Synthesis of GLP-1R / GIPR agonists and their precursors

Abstract

This disclosure provides a novel synthetic method for preparing GLP-1R / GIPR agonists. A synthetic method for preparing 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid is also provided.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in XML file format, the entirety of which is incorporated herein by reference. A copy of the XML was created on 19 June 2024, named 124921_WO006_SL.xml, and has a size of 49,813 bytes. [Background technology]

[0002] Background of the Invention Incretin hormones are hormones that provide blood glucose control in response to food intake. Gastric suppressor polypeptide ("GIP") and glucagon-like peptide-I ("GLP-1") are major incretin hormones secreted from L cells and K cells of the small intestine, respectively, in response to glucose or nutrient intake to stimulate insulin secretion from pancreatic cells. GIP and GLP-1 are degraded by dipeptidyl peptidase-4 (DPP-4), rapidly losing their biological activity (see, for example, Sieno et al., J Diab Invest. (2013) 4:108-30 (Non-Patent Literature 1)).

[0003] The actions of GIP and GLP-1 are thought to be mediated by their receptors, the GIP receptor (GIPR) and the GLP-1 receptor (GLP-1R), respectively. Both belong to the G protein-coupled receptor family and are expressed in pancreatic cells as well as in various tissues and organs. GLP-1 activity includes, but is not limited to, stimulation of insulin synthesis and secretion, inhibition of glucagon secretion, and inhibition of food intake. GIP activity includes, but is not limited to, stimulation of glucose-dependent insulin secretion, increased cell mass, stimulation of glucagon secretion, and decreased gastric acid secretion.

[0004] GLP-1 and GLP-1 analogs, which act as GLP-1R agonists, have been shown to be effective in blood glucose management, for example, in type 2 diabetes. See, for example, International Publication No. 2016 / 131893 (Patent Document 1). In addition to their insulin secretion effects, GIP and GLP-1 are thought to be involved in various biological processes in different tissues and organs that express GIPR and GLP-IR. Studies using mice lacking GIPR and / or GLP-1R, as well as mice lacking DPP-4, have shown the involvement of GIP and GLP-1 in diverse biological activities. The results of these studies point to the involvement of GIP and GLP-1 in treating and / or preventing diabetes-related microvascular complications (e.g., retinopathy, nephropathy, and neuropathy) and macrovascular complications (e.g., coronary artery disease, peripheral artery disease, and cerebrovascular disease), as well as diabetes-related comorbidities (e.g., obesity, non-alcoholic fatty liver disease, fractures, and cognitive impairment). For example, see page 108 of Sieno.

[0005] Chemical entities that modulate the activity of GLP-1R and / or GIPR, as well as improved methods for synthesizing such entities, are still needed. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2016 / 131893 [Non-patent literature]

[0007] [Non-Patent Document 1] Sieno et al., J Diab Invest.(2013)4:108-30 [Overview of the project]

[0008] This disclosure is based on formula (I): TIFF2026522422000001.tif16128 (In the formula, sequence Aa is a peptide) A method for synthesizing an N-terminal conjugate peptidyl compound, Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): TIFF2026522422000002.tif22128 was analyzed using a catalyst in the presence of H2 (hydrogen gas) to obtain 1-(2-aminoethyl)piperidine-2-one (XX): The present invention provides a method that includes a step of reducing to TIFF2026522422000003.tif24128. In some embodiments, sequence Aa is given by formula WR 5 The formula includes, where W is a peptide sequence, and R 5 It is conjugated to the C-terminus of W, and W has the following sequence: EGT(Xaa4)(Xaa5)SD(Xaa8)S(Xaa10)(Xaa11)(Xaa12)(Xaa13)(Xaa14)(Xaa15)(Xaa16)(Xaa17)( Xaa18)(Xaa19)(Xaa20)(Xaa21)(Xaa22)WL(Xaa25)(Xaa26)(Xaa27)GPSSGAPPP(Xaa37)(SEQ ID NO: 1); (Here, Xaa4 is F; Xaa5 is T or I (for example, T); Xaa8 is Y, V, L, or K * (For example, Y) Xaa10 is I or S (for example, I); Xaa11 is Y, Y * , Q, A, or (Aib) (for example, Y); Xaa12 is L, M, or L * (For example, L) Xaa13 is D or E (for example, D); Xaa14 is K, G, R, or E (for example, K); Xaa15 is Q or I (for example, Q); Xaa16 is A, H, or R (e.g., A); Xaa17 is A, Q, or V (e.g., A); Xaa18 is A, (Aib), K * , K, or Q (e.g., (Aib)); Xaa19 is A, D, E, (Aib), or L (e.g., A, D, E, or L (e.g., E)); Xaa20 is F or A (e.g., F); Xaa21 is V or I (e.g., V); Xaa22 is N, A, Q, K * , or E (e.g., N); Xaa25 is I, L, or V (e.g., L); Xaa26 is A, K, or I (e.g., A); Xaa27 is Q-R, G-R-G-K * , Q, or G (e.g., G); and Xaa37 is S or absent (e.g., S)). It includes R 5 is a C-terminal amino acid amide or C-terminal amino acid optionally substituted with one or two modifying groups selected from acyl groups and PEG groups. W is also the following sequence: EGTFTSDYSIYLDKQAA(Aib)EFVNWLLAGGPSSGAPPPS (SEQ ID NO: 4) It may include.

[0009] In some embodiments, R 5 is a C-terminal lysylamide residue optionally substituted with one or two modifying groups selected from acyl groups and PEG groups. R 5 is of formula (II): It may include TIFF2026522422000004.tif49128, wherein R * is structure (IV): It includes TIFF2026522422000005.tif18128. In some embodiments, WR 5 Structure (V): Includes TIFF2026522422000006.tif49155.

[0010] This method is (i) Equation (XXIII): (ii) a step of treating a resin-bound peptide with 20% piperidine in DMF or NMP, wherein optionally the resin-bound peptide is treated twice with 20% piperidine in DMF or NMP, and (ii) the product of step (i) is subjected to conditions that form an amide bond to 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)amino)ethyl)thio)acetic acid (XVII): This may further include a step of reacting with TIFF2026522422000008.tif24128.

[0011] Formula (XVI): A method for synthesizing the compound TIFF2026522422000009.tif45151, Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): TIFF2026522422000010.tif22128 was analyzed using a catalyst in the presence of H2 (hydrogen gas) to obtain 1-(2-aminoethyl)piperidine-2-one (XX): A method is also provided that includes a step of reducing to TIFF2026522422000011.tif24128. In some embodiments, the method is (i) Formula (XXII): The steps of TIFF2026522422000012.tif37154, (ii) treating the peptide bound to the resin with 20% piperidine in DMF, and (ii) the product of step (i) under conditions that form an amide bond, 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)amino)ethyl)thio)acetic acid (XVII): The process further includes reacting with TIFF2026522422000013.tif24128.

[0012] In some embodiments, the conditions for forming an amide bond include the use of a coupling reagent. The coupling reagent may be azabenzotriazoletetramethyluronium hexafluorophosphate (HATU).

[0013] 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): A method for synthesizing TIFF2026522422000014.tif24128, Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): TIFF2026522422000015.tif22128 was analyzed using a catalyst in the presence of H2 (hydrogen gas) to obtain 1-(2-aminoethyl)piperidine-2-one (XX): A method is also provided that includes a step of reducing it to TIFF2026522422000016.tif24128.

[0014] In some embodiments, the catalyst for reduction is palladium-on-carbon (Pd / C) or Pd(OH)2-on-carbon (Pd(OH)2 / C).

[0015] Equation (I): TIFF2026522422000017.tif16128 (In the formula, sequence Aa is a peptide) A method for synthesizing an N-terminal conjugate peptidyl compound, 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): To generate TIFF2026522422000018.tif24128, 1-(2-aminoethyl)piperidine-2-one (XX): A method is also provided which includes the step of reacting TIFF2026522422000019.tif21128 or a salt thereof with 1,4-oxatian-2,6-dione. In some embodiments, a hydrobromide of 1-(2-aminoethyl)piperidine-2-one(XX) is reacted with 1,4-oxatian-2,6-dione.

[0016] This disclosure also includes formula (XVI): A method for synthesizing the compound TIFF2026522422000020.tif45151, 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): To generate TIFF2026522422000021.tif24128, 1-(2-aminoethyl)piperidine-2-one (XX): The present invention also provides a method comprising the step of reacting TIFF2026522422000022.tif21128 or a salt thereof with 1,4-oxatian-2,6-dione. In some embodiments, a hydrobromide of 1-(2-aminoethyl)piperidine-2-one(XX) is reacted with 1,4-oxatian-2,6-dione.

[0017] 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): A method for synthesizing TIFF2026522422000023.tif24128, 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): To generate TIFF2026522422000024.tif24128, 1-(2-aminoethyl)piperidine-2-one (XX): A method is also provided which includes the step of reacting TIFF2026522422000025.tif21128 or a salt thereof with 1,4-oxatian-2,6-dione. In some embodiments, a hydrobromide of 1-(2-aminoethyl)piperidine-2-one(XX) is reacted with 1,4-oxatian-2,6-dione.

[0018] In some embodiments, this method uses benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): To generate TIFF2026522422000026.tif22128, pyridine-2(1H)-one (XVIII): The process further comprises reacting TIFF2026522422000027.tif19128 with benzyl(2-bromoethyl) carbamate in the presence of a strong non-nucleophilic base. A strong non-nucleophilic base can be sodium hydride.

[0019] In some embodiments, the method involves 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI): To generate TIFF2026522422000028.tif21128, 1-(2-aminoethyl)piperidine-2-one (XX): The process further includes reacting TIFF2026522422000029.tif21128 with hydrobromic acid. In some embodiments, the method involves 2-((2-(2-oxopiperidine-1-yl)ethylcarbamoyl)methylthio)acetic acid (XVII): To generate TIFF2026522422000030.tif23128, 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI): The process further includes a step of reacting TIFF2026522422000031.tif21128 with 1,4-oxatian-2,6-dione.

[0020] Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): The compound TIFF2026522422000032.tif20128 is also provided.

[0021] Other features, purposes, and advantages of the present invention will become apparent in the following detailed description. However, it should be understood that the detailed description illustrates, but is not limiting, embodiments and aspects of the present invention. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Modes for carrying out the invention]

[0022] Detailed description of the invention This disclosure provides a novel synthetic method for preparing the N-terminal chemical moiety of a GLP-1R / GIPR agonist peptide mimetic. This peptide mimetic is given by formula (I): Represented by TIFF2026522422000033.tif14128, where sequence Aa represents the peptidyl structure. This synthetic method enables the synthesis of chemical moieties conjugated to sequence Aa with improved handling, purity, scalability, reproducibility, and yield.

[0023] The agonists described herein stimulate the activity of GLP-1 and GIP. As used herein, the term “natural GLP-1” refers to a peptide containing the sequence of human GLP-1 (7-36 or 7-37), and the term “natural GIP” refers to a peptide containing the sequence of human GIP (1-42). As used herein, any general reference to “GLP-1” or “GIP” without further designation is intended to mean natural GLP-1 or natural GIP, respectively. In some embodiments, the agonists described herein have at least 50% (e.g., at least 60, 70, 80, 90, 95, or 99%) of the GLP-1R activating activity of natural GLP-1-OH or GLP-1-NH2 and / or at least 50% (e.g., at least 60, 70, 80, 90, 95, or 99%) of the GIPR activating activity of natural GIP.

[0024] I. Array Aa In some embodiments, the sequence Aa, which is the peptidyl moiety of the compound herein, is of formula WR 5 The formula includes, where W represents a peptidyl structure, and R 5 This represents the conjugated portion at the C-terminus of the peptidyl structure.

[0025] A. Peptidyl structure W Peptidyl structure W may include amino acid sequences present in natural GLP-1 (i.e., GLP-1-OH or GLP-1-NH2) having either an -OH group or an -NH2 group at its C-terminus. Peptidyl structures may also include amino acid sequences present in natural GIP. For example, a peptidyl structure may include a hybrid sequence having one or more amino acid sequence fragments (e.g., functional fragments) present in natural GLP-1 and one or more amino acid sequence fragments (e.g., functional fragments) present in natural GIP.

[0026] In some embodiments, W is given by the following formula: EGT(Xaa4)(Xaa5)SD(Xaa8)S(Xaa10)(Xaa11)(Xaa12)(Xaa13)(Xaa14)(Xaa15)(Xaa16)(Xaa17) (Xaa18)(Xaa19)(Xaa20)(Xaa21)(Xaa22)WL(Xaa25)(Xaa26)(Xaa27)GPSSGAPPP(Xaa37)(Sequence number 1) It has, Here, Xaa4 is F; Xaa5 is T or I (for example, T); Xaa8 is Y, V, L, or K * (For example, Y) Xaa10 is I or S (for example, I); Xaa11 is Y, Y * , Q, A, or (Aib) (for example, Y); Xaa12 is L, M, or L * (For example, L) Xaa13 is D or E (for example, D); Xaa14 is K, G, R, or E (for example, K); Xaa15 is Q or I (for example, Q); Xaa16 is A, H, or R (for example, A); Xaa17 is A, Q, or V (for example, A); Xaa18 is A, (Aib), K * , K, or Q (for example, (Aib)); Xaa19 is A, D, E, (Aib), or L (e.g., A, D, E, or L (e.g., E)); Xaa20 is F or A (for example, F); Xaa21 is V or I (for example, V); Xaa22 is N, A, Q, K * , or E (for example, N); Xaa25 is I, L, or V (for example, L); Xaa26 is A, K, or I (for example, A); Xaa27 is QR, GRGK * , Q, or G (for example, G); and Xaa37 is either S or non-existent (e.g., S). In this embodiment, the nitrogen atom directly bonded to sequence Aa in formula (I) is the amino group of the first glutamic acid (E) amino acid of W.

[0027] In some embodiments, W is given by the following formula: EGTF(Xaa5)SD(Xaa8)S(Xaa10)(Xaa11)(Xaa12)(Xaa13)(Xaa14)QA(Xaa17)(Xaa18)(Xaa19)F(Xaa21)(Xaa22)WL(Xaa25)(Xaa26)GGPSSGAPPPS(SEQ ID NO: 2) It has, Here, Xaa5 is T or I (for example, T); Xaa8 is Y, V, or L (for example, Y); Xaa10 is I or S (for example, I); Xaa11 is Y, Q, or A (for example, Y); Xaa12 is L, M, or L * (For example, L) Xaa13 is D or E (for example, D); Xaa14 is K, G, or E (for example, K); Xaa17 is A or V (for example, A); Xaa18 is (Aib) or K (for example, (Aib)); Xaa19 is E or L (for example, E); Xaa21 is V or I (for example, V); Xaa22 is N, A, or E (for example, N); Xaa25 is L or V (for example, L); and Xaa26 is either A or K (for example, A). In this embodiment, the nitrogen atom directly bonded to sequence Aa in formula (I) is the amino group of the first glutamic acid (E) amino acid of W.

[0028] In some embodiments, W is given by the following formula: EGTF(Xaa5)SD(Xaa8)S(Xaa10)(Xaa11)(Xaa12)(Xaa13)(Xaa14)QA(Xaa17)(Aib)(Xaa19)F(Xaa21)(Xaa22)WL(Xaa25)(Xaa26)GGPSSGAPPPS(SEQ ID NO: 3) It has such that each of the "Xaa" variables is as defined above. In this embodiment, the nitrogen atom directly bonded to sequence Aa in formula (I) is the amino group of the first glutamic acid (E) amino acid of W.

[0029] In some embodiments, W is given by the following formula: EGTFTSDYSIYLDKQAA(Aib)EFVNWLLAGGPSSGAPPPS(Sequence ID 4) It holds. In this embodiment, the nitrogen atom directly bonded to sequence Aa in formula (I) is the amino group of the first glutamic acid (E) amino acid of W.

[0030] As used herein, "(Aib)" refers to 2-aminoisobutyric acid (also known as α-aminoisobutyric acid, α-methylalanine, or 2-methylalanine).

[0031] When used herein, Y * This refers to 2-amino-3-(4-hydroxyphenyl)-2-methylpropanoic acid (for example, (S)-2-amino-3-(4-hydroxyphenyl)-2-methylpropanoic acid).

[0032] When used herein, L *This refers to 2-amino-2-methylpentanoic acid (e.g., (S)-2-amino-2-methylpentanoic acid) or its C-terminal amino acid, amino acid ester, or amino acid amide.

[0033] When used in this specification, K * This refers to a lysine residue substituted with a modifying group, or its C-terminal amino acid, amino acid ester, or amino acid amide.

[0034] A given amino acid can be replaced by residues having similar physicochemical properties, for example, by substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala with each other), or by substituting one polar residue for another polar residue (such as Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, such as substitution of an entire region having similar hydrophobic properties or substitution of residues having similar side-chain volumes, are also within the scope of this disclosure.

[0035] Amino acids can be grouped according to the similarity of the properties of their side chains (e.g., Allehninger, in Biochemistry 2) nd See ed., pp. 73-75, Worth Publishers, New York (1975): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Non-charged: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H).

[0036] Alternatively, naturally occurring residues can be grouped based on common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, Phe, Trp; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, Ala, Tyr, His, Pro, Gly; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe, Pro, His, or hydroxyproline. Non-conservative substitutions involve exchanging one member of one of these classes for another.

[0037] In some embodiments, the conservative substitutions for use in the variants described herein are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu or Asn; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr or Phe; Tyr to Phe or Trp; and / or Phe to Val, Tyr, Ile or Leu.

[0038] Generally, conservative substitutions include the exchange of residues with similar physicochemical properties (for example, the substitution of one hydrophobic amino acid residue with another hydrophobic amino acid residue).

[0039] In some embodiments, W comprises one or more naturally occurring amino acids found in polypeptides and / or proteins produced by living organisms, such as Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M), Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q), Asp(D), Glu(E), Lys(K), Arg(R), and His(H).

[0040] In some embodiments, W comprises one or more independently selected modifications present in the so-called modified peptide. Such modifications include, but are not limited to, (i) incorporation of lactam crosslinks; (ii) head-tail cyclization; (iii) one or more alternative or naturally non-existent (D- or L-) amino acids, such as synthetic non-natural amino acids, substituted amino acids, and D-amino acids; (iv) peptide bond substitution; and (v) targeting groups. In some embodiments, the peptide comprises W or R 5 The peptide contains one modification in either of the following: In other embodiments, the peptide is W and / or R 5 (For example, only to W; or R) 5 Only; or W and R 5 It includes more than one independently selected modifier present in both (for example, two independently selected modifiers, three independently selected modifiers, four independently selected modifiers, five independently selected modifiers, six independently selected modifiers, seven independently selected modifiers, eight independently selected modifiers, nine independently selected modifiers, or ten independently selected modifiers).

[0041] Non-limiting examples of alternative or naturally occurring amino acids include D-amino acids, β-amino acids, homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, γ-carboxyglutamic acid, hippuric acid, octahydroindole-2-carboxylic acid, statins, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citrulline, α-methylalanine, para-benzoylphenylalanine, para-ammophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine and tert-butylglycine), diaminobutyric acid, 7-hydroxytetrahydroisoquinolinecarboxylic acid, naphthylalanine, biphenylalanine, cyclohexyl This includes lanin, aminoisobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, aminobenzoic acid, aminonaphthoic acid, γ-aminobutyric acid, difluorophenylalanine, nipecotinic acid, α-aminobutyric acid, thienylalanine, t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogues; azide-modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof (each of which may independently be a D- or L-amino acid).

[0042] The peptidyl structure W may contain one or more non-natural peptide bonds. Non-limiting examples of peptide bond substitutions include urea, thiourea, carbamate, sulfonylurea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-s(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic acid ester, olefin group, and their derivatives. Unless otherwise indicated, peptide bonds as used herein are naturally occurring peptide bonds.

[0043] In some embodiments, W comprises only naturally occurring amino acids. In other embodiments, W comprises only alternative or non-naturally occurring amino acids. In yet another embodiment, W comprises one or more naturally occurring amino acids and one or more alternative or non-naturally occurring amino acids. In some of the embodiments described above, W comprises only L-amino acids, or W comprises both D-amino acids and L-amino acids, or W comprises only D-amino acids. While we do not wish to be bound by theory, it is thought that the incorporation of D-amino acids may confer enhanced in vivo or intracellular stability to the compounds described herein.

[0044] BR 5 portion In some embodiments, R 5 R is a C-terminal amino acid amide which may be substituted with one or two modifying groups (e.g., one or two groups selected from an acyl group and a PEG group). In other embodiments, R 5 This is a C-terminal amino acid that may be substituted with one or two modifying groups (e.g., one or two groups selected from an acyl group and a PEG group).

[0045] In some embodiments, R 5 R is a C-terminal lysylamide residue which may be substituted with one or two modifying groups (e.g., one or two groups selected from an acyl group and a PEG group). In some embodiments, R 5 This is a C-terminal L-lysylamide residue that may be substituted with one or two modifying groups (e.g., one or two groups selected from an acyl group and a PEG group).

[0046] In some embodiments, R 5 This is equation (II) or (III): It has TIFF2026522422000034.tif65128, In the formula, R *is H or a modifying group (e.g., an acyl group or a PEG group). In some embodiments, formula (II) or (III) represents an L-amino acid. In other embodiments, formula (II) or (III) represents a D-amino acid.

[0047] In some embodiments, R * H is H.

[0048] In some embodiments, the modifying group (i.e., R * ) is an acyl group. In further embodiments, R * R is one or two independently selected R f Even if replaced by C 2~30 (For example, C 2~20 , C 2~10 , C 2~6 ) It is an acyl group. f Each occurrence is -C(=O)(OH);-C(=O)(C 2~20 Alkyl);-C(=O)O(C 2~20 alkyl); -P(=O)(OH)2; and -S(O) 1~2 (C 1~6 Alkyl; Oxo; F; C 1~10 Alkoxy; C 1~10 Haloalkoxy; and -N(R g )(R h ) can be selected from the group consisting of ). In some embodiments, R f Each occurrence of -C(=O)(OH) and -N(R g )(R h It is independently selected from the group consisting of ). g and R h Each occurrence is H;C 1~4 Alkyl;-C(=O)(C 2~20 Alkyl);-C(=O)O(C 2~20 alkyl; and -S(O) 1~2 (C 1~6 It is independently selected from the group consisting of alkyls.

[0049] In some embodiments, the modifying group (i.e., R *) is equation (IV): It has TIFF2026522422000035.tif18128.

[0050] C. Example sequence Aa In some embodiments, WR in this specification 5 The structure is given by the following equation (V): It has TIFF2026522422000036.tif43131. In such embodiments, the nitrogen atom directly bonded to sequence Aa in formula (I) is the amino group of the first glutamic acid (E) amino acid of W.

[0051] In some embodiments, WR 5 The structure is as follows (VI~XV): TIFF2026522422000037.tif158158TIFF2026522422000038.tif191158 (Here, in formula (I), the nitrogen atom directly bonded to sequence Aa is the amino group of the first glutamic acid (E) amino acid of W.) and It may have one of the following: TIFF2026522422000039.tif38154. When used in sequence numbers 6-13 and 15, TIFF2026522422000040.tif7170 shows the points of attachment of the peptide to the chemical structure in formula (I).

[0052] Carboxylic acid (XVII) and WR 5 To illustrate the conjugation between them, a non-restrictive, exemplary dual GLP-1R / GIPR agonist is shown in the following structural formula (XVI): It has TIFF2026522422000041.tif37150.

[0053] Unless otherwise indicated, the single-letter abbreviations used to describe amino acid residues in Sequence IDs 4–15 represent amino acid residues in their native configurations linked by natural peptidyl bonds.

[0054] II. Synthesis of the N-terminal portion The double GLP-1R / GIPR agonists described herein are compounds of formula (XVII) with an amide bond at the N-terminus of sequence Aa: It can be synthesized by conjugating it into TIFF2026522422000042.tif24128. This compound, 2-((2-(2-oxopiperidine-1-yl)ethylcarbamoyl)methylthio)acetic acid (formula XVII), is a key intermediate in the synthesis of agonist compounds as described herein. This disclosure provides an improved synthetic method for the production of this intermediate using a novel synthetic procedure.

[0055] In some embodiments, the synthesis method comprises one or more steps as shown in the following synthesis scheme. TIFF2026522422000043.tif29153

[0056] In some embodiments, the method includes the step of reacting pyridine-2(1H)-one (XVIII) with benzyl(2-bromoethyl) carbamate in a polar aprotic solvent (e.g., dimethylformamide) in the presence of a strong non-nucleophilic base (e.g., sodium hydride) over an arbitrary temperature range (e.g., 0°C to 90°C) to produce benzyl 2-(2-oxopyridine-1(2H)-yl)ethyl carbamate (XIX). In some embodiments, the method optionally includes the step of reducing benzyl 2-(2-oxopyridine-1(2H)-yl)ethyl carbamate (XIX) with a catalyst (e.g., palladium-on-carbon or palladium hydroxide-on-carbon) under hydrogen gas in a polar aprotic solvent (e.g., THF) or a polar protic solvent (e.g., methanol or water or a mixture thereof) at a high temperature (e.g., 50°C) to produce 1-(2-aminoethyl)piperidine-2-one (XX). In some embodiments, the method optionally includes the step of reacting 1-(2-aminoethyl)piperidine-2-one (XX) with hydrobromic acid in a protic solvent (e.g., methanol) at a low temperature (e.g., 5°C to 10°C) to produce 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI). In some embodiments, the method involves reacting 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI) with 1,4-oxathian-2,6-dione in a weakly polar or nonpolar solvent (e.g., dichloromethane) in the presence of an organic base (e.g., triethylamine, N,N-diisopropylethylamine, or other trialkylamine) to produce 2-((2-(2-oxopiperidine-1-yl)ethylcarbamoyl)methylthio)acetic acid (XVII).

[0057] In some embodiments, the method includes the step of reacting pyridine-2(1H)-one (XVIII) with benzyl(2-bromoethyl)carbamate in a polar aprotic solvent (e.g., MeCN) in the presence of a non-nucleophilic base (e.g., Cs2CO3) to produce benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX).

[0058] In some embodiments, the method includes reducing benzyl 2-(2-oxopyridine-1(2H)-yl)ethyl carbamate (XIX) in a polar protic solvent (e.g., methanol) under hydrogen gas conditions using a catalyst (e.g., 20% Pd(OH)2·on·carbon) to produce 1-(2-aminoethyl)piperidine-2-one (XX).

[0059] In some embodiments, the synthesis method includes the steps shown in the following synthesis scheme. TIFF2026522422000044.tif14128

[0060] This processing method involves 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)amino)ethyl)thio)acetic acid (XVII) To generate TIFF2026522422000045.tif24128, 1-(2-aminoethyl)piperidine-2-one (XX): The reaction may include a step of reacting TIFF2026522422000046.tif21128 or a salt thereof with 1,4-oxatian-2,6-dione. In some embodiments, this step is carried out in a polar aprotic solvent (e.g., DCM, MeCN, and / or THF). The reaction may be carried out in the presence of a tertiary amine base (e.g., TEA or DIPEA), optionally a non-nucleophilic tertiary amine base (e.g., DIPEA). In some embodiments, the reaction is carried out in MeCN and DIPEA. The product may be recovered in a yield of 70% or more (e.g., 75% or more or 80% or more). In some embodiments, about 1.07 molar equivalents (e.g., about 1.01, 1.02, 1.03, 1.04, 1.05, 10.6, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, or 1.14 molar equivalents) of 1-(2-aminoethyl)piperidine-2-one or a salt thereof (e.g., its hydrobromide) is reacted with 1.0 molar equivalent of 1,4-oxatian-2,6-dione.

[0061] In some embodiments, the synthesis method comprises one or more steps as shown in the following synthesis scheme. TIFF2026522422000047.tif44128 For example, tert-butyl N-(2-aminoethyl)carbamate (XXIV): TIFF2026522422000048.tif11128 is reacted with 5-chlorovaleryl chloride (i.e., 5-chlorovaleroyl chloride) in a polar solvent (e.g., THF or H2O) in the presence of a base (e.g., NaHCO3) to produce tert-butyl N-[2-(5-chloropentanoylamino)ethyl]carbamate (XXV): TIFF2026522422000049.tif15128 can be produced. In some embodiments, tert-butyl N-[2-(5-chloropentanoylamino)ethyl]carbamate (XXV) is treated with a base (e.g., tBuOK) in a polar solvent (e.g., THF) to produce tert-butyl 2-(2-oxopiperidine-1-yl)ethylcarbamate (XXVI): TIFF2026522422000050.tif19128 is obtained. tert-butyl 2-(2-oxopiperidine-1-yl)ethyl carbamate (XXVI) is reacted with an acid (e.g., HBr) to obtain 1-(2-aminoethyl)piperidine-2-one (XX): TIFF2026522422000051.tif16128 or its hydrobromide (XXI): It can produce salts such as TIFF2026522422000052.tif19128.

[0062] In some embodiments, the method comprises one or more steps as shown in the following synthesis scheme. TIFF2026522422000053.tif36148

[0063] In some embodiments, this method uses a standard solid-phase synthesis procedure to construct (XXII): The process includes a step of generating a resin-bound peptide having TIFF2026522422000054.tif44156, which in some embodiments is (XXIII): It is referred to as TIFF2026522422000055.tif9128.

[0064] In some embodiments, the method includes a step of deprotecting a resin-bound peptide of formula (XXII) or (XXIII) using 20% ​​piperidine in DMF or NMP. In some embodiments, the deprotected, resin-bound peptide is reacted with intermediate (XVII) under coupling conditions that form an amide bond. The conditions for forming the amide bond may include treating intermediate (XVII) with a coupling reagent (e.g., azabenzotriazoletetramethyluronium hexafluorophosphate (HATU)) in a polar aprotic solvent (e.g., dimethylformamide) in the presence of a weak base (e.g., diisopropylethylamine). In some embodiments, after coupling intermediate (XVII) to the deprotected peptide bound to the resin via an amide bond, the conjugated peptide is cleaved from the resin using a strong acid (e.g., trifluoroacetic acid) in an aqueous solvent in the presence of a reducing agent (e.g., triisopropylsilane).

[0065] The carboxylic acid of formula (XVII) can be produced from commercially available starting materials in yields exceeding 5% (e.g., exceeding 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%).

[0066] The present method for synthesizing peptide (I), GLP-1R / GIPR agonists (VI) to (XVI), and their intermediates (XVII) offers several advantages over previously reported synthetic procedures, including increased yield and reduced number of synthesis and / or purification steps to reach the desired compounds. In some embodiments, the method also avoids the use of hazardous reagents (e.g., hydrazine or diisopropyl azodicarboxylate (DIAD)) or reagents that produce byproducts that are difficult to dispose of (e.g., triphenylphosphine and triphenylphosphine oxide). Furthermore, in some embodiments, the synthetic methods described herein involve the reduction of a pseudo-aromatic pyridine-2(1H)-one group with a mild and readily available reducing agent (hydrogen gas in the presence of catalytic Pd / C). Thus, the present invention streamlines the synthesis of peptide (I), GLP-1R / GIPR agonists (VI) to (XVI), and their intermediates (XVII) compared to previously reported synthetic procedures.

[0067] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of this disclosure. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, unless specifically required by context, singular terms shall include plural forms, and plural terms shall include singular forms. Throughout this specification and its embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” shall be understood to include the integer or set of integers described, but not to exclude any other integer or set of integers. All publications and other references referenced herein are incorporated by reference in whole, as if specifically and individually indicated that each individual reference is incorporated by whole. Numerous sources are cited herein, but this citation does not imply that any of these sources constitute common knowledge in the art. Where used herein, the terms “approximately” or “about” refer to a value similar to the given reference value when applied to one or more values ​​of interest. In some embodiments, unless otherwise specified or evident from the context, the terms refer to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (greater than or less than) the given reference value.

[0068] According to this disclosure, backreferences in dependent claims are intended as abbreviated descriptions for a direct and clear disclosure of any combination of claims indicated by the backreference. Furthermore, headings in this specification are created for ease of organization and are not intended in any way to limit the scope of the claimed invention.

[0069] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention. [Examples]

[0070] Example 1: Synthesis of 2-((2-(2-oxopiperidine-1-yl)ethylcarbamoyl)methylthio)acetic acid (XVII) The synthesis of 2-((2-(2-oxopiperidine-1-yl)ethylcarbamoyl)methylthio)acetic acid (XVII) was carried out in four steps with an overall yield of 14%. The overall scheme is shown below. TIFF2026522422000056.tif31164

[0071] Synthesis of benzyl(2-(2-oxopyridine-1(2H)-yl)ethyl)carbamate (XIX) TIFF2026522422000057.tif19133 To a solution of pyridine-2(1H)-one (XVIII) (CAS: 142-08-5, 10 g, 105.1 mmol) in anhydrous DMF (100 mL), NaH (60% dispersion in mineral oil, 5.1 g, 1.2 equivalents, 210.3 mmol) was gradually added under N2 at 0°C. The mixture was stirred for 0.5 hours. Then, benzyl(2-bromoethyl) carbamate (CAS: 53844-02-3, 33.92 g, 1.25 equivalents, 131.43 mmol) was added all at once. The reaction mixture was then heated to 90°C for a further 13 hours, at which point the reaction was considered complete, and was then quenched with water (50 mL) and extracted with siRNA (3 × 100 mL). The combined organic extracts were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude residue. The crude residue was purified using reversed-phase column chromatography (ACN / H2O, gradient: 25%~30%) to obtain 7 g of yellow solid (yield 27.9%). MS (ESI, positive ion) m / z: 273.1 (M+1).

[0072] Synthesis of 1-(2-aminoethyl)piperidine-2-one (XX) To a solution of benzyl (2-(2-oxopyridine-1(2H)-yl)ethyl) carbamate (XIX) (15 g, 55.08 mmol) in TIFF2026522422000058.tif19128THF (80 mL), Pd / C (3 g, 10 wt%) was added. The reaction mixture was heated at 50 °C under H2 (50 psi) for 5 hours, and then Celite (登録商標) It was filtered through Celite. (登録商標) The bed was washed with ELISA (2 × 100 mL). The filtrate and washing solution were combined and concentrated under reduced pressure to obtain a pale-colored oily substance (12 g, yield 153%, purity = 50%; impurities were solvent), which was used directly in the next step. MS (ESI, positive ion) m / z: 142.2 (M+1).

[0073] Synthesis of 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI) TIFF2026522422000059.tif19128 A solution of 1-(2-aminoethyl)piperidine-2-one (XX) (100 g, 704 mmol, 1.0 equivalent) in 500 mL of MeOH, pre-cooled using an ice bath, was mixed with 40% HBr aqueous solution (169 g, 845 mmol, 1.2 equivalents). The reaction mixture was stirred at 5°C for 2 hours. The solvent was then removed under reduced pressure, and EtOH (1 L) was added. The reaction mixture was concentrated again to remove water. The resulting residue was placed in an ice bath and slowly dissolved in MeOH (300 mL) and MTBE (600 mL). The solution was stirred at 5-10°C for 2 hours. The reaction mixture was filtered and washed with MTBE (1 L) to obtain 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI) (100 g, purity 98%, yield 64%) as a white solid. MS (ESI, positive ion) m / z: 143.2 (M+1). 1 H NMR (400MHz, D2O) δ3.61(t,J=5.9Hz,2H), 3.35(t,J=5.7Hz,2H), 3.17(t,J=5.9Hz,2H), 2.33(t,J=6.3Hz,2H), 1.80-1.68(m,4H).

[0074] 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI) (90 g, 1 equivalent) was added to a 500 mL glass flask containing 200 mL of DCM. After cooling to 0°C, TEA (85.72 g, 2.1 equivalents) was added. The reaction mixture was stirred at 0°C for 0.5 hours. Then, a solution of 1,4-oxatian-2,6-dione (CAS: 3261-87-8, 106.61 g, 2 equivalents) in 50 mL of THF was added dropwise to the mixture. The reaction mixture was stirred at 25°C for 12 hours under an N2 atmosphere. Ion-to-ion chromatography (IPC): HPLC showed complete consumption of 1-(2-aminoethyl)piperidine-2-one. Next, the reaction mixture was concentrated under reduced pressure, and the resulting residue was tritulated with Â(7.5V) for 1 hour. The slurry was then filtered. The filter cake was then tritulated with CH3CN(5V) for 1 hour and filtered. The filter cake was dried in vacuum to obtain the crude product (260g). 1 ¹H-NMR showed the absence of excess 1,4-oxatian-2,6-dione. Next, 260 g of the crude product was dissolved in water (2V). Two equivalents of saturated NaHCO3 solution were added, and the reaction mixture was stirred for 0.5 hours, after which another equivalent of solid Na2CO3 was added. The solution was then concentrated ( 1 TEA was removed (until it was no longer present by 1H NMR analysis). The residue was dissolved in water (2V), and the pH of the resulting solution was adjusted to pH 3 with 6N HCl. The reaction mixture was concentrated to obtain the residue. The residue was then triturated with DCM / MeOH (10 / 1 v / v, 5V) for 30 minutes. The slurry was then filtered, and the filter cake was washed with DCM / MeOH (10 / 1 v / v, 1V). The filtrate was concentrated to obtain 250 g of crude acid (XVII) as a yellow oily substance, which HPLC and LCMS showed to contain approximately 50% methyl ester byproducts.

[0075] Next, the product was purified by silica gel column chromatography (5-25% MeOH in DCM) to obtain a mixture of 128 g of the acid product and methyl ester byproduct. The mixture (128 g) was dissolved in THF (200 mL) before the addition of MeOH (200 mL) and H2O (200 mL). Then, NaOH (19.5 g, 1.1 equivalents) was added, and the mixture was stirred at 25°C for 12 hours under an N2 atmosphere. HPLC and LCMS showed that the methyl ester byproduct was completely consumed. The reaction mixture was concentrated under reduced pressure, and THF and MeOH were removed to obtain the residue. The residue was triturated with H2O (200 mL) at 0°C for 1 hour and filtered to obtain 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII) (87 g) as a white solid in 78.6% yield. MS (ESI, positive ion) m / z: 275.1 (M+1). 1 H-NMR (400MHz, CDCl3) δ7.53(s,1H), 3.60(t,2H), 3.52(dd,2H), 3.42-3.31(m,4H), 3.29(s,2H), 2.43(t,2H), 1.82(dd,4H).

[0076] Example 2: Synthesis of GPCR agonist peptide (I) Following a standard solid-phase peptide synthesis protocol, the amino acid peptide sequence Aa(XXIII) was prepared conjugated to a Rink amide resin. 20% v / v piperidine / DMF (2 mL) was added to a peptide-resin conjugate (XXIII) (0.125 mmol / g, 41 mg) in a 6 mL polypropylene tube with an end cap. The tube was capped and stirred at ambient temperature for 30 minutes, then drained. The resin was washed with DMF (5 × 3 mL). A solution of carboxylic acid component (XVII) (36.6 mg, 8.0 equivalents) in DMF (2.8 mL) was added to the resin, followed by the addition of DIPEA (61 μL, 20 equivalents). HATU (80 mg, 12.0 equivalents) was then added, and the reaction mixture was stirred at ambient temperature for 18 hours. The reaction mixture was drained, the resin was washed with DMF (5 × 3 mL) and DCM (5 × 3 mL), and dried in a vacuum for 30 minutes.

[0077] The resin was transferred to a 15 mL Falcon tube, and 3 mL of cutting reagent (95:2.5:2.5 v / v / v TFA / TIS / H2O) was added. The reaction mixture was stirred at ambient temperature for 1 hour. The resin was filtered and washed with TFA (2 × 3 mL). The combined filtrate and washing solution were concentrated under reduced pressure to obtain the residue, which was tritulated with Et2O (3 mL) to precipitate the peptide. The peptide was redissolved in ice AcOH (2 mL) and purified by preparative HPLC (Phenomenex Jupiter 10 μM Proteo 90 Å LC column, 250 × 21.2 mm, flow rate 15 mL / min, gradient of 0-100% acetonitrile in 25 mM ammonium acetate aqueous solution over 30 minutes) to obtain 4.1 mg of (I) as a white solid. ESI-MS (positive ionization) found 1153.3, and C 214 H 327 N 47 O 64 S(M+4H + ) requires 1152.8.

[0078] Example 3: Alternative synthesis of 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI) 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI) was synthesized according to the following scheme. TIFF2026522422000062.tif51143

[0079] The synthesis begins with commercially available pyridone (XVIII), which is treated with benzyl (2-bromoethyl) carbamate under basic conditions to alkylate pyridone (XIX). Catalytic hydrogenation of pyridone (XIX) with deprotection of the carbobenzyloxy group yields amine (XX). Amine (XX) is then subjected to hydrobromide formation, and recrystallization of this salt yields hydrobromide (XXI).

[0080] Synthesis of benzyl(2-(2-oxopyridine-1(2H)-yl)ethyl)carbamate (XIX) To a solution of pyridine-2(1H)-one (CAS: 142-08-5, 5.0 g, 52.1 mmol, 1.0 equivalent) in 50 mL of MeCN, pre-cooled in an ice bath, benzyl(2-bromoethyl) carbamate (CAS: 53844-02-3, 16.1 g, 62.5 mmol, 1.2 equivalents) and Cs2CO3 (33.9 g, 104.2 mmol, 2.0 equivalents) were added. The reaction mixture was stirred and stirred over 2 hours for 25 minutes. ℃ The mixture was heated to [temperature]. After completion, the reaction mixture was filtered. The filtrate was concentrated to obtain the crude product, which was purified by solvation gas chromatography (SGC) (Â1 / DCM = 1:1) to obtain benzyl(2-(2-oxopyridine-1(2H)-yl)ethyl) carbamate (XIX) (12 g, purity 90%, yield 86%) as a white solid. MS (ESI, positive ion) m / z: 273.1 (M+1).

[0081] The synthesis was also carried out on a larger scale. To a solution of pyridine-2(1H)-one (500 g, 5.2 mol, 1.0 equivalent) in 5 L of MeCN pre-cooled in an ice bath, benzyl(2-bromoethyl)carbamate (1620 g, 6.3 mol, 1.2 equivalents) and Cs2CO3 (2190 g, 6.8 mol, 1.3 equivalents) were added. The reaction mixture was 0-25 ℃The mixture was stirred for 16 hours and then filtered. The filtrate was concentrated to obtain the crude product, which was purified by SGC (Âxy / DCM = 1:1) to obtain benzyl(2-(2-oxopyridine-1(2H)-yl)ethyl) carbamate (XIX) (1200 g, purity 90%, yield 86%) as a white solid. MS (ESI, positive ion) m / z: 273.1 (M+1).

[0082] Synthesis of 1-(2-aminoethyl)piperidine-2-one (XX) To a solution of benzyl (2-(2-oxopyridine-1(2H)-yl)ethyl) carbamate (XIX) (100 g, 366 mmol, 1.0 equivalent) in 500 mL of MeOH (HPLC grade), 20% Pd(OH)2 / C (20 g) was added. Under H2 (0.4 MPa), 25 ℃ The reaction mixture was stirred for 24 hours. The mixture was filtered and concentrated under reduced pressure to obtain the crude product 1-(2-aminoethyl)piperidine-2-one (XX) (50 g, purity 80%) as a yellow oil. MS (ESI, positive ion) m / z: 143.2 (M+1). 1 H NMR (400MHz, DMSO) δ3.26(t,J=5.6Hz,3H), 3.21(d,J=7.0Hz,2H), 2.62(t,J=6.9Hz,2H), 2.51-2.49(m,2H), 2.18(t,J=6.1Hz,2H), 1.73-1.65(m,4H).

[0083] The synthesis was also carried out on a larger scale. 20% Pd(OH)2 / C (40g) was added to a solution of benzyl(2-(2-oxopyridine-1(2H)-yl)ethyl) carbamate (XIX) (200g, 735 mmol, 1.0 equivalent) in 1 L of MeOH (HPLC grade). Under H2 (0.4 MPa), 25 ℃ The mixture was stirred for 24 hours. The mixture was then filtered and concentrated under reduced pressure to obtain the crude product 1-(2-aminoethyl)piperidine-2-one (XX) (50 g, purity 80%) as a yellow oil. MS (ESI, positive ion) m / z: 143.2 (M+1).

[0084] Synthesis of 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI) To a solution of 1-(2-aminoethyl)piperidine-2-one (XX) (100 g, 704 mmol, 1.0 equivalent) in 500 mL of MeOH, which had been pre-cooled in an ice bath, 40% HBr aqueous solution (169 g, 845 mmol, 1.2 equivalents) was added. The reaction mixture was stirred at 5°C for 2 hours. Then, the solvent was removed under reduced pressure, and the reaction mixture was diluted with EtOH (1 L). The mixture was concentrated again to remove water. The resulting crude product was dissolved in MeOH (300 mL), and the solution was cooled in an ice bath. MTBE (600 ml) was slowly added, and the reaction mixture was stirred at 5-10°C for 2 hours. Then, the mixture was filtered, the filter cake was washed with MTBE (1 L), and dried to obtain the product 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI) (100 g, purity 98%, yield 64%) as a white solid. With a purity of >95% (HPLC 214 and 254 nm), 1 The chemical structure of the target compound was confirmed by 1H NMR and LC-MS. MS (ESI, positive ion) m / z: 143.2 (M+1). 1 H NMR (400MHz, D2O) δ3.61(t,J=5.9Hz,2H), 3.35(t,J=5.7Hz,2H), 3.17(t,J=5.9Hz,2H), 2.33(t,J=6.3Hz,2H), 1.80-1.68(m,4H).

[0085] The synthesis was also carried out on a larger scale. A solution of 1-(2-aminoethyl)piperidine-2-one (1000 g, 7.1 mol, 1.0 equivalent) in 3 L of MeOH, pre-cooled in an ice bath, was mixed with 40% HBr aqueous solution (1690 g, 8.5 mol, 1.2 equivalents). Reaction reaction 5 ℃ The mixture was stirred for 2 hours. Then, the solvent was removed under reduced pressure, and the reaction mixture was diluted with EtOH (3 L). The reaction mixture was concentrated again, and water was removed. The resulting crude product was dissolved in MeOH (2 L), and the solution was cooled in an ice bath. Then, MTBE (3 L) was slowly added. The reaction mixture was 5-10 ℃It was stirred for 2 hours. Subsequently, the reaction mixture was filtered, and the solid was washed with MTBE (2 L) and dried to obtain 1-(2-aminoethyl)piperidin-2-one hydrobromide (850 g, purity 99%, yield 54%) as a white solid. MS (ESI, positive ion) m / z: 143.2 (M+1). 1 H NMR (400 MHz, D2O) δ 3.56 (t, J = 5.9 Hz, 2H), 3.31 (t, J = 5.5 Hz, 2H), 3.13 (t, J = 5.7 Hz, 2H), 2.29 (t, J = 6.2 Hz, 2H), 1.79 - 1.62 (m, 4H).

[0086] Example 4: Alternative synthesis of 1-(2-aminoethyl)piperidin-2-one hydrobromide (XXI) As shown in the following scheme, the synthesis of 1-(2-aminoethyl)piperidin-2-one hydrobromide (XXI) was carried out. TIFF2026522422000063.tif46128

[0087] The synthesis started from the acylation of mono-protected ethylenediamine (XXIV) with an acid chloride to obtain chloropentanoyl amide (XXV), and the chloropentanoyl amide (XXV) was treated with a base to obtain piperidone (XXVI). Deprotection of the carbamate directly gave piperidone (XXI), which was purified by recrystallization as in the previous example.

[0088] Synthesis of tert-butyl (2-(5-chloropentanamide)ethyl)carbamate NaHCO3 (2.1 kg, 25 mol, 2.0 equivalents) was added to a solution of N-Boc-ethylenediamine (XXIV) (2.0 kg, 12.5 mol) in THF (10 L) and water (10 L). The reaction mixture was at 0 ℃The mixture was cooled to room temperature. 5-Chlorovaleryl chloride (2.3 kg, 15 mol, 1.2 equivalents) was added dropwise over 2 hours. The reaction mixture was stirred at room temperature for 1 hour, at which point the reaction was considered complete. The reaction mixture was filtered, and the filter cake was washed with water (15 L). The solid was slurried in a solution of butyl / petroleum ether (1:50 v / v, 5 L) for 1 hour, then filtered, and the filter cake was dried to obtain tert-butyl (2-(5-chloropentanamide)ethyl) carbamate (XXV) as a white solid in 86.2% yield (3.0 kg).

[0089] This process was carried out in two batches and combined for the next step. In the first batch, 2,000 g of starting material was converted to 3,000 g of product (yield 86.2%). In the second batch, 4,000 kg of starting material was converted to 6,500 g of product (yield 93.4%). Mass (m / z): 279.0 [M+H] + . 1 H NMR(400MHz,CDCl3)δ6.38(br,1H), 5.01(br,1H), 3.53-3.56(t,J=6,2H), 3.34-3 .37(m,2H), 3.28(br,2H)2.20-2.24(t,J=8,2H), 1.77-1.81(m,4H), 1.44(s,9H).

[0090] Synthesis of tert-butyl(2-(2-oxopiperidine-1-yl)ethyl)carbamate While maintaining the temperature at 0°C, t-BuOK (6.85 kg, 61.2 mol, 2.0 equivalents) was added to a solution of tert-butyl(2-(5-chloropentanamide)ethyl) carbamate (XXV) (8.5 kg, 30.6 mol) in THF (40 L). The reaction mixture was warmed to ambient temperature and stirred for 1 hour. The reaction mixture was then quenched with ice water and extracted with ethyl acetate (2 × 10 L). The organic phases were combined, washed with brine (20 L), dried over anhydrous Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by silica gel chromatography (Â:petroleum ether = 1:1) to obtain 7.16 kg (yield 96.5%) of tert-butyl(2-(2-oxopiperidine-1-yl)ethyl) carbamate (XXVI) as a yellow oil.

[0091] This process was carried out in two batches and combined for the next step. In the first batch, 1,000 g of starting material was converted to 840 g of product (yield 96.5%). In the second batch, 8,500 g of starting material was converted to 7,160 g of product (yield 96.5%). Mass (m / z): 243.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ5.01-5.13(m,1H), 3.44-3.47(m,2H), 3.26-3.33(m,4H), 2.34-2.37(m,2H), 1.73-1.79(m,4H), 1.40(s,9H).

[0092] Synthesis of 1-(2-aminoethyl)piperidine-2-one hydrobromide A stirred solution of tert-butyl(2-(2-oxopiperidine-1-yl)ethyl)carbamate (XXVI) (3.0 kg, 12.4 mol) in butyl (30 L) is heated under a nitrogen atmosphere at -10°C. ℃HBr (7.6 kg, 31 mol, 2.5 equivalents) was added. The reaction mixture was warmed to room temperature and stirred for 16 hours, at which point the reaction was considered complete. The reaction mixture was filtered, and then the filter cake was washed with EtOAc (15 L). The filter cake was dissolved in MeOH (10 L) and concentrated under reduced pressure to obtain the crude product (4 kg). The crude product was dissolved in MeOH (8 L), and the solution was cooled in an ice bath. Then, MTBE (12 L) was slowly added to the solution. The reaction mixture was stirred at 5 - 10 ℃ for 2 hours, then filtered, the filter cake was washed with MTBE (5 L), and dried to obtain 1-(2-aminoethyl)piperidin-2-one hydrobromide (XXI) as a white solid in a 101% yield (2.79 kg).

[0093] This step was carried out in two batches combined together for the next step. In the first batch, 3,000 g of starting material was converted to 2,790 g of product (yield 101%). In the second batch, 4,820 g of starting material was converted to 4,600 g of product (yield 104%). Mass (m / z): 143.1 [M + H] + . 1 H NMR (400 MHz, D2O) δ 3.54 - 3.57 (t, J = 6, 2H), 3.28 - 3.31 (t, J = 6, 2H), 3.10 - 3.13 (t, J = 6, 2H), 2.26 - 2.29 (t, J = 6, 2H), 1.64 - 1.74 (m, 4H).

[0094] The two batches were combined together and recrystallized with MTBE (15 L) to obtain 1-(2-aminoethyl)piperidin-2-one hydrobromide (7.3 kg, purity 99.3%, yield 101.7%) as a white solid. Mass (m / z): 143.1 [M + H] + . 1¹H NMR (400MHz, D2O) δ 3.54-3.57 (t, J=6, 2H), 3.29-3.32 (t, J=6, 2H), 3.11-3.14 (t, J=6, 2H), 2.27-2.30 (t, J=6, 2H), 1.67-1.74 (m, 4H). The estimated HBr content was 46%, indicating approximately 1.5 moles of HBr per mole of product.

[0095] Example 5: Alternative synthesis of 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)amino)ethyl)thio)acetic acid (XVII) A solution of 1-(2-aminoethyl)piperidine-2-one (4 g, 28.17 mmol) and 1,4-oxatian-2,6-dione (CAS: 3261-87-8, 4.6 g, 1.2 equivalents) in 40 mL of DCM was stirred at 20°C for 0.5 hours, at which point the reaction was considered complete. The reaction mixture was concentrated under reduced pressure to obtain a pale oily substance, which was purified by reverse-phase column chromatography (MeCN / H2O, both containing 0.5% FA; 6%-8% gradient) to obtain 2.69 g of product (XVII) as a white solid (yield 35%). MS (ESI, positive ion) m / z: 275.1 (M+1H + ). 1 H NMR (400MHz, CDCl3) δ7.53(s,1H), 3.60(t,2H), 3.52(dd,2H), 3.42-3.31(m,4H), 3.29(s,2H), 2.43(t,2H), 1.82(dd,4H).

[0096] Example 6: Alternative synthesis of 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)amino)ethyl)thio)acetic acid (XVII) The synthesis of TIFF2026522422000065.tif20150 was carried out on a molar scale of 0.283 mol. The theoretical yield was 77.6 g (0.283 mol × 274.34 g / mol). Cation exchange resin AG50W-X8 H +A type (20-50 mesh) was used. For analysis, YMC Pro Analytical HPLC (C18 column, 3 μm, 120, 4.6 × 150 mm) was used, and eluate A was 0.1% TFA in H2O, and eluate B was 0.1% TFA in ACN. Flow rate of 1.5 mL / min, injection volume of 10 μL, and 30 ℃ A gradient of 5–35%B over 20 minutes was used at the specified column temperature. Absorbance was measured at 214 nm and 254 nm.

[0097] In a round-bottom flask (3) equipped with a magnetic stirrer / overhead stirrer, 1-(2-aminoethyl)-2-piperidinone hydrobromide (XXI) (75.8 g, 1.2 equivalents) and thiodiglycolic acid anhydride (37.4 g, 283 mmol) were added. Then, DCM (2830 mL) was added, and the suspension was stirred for 3 to 7 minutes. Subsequently, DIPEA (208 mL, 4.2 equivalents) was added to the reaction mixture over 1 to 3 minutes. The temperature after the addition of DIPEA was 25.3°C. ℃ The reaction was stirred for at least 30 minutes and up to 4 hours. The completion of the reaction was monitored by HPLC at 214 nm and 254 nm.

[0098] The product peak areas at a given time were as follows: 30 min: 34.2, 33.1% area; 60 min: 57.4, 69.9% area; 90 min: 56.7, 70.1% area; 2 hours: 56.4, 70.1% area. Based on the peak area, the reaction was considered complete after 2.5 hours. Subsequently, the mixture was allowed to stand for 20-30 minutes until no further condensation formed and the residue appeared visually dry. ℃ The reaction mixture was then concentrated under reduced pressure. Next, water (2264 mL) was added, and the mixture was stirred until a solution was obtained.

[0099] Before using the ion exchange resin, it was washed with water (2-3 mL of USP water per gram of resin). The suspension was stirred for 5 minutes, and then the water was drained through a coarse funnel. The washing was repeated 5 times, using fresh USP water each time. 948 grams of resin was used, and 1900 mL of water was used for each wash.

[0100] Next, the pre-washed ion exchange resin (H+) was divided into four portions (189.6 g each) and added to the product solution. After each addition, the slurry was stirred for 5 minutes, and then the pH was checked. The pH after 5 minutes following the first addition was 10, after the second it was 4, and after the third it was 1. If the pH was still greater than 2, a fifth ion exchange resin treatment could be applied. The desired pH was 2 or less.

[0101] After the final treatment of the dissolved product in the ion exchange resin, the suspension was filtered through a coarse funnel and the filtrate was collected. The ion exchange resin was then washed with 500 mL of USP water to remove any remaining product.

[0102] All sublots were transferred to glass flasks or containers. After all product-containing solutions had been transferred, the solutions were mixed using a stirring rod and then covered with red plastic caps. The total volume was 2764 mL. The solutions were freeze-dried using a bottle freeze-dryer with a condenser temperature of -90.6 to -96°C and a vacuum of 50 to 80 mTorr.

[0103] Two 2.5L lyophilized bottles were used. 64.0g of product was recovered from the first bottle and 64.6g from the second bottle, for a total of 128.6g. The HPLC purity at 214nm was 84.0% area, and the HPLC purity at 254nm was 75.3% area. The product was recovered in 165.6% yield.

[0104] Example 7: Alternative synthesis of 2-(2-piperidon-1-yl)-ethylcarbamoylmethylthioacetic acid This example describes a one-step manufacturing process successfully applied in a production campaign of 1.7 kg of 2-(2-piperidon-1-yl)-ethylcarbamoylmethylthioacetic acid (XVII). Compared to previous synthesis schemes, the production yield increased from 12% to 81%, with comparable, or even slightly improved, product quality.

[0105] As described in the following scheme, 2-(2-piperidon-1-yl)-ethylcarbamoylmethylthioacetic acid (XVII) was previously prepared twice in batch sizes of 324 g and 458 g. TIFF2026522422000066.tif58139

[0106] From a scalability standpoint, this process has two major limitations. Firstly, it is a three-step process with a very low overall yield of 12%. Due to selectivity issues and extraction losses, the yield in step 1 is only about 30%, which further negatively impacts the volume yield of the process. The yield in step 2 is 44%. This also results in a significant loss of the rather expensive reagent 1-(2-aminoethyl)piperidine-2-one hydrobromide in this step.

[0107] The second constraint is the purification of the intermediate in step 2 by countercurrent partitioning (CCD). This purification technique limits the maximum batch size to 1-2 kg of material, significantly increases manufacturing costs, and can result in very long lead times due to the limited availability of specialized equipment for CCD.

[0108] In light of these challenges, we developed a manufacturing process with the aim of establishing a scalable process. The success of the process development resulted in a one-step manufacturing process that does not use a CCD purification step, as shown in the following scheme, which was subsequently applied to 1650g production activities. TIFF2026522422000067.tif16139

[0109] Tests used for determining stoichiometry A clean reaction with minimal byproducts is essential for the efficient production of 2-(2-piperidone-1-yl)-ethylcarbamoylmethylthioacetic acid and the isolation of its components by crystallization. Therefore, a usage test was performed using 1-(2-aminoethyl)piperidine-2-one hydrobromide and thiodiglycolic acid anhydride to correct for inaccuracies in the starting material assay. The optimal stoichiometry was found to be 1.07 equivalents of 1-(2-aminoethyl)piperidine-2-one hydrobromide and 1.00 equivalent of thiodiglycolic acid anhydride.

[0110] Preparation of 2-(2-piperidon-1-yl)-ethylcarbamoylmethylthioacetic acid In a 90 L glass reactor, 2.00 kg of 1-(2-aminoethyl)piperidine-2-one hydrobromide (8.15 mol, 1.07 equivalents) was dissolved in 40 L of ACN at 15°C (ambient temperature set to 15°C) under a nitrogen atmosphere. Then, while maintaining the internal temperature at 15°C, 1.96 L of DIPEA (11.4 mol, 1.50 equivalents) was added. To the resulting white suspension, another 10 L of ACN was added, and stirring was continued for approximately 30 minutes. Meanwhile, a solution of 1.01 kg of thiodiglycolic acid anhydride (7.62 mol, 1.00 equivalent) in 4 L of ACN was prepared in a 10 L flask. While maintaining the internal temperature below 20°C (ambient temperature was reduced to a minimum of 5°C to counteract the exothermic reaction), the thiodiglycolic acid anhydride solution was added to the reaction mixture over 30 minutes using a peristaltic pump. Next, the 10 L flask was rinsed into the reactor via a pump containing an additional 0.6 L of ACN. The reaction mixture was stirred for 2 hours at an internal temperature of 20–22°C, first yielding a yellowish solution, and then a white suspension.

[0111] The suspension was transferred to a 50 L flask and concentrated under vacuum (80 mbar) using a 50 L rotary evaporator. After evaporating 44 L of ACN, 10.4 kg of white suspension was obtained and filtered under vacuum. The white solid was washed with ACN (2 × 4 L) and IPE (2 × 4 L) and dried under vacuum at 40°C to obtain 1.87 kg of the crude product 2-(2-piperidon-1-yl)-ethylcarbamoylmethylthioacetic acid.

[0112] The crude product was suspended in ACN (20 L) and 1.5 L of deionized water was added. The reaction mixture was heated to 40°C to obtain a yellowish, turbid solution, which was then filtered for clarification. The filter was washed with ACN (2 L), and the combined filtrate was concentrated under vacuum at 40°C. After collecting 10 L of distillate, an additional 20 L of ACN was added to the resulting white suspension, and distillation was continued until an additional 18 L of distillate was collected. The suspension was then rotated at room temperature for 16 hours and filtered under vacuum to obtain a white solid. The white solid was washed with 2 × 3.8 L of ACN and 2 × 3.8 L of IPE, and dried under vacuum at 40°C to obtain 1698 g (6.19 mol, yield 81%) of the product 2-(2-piperidon-1-yl)-ethylcarbamoylmethylthioacetic acid as a white solid.

[0113] Summary and Conclusion The manufacturing process for 2-(2-piperidon-1-yl)-ethylcarbamoylmethylthioacetic acid was developed with the aim of creating a scale-scalable process suitable for future production on a multi-kg scale. The process development resulted in a one-step manufacturing process that does not require purification by CCD. This new process was successfully applied to the production activity of 1.7 kg of 2-(2-piperidon-1-yl)-ethylcarbamoylmethylthioacetic acid.

[0114] Batch comparison Table 1 shows a comparison of three batches of 2-(2-piperidone-1-yl)-ethylcarbamoyl-methylthioacetic acid produced. The first two batches (1000013161 and 1000032416) were produced using the original three-step process, while the third batch (1000074879) was produced according to the developed efficient and scalable one-step process.

[0115] (Table 1) Comparison of data for three batches of 2-(2-piperidon-1-yl)-ethylcarbamoyl-methylthioacetic acid TIFF2026522422000068.tif61160

[0116] The yield of the new one-step process was 81%, a 575% increase compared to the three-step process (12% yield). The product quality of the one-step process was comparable to, and even slightly better than, that of the three-step process. The product did not contain a significant amount of water, whereas in the original process, the material was isolated as a hydrate (approximately 6% water content). The assay rate of the developed process batch was nearly 100%, compared to only about 80% for the previous batch.

[0117] Example 8: HPLC purification of 2-((2-(2-oxopiperidine-1-yl)ethylcarbamoyl)methylthio)acetic acid (XVII) 2-((2-(2-oxopiperidine-1-yl)ethylcarbamoyl)methylthio)acetic acid (XVII) was purified by RP-UPLC in a ULC equipped with a gradient system, autosampler, and UV detection. The solvent was acetonitrile / water (10 / 90 (v / v)). The test substance was present at 0.5% (e.g., 50 mg in 10 mL) during injection. Elution A was TFA / acetonitrile / water (0.05 / 1 / 99 (v / v / v)). Elution B was TFA / acetonitrile (0.05 / 100 (v / v)).

[0118] A USP L1, RP C18 Aeris Peptide XB-C18 column (2.6 u; 250 × 2.1 mm) was used. The gradient program is shown in Table 2.

[0119] (Table 2) Gradient Program TIFF2026522422000069.tif41133

[0120] The data acquisition time was 16.0 minutes, and the runtime was 26.0 minutes. Detection was performed using UV absorbance (λ=240nm). The column temperature was 25°C, the autosampler temperature was 5°C, and the injection volume was 5 μl.

[0121] The impurity 2,2-thiodiacetic acid eluted after a retention time of 3.407 minutes. The desired product eluted after a retention time of 6.662 minutes.

Claims

1. Equation (I): (In the formula, sequence Aa is a peptide.) A method for synthesizing an N-terminal conjugate peptidyl compound, Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): H ２ In the presence of , using a catalyst, 1-(2-aminoethyl)piperidine-2-one (XX): A method that includes a step of reducing to a certain state.

2. Formula (XVI): A method for synthesizing the compound, Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): H ２ In the presence of , using a catalyst, 1-(2-aminoethyl)piperidine-2-one (XX): A method that includes a step of reducing to a certain state.

3. 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): A method for synthesizing, Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): H ２ In the presence of , using a catalyst, 1-(2-aminoethyl)piperidine-2-one (XX): A method that includes a step of reducing to a certain state.

4. The method according to any one of claims 1 to 3, wherein the catalyst for reduction is palladium-on-carbon (Pd / C).

5. The catalyst for reduction is palladium hydroxide on carbon (Pd(OH) ２ The method according to any one of claims 1 to 3, wherein C)

6. Equation (I): (In the formula, sequence Aa is a peptide.) A method for synthesizing an N-terminal conjugate peptidyl compound, 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): To produce 1-(2-aminoethyl)piperidine-2-one (XX): A method comprising the step of reacting a salt thereof with 1,4-oxatian-2,6-dione.

7. Formula (XVI): A method for synthesizing the compound, 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): To produce 1-(2-aminoethyl)piperidine-2-one (XX): A method comprising the step of reacting a salt thereof with 1,4-oxatian-2,6-dione.

8. 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): A method for synthesizing, 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)aminoethyl)thio)acetic acid (XVII): To produce 1-(2-aminoethyl)piperidine-2-one (XX): A method comprising the step of reacting a salt thereof with 1,4-oxatian-2,6-dione.

9. The method according to any one of claims 6 to 8, wherein a hydrobromide salt of 1-(2-aminoethyl)piperidine-2-one (XX) is reacted with 1,4-oxatian-2,6-dione.

10. Array Aa is given by formula W-R ５ Includes, In the formula, W is the peptide sequence, and R ５ It is conjugated to the C-terminus of W, W is the following array: EGT (Xaa4) (Xaa5) SD (Xaa8) S (Xaa10) (Xaa11) (Xaa12) (Xaa13) (Xaa14) (Xaa15) (Xaa16) (Xaa17) ( Xaa18) (Xaa19) (Xaa20) (Xaa21) (Xaa22) WL (Xaa25) (Xaa26) (Xaa27) GPSSGAPPP (Xaa37) (SEQ ID NO: 1); (Here, Xaa4 is F; Xaa5 is T or I; Xaa8 is Y, V, L, or K ＊ And; Xaa10 is I or S; Xaa11 is Y, Y ＊ , Q, A, or (Aib); Xaa12 is L, M, or L ＊ and is; Xaa13 is either D or E; Xaa14 is K, G, R, or E; Xaa15 is Q or I; Xaa16 is A, H, or R; Xaa17 is A, Q, or V; Xaa18 is A, (Aib), K ＊ , K, or Q; Xaa19 is A, D, E, (Aib), or L; Xaa20 is F or A; Xaa21 is V or I; Xaa22 is N, A, Q, K ＊ , or E; Xaa25 is I, L, or V; Xaa26 is A, K, or I; Xaa27 is Q-R, G-R-G-K ＊ , Q, or G; and Xaa37 is either S or non-existent. Includes, R ５ The method according to claim 1 or 6, wherein is a C-terminal amino acid amide or C-terminal amino acid which may be substituted with one or two modifying groups selected from an acyl group and a PEG group.

11. W is the following array: EGTFTSDYSIYLDKQAA(Aib)EFVNWLLAGGPSSGAPPPS(Sequence ID 4) The method according to claim 10, including the method described in claim 10.

12. R ５ The method according to claim 10 or 11, wherein the C-terminal lysylamide residue may be substituted with one or two modifying groups selected from an acyl group and a PEG group.

13. R ５ However, equation (II): Includes, In the formula, R ＊ Structure (IV): The method according to any one of claims 10 to 12, including the method described in any one of claims 10 to 12.

14. W-R ５ However, structure (V): The method according to any one of claims 10 to 13, including the method described in any one of claims 10 to 13.

15. (i) Formula (XXIII): The process involves treating the peptide bound to the resin with 20% piperidine in DMF, and (ii) The product of step (i) is subjected to conditions that form an amide bond, and 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)amino)ethyl)thio)acetic acid (XVII): The process of reacting with The method according to any one of claims 1, 6 and 10 to 14, further comprising:

16. (i) Formula (XXII): The process involves treating the peptide bound to the resin with 20% piperidine in DMF, and (ii) The product of step (i) is subjected to conditions that form an amide bond, and 2-((2-oxo-2-((2-(2-oxopiperidine-1-yl)ethyl)amino)ethyl)thio)acetic acid (XVII): The process of reacting with The method according to any one of claims 2, 7, and 14, further comprising:

17. The method according to claim 15 or 16, wherein the conditions for forming the amide bond include the use of a coupling reagent.

18. The method according to claim 17, wherein the coupling reagent is azabenzotriazoletetramethyluronium hexafluorophosphate (HATU).

19. Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): To generate pyridine-2(1H)-one (XVIII): The process involves reacting it with benzyl(2-bromoethyl) carbamate in the presence of a non-nucleophilic base. The method according to any one of claims 1 to 18, further comprising:

20. The method according to claim 19, wherein the non-nucleophilic base is sodium hydride.

21. 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI): To produce 1-(2-aminoethyl)piperidine-2-one (XX): The process of reacting it with hydrobromic acid. The method according to any one of claims 1 to 20, further comprising:

22. 2-((2-(2-oxopiperidine-1-yl)ethylcarbamoyl)methylthio)acetic acid (XVII): To produce 1-(2-aminoethyl)piperidine-2-one hydrobromide (XXI): The process of reacting with 1,4-oxatian-2,6-dione. The method according to any one of claims 1 to 5 and 11 to 21, further comprising:

23. Benzyl 2-(2-oxopyridine-1(2H)-yl)ethylcarbamate (XIX): It is a compound.

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