were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, and maintained at about 37 °C in a humidified atmosphere of 5% CO2/95% air.
Membranes were prepared for radioligand binding studies by homogenization of the RIN cells in 20 ml of ice-cold 50 mM Tris-HCl with a Brinkman Polytron (Westbury, NY) (setting 6, 15 sec). The homogenates were washed twice by centrifugation (39,000 g / 10 min), and the final pellets were re-suspended in 50 mM Tris-HCl, containing 2.5 mM MgCl2, 0.1 mg/ml bacitracin (Sigma Chemical, St. Louis, MO), and 0.1% BSA. For assay, aliquots (0.4 ml) were incubated with 0.05 nM [125I]GLP-1(7-36) (∼2200 Ci/mmol, New England Nuclear, Boston, MA), with and without 0.05 ml of unlabeled competing test peptides. After a 100 min incubation (25 °C), the bound [125I]GLP-1(7-36) was separated from the free by rapid filtration through GF/C filters (Brandel, Gaithersburg, MD), which had been previously soaked in 0.5% polyethyleneimine. The filters were then washed three times with 5 ml aliquots of ice-cold 50 mM Tris-HCl, and the bound radioactivity trapped on the filters was counted by gamma spectrometry (Wallac LKB, Gaithersburg, MD). Specific binding was defined as the total [125I]GLP-1(7-36) bound minus that bound in the presence of 1000 nM GLP1 (7-36) (Bachem, Torrence, CA).
The peptides of this invention can be provided in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids). A typical method of making a salt of a peptide of the present invention is well known in the art and can be accomplished by standard methods of salt exchange. Accordingly, the TFA salt of a peptide of the present invention (the TFA salt results from the purification of the peptide by using preparative HPLC, eluting with TFA containing buffer solutions) can be converted into another salt, such as an acetate salt by dissolving the peptide in a small amount of 0.25 N acetic acid aqueous solution. The resulting solution is applied to a semi-prep HPLC column (Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1N ammonium acetate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueous solution 0.5 hrs. and (3) a linear gradient (20% to 100% of solution B over 30 min.) at a flow rate of 4 ml/min (solution A is 0.25N acetic acid aqueous solution; solution B is 0.25N acetic acid in acetonitrile/water, 80:20). The fractions containing the peptide are collected and lyophilized to dryness.
As is well known to those skilled in the art, the known and potential uses of GLP-1 is varied and multitudinous [See,
Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of formula (I) in association with a pharmaceutically acceptable carrier or diluent.
The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. In general, an effective dosage for the activities of this invention is in the range of 1x10-7 to 200 mg/kg/day, preferably 1x10-4 to 100 mg/kg/day, which can be administered as a single dose or divided into multiple doses.
The compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.
Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
Further, a compound of this invention can be administered in a sustained release composition such as those described in the following patents and patent applications.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The following examples describe synthetic methods for making a peptide of this invention, which methods are well-known to those skilled in the art. Other methods are also known to those skilled in the art. The examples are provided for the purpose of illustration.
Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc. Louisville, KY) (0.9 g, 0.3 mmole) in the chloride ion form was placed in a reaction vessel of an Advanced ChemTech peptide synthesizer Model 200 programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 15 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f) 10% diisopropylethylamine in methylene chloride.
The neutralized resin was stirred with Boc-Gaba and diisopropylcarbodiimide (3 mmole each) in methylene chloride for 1 hour and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash program. The following amino acids (3 mmole) were then coupled successively by the same procedure: Boc-Val, Boc-Leu, Boc-3-Pal, Boc-Ala, Boc-Ile, Boc-Phe, Boc-Ala, Boc-Lys(2-Cl-Z), Boc-Ala, Boc-Ala, Boc-Ala, Boc-Ala, Boc-Glu(Bzl), Boc-Leu, Boc-3-Pal, Boc-Ser(Bzl), Boc-Ala, Boc-Val, Boc-Asp(Bzl), Boc-Ser(Bzl), Boc-Thr(Bzl), Boc-Phe, Boc-Thr(Bzl), Boc-Gly, Boc-Glu(Bzl), Boc-D-Ala, Boc-His(Bom).
The resin with the completed peptide sequence was mixed with anisole (5 ml), dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at about 0 °C and stirred for about 45 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide was then dissolved in a minimum volume of dilute acetic acid and eluted on a column (2.5 x 25 cm) of VYDAC® octadecylsilane silica (10 mM) and eluted with a linear gradient of 20-60% acetonitrile over about 1 h in 0.1% trifluoroacetic acid in water. Fractions were examined by thin layer chromatography and analytical high performance liquid chromatography (40-70% B at 1%/main: r.t.: 14.1 min) and pooled to give maximum purity rather than yield. Repeated lyophilization of the solution from water gives the product (49.9 mg) as a white, fluffy powder.
The product was found to be homogeneous by HPLC and tlc. Amino acid analysis of an acid hydrolysate confirms the composition of the peptide. Laser desorption MS gave a MW of 2880 (Calc. M+H 2873).
Peptides are assembled on O-benzyl-polystyrene resin (often referred to as Merrifield resin) using the Boc amino acid protocol described in Example 1, except that Asp and Glu amino acid carboxyl side-chains are protected with an Fm (fluorenylmethyl ester) group. Completed peptide-resins are suspended in dilute DMF solutions of an appropriate lower alkylamine (such as ethylamine, propylamine, phenethylamine, 1,2-diaminoethane, etc.) and stirred at about 60 °C (for about 18 hrs) whereupon filtration, removal of solvents under reduced pressure and trituration of cleaved peptide oil with ether gives a solid, protected alkylamide peptide. This is then subjected to HF cleavage to remove additional side chain protecting groups and HPLC purification as described in Example 1.
Examples 11 as made substantially according to the procedure described for Example 1 but using the appropriate protected amino acid to yield the noted peptide. MS were obtained by laser desorption MS (NA means not available).
Example 11: [Hppa7]hGLP-1 (7-36)-NH2; MS = NA
The title peptide was synthesized on an Applied Biosystems (Foster City, CA) model 430A peptide synthesizer which was modified to do accelerated Boc-chemistry solid phase peptide synthesis. See
At the end of the assembly of the peptide chain, the resin was treated with a solution of 20% mercaptoethanol/10% DIEA in DMF for 2 x 30 min to remove the DNP group on the His side chain. The N-terminal Boc group was then removed by treatment with 100% TFA for 2 x 2 min. After neutralization of the peptide-resin with 10% DIEA in DMF (1 x 1 min), the formal group on the side of the chain of Trp was removed by treatment with a solution of 15% ethanolamine/ 15% water/ 70% DMF for 2 x 30 min. The partially-deprotected peptide-resin was washed with DMF and DCM and dried under reduced pressure. The final cleavage was done by stirring the peptide-resin in 10 ml of HF containing 1 ml of anisole and dithiothreitol (24 mg) at 0 °C for about 75 min. HF was removed with a flow of nitrogen. The residue was washed with ether (6 x 10 ml) and extracted with 4N HOAc (6 x 10 ml).
The peptide mixture in the aqueous extract was purified on a reverse-phase preparative high pressure liquid chromatography (HPLC) using a reverse phase VYDAC® C18 column (Nest Group, South borough, MA). The column was eluted with a linear gradient (20% to 50% of solution B over 105 min) at a flow rate of 10 ml/min (Solution A = water containing 0.1% TFA; Solution B = acetonitrile containing 0.1% of TFA). Fractions were collected and checked on analytical HPLC. Those containing pure product were combined and lyophilized to dryness. 92 mg of a white solid was obtained. Purity was >99% based on analytical HPLC analysis. Electro-spray mass spectrometer analysis gave the molecular weight at 3324.2 (the calculated molecular weight is 3323.7).
The synthesis of other compounds of the present invention can be carried out in the same manner as described for the synthesis of [Aib8, A6c32]hGLP-1(7-36)NH2 in Example 12 above but using the appropriate protected amino acids depending on the desired peptide.
[(Nα-HEPES-His)7]hGLP-1(7-36)NH2 {HEPES is (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)} can be synthesized as follows: After assembly of the peptide long chain on MBHA resin (0.20 mmol), the peptide-resin is treated with 100% TFA (2 x 2 min.) and washed with DMF and DCM. The resin is then neutralized with 10% DIEA in DMF for about 2 min. After washing with DMF and DCM, the resin is treated with 0.23 mmol of 2-chloro-1-ethanesulfonyl chloride and 0.7 mmol of DIEA in DMF for about 1 hour. The resin is washed with DMF and DCM and treated with 1.2 mmol of 2-hydroxyethylpiperazine for about 2 hours. The resin is washed with DMF and DCM and treated with different reagents ((1) 20% mercaptoethanol / 10% DIEA in DMF and (2) 15% ethanolamine / 15% water / 70% DMF) to remove the DNP group from the His side chain and formyl group on the Trp side chain as described above before the final HF cleavage of the peptide from the resin.
[(Nα-HEPA-His)7]hGLP-1(7-36)NH2, ([(4-(2-hydroxyethyl)-1-piperazineacetyl)-His7]hGLP-1(7-36)NH2), can be made substantially according to the procedure described immediately above for making [(Nα-HEPES-His)7]hGLP-1(7-36)NH2 except that 2-bromo-acetic anhydride is used in place of 2-chloro-1-ethanesulfonyl chloride.
Examples 13-15 were made substantially according to Example 12 but using the appropriate protected amino acid.
Example 13 : [Ura7]hGLP-1(7-36)-NH2; MS = 3279.5; Calc. MW = 3280.7.
Example 14 : [Paa7]hGLP-1(7-36)-NH2; MS = 3290.9; Calc. MW = 3291.8.
Example 15 : [Pta7]hGLP-1(7-36)-NH2; MS = 3311.2; Calc. MW = 3311.8.
The Boc amino acids to be used are the same as those in the synthesis of [Aib8, A6c32]hGLP-1(7-36)NH2 (Example 12) except that Fmoc-Lys(Boc)-OH is used here for the Lys36(Nε-tetradecanoyl) residue. The first amino acid residue is coupled to the resin manually on a shaker. 2.5 mmol of Fmoc-Lys(Boc)-OH is dissolved in 4 ml of 0.5N HBTU in DMF. To the solution is added 1 ml of DIEA. The mixture is shaken for about 2 min. To the solution is then added 0.2 mmol of MBHA resin (substitution = 0.91 mmol/g). The mixture is shaken for about 1 hr. The resin is washed with DMF and treated with 100% TFA for 2x2 min to remove the Boc protecting group. The resin is washed with DMF. Myristic acid (2.5 mmol) is pre-activated with HBTU (2.0 mmol) and DIEA (1.0 ml) in 4 ml of DMF for 2 min and is coupled to the Fmoc-Lys-resin. The coupling time is about 1 hr. The resin is washed with DMF and treated with 25% piperidine in DMF for 2x20 min to remove the Fmoc protecting group. The resin is washed with DMF and transferred to the reaction vessel of the peptide synthesizer. The remainder of the synthesis and purification procedures of the peptide are the same as those in the synthesis of [Aib8, A6c32]hGLP-1(7-36)NH2.
The syntheses of other compounds containing Lys(Ne-alkanoyl) residue are carried out in an analogous manner as described for the synthesis of [Aib8, A6c32, Lys36(Nε-octanoyl)]hGLP-1(7-36)NH2. Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(Nε -alkanoyl) in the peptide, while Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys. If the Lys(Nε-alkanoyl) residue is not at the C-terminus, the peptide fragment immediately prior to the Lys(Nε-alkanoyl) residue is assembled on the resin on the peptide synthesizer first.
The Boc amino acids to be used are the same as those used in the synthesis of [Aib8, A6c32, Lys36(Nε-tetradecanoyl)]hGLP-1(7-36)NH2 (Example 16). Fmoc -Lys(Boc)-OH (2.5 mmol) is pre-activated with HBTU (2.0 mmol), HOBt (2.0 mmol) and DIEA (2.5 ml) in DMF (4 ml) for about 2 min. This amino acid is coupled to 235 mg of PAM resin (Chem-Impex, Wood Dale, IL; substitution = 0.85 mmol/g) manually on a shaker. The coupling time is about 8 hrs. The remainder of the synthesis and purification procedures for making the peptide are the same as those described in Example 52.
The syntheses of other analogs of hGLP-1(7-36)-OH and hGLP-1(7-37)-OH, which contain Lys(Nε-alkanoyl) residue, are carried out in an analogous manner as described for the synthesis of (Aib8, Arg26.34, A6c32, Lys36(Nε-tetradecanoyl)]hGLP-1(7-36)-OH. Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(Ne-alkanoyl) in the peptide, while Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys.