WO2015149627A1 - Analogue du glp-1 à structure modifiée et son procédé de préparation - Google Patents

Analogue du glp-1 à structure modifiée et son procédé de préparation Download PDF

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WO2015149627A1
WO2015149627A1 PCT/CN2015/074560 CN2015074560W WO2015149627A1 WO 2015149627 A1 WO2015149627 A1 WO 2015149627A1 CN 2015074560 W CN2015074560 W CN 2015074560W WO 2015149627 A1 WO2015149627 A1 WO 2015149627A1
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glp
lys
val
glu
arg
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袁建栋
黄仰青
宋云松
顾家宁
杨晴铖
朱锐
季南南
方程
姚翠萍
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博瑞生物医药技术(苏州)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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 the field of therapeutic peptides, in particular to structurally modified GLP-1 analogs and methods for their preparation, and further to structurally modified exenatide derivatives, and to exenatide-derived structurally modified exenatide The preparation method of the substance.
  • Exenatide (also known as exenatide or exenatide, Exenatide, or Exendin-4, trade name Byetta) is a polypeptide consisting of 39 amino acids with a molecular weight of 4186.6 and a molecular formula of C 184 H 282 N 50 O 60 S, CAS Registry Number is 141758-74-9, amino acid sequence is: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu- Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 ; Produced and sold by Amylin Pharmaceuticals and Eli Lilly Company (Eli Lillyand Company).
  • Exenatide has been approved by the FDA in April 2005. It is a subcutaneous injection preparation that promotes glucose-dependent insulin secretion, restores first-phase insulin secretion, inhibits glucagon secretion, and slows gastric contents.
  • the emptying, improving the function of pancreatic beta cells, is very suitable for the treatment of type 2 diabetes, for example, to improve and control the blood glucose of patients with type 2 diabetes who are not ideal for metformin and sulfonylureas.
  • Exenatide is a synthetic form of the hormone exendin-4 in the saliva of the lizard Gilamonster grown in several states in the southwestern United States (J. Biol. Chem. 1990, 265, 20259-20262; J. Biol. .1992, 267, 7402-7405), is an analog of human glucagon-like peptide-1 (GLP-1), whose amino acid sequence partially overlaps with the amino acid sequence of GLP-1, and is a relatively effective GLP- A receptor agonist, also known as an incretin agonist, because exenatide mimics the glucose-regulating action of GLP-1.
  • GLP-1 human glucagon-like peptide-1
  • GLP-1 glucagon-like peptide-1
  • exenatide Unlike sulfonylureas and meglitinides, exenatide increases insulin synthesis and secretion only in the presence of glucose, reducing the risk of hypoglycemia. Some physicians also use Byetta for the treatment of insulin
  • Chinese Patent No. CN00804847.9 discloses a novel exendin agonist preparation and a method of administration thereof, in which a compound structure of exenatide and a preparation method thereof are disclosed.
  • Chinese patent CN102532303 discloses the use of a methoxypolyethylene glycol residue conjugated to the amino group of the lysine residue in the exenatide molecule or the amino group of the N-terminal histidine residue to synthesize polyethylene glycol conjugated
  • the method of exenatide; CN200980111088 also explicitly discloses the structure of fatty acid-PEG-exenatide, and the modification site of PEG is on the N-terminal His; WO2005028516 and WO2012035139 also disclose fatty acid-PEG-exenatide structure.
  • Chinese patent CN101215324B discloses an exenatide short peptide mimetic peptide obtained by structurally modifying exenatide.
  • the object of the present invention is to overcome the shortcomings of the prior art, to provide exenatide derivatives which have long half-life, good stability, and can simultaneously maintain good hypoglycemic effect and low toxicity, and at the same time, the present invention also provides exenatide derivatives. Methods of preparation, and the use of these derivatives in the manufacture of a medicament for the treatment and/or prevention of diabetes.
  • the present invention provides a compound having the structure: Compound: D-A-P-L
  • D is a GLP-1 analog moiety
  • A is of the following structure: -NR 1 -X-NR 2 -,
  • R 1 and R 2 are independently selected from the group consisting of H and C 1-6 alkyl
  • X is selected from:
  • n is an integer from 2 to 10;
  • an alkylene group of C 1-10 is substituted by one or more of the following substituents selected from the group consisting of: a linear or branched alkyl group of C 1 -C 4 , halogen, NHR 5 , CN, carboxyl, nitro and C 1 -C 4 alkyl alcohol; wherein R 5 is H or C 1 -C 4 straight Chain or branched alkyl group;
  • P is a linking moiety containing an oligoethylene glycol
  • L is a lipophilic linking moiety
  • the carbon terminal of the terminal amino acid residue of D is linked to NR 1 of A through an amide bond
  • NR 2 of A is linked to P via an amide bond
  • P and L are linked by an amide bond.
  • P in the compound is a moiety having the following structure:
  • the L has the following structure:
  • n is an integer from 1 to 20; k is an integer from 6 to 20; wavy lines Indicates a linking site; further preferably, m is 2 or 5.
  • the A has the following structure: -NR 1 -X-NR 2 -,
  • the X is -(CH 2 ) n -, wherein the -(CH 2 ) n - represents an alkylene structure;
  • the compound DAPL has the following structure:
  • R 1 and R 2 are independently selected from the group consisting of H and C 1-6 alkyl groups, preferably R 1 and R 2 are both H; preferably n is an integer of 2 to 6, more preferably n is 2 or 4; m is an integer of 1-20, and k is an integer of 6-20.
  • the A has the following structure: -NR 1 -X-NR 2 -,
  • the X is -[(CH 2 ) n Z] p (CH 2 ) n -,
  • the compound D-A-P-L preferably has the following structure:
  • Z is O or NR 3
  • n is independently selected from an integer of 1 to 10
  • p is an integer of 0 to 10
  • p is an integer of 2 to 5
  • m is an integer of 1 to 20, and k is 6 to 20.
  • the A is a structure having: -NR 1 -X-NR 2 -, wherein the X is an amino acid residue or an amino acid residue substituted with one or more substituents, and the amino acid
  • the A is a structure having the following structure: -NR 1 -X-NR 2 -, wherein X is -(CH 2 ) n C(O)Z(CH 2 ) n -, preferably Z is O Or a single bond, n independently selected from an integer from 1 to 10, wherein R 3 is selected from the group consisting of H and C 1-6 alkyl; further preferably X has the structure: -CH 2 CH 2 -C(O)- CH 2 CH 2 - or -CH 2 CH 2 -C(O)-NH-CH 2 CH 2 -; preferably A has the structure -NR 1 -CH 2 CH 2 -C(O)-CH 2 CH 2 -NR 2 - or -NR 1 -CH 2 CH 2 -C(O)-NH-CH 2 CH 2 -NR 2 -;
  • the D-A-P-L has a compound having the following structure:
  • Z is preferably O or a single bond
  • R 1 and R 2 are H.
  • the A is a structure having the following structure: -NR 1 -X-NR 2 -, the X is a C 1-10 alkylene group, preferably the H in the alkylene group is one or Substituted by a plurality of substituents selected from the group consisting of: A linear or branched alkyl group of C 1 -C 4, NHR 5, carboxy and C 1 -C 4 alkyl alcohols; wherein, R 5 is H or a straight-chain or branched-chain C 1 -C 4 alkyl group; and Further preferably, the substituent is Also, R 5 is H; preferably A has the following structure:
  • the compound D-A-P-L has the following structure:
  • D of the present invention is a GLP-1 analog moiety
  • GLP-1 analogs are preferably: GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-37), GLP-1 (7-38), GLP-1 (7) -39), GLP-1 (7-40), GLP-1 (7-41), GLP-1 (7-42), GLP-1 (7-43), GLP-1 (7-44), GLP -1 (7-45), GLP-1 (7-46) or an analogue thereof; still more preferably the GLP-1 analogue is a peptide comprising the following amino acid sequence:
  • aa7 is L-His, D-His, 2-aminohistidine, ⁇ -histidine or ⁇ -methylhistidine;
  • the GLP-1 analogue is selected from the group consisting of: Arg 34 GLP-1 (7-37), Lys 38 Arg 26, 34 GLP-1 (7-38), Lys 38 Arg 26, 34 GLP-1 (7- 37) -OH, Lys 36 Arg 26,34 GLP-1 (7-36), Aib 8,22,35 GLP-1 (7-37), Aib 8,35 GLP-1 (7-37), Aib 8 , 22 GLP-1 (7-37) , Aib 8,22,35 Arg 26,34 GLP-1 (7-38), Aib 8,35 Arg 26,34 Lys 38 GLP-1 (7-38), Aib 8,35 Arg 26,34 Lys 38 GLP-1 (7-38), Aib 8,22 Arg 26,34 Lys 38 GLP-1(7-38),Aib 8,22,35 Arg 26 Lys 38 GLP-1(7-38),Aib 8 Arg 26 Lys 38 GLP-1(7-38),Aib 8 Arg 26 Lys 38 GLP-1(7-38), Aib 8,35 Arg
  • the GLP-1 analog is Exendin-3, Exendin-4 or a derivative thereof; most preferably the GLP-1 analog is Exendin-4.
  • Exendin-4 is a peptide having the following amino acid sequence:
  • Exendin-3 is a peptide having the following amino acid sequence:
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound D-A-P-L provided herein, and optionally a pharmaceutically acceptable carrier.
  • “Pharmaceutical composition” means one or more active ingredients and optionally one or more inert ingredients, as well as any product, which is obtained directly or indirectly from a combination of any two or more ingredients, Compounding or agglomeration, or dissociation from one or more components, or other types of reactions or interactions from one or more components. Accordingly, the pharmaceutical compositions of the present invention include any composition prepared by admixing a compound of the present invention and a pharmaceutically acceptable excipient (pharmaceutically acceptable carrier).
  • excipient refers to a diluent, adjuvant or carrier that is administered with a therapeutic agent.
  • the pharmaceutical excipient can be a sterile liquid, such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred excipients when the pharmaceutical composition is administered intravenously. Preferably, a saline solution and an aqueous dextrose solution and a glycerol solution are used as liquid excipients for the injectable solution.
  • compositions include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, natural calcium carbonate, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, Skimmed milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc.
  • the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • the composition can be formulated as a suppository with conventional binders and excipients such as triglycerides.
  • Oral formulations may contain standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • Suitable pharmaceutical excipients are described in "Remington's Pharmaceutical Sciences” by EW Martin, which contains a therapeutically effective amount of a therapeutic agent (preferably in purified form) and an appropriate amount of excipient, Thereby providing a form suitable for administration to a patient.
  • the formulation should suit the mode of administration.
  • the invention relates to the use of any of the compounds provided for the manufacture of a medicament for delaying or treating the development of diabetes.
  • the therapeutically effective amount of the Exenatide derivative provided by the present invention will depend on the route of administration, the type of subject and the physical characteristics of the particular mammal in question. These factors and their relationship to determining this amount are well known to those skilled in the medical arts. This amount and method of use can be adjusted to achieve optimal efficacy to deliver the peptide to the subject, but will depend on factors well known to those skilled in the medical arts such as body weight, diet, concomitant medication, and other factors.
  • Glucagon-like peptide (GLP) Glucagon-like peptide (GLP) and GLP derivatives are enteric hormones, which generally stimulate insulin secretion in hyperglycemia, inhibit glucagon secretion, and promote insulin ( Biosynthesis of the original) and slowing gastric emptying and gastric acid secretion. Some GLP and GLP derivatives promote cellular uptake of glucose but do not promote insulin expression as described in U.S. Patent 5,574,008.
  • Glucagon-like peptide refers to glucagon elongation protein (exendin) and a homologous peptide derived from the proglucagon gene other than glucagon, ie, glucagon-like peptide- 1 (GLP-1), glucagon-like peptide 2 (GLP-2) HE oxynthomodulin (OXM) and their analogs and derivatives.
  • GLP-1 and GLP-2 have the following amino acid sequences, respectively:
  • GLP-1 (7-36)-NH 2 HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR-NH 2 ;
  • GLP-1 (7-37)-OH HAEGT FTSDV SSYLE GQAAK EFIAW LVKGRG-COOH;
  • GLP-2 HADGS FSDEM NTILD NLAAR DFINW LIQTK ITD.
  • GLP-L and its derivatives the known synthetic hormone glucagon is a large molecular weight precursor molecule which is subsequently cleaved under proteolysis into three peptides: glucagon, glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2).
  • Exendin-4 39 amino acid peptide, about 53% homologous to GLP-1, and has insulinotropic activity.
  • a GLP-1 analog refers to a modified peptide in which one living multiple amino acid residue of the peptide is replaced by another amino acid residue, and/or one or more amino acid residues are deleted from the peptide, And/or one of the living multiple amino acid residues is added to the peptide.
  • GLP-1 (7-38) refers to a GLP-1 analogue, wherein Adding lysine 38, the naturally occurring lysine at positions 26 and 34 is replaced by arginine; Aib 8,22,35 GLP-1 (7-37) refers to a GLP-1 analogue, wherein position 8 The naturally occurring alanine and the glycine at positions 22 and 35 are replaced by alpha-aminoisobutyric acid.
  • exenatide, Exendin-4, and Exenatide mentioned in the present invention all refer to the same substance, and the CAS Registry Number thereof is 141758-74-9.
  • a linking moiety refers to a fatty long-chain carboxylic acid structure or a polyethoxy-containing structure capable of reacting with an amino group on an amino acid to form an amide bond, such as palmitic acid or (2-amino)ethoxyacetic acid.
  • a protecting group is a chemical group used to protect a peptide derivative from self-reaction. Including acetyl, decylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), etc., the specifically protected amino acids and abbreviations and abbreviations used in the present invention are shown in Table 1:
  • Ts-Cl or TsCl mentioned in the present invention means p-toluenesulfonyl chloride
  • EA means ethyl acetate
  • (Boc) 2 O means di-tert-butyl dicarbonate
  • “Boc” means t-butoxycarbonyl
  • DIC means N,N'-diisopropylcarbodiimide
  • Fmoc means fluorenylmethoxycarbonyl
  • HOBT means 1-hydroxybenzotriazole
  • OtBu means tert-butoxy NMM refers to N-methylmorpholine
  • AC or HOAC refers to acetic acid
  • HBTU refers to O-benzotriazol-1-yl-N,N,N',N'-tetramethylurea-hexafluorophosphate Salt
  • Alloc means allyloxycarbonyl
  • Aib means ⁇ -aminoisobutyric acid.
  • Exendin-4 refers to all or part of the amino acid structural sequence of Exendin, which shares Exendin A substantially homologous or similarly sized GLP-1 fragment of -4; a class of polypeptide that binds to the glucagon-like peptide-1 receptor and produces a cellular signaling cascade.
  • Exendin or analogs thereof according to the invention include, but are not limited to, those similar polypeptides obtained by amino acid substitution, addition and deletion of the amino acid sequence of native Exendin-3 or Exendin-4.
  • the invention relates to a process for the preparation of the provided compounds.
  • the general preparation method of the compound D-A-P-L provided by the invention is to separately prepare the polypeptide moiety D and the linking moiety H-A-P-L, and then couple the C-terminus of the D-terminal amino acid with the amino group of the linker moiety H-A-P-L, the specific route is as follows:
  • the GLP-1 analogue (D) can be expressed by chemical solid phase synthesis or genetically engineered host bacteria, and then purified by one or more steps.
  • the "Fmoc strategy” is used herein, and the “Fmoc strategy” refers to a method for synthesizing a polypeptide by sequentially condensing an amino terminal Fmoc-protected amino acid in the presence of a coupling reagent by using a polymer resin as a solid phase reaction substrate.
  • Fmoc solid phase peptide synthesis a practical approach, 2000, Oxford University Press. J. M. Stewart 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. Portions of these documents are incorporated herein by reference.
  • the method involves the sequential addition of one or more amino acids or appropriately protected amino acids to lengthen the peptide chain.
  • the amino or carboxyl group of the first amino acid is normally protected by a suitable protecting group.
  • the protecting group or derivatized amino acid is attached to an inert solid support, or by adding the next suitably protected amino acid having a complementary (amino or carboxyl) group in the sequence and under conditions suitable for the formation of an amide bond
  • the solution form is utilized.
  • the protecting group is then removed from the newly added amino acid residue and the next amino acid is added and continues as such.
  • any remaining protecting groups are removed sequentially or simultaneously to yield the final polypeptide.
  • X is selected from -(CH 2 ) n -, wherein n is an integer of 2 to 10:
  • R 1 NH-(CH 2 ) n -NHR 2 can be obtained by the following formula,
  • X is selected from -[(CH 2 ) n Z] p (CH 2 ) n -, and when Z is O, NH 2 -[(CH 2 ) 2 O] n (CH 2 ) 2 - NH 2 can be prepared as follows:
  • X is an amino acid residue or an amino acid residue substituted by one or more substituents selected from the group consisting of H, C 1-6 alkyl, carbonyl and -NR 2 R 3 and combinations thereof Wherein R 2 and R 3 are independently selected from the group consisting of H and C 1-6 alkyl; the specific preparation method of HNR 1 -X-NR 2 H may be based on the specific structure of the selected amino acid residue or substituted amino acid residue. It is prepared according to the specific molecular structure according to methods known in the art.
  • n is independently selected from an integer from 1 to 10; when Z is a single bond,
  • NHR 1 -(CH 2 ) n C(O)(CH 2 ) n -NHR 2 please refer to the following literature: Structure-activity relationship studies of 1-(4-chloro-2,5-dimethoxyphenyl)-3- (3-propoxypropyl)thiourea, a nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type-1 (European Journal of Medicinal Chemistry, Volume: 46, Issue: 2, Pages: 447-467, 2011); Imidazolic H2-agonists.
  • X is a C 1-10 alkylene group
  • H in the alkylene group is substituted with one or more of the following substituents selected from the group consisting of: a linear or branched alkyl group of C 1 -C 4 , halogen, NHR 5 , CN, carboxyl, nitro and C 1 -C 4 alkyl alcohol; wherein R 5 is H or C 1 -C 4 straight Chain or branched alkyl; may be prepared according to specific structures by methods known in the art, or commercially available.
  • HAH has the following structure:
  • R 1 and R 2 are H, which can be prepared by the method disclosed in Chemical Communications (Cambridge, United Kingdom), 49 (91), 10703-10705;
  • the reaction process is as follows:
  • the compound of the formula 8 is commercially available.
  • the fully protected polypeptide was reacted with the side chain HAPL at a molar ratio of 1.0:1.5, using DMF as a solvent, and stirred at room temperature overnight in the presence of HOAT and DIC. The reaction was monitored by TLC. After the reaction was completed, water was added and extracted with DCM.
  • Preparative HPLC was performed on Beijing Xintongtong liquid chromatography.
  • a solution of various crude peptides was injected into a Beijing Innovation Tongheng ODS (50*150 mm) column and eluted with a gradient of acetonitrile and 0.1% TFA.
  • the typical gradient used was 60 min acetonitrile increased from 30% to 40%, the detection wavelength was 210 nm, the flow rate was 50 ml/min, the fraction was collected in stages, and the fraction was lyophilized to obtain a purified target peptide.
  • a YMC ODS (4.6*150mm) column was used and eluted using the following gradient: B phase increased from 25% to 65% over 20 min; B phase increased from 65% to 95% over a period of 5 min; Phase B is stable at 95%; last 5 minutes, phase B is stable at 25%, detection wavelength is 215 nm, flow rate is 1.0 ml/min, phase A is 0.1% TFA, and phase B is acetonitrile.
  • A is an amino acid residue or a substituted amino acid residue
  • the compound of the present invention has the following structure:
  • the specific method includes the following methods: (1) solid phase synthesis of D-Lys(Alloc)-OH by Fmoc method; (2) selective removal of Lys(Alloc) side chain protecting group to obtain D-Lys-NH 2 ; (3) attaching compound 10 to the target peptide D-Lys-NH 2 ; (4) cleavage of the resin to obtain the target compound.
  • Exendin-4 can be synthesized by Fmoc solid phase synthesis method well known to those skilled in the art, such as Fmoc-Rink MBHA Amide resin, Fmoc with 20% piperidine/DMF, HOBT/DIC coupling reagent, DMF as reaction solvent. The reaction was monitored by ninhydrin detection method, and the protected amino acid was sequentially attached to Rink MBHA Amide resin to obtain a fully protected Exendin-4 resin. The resin was cleaved using 85.2% TFA/5% phenol/5% water/5.
  • the Exenatide derivative provided by the present invention is capable of significantly increasing intracellular cAMP content, and the inventors have unexpectedly discovered that the Exenatide derivative provided by the present invention retains GLP-1 compared to Exenatide. At the same time as the receptor agonist activity, the in vivo biological half-life and/or plasma half-life is significantly prolonged. Since the exenatide derivative provided by the present invention has a long half-life and good stability in vivo, the frequency of administration can be reduced, and patient compliance can be remarkably improved. Moreover, the preparation method of the Exenatide derivative provided by the invention is simple and effective, and is suitable for industrial production.
  • 1 to 10 show mass spectra of the compounds pp01a03, pp01b, pp01c, pp01d, pp01e, pp01f, pp01g, pp01h, pp01j, and pp01k, respectively.
  • Figure 11 shows a standard curve for a non-interfering protein concentration assay kit.
  • Figure 12 shows the Mouse/Rat cAMP Assay kit standard curve.
  • Figure 13 is a dose-effect relationship of each test drug to stimulate c12 production by PC12 cells.
  • k is an integer from 6 to 20
  • m is an integer of from 1 to 6, which is commercially available by a conventional method.
  • reaction solution is poured into iced dilute hydrochloric acid solution, solid is produced, extracted with EA, and the EA layer is washed once with dilute hydrochloric acid, washed with saturated sodium hydrogencarbonate, washed with saturated brine, dried over anhydrous Na 2 SO 4
  • N-hydroxysuccinimide HOSU
  • 50 g of the compound of formula 8 and 300 ml of DMF were added to a 500 mL three-necked flask, the temperature was controlled to about -10 to 0 ° C, and 44.2 g of N,N'-dicyclohexyl carbon was added.
  • Diimine (DCC) was stirred at room temperature overnight, and the reaction was completed by TLC. Water and EA was evaporated, washed with brine, and then evaporated.
  • Compound 12 was prepared in a similar manner to compound 9. To a 100 mL three-necked flask, 1.0 g of Compound 7, 15 ml of water, 0.7 g of NaHCO 3 was added, and 1.32 g of Compound 12 and 15 ml of a DME solution were added dropwise, stirred overnight, and the progress of the reaction was examined by TLC.
  • Post-reaction treatment suction filtration, addition of water to the filtrate, extraction with EA, washing with water, washing with saturated brine, dried over anhydrous sodium sulfate,
  • Example 4 Compound 19, Compound 22 was prepared according to the general procedure of scheme 2.
  • the solid phase peptide synthesis of the target peptide was carried out by solid phase synthesis using Fmoc method, Fmoc-Rink MBHA Amide resin, Fmoc with 20% piperidine/DMF, HOBT/DIC with coupling reagent, DMF as reaction solvent, and reaction monitoring.
  • the ninhydrin assay linked the following protected amino acids to Rink MBHA Amide resin: Fmoc-Lys(Alloc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly -OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH , Fmoc-Phe-
  • Post-treatment Transfer to 500ml reaction column, DMF cleaning 3 times, add 50ml 20% piperidine DMF solution, nitrogen bubbling for 20min, DMF cleaning 5 times, ninhydrin monitoring reaction after adding 3.0eqiv amino acid 3.3eq HOBT , 3.3 eq DIC, after 1 h of reaction, the ninhydrin monitoring was negative, DMF was washed, and 20% piperidine DMF solution was added. This step is repeated to condense the subsequent amino acids in sequence until condensation to the last amino acid (as is the solid phase synthesis of the fragments of the polypeptide).
  • the solid phase peptide synthesis of the target peptide was carried out by solid phase synthesis using Fmoc method, Fmoc-Rink MBHA Amide resin, Fmoc with 20% piperidine/DMF, HOBT/DIC with coupling reagent, DMF as reaction solvent, and reaction monitoring.
  • the ninhydrin assay linked the following protected amino acids to Rink MBHA Amide resin: Fmoc-Lys(Alloc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly -OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH , Fmoc-Phe-
  • the cracking of the resin was carried out using 82.5% TFA / 5% phenol / 5% water / 2.5% EDT / 5% thioanisole, followed by precipitation with ice methyl tert-butyl ether (MTBE) and washing.
  • the crude product was purified by HPLC to give the title compound pp01b.
  • the purity of the target compound was determined by RP-HPLC to be >96%.
  • Post-treatment Transfer the above reaction mixture to 50ml reaction column, DMF clean 3 times, add 10ml 20% piperidine DMF solution, nitrogen bubbling for 20min, DMF cleaning 5 times, ninhydrin monitoring reaction, add 3.0eqiv
  • the ninhydrin monitoring was negative, DMF was washed, and 20% piperidine DMF solution was added. This step is repeated to condense the subsequent amino acids in sequence until condensation to the last amino acid (as is the solid phase synthesis of the fragments of the polypeptide).
  • the cleavage reagent 82.5% TFA / 5% phenol / 5% water / 2.5% EDT / 5% thioanisole was added, followed by precipitation, washing with free methyl tert-butyl ether (MTBE), free peptide, 890 mg of the target compound pp01b, HPLC: 96%, and the mass spectrum is shown in Fig. 2.
  • Test sample Compound pp01b and compound pp01c prepared according to the present invention and reference substance Exendin-4.
  • Tumor cell line PC12 (rat adrenal pheochromocytoma) from the cell bank of Zhongshan University College of Science and Technology;
  • the 25 cm 2 flasks, 24-well plates, 96-well plates used were purchased from Corning; DMEM high-sugar medium (Cat no. SH30022.01), fetal bovine serum (Cat no. SV30087), purchased from Hyclone; Serum (Cat no. 16050-122), trypsin purchased from Gibco; 3-isobutyl-1-methylxanthine (IBMX) purchased from Sigma, Cat no. I5879-100 mg; non-interfering protein concentration determination
  • the kit (Cat no. SK3071) was purchased from Biotech (Shanghai) Co., Ltd.; the Mouse/Rat cAMP Assay kit (Cat no. KGE012) was purchased from R&D Systems.
  • Test equipment Sujing ultra-clean workbench, Themro carbon dioxide incubator, BIO-RAD 680 microplate reader.
  • Sample processing method Each test sample was dissolved in double distilled water at a final concentration of 1.0 ⁇ 10 -2 mol/L, and stored at 4 °C.
  • PC12 cells were cultured in 25 cm 2 flasks and placed in a CO 2 incubator (37 ° C, 95% air, 5% CO 2 ).
  • the medium was DMEM (pH 7.4, High sugar), adding 5% fetal bovine serum and 10% horse serum.
  • PC12 cells with good growth state were digested with 0.25% trypsin, adjusted to a cell concentration of 1.0 ⁇ 10 5 /ml, and seeded in 24-well plates. When the cells were grown to a density of 60-70%, they were washed twice with PBS and added.
  • each of the three test drugs were divided into 5 gradients (10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 mol / L ) and IBMX (100 ⁇ mol / L) A total of 30 min incubation, 3 replicate wells per sample.
  • This test uses a non-interfering protein concentration determination kit.
  • the special precipitation reagent in the kit can remove most interfering substances.
  • the precipitated protein is mixed with the alkaline substance containing copper.
  • the copper that is not combined with the protein is being Reducing to a monovalent value and reacting with a specific chromogenic reagent to produce a specific absorption wavelength of 480 nm, so as the concentration of the protein increases, the copper that is not bound to the protein is correspondingly reduced, so that the absorption value of the specific absorption wavelength and the protein in the solution Concentration and inverse ratio.
  • the content of cAMP in the cell lysate was determined by R&D Systems' cAMP detection kit (Mouse/Rat cAMP Assay kit (Cat no. KGE012)), ELISA method, according to the instructions.
  • Curves were calculated using CurveExpert 1.3 software and the standard curve formula was calculated to calculate the concentration of each sample. (The sample was diluted 10 times and its actual concentration should be ⁇ 10); data processing and mapping were performed using the computer program Microsoft Excel and GraphPad Prism 5 software, and the EC50 of each test drug was calculated.
  • the reliable quantitative range of the non-interfering protein concentration assay kit is 1-50 ⁇ g.
  • the standard BSA is concentration dependent on the absorbance value
  • the total protein concentration of each sample was calculated from the standard curve and the 480 nm absorption value of the protein sample solution to be quantified, as shown in Table 1.
  • test drugs pp01b, pp01c, and Exendin-4 were diluted to 5 gradient concentrations (10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 mol/L ) and PC12 was co-incubated with IBMX (100 ⁇ mol/L). The cells were incubated for 30 min, and the biological activities of the respective test drugs are shown in Table 2.
  • P is the value of the sample group compared to the control group.
  • the concentration of each test drug is in a concentration-dependent manner with the amount of cAMP in the range of 10 -10 ⁇ 10 -6 mol/L drug concentration.
  • concentration of the test drug was in the range of 10 -9 to 10 -6 mol/L, the amount of intracellular cAMP in the sample group was significantly different from that in the blank control group (p ⁇ 0.05).
  • concentration of the test drug increased to a certain extent, the amount of intracellular cAMP no longer increased, which may be due to the saturation of the binding of the test drug to the GLP-1 receptor of PC12 cells.
  • P is the value of the sample group compared to the control group.
  • the GraphPad Prism 5 software was used for mapping and calculating the half-effect dose (EC50) of the drug, as shown in FIG.
  • the EC50 of pp01b is 5.098 nmol/L
  • the EC50 of pp01c is 7.210 nmol/L
  • the EC50 of Exendin-4 is 5.096 nmol/L.
  • the present invention provides the in vivo activity of the compound:
  • the compounds pp01b, pp01c and pp01k provided by the present invention were used as experimental drugs, and male rats with the same physiological state were divided into 5 groups (4 in each group) with fasting for 24 hours, using exenatide and physiological saline as controls.
  • Glucose (20 mmol/kg, body weight) was injected into the abdominal cavity of the five groups of rats, and then treated as follows:
  • Control group 1 exenatide was injected (same dose as the experimental group);
  • Control group 2 saline was injected (volume was the same as the experimental group). After the injection, blood was collected from the tail vein at 0, 15 min, 30 min, 60 min, 240 min, and 480 min. The glucose content in the blood was measured by the glucose oxidase method (GOD method). The experimental results are shown in Table 4 below:
  • exenatide achieves a stable hypoglycemic effect at about 0.5, and blood glucose is reduced by about 16% compared with physiological saline; the compounds pp01b, pp01c and pp01k provided by the present invention are also realized at about 0.5 h.
  • blood glucose was reduced by about 70%, and the hypoglycemic effect was maintained for more than 8 hours.
  • the insulin content in the blood was examined by an enzyme-linked immunosorbent kit for insulin, and the results are shown in Table 5:
  • the blood of the experimental mice was taken every hour, and the content of exenatide was measured. It was determined that the half-lives of pp01b, pp01c and pp01k in the rats were significantly prolonged compared with exenatide.
  • exenatide derivatives provided by the present invention have improved pharmacokinetic properties, can significantly lower blood glucose, and have comparable or superior biological activity to exenatide. Moreover, the Exenatide derivative provided by the invention significantly prolongs the half-life, and the stability in vivo is much better than that of Exenatide, and the hypoglycemic effect is more obvious.

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Abstract

La présente invention concerne un analogue de GLP-1 structurellement modifié et un procédé pour le préparer, et elle concerne plus particulièrement un dérivé avec une extrémité C d'exénatide structurellement modifié et un procédé pour le préparer, ainsi que des compositions pharmaceutiques le comprenant et leurs utilisations dans le traitement de maladies telles que le diabète sucré de type II.
PCT/CN2015/074560 2014-03-31 2015-03-19 Analogue du glp-1 à structure modifiée et son procédé de préparation WO2015149627A1 (fr)

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AU2022245736A1 (en) * 2021-03-25 2023-10-12 Brightgene Bio-Medical Technology Co., Ltd. Gip and glp-1 dual receptor agonist, pharmaceutical composition, and use

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CN101980725A (zh) * 2008-02-01 2011-02-23 阿森迪斯药物股份有限公司 包含可自裂解的连接体的前药
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