CRF2 receptor agonists and their therapeutic use

Abstract

The present invention provides a CRF2 agonist having desirable efficacy, pharmacokinetic properties (e.g., improved half-life), and / or physicochemical properties (e.g., improved stability and / or solubility). [Solution] Provided are compounds that are agonists of corticotropin-releasing factor receptor 2 (CRF2), which are peptides containing a specific amino acid sequence, or pharmaceutically acceptable salts thereof, and the use thereof in the treatment, particularly in the treatment or prevention of cardiovascular disease, obesity and diabetes.

Description

[Technical Field]

[0001] The present invention relates to compounds that are agonists of corticotropin-releasing factor receptor 2 (CRF2), and to their use in the treatment, particularly in the treatment or prevention of cardiovascular disease, obesity, and diabetes. [Background technology]

[0002] Urocortin (UCN) is an endogenous peptide that acts through the corticotropin-releasing factor (CRF) receptor, a type 2 G protein-coupled receptor (GPCR). The CRF receptor family includes the CRF1 receptor, encoded by the CRHR1 gene, and the CRF2 receptor, encoded by the CRHR2 gene.

[0003] Three known endogenous urocortins exist in mammals: UCN1, UCN2, and UCN3. Despite their high degree of sequence homology, the binding of these peptides to CRF1 and CRF2 differs. CRF1 and CRF2 are non-selectively activated by CRF and UCN1, respectively, while UCN2 and UCN3 are CRF2-selective agonists. In particular, UCN2 is a 38-amino acid peptide that selectively activates the CRF2 receptor and includes the known isoforms CRF2-α, -β, and -γ.

[0004] Urocortin and its receptors are involved in neurohumoral responses to various stress and pathological conditions. In particular, urocortin induces positive hemodynamic effects in both preclinical models and patients with heart failure or hypertension. Urocortin and CRF receptors are expressed in the heart and blood vessels, with CRF2 being very strongly expressed and CRF1 being expressed minimally, if at all (see Waser et al., Peptides, 2006, 27, 3029-3038). In experimental models, urocortin acting via CRF2 activation has been shown to improve cardiovascular function through relaxation of vascular resistance and through cardiac inotropic (cardiac contraction) and lucitropic action. In clinical trials, UCN2 and UCN3 have been shown to have direct vasodilatory effects in healthy volunteers and heart failure patients (see Stirrat et al., Br. J. Clin. Pharmacol., 2016, 82, 974-982). Meanwhile, UCN2 has also been shown to increase cardiac output and decrease vascular resistance in heart failure patients (see Davis et al., Eur. Heart J., 2007, 28, 2589-2597; and Chan et al., JACC: Heart Failure, 2013). Recombinant UCN3 acetate has been shown to improve cardiac output and reduce vascular resistance in a multicenter study of patients with chronic stable heart failure (see Gheorghiade et al., Eur. J. Heart Fail., 2013, 15, 679-89).

[0005] Recent studies have demonstrated that transgenicity of UCN2 and / or UCN3 genes in mice improves not only cardiac function but also glucose processing (see Giamouridis et al., JACC:Basic to Translational Science, 2018, 3, 2). In addition, subcutaneous administration of pegylated UCN2 has been shown to improve glucose tolerance and increase glucose uptake in skeletal muscle while reducing body weight through restriction of food intake (see Borg et al., Diabetes, 2019, 1403-1414). These findings suggest that urocortin may be useful not only in the treatment of cardiovascular disease but also in the treatment of diseases such as diabetes and obesity.

[0006] Nevertheless, the extremely short half-life of urocortin remains a major limitation to its therapeutic use (see Davies et al., JACC, 2007, 49, 461-471). To date, urocortin treatment cannot be sustained without long-term intravenous administration. However, the management of patients with chronic diabetes or heart failure requires treatment suitable for long-term home self-administration.

[0007] In attempts to overcome such limitations, various analogues of urocortin have been proposed. For example, WO2018 / 013803 (Alsina-Fernandez; Eli Lilly and Company) discloses an analogue of UCN2 that has been taught to be useful in the treatment of diseases such as chronic kidney disease and type II diabetes.

[0008] However, there is still a need for improved CRF2 receptor agonists that are useful as therapeutic agents, particularly in the treatment and prevention of cardiovascular disease, obesity, and diabetes. In particular, there is a need for CRF2 agonists with desirable efficacy, pharmacokinetic properties (e.g., improved half-life), and / or physicochemical properties (e.g., improved stability and / or solubility). [Overview of the Initiative]

[0009] In a first aspect, the present invention provides a compound that is a peptide comprising the amino acid sequence of formula (I) (SEQ ID NO: 186): Equation (I) X1-X2-X3-X4-X5-X6-X7-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X2 1-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38, During the ceremony X1 is isoleucine (I) or phenylalanine (F); X2 is either valine (V) or threonine (T); X3 is leucine (L); X4 is serine (S); X5 is leucine (L); X6 is aspartic acid (D); X7 is valine(V) or D-valine(v); X8 is proline (P); X9 is isoleucine (I) or threonine (T); X10 is lysine (K), glutamic acid (E), histidine (H), or glycine (G); X11 is isoleucine(I) or leucine(L); X12 is lysine (K), where the epsilon-amino group of the lysine side chain is covalently bonded to the albumin-binding site; X13 is glutamine (Q) or lysine (K); X14 is isoleucine (I), lysine (K), or 2-aminoisobutyric acid (Aib); X15 is leucine (L); X16 is either leucine (L) or phenylalanine (F); X17 is glutamic acid (E) or lysine (K); X18 is glutamine (Q); X19 is alanine (A), glutamic acid (E), or glutamine (Q); X20 is either lysine (K) or arginine (R); X21 is glutamine (Q) or lysine (K); X22 is lysine (K), arginine (R), or glutamic acid (E); X23 is lysine (K) or 2-aminoisobutyric acid (Aib); X24 is glutamine (Q), 2-aminoisobutyric acid (Aib), leucine (L), or glutamic acid (E); X25 is arginine (R), lysine (K), or 2-aminoisobutyric acid (Aib); X26 is alanine (A), glutamic acid (E), 2-aminoisobutyric acid (Aib), or glutamine (Q); X27 is glutamine (Q), 2-aminoisobutyric acid (Aib), or lysine (K); X28 is alanine (A); X29 is glutamic acid (E) or lysine (K); X30 is either lysine (K) or threonine (T); X31 is either asparagine (N) or alanine (A); X32 is lysine (K), alanine (A), valine (V), threonine (T), glutamic acid (E), or 2-aminoisobutyric acid (Aib); X33 is arginine (R), lysine (K), or glutamine (Q); X34 is isoleucine(I) or leucine(L); X35 is leucine (L); X36 is either alanine (A) or glutamic acid (E); X37 is glutamine (Q) or arginine (R); and X38 is isoleucine(I) or valine(V); or a pharmaceutically acceptable salt thereof.

[0010] A second aspect of the present invention provides a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable excipient, diluent, or carrier.

[0011] The compounds and pharmaceutical compositions disclosed herein are therapeutically useful and can be used to treat or prevent various diseases via CRF2 receptor agonism. Accordingly, in other embodiments, the present invention relates to the use of compounds and pharmaceutical compositions in the treatment; in particular, in the treatment or prevention of cardiovascular diseases, obesity, diabetes, sarcopenia, muscular dystrophy, kidney disease, pulmonary hypertension, peripheral artery disease, inflammation, allergies, and tissue ischemia; in particular, in the treatment or prevention of cardiovascular diseases including heart failure, obesity, and diabetes. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a graph showing the food intake throughout the treatment period of high-fat C57BL / 6N mice (DIO) subcutaneously treated every two days with compound SEQ ID NO: 35 (black circles), a reference compound (gray circles), or a vehicle (white circles). The x-axis represents the test day, and the y-axis represents the food intake (grams). Values ​​are expressed as mean ± standard error of the mean (SEM). [Figure 2] Figure 2 is a graph showing the relative body weight change over the course of treatment in C57BL / 6N mice (DIO) subcutaneously treated with compound SEQ ID NO:35 (black circles), a reference compound (gray circles), or a vehicle (white circles) on a high-fat diet. The x-axis represents the test day, and the y-axis represents the relative body weight change (%). Values ​​are expressed as mean ± SEM.

[0013] [Figure 3] Figure 3 shows a representative sequence from the peptide according to the present invention having an optimized stability profile, where K[sema] represents K[gGlu-C(O)(CH2)14CH3]. [Modes for carrying out the invention]

[0014] The present invention relates to a compound that is a derivative of UCN2 and is useful as a CRF2 receptor agonist. The compound exhibits desirable activity and selective specificity, along with improved pharmacokinetic properties and beneficial in vivo effects, in relevant animal models with respect to the CRF2 receptor, particularly the CRF1 receptor. In addition, the compound exhibits desirable physicochemical properties such as desirable solubility and stability, making it an excellent candidate for solution formulations for subcutaneous administration. The compound of the present invention is useful for the treatment or prevention of cardiovascular diseases, particularly obesity, diabetes, sarcopenia, muscular dystrophy, renal diseases, pulmonary hypertension, peripheral artery disease, inflammation, allergies, and tissue ischemia, especially for the treatment or prevention of cardiovascular diseases including heart failure, obesity, and diabetes.

[0015] compound The compounds of the present invention are peptides containing the amino acid sequence of formula (I) above, or pharmaceutically acceptable salts of said peptides.

[0016] In the present invention, amino acids are referred to by their names, their commonly known three-letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Commonly accepted three-letter codes for other amino acids, such as Aib for 2-aminoisobutyric acid, may also be used. Unless otherwise specified, all amino acids used in the compounds of the present invention are L-amino acids. Thus, for example, L-valine is called "valine," "V," or "Val," while D-valine is specifically identified as such.

[0017] In one embodiment, X1 is isoleucine(I). In one embodiment, X2 is valine (V). In one embodiment, X9 is isoleucine(I). In one embodiment, X10 is lysine (K) or glycine (G). In one embodiment, X11 is leucine (L). In one embodiment, X14 is isoleucine(I). In one embodiment, X16 is leucine (L). In one embodiment, X19 is alanine (A) or glutamic acid (E). In one embodiment, X21 is glutamine (Q). In one embodiment, X22 is lysine (K). In one embodiment, X24 is glutamine (Q) or 2-aminoisobutyric acid (Aib). In one embodiment, X25 is arginine(R). In one embodiment, X26 is alanine (A), glutamic acid (E), or glutamine (Q). In one embodiment, X27 is glutamine (Q). In one embodiment, X29 is glutamic acid (E). In one embodiment, X31 is asparagine (N). In one embodiment, X32 is lysine (K). In one embodiment, X33 is arginine (R) or glutamine (Q). In one embodiment, X34 is isoleucine(I). In one embodiment, X35 is leucine (L). In one embodiment, X38 is valin(V). In one embodiment, the amino acid residue of X38 is amidated. In one embodiment, the amino acid residue of X1 is acetylated.

[0018] In one embodiment: X1 is isoleucine(I); X2 is valine (V); X9 is isoleucine(I); X10 is either lysine (K) or glycine (G); X11 is leucine (L); X14 is isoleucine(I); X16 is leucine (L); X19 is either alanine (A) or glutamic acid (E); X21 is glutamine (Q); X22 is lysine (K); X24 is glutamine (Q) or 2-aminoisobutyric acid (Aib); X25 is arginine(R); X26 is alanine (A), glutamic acid (E), or glutamine (Q); X27 is glutamine (Q); X29 is glutamic acid (E); X31 is asparagine(N); X32 is lysine (K); X33 is either arginine (R) or glutamine (Q); X34 is isoleucine(I); X35 is leucine (L); and X38 is a Valine (V) tube.

[0019] In one embodiment: X10 is lysine (K); X19 is glutamic acid (E); X24 is glutamine (Q); and X26 is glutamic acid (E).

[0020] In one embodiment: X33 is glutamine (Q); and X37 is glutamine (Q).

[0021] In one embodiment: X1 is isoleucine(I); X2 is valine (V); X9 is isoleucine(I); X10 is lysine (K); X11 is leucine (L); X14 is isoleucine(I); X16 is leucine (L); X19 is glutamic acid (E); X21 is glutamine (Q); X22 is lysine (K); X24 is glutamine (Q); X25 is arginine(R); X26 is glutamic acid (E); X27 is glutamine (Q); X29 is glutamic acid (E); X31 is asparagine(N); X32 is lysine (K); X33 is glutamine (Q); X34 is isoleucine(I); X35 is leucine (L); X37 is glutamine (Q); and X38 is a Valine (V) tube.

[0022] As described above, the compound of the present invention contains a modified lysine (K) residue at the position indicated by X12 in formula (I), where the albumin-binding moiety is covalently bonded to the epsilon-amino group of the lysine side chain.

[0023] As used herein, the term “albumin-binding moiety” refers to a moiety that can be bound to albumin by covalent or non-covalent bonds. In embodiments, the albumin-binding moiety can be bound to albumin by non-covalent bonds. The albumin-binding moiety includes, or may consist of, a group selected from fatty acids, phthalocyanines, coumarins, flavonoids, tetracyclines, naphthalenes, arylcarboxylic acids, heteroarylcarboxylic acids, lipids, alkylamines, cyclic or linear tetrapyrroles and their organometallic compounds, halo-substituted aromatic acid derivatives, organic dyes, and derivatives of tryptophan and thyroxine.

[0024] In certain embodiments, the albumin-binding portion comprises a C14-C24 fatty acid group conjugated to the epsilon-amino group of the lysine side chain either by direct bonding or a linker. As used herein, the term “C14-C24 fatty acid” means a carboxylic acid having 14 to 24 carbon atoms. The C14-C24 fatty acid may be a saturated monoacid or a saturated diacid. “Saturated” means that the fatty acid does not contain a carbon-carbon double bond or a carbon-carbon triple bond.

[0025] Examples of saturated C14-C24 fatty acids include myristic acid (tetradecanoic acid), tetradecane dioic acid (C14 dioic acid), pentadecyl acid (pentadecanoic acid; C15 monoic acid), pentadecanoic acid (C15 dioic acid), palmitic acid (hexadecanoic acid; C16 monoic acid), hexadecanedioic acid (C16 dioic acid), margaric acid (heptadecanoic acid; C17 monoic acid), heptadecanedioic acid (C17 dioic acid), stearic acid (octadecanoic acid; C18 monoic acid), octadecane dioic acid (C18 dioic acid), and nonadecy This product contains nonadecanoic acid (C19 monoacid), nonadecanedioic acid (C19 diacid), aracadic acid (eicosanoic acid; C20 monoacid), eicosanedioic acid (C20 diacid), henicosylic acid (henicosanoic acid; C21 monoacid), henicosandioic acid (C21 diacid), behenic acid (docosanoic acid; C22 monoacid), docosanedioic acid (C22 diacid), lignoceric acid (tetracosanoic acid; C24 monoacid), tricosanoic acid (C23 monoacid), tricosanoic acid (C23 diacid), and tetracosanedioic acid (C24 diacid).

[0026] In one embodiment, the C14-C24 fatty acid group is selected from the group consisting of palmitic acid (hexadecanoic acid; C16 monoacid), hexadecanedioic acid (C16 diacid), stearic acid (octadecanoic acid; C18 monoacid), octadecanodioic acid (C18 diacid), aranoic acid (eicosanoic acid; C20 monoacid), and eicosanedioic acid (C20 diacid).

[0027] C14-C24 fatty acids can bind directly to the epsilon-amino group of the lysine side chain. Alternatively, the C14-C24 fatty acids can be linked to the epsilon-amino group of the lysine side chain via a linker. The linker may comprise one or more groups selected from [2-(2-aminoethoxy)ethoxy]acetyl (herein referred to as "AEEA"), glycine (Gly), N-methylglycine (N-MeGly), 3-[2-[2-[2-(2-aminoethoxy)-ethoxy]-ethoxy]-ethoxy]-propionic acid (CAS N°663921-15-1, also known as amino-PEG4-acid or (PEO)4-aminopropionic acid), amino-PEG6-acid (CAS N°905954-28-1), amino-PEG8-acid (CAS N°756526-04-2) and gamma-glutamate (gGlu); particularly, selected from AEEA, Gly, N-MeGly, and gGlu; more preferably, selected from AEEA and Glu.

[0028] In one embodiment, the albumin-binding moiety is a group of formula (II):: -Z 1 -Y-Z 2 -C(O)R 1 (Formula II) Wherein Y is AEEA, {AEEA}2, {AEEA}3, Gly, {Gly}2, {Gly}3, N-MeGly, {N-MeGly}2, {N-MeGly}3 or absent; Z 1 and Z 2 are each independently selected from gGlu, {gGlu}2, {gGlu}3, {gGlu}4 or absent; and R 1 is -(CH2) x COOH or -(CH2) x CH3; particularly, x is an integer from 12 to 22 of -(CH2) x COOH.

[0029] In one embodiment, Z 1 is absent, Y is {AEEA}2, and Z 2gGlu or {gGlu}2. In one embodiment, Z 1 is gGlu or {gGlu}2, Y is {AEEA}2, Z 2 gGlu or {gGlu}2. In one embodiment, Z 1 Y does not exist, Y does not exist, Z 2 gGlu or {gGlu}2. In one embodiment, Y and Z 1 and Z 2 None of them exist.

[0030] Surprisingly, the albumin-binding moiety containing the free carboxylic acid was found to have improved CRF2 selectivity, as demonstrated in the following examples. Therefore, in certain embodiments, R 1 ga-(CH2) x COOH, where x is 14, 16, or 18; particularly 16 or 18; more preferably 14 or 16; even more preferably 14.

[0031] In one embodiment, R1 is -(CH2) x CH3, where x is 14, 16, or 18; especially 14 and 16.

[0032] In one embodiment, the albumin-binding moiety is selected from the following groups: -(gGlu) n=1-4 -C(O)(CH2) 14 CH3 -(gGlu) n=1-4 -C(O)(CH2) 14 COOH -(gGlu) n=1-4 -C(O)(CH2) 16 CH3 -(gGlu) n=1-4 -C(O)(CH2) 16 COOH -(gGlu) n=1-4 -C(O)(CH2) 18 CH3 -(gGlu) n=1-4 -C(O)(CH2) 18COOH -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 14 CH3 -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 14 COOH -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 16 CH3 -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 16 COOH -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 18 CH3 -(AEEA) n=1,2 -(gGlu) n=1-4 -C(O)(CH2) 18 COOH -(gGlu) n=1-4 -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 14 CH3 -(gGlu) n=1-4 -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 14 COOH -(gGlu) n=1-4 (EEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 16 CH3 -(gGlu) n=1-4 -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 16 COOH -(gGlu) n=1-4 -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 18 CH3 -(gGlu) n=1-4 -(AEEA) n=1,2 -(gGlu) n=1-4 -C(O)(CH2) 18 COOH

[0033] In one embodiment, the albumin-binding moiety is selected from the following groups: -(gGlu) n=1-4 -C(O)(CH2) 14 COOH -(gGlu) n=1-4 -C(O)(CH2) 16 COOH -(gGlu) n=1-4 -C(O)(CH2) 18 COOH -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 14 COOH -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 16 COOH -(AEEA) n=1,2 -(gGlu) n=1-4 -C(O)(CH2) 18 COOH -(gGlu) n=1-4 -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 14 COOH -(gGlu) n=1-4 -(AEEA) n=1,2 --(gGlu) n=1-4 -C(O)(CH2) 16 COOH -(gGlu) n=1-4 -(AEEA) n=1,2 -(gGlu) n=1-4 -C(O)(CH2) 18 COOH

[0034] In one embodiment, the albumin-binding moiety is selected from the following groups: -{AEEA}2-gGlu-C(O)(CH2) 16COOH; -{AEEA}2-gGlu-C(O)(CH2) 18 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 16 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 18 COOH; -C(O)(CH2) 16 COOH; -gGlu-C(O)(CH2) 16 COOH; -gGlu-C(O)(CH2) 14 CH3; -gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 14 COOH; -{AEEA}2-gGlu-C(O)(CH2) 14 CH3; and -{AEEA}2-{gGlu}2-C(O)(CH2) 14 CH3.

[0035] In one embodiment, the albumin-binding moiety is selected from the following groups: -{AEEA}2-gGlu-C(O)(CH2) 16 COOH; -{AEEA}2-gGlu-C(O)(CH2) 18 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 16 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 18 COOH; -C(O)(CH2) 16 COOH; -gGlu-C(O)(CH2) 16 COOH; -gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-gGlu-C(O)(CH2) 14 COOH; and -{AEEA}2-{gGlu}2-C(O)(CH2) 14 COOH.

[0036] In one embodiment, the albumin-binding moiety is selected from the following groups: -(gGlu) n=1-4 -C(O)(CH2) 14 COOH; -(gGlu) n=1-4 -C(O)(CH2) 16 COOH; -(AEEA) n=1,2 --(gDi) n=1-4 -C(O)(CH2) 14 COOH; -(AEEA) n=1,2 --(gDi) n=1-4 -C(O)(CH2) 16 COOH; -(gGlu) n=1-4 -(AEEA) n=1,2 --(gDi) n=1-4 -C(O)(CH2) 14 COOH; and -(gGlu) n=1-4 -(AEEA) n=1,2 --(gDi) n=1-4 -C(O)(CH2) 16 COOH.

[0037] In one embodiment, the albumin-binding moiety is selected from the following groups: -{AEEA}2-gGlu-C(O)(CH2) 16 COOH; -{AEEA}2-gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 16 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 14 COOH; -C(O)(CH2) 16COOH; -C(O)(CH2) 14 COOH; -gGlu-C(O)(CH2) 16 COOH; and -gGlu-C(O)(CH2) 14 COOH.

[0038] In one embodiment, the albumin-binding moiety is selected from the following groups: -(gGlu) n=1-4 -C(O)(CH2) 14 COOH; -(AEEA) n=1,2 --(gDi) n=1-4 -C(O)(CH2) 14 COOH; and -(gGlu) n=1-4 -(AEEA) n=1,2 --(gDi) n=1-4 -C(O)(CH2) 14 COOH.

[0039] In one embodiment, the albumin-binding moiety is selected from the following groups: -{AEEA}2-gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 14 COOH; -C(O)(CH2) 14 COOH; and -gGlu-C(O)(CH2) 14 COOH.

[0040] In one embodiment, the albumin-binding portion is -{AEEA}2-gGlu-C(O)(CH2) 14 COOH; or -{AEEA}2-gGlu-C(O)(CH2) 16 It is COOH.

[0041] The chemical structures and IUPAC names of these groups are shown in Table 1 below, where R indicates the binding site of each group to the epsilon-amino group of the lysine residue in X12. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0042] In a particular embodiment, the albumin-binding portion is -{AEEA}2-gGlu-C(O)(CH2) 16 COOH;-{AEEA}2-gGlu-C(O)(CH2) 14 COOH; or -{AEEA}2-{gGlu}2-C(O)(CH2) 14 It is COOH. In a more specific embodiment, the albumin-binding moiety is -{AEEA}2-gGlu-C(O)(CH2) 16 It is COOH.

[0043] In one embodiment, the compound is a peptide containing any one amino acid sequence of SEQ ID NOs (also known as sequence number) 1 to 227, or a pharmaceutically acceptable salt thereof.

[0044] In one embodiment, the compound is a peptide containing any one of the amino acid sequences of SEQ ID NOs: 3, 7, 35, 83, 130, 135, 136, 137, 138, 139, 140, 141, 142, 147, 149, 151, 152, 171, 172, 173, and 174, or a pharmaceutically acceptable salt thereof. In another embodiment, the compound is a peptide comprising one of the amino acid sequences of SEQ ID NOs: 35, 130, 135, 137, 140, 142, 149, 151, and 152. In one embodiment, the compound is a peptide containing one of the amino acid sequences of SEQ ID NOs: 35, 130, 83, 135, 136, 137, 138, 139, 147, 151, and 174. In a further embodiment, the compound is a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 35, 130, 135, 137, 138, 139, 140, 141, 142, 149, 151, and 152, or a pharmaceutically acceptable salt thereof.

[0045] In one embodiment, the compound is a peptide having the amino acid sequence of formula (I), wherein the amino acid residue X1 is optionally acetylated, and the amino acid residue X38 is optionally amidated; or a pharmaceutically acceptable salt thereof.

[0046] In one embodiment, the compound is a peptide of the amino acid sequence of formula (I), or a pharmaceutically acceptable salt thereof; where: X1 is isoleucine(I); X2 is valine (V); X7 is valine(V) or D-valine(v); X9 is isoleucine(I) or threonine(T); in particular is isoleucine(I); X10 is either lysine (K) or glycine (G); X11 is leucine (L); X13 is either lysine (K) or glutamine (Q); X14 is lysine (K) or isoleucine (I); in particular isoleucine (I). X16 is leucine (L); X19 is either alanine (A) or glutamic acid (E); X21 is glutamine (Q); X22 is glutamic acid (E), arginine (R), or lysine (K); particularly arginine (R) or lysine (K); more preferably lysine (K); X23 is lysine (K) or 2-aminoisobutyric acid (Aib); in particular, lysine (K). X24 is glutamic acid (E), glutamine (Q), or 2-aminoisobutyric acid (Aib); in particular, glutamine (Q) or 2-aminoisobutyric acid (Aib); X25 is lysine (K) or arginine (R); in particular, arginine (R); X26 is alanine (A), glutamic acid (E), or glutamine (Q); in particular, glutamic acid (E) or glutamine (Q); X27 is lysine (K) or glutamine (Q); particularly lysine (K) or glutamine (Q); more preferably glutamine (Q); X29 is glutamic acid (E); X30 is lysine (K) or threonine (T); in particular, lysine (K); X31 is asparagine(N); X32 is lysine (K) or alanine (A); in particular, lysine (K). X33 is either arginine (R) or glutamine (Q). X34 is isoleucine(I); X35 is leucine (L); X36 is alanine (A) or glutamic acid (E); in particular, glutamic acid (E); X37 is arginine (R) or glutamine (Q); in particular glutamine (Q); and X38 is Valine (V); Furthermore, selectively, the amino acid residue of X1 may be acetylated, and selectively, the amino acid residue of X38 may be amidated.

[0047] In one embodiment, the compound is a peptide of the amino acid sequence of formula (I), or a pharmaceutically acceptable salt thereof; where X1 is isoleucine(I); X2 is valine (V); X7 is D-valine(v); X9 is isoleucine(I); X10 is either lysine (K) or glycine (G); X11 is leucine (L); X13 is glutamine (Q); X14 is isoleucine(I); X16 is leucine (L); X19 is either alanine (A) or glutamic acid (E); X21 is glutamine (Q); X22 is lysine (K); X23 is lysine (K) or 2-aminoisobutyric acid (Aib); X24 is glutamine (Q) or 2-aminoisobutyric acid (Aib); X25 is arginine(R); X26 is glutamic acid (E), glutamine (Q), or alanine (A); X27 is lysine (K) or glutamine (Q); in particular, glutamine (Q); X29 is glutamic acid (E); X30 is either lysine (K) or threonine (T); X31 is asparagine(N); X32 is lysine (K); X33 is either arginine (R) or glutamine (Q); X34 is isoleucine(I); X35 is leucine (L); X36 is glutamic acid (E) or alanine (A); X37 is glutamine (Q) or arginine (R); X38 is valine (V); and The amino acid residue of X1 may be selectively acetylated, and the amino acid residue of X38 may be selectively amidated.

[0048] In one embodiment, the compound is a peptide of the amino acid sequence of formula (I), or a pharmaceutically acceptable salt thereof; where X1 is isoleucine(I); X2 is valine (V); X9 is isoleucine(I); X10 is lysine (K); X11 is leucine (L); X14 is isoleucine(I); X16 is leucine (L); X19 is glutamic acid (E); X21 is glutamine (Q); X22 is lysine (K); X24 is glutamine (Q); X25 is arginine(R); X26 is glutamic acid (E); X27 is glutamine (Q); X29 is glutamic acid (E); X31 is asparagine(N); X32 is lysine (K); X33 is glutamine (Q); X34 is isoleucine(I); X35 is leucine (L); X37 is glutamine (Q); and X38 is valine (V); and Selectively, the amino acid residue of X1 may be acetylated, and selectively, the amino acid residue of X38 may be amidated.

[0049] In one embodiment, the compound is a peptide of the amino acid sequence of formula (I), or a pharmaceutically acceptable salt thereof; where X7 is D-valine (V); X10 is ricin; X19 is glutamic acid (E); X24 is glutamine (Q); X25 is arginine(R); X26 is glutamic acid (E); X27 is glutamine (Q). X30 is lysine (K); X33 is glutamine (Q); X36 is alanine (A); X37 is glutamine (Q); and Selectively, the amino acid residue of X1 may be acetylated, and selectively, the amino acid residue of X38 may be amidated.

[0050] In one embodiment, the compound is a peptide of the amino acid sequence of formula (I), or a pharmaceutically acceptable salt thereof; where the amino acid residue X38 is amidated as a primary amide.

[0051] In one embodiment, the compound is a peptide of the amino acid sequence of formula (I), or a pharmaceutically acceptable salt thereof; where X1 is isoleucine(I); X2 is valine (V); X9 is isoleucine(I); X10 is lysine (K) or glycine (G); X11 is leucine (L); X14 is isoleucine(I); X16 is leucine (L); X19 is either alanine (A) or glutamic acid (E); X21 is glutamine (Q); X22 is lysine (K); X24 is glutamine (Q) or 2-aminoisobutyric acid (Aib); X25 is arginine(R); X26 is alanine (A), glutamic acid (E), or glutamine (Q); X27 is glutamine (Q); X29 is glutamic acid (E); X31 is asparagine(N); X32 is lysine (K); X33 is either arginine (R) or glutamine (Q); X34 is isoleucine(I); X35 is leucine (L); X38 is valine (V); and Here, the amino acid residue of X38 is amidated as a primary amide.

[0052] In one embodiment, the compound is a peptide of the amino acid sequence of formula (I), or a pharmaceutically acceptable salt thereof; where X1 is isoleucine(I); X2 is valine (V); X9 is isoleucine(I); X10 is lysine (K), glutamic acid (E), or glycine (G); X11 is leucine (L); X14 is isoleucine(I); X15 is leucine (L). X16 is leucine (L); X19 is alanine (A) or glutamic acid (E); in particular, glutamic acid (E); X21 is glutamine (Q); X22 is either lysine (K) or glutamic acid (E); X23 is 2-aminoisobutyric acid (Aib); X24 is glutamine (Q); X25 is arginine(R); X26 is glutamic acid (E); X27 is glutamine (Q); X29 is glutamic acid (E); X31 is asparagine(N); X32 is lysine (K) or glutamic acid (E); in particular, glutamic acid (E); X33 is glutamine (Q); X34 is isoleucine(I); X35 is leucine (L); X38 is valine (V); and Here, the amino acid residue of X1 is acetylated, and the amino acid residue of X38 is amidated as a primary amide.

[0053] In one embodiment, the compound is a peptide of the amino acid sequence of formula (I), or a pharmaceutically acceptable salt thereof; where X1 is isoleucine(I); X2 is valine (V); X9 is isoleucine(I); X10 is lysine (K); X11 is leucine (L); X14 is isoleucine(I); X16 is leucine (L); X19 is glutamic acid (E); X21 is glutamine (Q); X22 is lysine (K); X24 is glutamine (Q); X25 is arginine(R); X26 is glutamic acid (E); X27 is glutamine (Q); X29 is glutamic acid (E); X31 is asparagine(N); X32 is lysine (K); X33 is glutamine (Q); X34 is isoleucine(I); X35 is leucine (L); X37 is glutamine (Q); X38 is valine (V); and Here, the amino acid residue of X38 is amidated as a primary amide.

[0054] In one embodiment, the compound is any one of the peptides of SEQ ID NOs: 1 to 227 or a pharmaceutically acceptable salt thereof. These peptides are listed in Table 2 below, where K * represents a modified lysine residue at X12, R a represents an albumin-binding moiety, Ac indicates that the N-terminus is acetylated, and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13]

[0055] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 7): IVLSLDvPTKLK * QKLLKQERQRKEREQAEKNARILARV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -gGlu-C(O)(CH2) 14 Chemically modified to form a covalent bond with the COOH group; and, -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; Alternatively, it is a pharmaceutically acceptable salt thereof.

[0056] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 32): IVLSLDvPTKLK * QKLLKQERQRKEREQAEKNVRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -gGlu-C(O) (CH2) 14 Chemically modified to form a covalent bond with the COOH group; and, -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; Alternatively, it is a pharmaceutically acceptable salt thereof.

[0057] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 35): IVLSLDVPIKLK * KILLEQEKQKKQREQAETNKQILAQV-NH2 During the ceremony The K residue at position 12 (K *(represented as) has an epsilon-amino group in its side chain, -{AEEA}2-gGlu-C(O)(CH2) 16 Chemically modified to covalently bond to COOH; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; Alternatively, it is a pharmaceutically acceptable salt thereof.

[0058] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 83): IVLSLDvPIGLK * QILLKQERQKKAibREQAETNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -gGlu-C(O)(CH2) 14 COOH; and gGlu-C(O)(CH2) 14 Chemically modified to covalently bond to an albumin-binding site selected from CH3; and, -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; Alternatively, it is a pharmaceutically acceptable salt thereof.

[0059] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 130): IVLSLDvPIKLK * QILLKQERQKAibQREQAEKNKQILAQV-NH2 During the ceremony The K residue at position 12 (K * (As indicated) The epsilon-amino group of its side chain is -{AEEA}2-gGlu-C(O)(CH2) 16 COOH; -gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 14 COOH; -gGlu-C(O)(CH2) 14 CH3; -{AEEA}2-gGlu-C(O)(CH2) 14 CH3; and -{AEEA}2-{gGlu}2-C(O)(CH2) 14 CH3; particularly -{AEEA}2-gGlu-C(O)(CH2) 16 COOH; is covalently bonded to an albumin-binding moiety selected from; and, -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0060] In one embodiment, the compound is a peptide having the following amino acid sequence (SEQ ID NO: 135): IVLSLDvPIKLK * QILLKQERQKAibQRQQAEKNKQILAQV-NH2 wherein the K residue at position 12 (K * shown as) has its epsilon-amino group of the side chain being -{AEEA}2-gGlu-C(O)(CH2) 16 COOH; and -gGlu-C(O)(CH2) 14 COOH; particularly -{AEEA}2-gGlu-C(O)(CH2) 16 COOH is covalently bonded to an albumin-binding moiety selected from; and, -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0061] In one embodiment, the compound is a peptide having the following amino acid sequence (SEQ ID NO: 136): IVLSLDvPIKLK * QILLKQERQKKAibRQKAEKNKQILAQV-NH2 wherein the K residue at position 12 (K * shown as) has its epsilon-amino group of the side chain being -gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-gGlu-C(O)(CH2)14 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 14 COOH; -gGlu-C(O)(CH2) 14 CH3; -{AEEA}2-gGlu-C(O)(CH2) 14 CH3; and -{AEEA}2-{gGlu}2-C(O)(CH2) 14 CH 3; Chemically modified to covalently bond to an albumin-binding site selected from; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0062] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 137): IVLSLDvPIKLK * QILLKQERQKKQREQAEKNKQILEQV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -gGlu-C(O)(CH2) 14 COOH; -gGlu-C(O)(CH2) 14 CH3; and -{AEEA}2-gGlu-C(O)(CH2) 16 COOH; especially -{AEEA}2-gGlu-C(O)(CH2) 16 Chemically modified to covalently bond to an albumin-binding site selected from COOH; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0063] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 138): IVLSLDvPIKLK * QILLKQERQKKQREQAEKNKQILEQV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -gGlu-C(O)(CH2) 14 COOH; and -gGlu-C(O)(CH2) 14 Chemically modified to covalently bond to an albumin-binding site selected from CH3; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0064] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 139): IVLSLDvPIKLK * QILLKQERQKKQRQQAEKNKQILAQV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -gGlu-C(O)(CH2) 14 COOH; and -gGlu-C(O)(CH2) 14 Chemically modified to covalently bond to an albumin-binding site selected from CH3; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0065] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 140): IVLSLDvPIKLK * QILLEQARQKAibQRAQAEKNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -{AEEA}2-gGlu-C(O)(CH2) 16 Chemically modified to covalently bond to COOH; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0066] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 141): IVLSLDvPIKLK * QILLKQARQKAibQRAQAEKNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -{AEEA}2-gGlu-C(O)(CH2) 16 Chemically modified to covalently bond to COOH; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0067] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 142): IVLSLDvPIKLK * QILLEQARQKAibQREQAEKNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -{AEEA}2-gGlu-C(O)(CH2) 16 Chemically modified to covalently bond to COOH; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0068] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 147): IVLSLDvPIGLK * QILLKQERQKKAibREQAETNKQILAQV-NH2 During the ceremony The K residue at position 12 (K *(represented as) has an epsilon-amino group in its side chain, -gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-gGlu-C(O)(CH2) 14 COOH; -{AEEA}2-{gGlu}2-C(O)(CH2) 14 COOH; -gGlu-C(O)(CH2) 14 CH3; -{AEEA}2-gGlu-C(O)(CH2) 14 CH3; and -{AEEA}2-{gGlu}2-C(O)(CH2) 14 Chemically modified to covalently bond to an albumin-binding portion selected from CH3; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0069] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 149): IVLSLDvPIKLK * QILLKQERQKKAibREQAETNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -{AEEA}2-gGlu-C(O)(CH2) 16 Chemically modified to covalently bond to COOH; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0070] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 150): IVLSLDvPIGLK * QILLKQERQKKAibRQQAETNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -gGlu-C(O)(CH2)14 COOH; and -gGlu-C(O)(CH2) 14 Chemically modified to covalently bond to an albumin-binding site selected from CH3; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0071] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 151): IVLSLDvPIGLK * QILLKQERQKKAibREQAEKNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, gGlu-C(O)(CH2) 14 COOH; -gGlu-C(O)(CH2) 14 CH3 and -{AEEA}2-gGlu-C(O)(CH2) 16 COOH; especially -{AEEA}2-gGlu-C(O)(CH2) 16 Chemically modified to covalently bond to an albumin-binding site selected from COOH; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0072] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 152): IVLSLDVPIKLK * QILLKQERQKKAibREQAETNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -{AEEA}2-gGlu-C(O)(CH2) 16 Chemically modified to covalently bond to COOH; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0073] In one embodiment, the compound is a peptide with the following amino acid sequence (SEQ ID NO: 174): IVLSLDvPIKLK * QILLKQARQKAibQRAQAEKNKRILERV-NH2 During the ceremony The K residue at position 12 (K * (represented as) has an epsilon-amino group in its side chain, -gGlu-C(O)(CH2) 14 COOH; and -gGlu-C(O)(CH2) 14 Chemically modified to covalently bond to an albumin-binding site selected from CH3; and -NH2 indicates that the C-terminal amino acid residue is amidated as a primary amide; or a pharmaceutically acceptable salt thereof.

[0074] The compounds disclosed in this invention can be prepared and used in the form of pharmaceutically acceptable salts. Pharmacopoeia for such preparation and methods are well known in the art (see, for example, Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use," 2nd edition, Wiley-VCH, 2011; and Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 1977, 66, 1). Examples of pharmaceutically acceptable salts include trifluoroacetate, acetate, and hydrochloride.

[0075] compound synthesis The compounds disclosed in this invention can be prepared using a variety of methods. These compounds can be prepared by synthesis in solution or on a solid support, followed by isolation and purification. Alternatively, the peptides can be prepared by gene expression in host cells into which the DNA sequence encoding the peptide has been introduced. Gene expression can also be achieved without the use of a cell system. Combinations of these methods may also be used.

[0076] In particular, the compound can be prepared by solid-phase synthesis on a suitable resin. Solid-phase synthesis is a well-established methodology (see, for example, Stewart and Young, "Solid-Phase Peptide Synthesis," Pierce Chemical Co., Ill, 1984, Rockford; and Atherton and Sheppard, "Solid-Phase Peptide Synthesis: A Practical Approach," Oxford-IRL Press, New York, 1989).

[0077] Compounds can be prepared using standard manual or automated solid-phase synthesis procedures. Automated peptide synthesizers are commercially available, for example, from Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid-phase synthesis are readily available from commercial sources. Solid-phase synthesizers can be used according to the manufacturer's instructions for coupling, deprotection, and capping of unreacted amino acids to block interfering groups and protect amino acids during the reaction.

[0078] Solid-phase synthesis can be initiated by bonding an N-terminally protected amino acid having a carboxyl terminus to an inert solid support having a cleavable linker. The solid support may be any polymer that allows the coupling of the first amino acid when the bonding of the carboxyl group (or carboxamide group for Rink resins) to the resin is acid-sensitive (when the Fmoc strategy is used), such as trityl resin, chlorotrityl resin, Wang resin, or Rink resin. The support must be stable under the conditions used to deprotect the α-amino group during peptide synthesis.

[0079] After the N-terminally protected first amino acid is coupled to a solid support, the α-amino protecting group of this amino acid is removed using a reagent such as trifluoroacetic acid (TFA) or piperidine. The remaining protected amino acids are then coupled sequentially in the order represented by the peptide sequence using a suitable amide coupling reagent, or added as pre-formed dipeptides, tripeptides, or tetrapeptides. Examples of coupling reagents include benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), hexafluorophosphate benzotriazole tetramethyluronium (HBTU), hexafluorophosphate azabenzotriazole tetramethyluronium (HATU), diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), and 1-hydroxy-7-azabenzotriazole, as well as combinations thereof. Typically, the coupling is carried out at room temperature in an inert solvent such as dimethylformamide (DMF), N-methylpyrrolidone (NMP), or dichloromethane (DCM).

[0080] Typically, the reactive side chain groups of amino acids are protected with appropriate blocking groups. These protecting groups are removed after the desired peptide has been assembled, and they can be removed simultaneously with the cleavage of the desired product from the resin under the same conditions. Protecting groups and procedures for their introduction are well known in the art (see, for example, Greene and Wuts, "Protective Groups in Organic Synthesis", 3rd ed., 1999, Wiley & Sons). Examples of protecting groups include tert-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc).

[0081] The albumin-binding moiety can be introduced by selectively functionalizing a lysine (K) residue at the position indicated by X12. Thus, the lysine residue may contain a side-chain protecting group that can be selectively removed, while other side-chain protecting groups remain intact, and the deprotected lysine residue can be selectively functionalized by the albumin-binding moiety. The binding of the albumin-binding moiety to the epsilon-amino group of the lysine side chain can be achieved via an acylation reaction or other suitable reactions known in the art.

[0082] For example, lysine residues can be protected with a 1-(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)-3-methylbutyl ("ivDde") protecting group, which is unstable to highly nucleophilic bases such as 4% hydrazine in DMF (see Chhabra et al., Tetrahedron Lett. 1998, 39, 1603). Therefore, if the N-terminal amino group and all side-chain functional groups are protected with acid-unstable protecting groups, the ivDde group can be selectively removed using a highly nucleophilic base. The resulting free amino group can then be attached to the albumin-binding site, for example, by acylation. Alternatively, the lysine residue may be protected with a (4-methoxyphenyl)diphenylmethyl ("Mmt") protecting group, which is unstable in very mild acids such as acetic acid and trifluoroethanol in dichloromethane (see Dubowchik et al., Tetrahedron Lett., 1997, 38(30), 5257). Therefore, if the N-terminal amino group and all side-chain functional groups are protected only with protecting groups unstable in strong acids, the Mmt group can be selectively removed, for example, using a mixture of acetic acid and trifluoroethanol in dichloromethane (e.g., in a ratio of 1:2:7). The resulting free amino group can then be attached to the albumin-binding moiety, for example, by acylation.

[0083] Alternatively, the albumin-binding moiety can be introduced together with lysine during peptide synthesis by using a pre-functionalized building block as a coupling partner. Examples of such pre-functionalized building blocks include Fmoc-L-Lys(Palm-L-Glu-OtBu)-OH and Fmoc-L-Lys[{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16 -C(O)OtBu]-OH is one example.

[0084] If necessary, the N-terminus of the peptide chain can be modified, for example, by acetylation. For the synthesis of C-terminal amide peptides, resins incorporating link amide 4-methylbenzhydrylamine (MBHA) or link amide AM linkers are typically used in conjunction with Fmoc synthesis, while MBHA resins are generally used in conjunction with tBoc synthesis.

[0085] After synthesis is complete, the peptide is cleaved from the solid support, and simultaneously, side chain deprotection is performed using a standard procedure. This can be achieved by using the King's cocktail (King et al., Int. J. Peptide Protein Res., 1990, 36, 255-266) or a similar cleavage cocktail known in the art.

[0086] The raw materials can be purified by chromatography (e.g., preparative RP-HPLC) as needed. Crude peptides are typically purified using RP-HPLC on a C8 or C18 column with a water-acetonitrile gradient in 0.05–0.1% trifluoroacetic acid (TFA). Peptide purity can be verified by analytical RP-HPLC. Peptide identity can be verified by mass spectrometry. Compounds can be isolated in solid form (e.g., as a dry powder) using techniques such as lyophilization.

[0087] The disclosure of the present invention also relates to intermediate compounds for use in the synthesis of the present compound. In particular, compounds or salts thereof are provided, which are peptides comprising the amino acid sequence of formula (I) disclosed herein, wherein residues X1-X11 and X13-X38 have the meanings enumerated in relation to formula (I), and X12 is lysine (K). The compound can be used as an intermediate in the preparation of the compounds disclosed herein, which can be obtained by conjugating an albumin-binding moiety to the epsilon-amino group of the lysine side chain at X12. The addition of the albumin-binding moiety can be carried out while the peptide is still bound to the solid phase. After the addition of the albumin-binding moiety, the peptide can be released from the resin and purified.

[0088] Specific methods for preparing the compounds disclosed in this invention are described in the following examples. The specific synthesis steps for each of the described pathways can be combined in different ways to prepare the compounds. Reagents and starting materials are readily available or can be prepared by methods known in the art.

[0089] Pharmaceutical composition The present invention also discloses pharmaceutical compositions comprising the compounds disclosed herein and pharmaceutically acceptable carriers or excipients.

[0090] A pharmaceutical composition may contain about 0.1% to about 99.9% by weight of the compounds disclosed in this invention, and about 99.9% to about 0.1% by weight of one or more pharmaceutically acceptable carriers, excipients, or diluents. For example, a pharmaceutical composition may consist of about 5% to about 75% by weight of the compounds disclosed in this invention, with the remainder being a suitable pharmaceutical carrier, diluent, or excipient. Methods for preparing pharmaceutical compositions are known or obvious to those skilled in the art from literature such as, for example, Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press.

[0091] In one embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0092] The pharmaceutical composition may be suitable for parenteral administration, such as oral, subcutaneous, intravenous, intraperitoneal, intramuscular, pulmonary, or transdermal administration. In particular, the pharmaceutical composition may be suitable for subcutaneous administration. In one embodiment, the pharmaceutical composition is a ready-to-use composition suitable for administration by pen or auto-injector device.

[0093] The compound may exhibit desirable solubility, chemical stability, and / or physical stability, particularly in solvents with physiological pH values ​​and solvents containing antimicrobial preservatives such as phenol or metacresol. As a result, the compound is particularly suitable for use in pharmaceutical compositions in solution form.

[0094] In certain embodiments, the pharmaceutical composition is a solution comprising a solvent, the compound disclosed herein dissolved therein, and an antimicrobial preservative selected from phenol and metacresol, wherein the compound disclosed herein is present in an amount of at least 1 mg / ml, at least 5 mg / ml, at least 10 mg / ml, or at least 20 mg / ml, and the solution has a pH of 6 to 8 (e.g., pH 7.0 or pH 7.4) as measured at 25°C.

[0095] Use in treatment The compounds disclosed in this invention are therapeutically useful and can be used to treat or prevent a variety of diseases. Accordingly, in other embodiments, the disclosure of this invention relates to the use of the compounds in therapy and to therapeutic methods in which an effective amount of the compounds disclosed in this invention is administered to a patient. The disclosure of this invention also relates to the use of the compounds for the manufacture of pharmaceuticals for therapeutic use. The compounds are particularly useful in treating diseases that can be treated or prevented by agonism of the CRF2 receptor.

[0096] As used herein, the term "therapy" refers to the treatment or prevention of a disease in a patient.

[0097] As used herein, the terms “treat” or “treating” include prohibiting, inhibiting, slowing, stopping, or reversing the progression or severity of an existing disease in a patient. Treatment may eliminate a disease; stop or delay a disease in a patient; inhibit or delay the onset of a new disease in a patient; reduce the frequency or severity of symptoms and / or relapses in a patient who currently has or has previously had a disease; and / or extend, i.e., increase, the patient’s lifespan. In particular, treatment of a disease may result in a cure of the disease or its symptoms, a reduction in duration, improvement, slowing, or inhibition of progression or worsening.

[0098] As used herein, the terms “prevent” or “preventing” refer to inhibiting or delaying a disease or the onset of a disease in a patient.

[0099] As used herein, the term “disease” refers to any condition or disorder that impairs or interferes with the normal function of a cell, tissue, or organ.

[0100] As used herein, the term “patient” refers to mammals such as humans, mice, guinea pigs, rats, dogs, or cats. In certain embodiments, the patient is a human patient.

[0101] The term "effective amount," as used herein, refers to the amount or dose of a compound disclosed herein that provides a desired effect in a patient in a single or multiple administration. The effective amount can be readily determined by the attending physician by the use of known techniques and by observing results obtained under similar circumstances. Many factors are considered by the attending physician when determining the effective amount for a patient, including, but are not limited to, the mammalian species; its size, age, and general health; the specific disease or disease involved; the extent or involvement or severity of the disease; the individual patient's response; the specific compound administered; the mode of administration; the bioavailability characteristics of the administered preparation; the dose regimen chosen; the use of concomitant medications; and other relevant circumstances.

[0102] The compounds disclosed in this invention may be effective over a wide dose range. For example, the daily dose may range from about 0.01 to about 50 mg / kg body weight.

[0103] The compounds disclosed in this invention have been observed to have excellent stability in different environments in which they have been tested. Accordingly, the compounds disclosed in this invention are particularly suitable for embodiments in which the compound is administered by once-daily, once-weekly, every other month, or once-monthly doses. This is especially true for the compounds disclosed in this invention that have lysine derivatized with D-valine at position 7 and a fatty acid as provided herein for half-life extension at position 12, and have excellent potency against CRF2 and a considerable selectivity profile over CRF1.

[0104] The compound may be administered in combination with one or more additional therapeutic agents. As used herein, the term "in combination with" means the administration of the compound disclosed herein simultaneously with, sequentially with, or in a single combination formulation of one or more additional therapeutic agents.

[0105] The compounds disclosed in this invention may be administered by parenteral routes, for example, by inhalation, subcutaneous, intravenous, intraperitoneal, intramuscular, pulmonary, or transdermal administration.

[0106] In certain embodiments, the compound is administered subcutaneously. The compound may be administered by a physician or self-administered using an injection device. It is understood that the gauge size and injection volume are determined by those skilled in the art. In one embodiment, the injection volume is 2 ml or less, for example, 1 ml or less. In another embodiment, needle gauges of 27 or more, for example, 29 or more, are used. Administration can be achieved using an autoinjector or a multi-dose delivery device.

[0107] The compounds disclosed in this invention may be useful in treating diseases that can be treated or prevented by agonism of the CRF2 receptor.

[0108] The compounds of the present invention are particularly useful for the treatment or prevention of sarcopenia (muscle loss), pulmonary hypertension, muscular dystrophy, renal disease, peripheral artery disease (PAD), and cardiovascular disease (particularly heart failure, obesity, and diabetes). Accordingly, embodiments disclosed herein relate to the use of the compounds in the treatment or prevention of sarcopenia, pulmonary hypertension, muscular dystrophy, renal disease, peripheral artery disease (PAD), and cardiovascular disease, particularly cardiovascular disease including heart failure, obesity, or diabetes, in patients. Furthermore, disclosures relating to methods for treating or preventing sarcopenia, pulmonary hypertension, muscular dystrophy, renal disease, peripheral artery disease (PAD), and cardiovascular disease (particularly heart failure, obesity, or diabetes) in patients, comprising administering an effective amount of the compounds disclosed herein to the patient. Moreover, disclosures relating to the use of the compounds in the manufacture of pharmaceuticals for the treatment or prevention of cardiovascular disease, including heart failure, obesity, or diabetes, in patients.

[0109] Examples of cardiovascular diseases that can be treated or prevented using the compounds of the present invention include heart failure, hypertension, dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, and stroke. The effects of the compounds in these conditions may be a result of, related to, or independent of, their effects on body weight. In certain embodiments, the compounds are used to treat or prevent heart failure. The compounds can be administered in combination with one or more additional therapeutic agents useful for treating heart failure, such as angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin II receptor blockers (ARBs), diuretics, sodium-glucose cotransporter inhibitors (SGLTi), beta-blockers, mineralocorticoid antagonists, or neprilysin inhibitors.

[0110] Furthermore, the compounds of the present invention may be useful in the treatment or prevention of obesity and other diseases caused or characterized by excessive weight, such as obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea. In adult human patients, obesity is defined as 30 kg / m². 2 The above can be defined as the Body Mass Index (BMI). BMI is a simple ratio of weight to height commonly used to classify overweight and obese adults. It is defined as a person's weight (kilograms) divided by the square of their height (meters), and therefore kg / m 2 It is expressed in units of . The compound can be administered in combination with one or more additional therapeutic agents useful for the treatment of obesity. Alternatively or additionally, the treatment can be combined with diet and exercise.

[0111] The compounds of the present invention can also be used for the treatment or prevention of diabetes, particularly type II diabetes. The compounds can be administered alone or in combination with one or more additional therapeutic agents useful for the treatment of diabetes, such as one or more agents selected from metformin, thiazolidinedione (TZD), sulfonylurea (SU), dipeptidyl peptidase-IV (DPP-IV) inhibitors, glucagon-like peptide-1 (GLP1) agonists, and sodium glucose cotransporters (SGLT). Alternatively or additionally, treatment can be combined with diet and exercise. The compounds can also be used to treat or prevent hyperglycemia, type I diabetes, and impaired glucose tolerance. Furthermore, the compounds of the present invention may be useful for the treatment or prevention of diabetes.

[0112] Furthermore, the compounds of the present invention may be useful in the treatment or prevention of other diseases such as sarcopenia, pulmonary hypertension, kidney disease, peripheral artery disease (PAD), metabolic syndrome, chronic kidney disease, degenerative diseases (e.g., neurodegenerative diseases), or diseases accompanied by nausea or vomiting.

[0113] The present invention is further illustrated by the following embodiments, which are provided for illustrative purposes only. The embodiments should not be construed as limiting the scope or content of the disclosures herein. [Examples]

[0114] Abbreviation Certain abbreviations are used in the examples and elsewhere in this specification: "AA" refers to amino acids; "AEEA" refers to [2-(2-aminoethoxy)ethoxy]acetyl; "Aib" refers to 2-aminoisobutyric acid; "AUC" refers to the area under the curve; "cAMP" refers to cyclic adenosine monophosphate; "Boc" refers to tert-butyloxycarbonyl; "BOP" refers to (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; "BSA" refers to bovine serum albumin; "tBu" refers to tertiary butyl; "DCM" refers to dichloromethane; "Dde" refers to 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl; "IvDde" refers to 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl; "DIC" refers to N,N'-diisopropylcarbodiimide; "DIPEA" refers to N,N-diisopropylethylamine; "DMEM" refers to Dulbecco's modified Eagle medium; "DMF" refers to dimethylformamide; "DMSO" refers to dimethyl sulfoxide; "EDT" refers to ethanedithiol; "FA" refers to formic acid; "FBS" refers to fetal bovine serum; "Fmoc" refers to fluorenylmethyloxycarbonyl; "gGlu" refers to gamma-glutamic acid (γE); "HATU" refers to O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; "HBSS" refers to Hanks equilibrium salt solution; "HBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; "HEPES" refers to 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; "HOAt" refers to 1-hydroxy-7-azabenzotriazole; "HOBt" refers to 1-hydroxybenzotriazole; "HOSu" refers to N-hydroxysuccinimide; "HPLC" refers to high-performance liquid chromatography. "hr" refers to time; "HTRF" refers to uniform time-resolved fluorescence; "IBMX" refers to 3-isobutyl-1-methylxanthine; "IV" refers to inside a vein; "kDa" refers to the kilodalton; "LC / MS" refers to liquid chromatography / mass spectrometry; "Mmt" refers to monomethoxytrityl; "MS" refers to mass spectrometry; "OtBu" refers to o-tert-butyl; "Palm" refers to palmitoyl; "Pbf" refers to 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; "PBS" refers to phosphate-buffered saline; "PK" refers to pharmacokinetics; "RP-HPLC" refers to reverse-phase high-performance liquid chromatography. "sc" refers to the subcutaneous tissue; "SEM" refers to the standard error of the mean; "Stea" refers to stearyl; "TIPS" refers to triisopropylsilane; "TFA" refers to trifluoroacetic acid; "Trt" refers to tritil; and "UV" refers to ultraviolet light.

[0115] Materials and methods The following starting materials and methods were used in the synthesis procedure described in the Examples.

[0116] Link amide resins (e.g., 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin, Merck Biosciences; 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetamidemethyl resin, Agilent Technologies) were used for the synthesis of peptide amides with loadings in the range of 0.2 to 0.7 mmol / g. Alternatively, pre-loaded Wang resins (e.g., ((S)-(9H-fluoren-9-yl)methyl(1-(tert-butoxy)-3-oxopropan-2-yl)carbamate resin, Fmoc-Ser(tBu)-Wang resin, Bachem) were used for the synthesis of peptide acids with loadings in the range of 0.2 to 0.7 mmol / g. Fmoc-protected natural amino acids were purchased from Protein Technologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, Iris Biotech, Bachem, Chem-Impex International, or MATRIX Innovation. The following standard amino acids were used in the synthesis: Fmoc-L-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH.Furthermore, the following amino acids were purchased from the same supplier as above: Fmoc-L-Lys(ivDde)-OH, Fmoc-L-Lys(Dde)-OH, Fmoc-L-Lys(Mmt)-OH, Fmoc-Aib-OH, Fmoc-D-Ser(tBu)-OH, Fmoc-D-Ala-OH, and Boc-L-Tyr(tBu)-OH.

[0117] The following side-chain structural units were obtained from commercial sources or synthesized by stepwise synthesis or solid-phase synthesis, for example, as described in CN104356224 (Liu; Hangzhou Adlai Nortye Pharmaceutical Technology Co. Ltd.): Fmoc-L-Lys(Palm-L-Glu-OtBu)-OH; Fmoc-L-Lys[{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16 C(O)OtBu]-OH; Fmoc-AEEA-OH; Fmoc-AEEA-AEEA-OH; Fmoc-L-Ile-Aib-OH; and Boc-L-Tyr-Aib-OH.

[0118] The following side-chain structural units were obtained from a commercial source (e.g., Chengdu Pukang) or synthesized by stepwise synthesis or solid-phase synthesis as described in WO2009 / 022006 (Madsen; Novo Nordisk A / S), WO2009 / 115469 (Madsen; Novo Nordisk A / S) or WO2015 / 028966 (Barlos; Chemical & Biopharmaceutical Laboratories of Patras SA): HO-{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16 C(O)OtBu; HO-{AEEA}2-gGlu(OtBu)-C(O)-(CH2) 18 C(O)OtBu; HO-{AEEA}2-{gGlu(OtBu)}2C(O)(CH2) 16 C(O)OtBu; HO-{AEEA}2-{gGlu(OtBu)}2-C(O)(CH2) 18-C(O)OtBu; HO-C(O)(CH2) 18 C(O)OtBu; HO-C(O)(CH2) 16 C(O)OtBu; and HO-gGlu(OtBu)-C(O)(CH2) 18 C(O)OtBu.

[0119] Crude peptides were purified using either an Akta purification system, a Jusco semiprep HPLC system, an Agilent 1100 HPLC system, or a similar HPLC system. Depending on the amount of crude peptide to be purified, preparative RP-C18-HPLC columns of different sizes and flow rates were used. Specifically, the following columns were used: Waters XSelect CSH C18 OBD Prep 5μm 30x250mm, Waters SunFire C18 OBD Prep 5μm 30x250mm, Waters SunFire C18 OBD Prep 5μm 50x150mm, and Phenomenex Luna Prep C18 5μm 21.2x250mm. Acetonitrile (B) and water + 0.1% TFA (A) or water + 0.1% FA (A) were used as eluents. The product-containing fractions were collected and lyophilized to obtain the purified product, typically as a TFA salt.

[0120] Alternatively, the compound was isolated as an acetate by the following procedure: The compound was dissolved in water, and the solution was adjusted to pH 7.05 with NaHCO3. The dissolved compound was then purified using an RP Kinetex 21.2x250mm column (column volume CV 88ml, 5μm, C18, 100A, Akta avant 25). The column was equilibrated with solvent A (3×CV), the compound was injected, and then washed at 3 CV with a mixture of solvent A (95%) and solvent B (5%). Next, a gradient of solvents A:B (95:5) to A:B (20:80) was run at 15 CV. The purified peptide was recovered and lyophilized. Column: Kinetex AXIA 5μm C18 21.2x250mm; Solvent: A (H2O + 0.5% acetic acid): B (ACN + H2O + 0.5% acetic acid) (flow rate 7ml / min); Gradient: 95:5 (0 min) ~ 95:5 (37 min) ~ 20:80 (180 min) ~ 0:100 (6 min)

[0121] Analytical HPLC / UHPLC was performed according to one of the following methods: Method A: Detection at 214nm Column: Waters ACQUITY UPLC 登録商標 CSH TM C18 1.7μm (150 x 2.1mm) 50℃ Solvent: H2O + 0.05% TFA : ACN + 0.045% TFA (flow rate 0.5 ml / min) Gradient: 80:20 (0 min) ~ 80:20 (3 min) ~ 25:75 (23 min) ~ 5:95 (23.5 min) ~ 5:95 (26.5 min) ~ 80:20 (27 min) ~ 80:20 (33 min) If necessary, use the following mass spectrometer: LCT Premier, electrospray positive ion mode.

[0122] Method B: Detection at 214nm Column: Water ACQUITY UPLC (登録商標) CSH (商標) C18 1.7μm(150×2.1mm), 50℃ Solvent: H2O + 0.05% TFA : ACN + 0.035% TFA (flow rate 0.5 ml / min) Gradient: 80:20 (0 min) ~ 80:20 (3 min) ~ 25:75 (23 min) ~ 2:98 (23.5 min) ~ 2:98 (30.5 min) ~ 80:20 (31 min) ~ 80:20 (37 min) Mass spectrometer: Agilent 6230 Accurate-Mass TOF or Agilent 6550 iFunnel Q-TOF; both feature a Dual Agilent Jet Stream ESI ion source.

[0123] Method C: Detection at 214nm Column: Waters ACQUITY UPLC 登録商標 CSH TM C18 1.7μm (150 x 2.1mm) 70℃ Solvent: H2O + 0.05% TFA : ACN + 0.035% TFA (flow rate 0.5 ml / min) Gradient: 63:37 (0 min) ~ 63:37 (3 min) ~ 45:55 (23 min) ~ 2:98 (23.5 min) ~ 2:98 (30.5 min) ~ 63:37 (31 min) ~ 63:37 (38 min) Mass spectrometer: Agilent 6230 Accurate-Mass TOF, Agilent Jet Stream ESI

[0124] Example 1: Synthesis of compounds by automated solid-phase peptide procedure Compounds were prepared using solid-phase synthesis with a Prelude Peptide Synthesizer (Mesa Laboratories / Gyros Protein Technologies) or a CS Bio automated synthesizer, employing standard Fmoc chemistry and HBTU / DIPEA or HATU / DIPEA activation. DMF was used as the solvent.

[0125] The following conditions were adopted: Deprotection: 20% piperidine / DMF for 2 x 2.5 minutes. Washing: 7 x DMF. 2:5:10 200mM AA / 500mM HBTU / 2M DIPEA coupled for 20 minutes in DMF 2x. Washing: 5 x DMF.

[0126] HBTU / DIPEA activation was used in all standard couplings. HBTU / DIPEA activation was used in the following couplings: Ile-Aib, Aib-Lys[{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16 C(O)OtBu], Lys[{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16 C(O)OtBu]-Asp, Gln-Aib, and Leu-Leu. HATU couplings were generally allowed to react for 2 × 40 minutes, sometimes 2 × 1 hour, and even up to 12 hours.

[0127] For the modified lysine side chain, Fmoc-L-Lys(Mmt)-OH was used at the position indicated as X12 in formula (I). After the synthesis was complete, the Mmt group was removed by repeatedly treating it with AcOH / TFE / DCM(1 / 2 / 7) at room temperature for 15 minutes, and then the resin was repeatedly washed with DCM, 5% DIPEA in DCM, and 5% DIPEA in DCM / DMF.

[0128] After removal of the Mmt group, the resin was treated with a solution of the protected form of albumin-binding moiety. For example, -{AEEA}2-gGlu-C(O)(CH2) 16 Peptides containing the COOH moiety are HO-{AEEA}2-gGlu(OtBu)-C(O)(CH2) in DMF pre-activated with HATU (3 equivalents), HOAt (3 equivalents), and DIPEA (4 equivalents). 16 The resin was prepared by treating it with a solution of C(O)OtBu (1 equivalent). The resin was then washed as described above. The OtBu protecting group was cleaved during the final peptide cleavage from the resin.

[0129] The peptide was cleaved from the resin using a King cleavage cocktail consisting of 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, and 2.5% EDT, or a modified cleavage cocktail consisting of 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, and 2.5% DODT. The resin used in the synthesis was such that the C-terminus was cleaved from the resin as a primary amide.

[0130] Next, the crude peptide was precipitated in diethyl or diisopropyl ether, centrifuged, and freeze-dried. The peptide was analyzed by analytical HPLC and verified by ESI mass spectrometry. The crude peptide was purified using a conventional preparative RP-HPLC purification procedure.

[0131] Example 2: Synthesis of compounds by manual solid-phase synthesis procedure The compounds were also prepared by manual synthesis procedures. An exemplary procedure is described below.

[0132] Dried Rink amide MBHA resin (0.3 g; 0.5-0.8 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter. The resin was swollen in DCM (15 ml) for 1 hour and in DMF (15 ml) for 1 hour. The Fmoc groups on the resin were deprotected by treating them twice with a 20% (v / v) piperidine / DMF solution for 5 minutes and 15 minutes. The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test (quantitative method; see Kaiser et al., Anal. Biochem., 1970, 34, 595-598) was used to confirm the removal of Fmoc from the solid support. The C-terminal Fmoc amino acid (5 equivalents excess, corresponding to resin loading) in dried DMF was added to the deprotected resin, and the coupling of the next Fmoc amino acid was initiated using 5 equivalents excess DIC and HOBT in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquots at the completion of coupling was negative (i.e., there was no color on the resin). After the first amino acid bond, if there were any unreacted amino groups in the resin, it was capped with acetic anhydride / pyridine / DCM (1 / 8 / 8) for 20 minutes to avoid sequence deletion. After capping, the resin was washed with DCM / DMF / DCM / DMF (6 / 6 / 6 / 6 times each). The Fmoc group on the C-terminal amino acid-bonded peptidyl resin was deprotected by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes and 15 minutes. The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on peptide resin aliquots was positive upon completion of Fmoc deprotection.

[0133] The remaining amino acids in the target sequence on the Rink amide MBHA resin were sequentially coupled using the Fmoc AA / DIC / HOBt method, with a 5-equivalent excess in DMF corresponding to the resin loading. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). After each coupling step and Fmoc deprotection step, a Kaiser test was performed to confirm the integrity of the reaction.

[0134] After linear arrangement was complete, the ε-amino group of lysine (protected with Dde) was deprotected with 2.5% hydrazine hydrate in DMF for 15 minutes x 2, and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The γ-carboxyl terminus of glutamic acid was bonded to the ε-amino group of Lysine using Fmoc-Glu(OH)-OtBu in DMF using the DIC / HOBt method (5 equivalents excess relative to the resin loading). The mixture was rotated on a rotor at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each, 30 ml each). The Fmoc group on glutamic acid was deprotected by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes and 15 minutes (25 ml each). The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquots after completion of Fmoc deprotection was positive.

[0135] For peptides containing additional γ-glutamic acid due to side chain branching, a second Fmoc-Glu(OH)-OtBu was used to bind to the free amino group of γ-glutamic acid using the DIC / HOBt method in DMF (5 equivalents in excess of resin loading). The mixture was rotated on a rotor at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each, 30 ml each). The Fmoc group on γ-glutamic acid was deprotected by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes and 15 minutes (25 ml each). The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquots at the completion of Fmoc deprotection was positive.

[0136] The albumin-binding moiety was formed as described in Example 1. The t-butyl ester protecting group was cleaved during the final peptide cleavage from the resin.

[0137] Alternatively, the albumin-binding moiety was introduced using a pre-functioned building block (constituent unit) in which the moiety is already bound to lysine as a coupling partner in peptide synthesis. This procedure avoids the need for selective deprotection steps and selective binding of side-chain constituent units on highly advanced synthetic intermediates. As an example, the following procedure was used to introduce Fmoc-L-Lys[{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16 The C(O)OtBu]-OH group was incorporated into the peptide. A 0.67 mmol peptide resin containing an amino group was washed with 20 ml of dimethylformamide. 2.93 g of Fmoc-L-Lys[{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16-C(O)OtBu]-OH is dissolved in 20 ml of dimethylformamide together with 310 mg of hydroxybenzotriazole hydrate and 0.32 ml of diisopropylcarbodiimide. After stirring for 5 minutes, the solution is added to the resin. The resin is stirred for 20 hours, and then washed three times with 20 ml of dimethylformamide each time. Small resin samples are taken and subjected to the Kaiser test and chloranil test (see Vojkovsky, Peptide Research 1995, 8, 236-237).

[0138] After coupling of the albumin-binding site, the peptidyl resin was washed with DCM (6 × 10 ml), MeOH (6 × 10 ml), and ether (6 × 10 ml) and dried overnight in a vacuum desiccator. Cleavage of the peptide from the solid support was achieved by treating the peptide resin with a reagent cocktail (92% TFA, 2% thioanisole, 2% phenol, 2% water, and 2% TIPS) at room temperature for 3–4 hours. The cleavage mixture was recovered by filtration, and the resin was washed with TFA (2 ml) and DCM (2 × 5 ml). Excess TFA and DCM were concentrated to a small amount under nitrogen, a small amount of DCM (5–10 ml) was added to the residue, and evaporated under nitrogen. This process was repeated 3–4 times to remove most of the volatile impurities. The residue was cooled to 0°C, and anhydrous ether was added to precipitate the peptide. The precipitated peptide was centrifuged, the supernatant ether was removed, fresh ether was added to the peptide, and recentrifugation was performed. The crude sample was purified by preparative HPLC and lyophilized. The identity of the peptide was confirmed by LC-MS.

[0139] Example 3: Synthesis of the peptide of SEQ ID NO: 35 The compound of SEQ ID NO: 35 was prepared according to the procedure described in Example 1. Novabiochem Rink-Amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh, 0.36 mmol / g loading was used. An automated Fmoc synthesis method was applied using either HBTU / DIPEA activation or HATU / DIPEA activation, depending on the amino acid sequence. At position 12, Fmoc-Lys(Mmt)-OH was used in a solid-phase synthesis protocol. The Mmt group was cleaved from the peptide as described in Example 1. Subsequently, HO-{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16 C(O)OtBu was coupled to the free amino group using DIPEA as the base and HATU / HOAt as the coupling reagent. The peptide was cleaved from the resin using a Kings cocktail. The crude product was purified by preparative HPLC using an acetonitrile / water gradient (water containing 0.1% TFA) on a Waters column (Waters SunFire C18 OBD Prep 5μm 50x150mm). Analysis of the purified peptide by LC-MS (Method B) revealed that the peptide mass of 5154.09 was consistent with the expected value of 5154.06, based on deconvolution of the mass signal detected under a peak with a retention time of 11.19 minutes.

[0140] Example 4: Synthesis of the peptide of SEQ ID NO: 141 The compound of Sequence ID No. 141 was prepared according to the procedure described in Example 1. Novabiochem Rink-Amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh, 0.34 mmol / g loading was used. Automated Fmoc synthesis was applied using HBTU / DIPEA activation or HATU / DIPEA activation depending on the amino acid sequence. At position 12, Fmoc-L-Lys[{AEEA}2-gGlu(OtBu)-C(O)(CH2) 16-C(O)OtBu] was used in the solid-phase synthesis protocol. The peptide was cleaved from the resin using the Kings cocktail. The crude product was first purified by preparative HPLC using an acetonitrile / water gradient (water containing 0.1% TFA) on a Waters column (Waters SunFire C18 OBD Prep 5μm 50x150mm), and then purified again by preparative HPLC using an acetonitrile / water gradient (water containing 0.1% formic acid) on a Waters column (Waters Xselect CSH Prep C18 5μm 30x250mm). The purified peptide was recovered and lyophilized. Analysis of the purified peptide by LC-MS (Method B) revealed that the peptide mass of 5163.18 was consistent with the expected value of 5163.17, based on deconvolution of the mass signal detected under the peak at a retention time of 9.97 minutes.

[0141] Example 5: Synthesis of the peptide of SEQ ID NO: 171 The compound of Sequence ID No. 171 was prepared according to the procedure described in Example 1. Novabiochem Rink-Amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh, 0.35 mmol / g loading was used. Automated Fmoc synthesis was applied using HBTU / DIPEA activation or HATU / DIPEA activation depending on the amino acid sequence. At position 12, Fmoc-Lys(Mmt)-OH was used in a solid-phase synthesis protocol. The Mmt group was cleaved from the peptide as described in Example 1. Subsequently, HO-{AEEA}2-{gGlu(OtBu)}2-C(O)(CH2) 16C(O)OtBu was coupled to the free amino group using DIPEA as the base and HATU / HOAt as the coupling reagent. The peptide was cleaved from the resin using a Kings cocktail. The crude product was purified by preparative HPLC on a Waters column (Waters SunFire C18 OBD Prep 5μm 50x150mm) using an acetonitrile / water gradient (water containing 0.1% TFA). Analysis of the purified peptide by LC-MS (Method B) revealed that the peptide mass of 5294.21 was consistent with the expected value of 5294.13, based on deconvolution of the mass signal detected under the peak at a retention time of 9.94 minutes.

[0142] Example 6: Further peptide synthesis The following peptides were synthesized according to the procedures described in Examples 1-5. Calculations were performed for these peptides, and the resulting masses and retention times, along with those of the compounds in Examples 3-5, are shown in Table 3 below: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0143] Example 7: Evaluation of activity in the human CRF2α receptor The agonism of compounds against human corticotropin-releasing factor 2α (CRF2α receptor) was determined by a functional assay measuring the cAMP response in TeloHEAC cell lines that stably express the human CRF2α receptor.

[0144] Cells were grown in T175 culture flasks at 37°C in medium (DMEM / 10% FBS) until near confluence, and then harvested into 2 ml vials in cell culture medium containing 10% DMSO at a concentration of 10 to 50 million cells / ml. Each vial contained 1.8 ml of cells. The vials were slowly frozen to -80°C in isopropanol and then transferred to liquid nitrogen for storage. Before use, the frozen cells were rapidly thawed at 37°C and washed with 20 ml of cell buffer (1x HBSS; 20 mM HEPES; 0% or 0.1% HSA) for 5 minutes at 900 rpm. The cells were resuspended in assay buffer (cell buffer + 2 mM IBMX) and adjusted to a cell density of 1 million cells / ml. For measurement, 5 μl of cells (final 2000 cells / well) and 5 μl of the test compound were added to a 384-well plate and then incubated at room temperature for 30 minutes.

[0145] Cellular cAMP content was determined based on HTRF (Hypothetical Time-Resolved Fluorescence) using a kit from Cisbio Corp (catalog number 62AM4PEC). After adding the HTRF reagent diluted with lysis buffer (kit component), the plate was incubated for 1 hour, followed by measurement of the fluorescence ratio at 665 / 620 nm. Percent activity (E%) was calculated by setting 1000 nM urocortin 2 (UCN2) as 100%. In vitro potency of the compound was quantified by determining the concentration that elicited 50% activation of the maximum response (EC50). Representative EC50 values ​​are provided in Table 4 below: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0146] Example 8: Evaluation of activity in rat CRF2α receptor The activity of the compound in the rat CRF2Rα receptor was evaluated using the procedure described in Example 7. The A7R5 rat aortic smooth muscle cell line was used in this study. Typical EC50 values ​​are provided in Table 5 below: [Table 5-1] [Table 5-2] [Table 5-3]

[0147] Example 9: Evaluation of activity at anti-target CFR1R receptors The activity of the compound at the anti-target CRF1R receptor was evaluated using the procedure described in Example 7. A CRF1R overexpressing CHO cell line was used in this study. Representative EC50 and Emax values ​​are provided in Table 6 below: [Table 6-1] [Table 6-2] [Table 6-3]

[0148] Example 10: Evaluation of activity in cells expressing endogenous human CRF2 The agonist effect of the compound was evaluated in NCI-H82 cells expressing endogenous human CRF2. NCI-H82 cells in suspension were seeded at a density of 30,000 cells / well in 5 μl of test medium (RPMI + 1 mM IBMX) into a low-volume 384-well plate. The plate was briefly centrifuged at 800 rpm to move the cell suspension to the bottom of the wells, and then incubated at 37°C in 5% CO2 for 2 hours. Subsequently, the cells were diluted to an appropriate concentration (10) in the test medium. -11 ~3x10 -7 Five μl of the test compound was transferred to a 384-well plate. After 5 minutes of activation, the reaction was stopped by adding HTRF expression reagents using a multi-drop dispenser. The two reagents were 5 μl / well of anti-cAMP-cryptate diluted 20-fold with conjugate and lysis buffer, and 25 μl / well of cAMP-D diluted 20-fold with conjugate and lysis buffer. After incubation at room temperature in the dark for 1 hour, the plate was read using a Clariostar instrument. The HTRF signals were read at 620 and 665 nm and calculated as follows: Ratio HTRF = [(signal at 665 nm) / (signal at 620 nm)] x 10 4 cAMP measurements were calculated using standard cAMP curves (0.17 nM to 712 nM) drawn on each plate, and the EC50 value was determined from these curves. Representative EC50 values ​​are provided in Table 7 below: [Table 7]

[0149] Example 11: Binding test in HEK cells expressing recombinant CRF2 The receptor binding of the compound was evaluated using the CRF2α(h) (agonist radioligand) assay. HEK cells expressing recombinant human CRF2 were used as a receptor source for membrane preparation. [125I]sauvazine was used as a competing ligand for the CRF2 receptor. Test compounds were incubated at several concentrations for 1 hour competing with [125I]sauvazine (0.1 nM) to determine residual binding. Compound binding was calculated as the percentage inhibition of binding to [125I]sauvazine, which is specific to CRF2. The IC50 value (the concentration that causes semi-maximal inhibition of control-specific binding) and the Hill coefficient (nH) were determined by nonlinear regression analysis of competitive curves generated using mean replicates with Hill equation curve fitting. The inhibition constant (Ki) was calculated using the Cheng-Prusoff equation [Ki = IC50 / (1+L / KD), where L is the concentration of the radioactive ligand in the assay and KD is the affinity of the radioactive ligand to the receptor]. KD was determined using a scatchard plot. Representative Ki and nH values ​​are provided in Table 8 below: [Table 8]

[0150] Example 12: Evaluation of chemical stability The chemical stability of the compounds was evaluated by storing them under various conditions and then determining the loss of purity by ULC-UV. Before measuring the chemical stability of batches of the test compound, the purity of the compound was measured by UPLC / MS. For stability testing, the target concentration was 300 μM of pure compound. Therefore, solutions from the solid sample were prepared at a concentration of 300 μM of the compound, based on the previously determined % purity, in a pH 4.5, 20 mM acetate buffer system: A solution of the test compound was filtered through a filter (0.22 μM pore size) and packed into aliquots under sterile conditions. At the start, UPLC-UV was performed by injecting 2 μl of undiluted sample. The aliquots were then stored at temperatures of 5 and 40 °C for 28 days. After this time, the samples were centrifuged at 2500 RCF for 15 minutes. Then, 2 μl of the undiluted supernatant was analyzed by UPLC-UV. Chemical stability was calculated using the formula [(purity after 28 days at 5 °C) - (purity after 28 days at 40 °C) / (purity after 28 days at 5 °C)] × 100%. Purity was calculated as [(peak area of ​​compound) / (total peak area)] × 100%. Representative stability data is provided in Table 9 below: [Table 9]

[0151] Example 13: Evaluation of solubility The solubility of the compound was evaluated using the following buffer systems: 100 mM acetate buffer, pH 4.5 (Buffer A); 100 mM acetate buffer, pH 4.5, 2.7 mg / ml m-cresol (Buffer B); and 100 mM phosphate buffer, pH 7.4 (Buffer C).

[0152] Before performing the solubility measurement, the purity of the compound was measured by UPLC / MS. For the solubility test, the target concentration was set to 10 mg of pure compound / ml. Therefore, a solution from the solid sample was prepared in a buffer system with a compound concentration of 10 mg / mL based on the previously determined % purity.

[0153] UPLC-UV was performed after 1 hour of gentle stirring of the supernatant obtained after 15 minutes of centrifugation (relative centrifugal acceleration) at 2500 RCF. Solubility was determined by comparing the UV peak area of ​​a 2 μL injection of a 1:10 diluted buffered sample with the standard curve of a reference compound with a known concentration. Different UV extinction coefficients for the sample and reference compound were calculated based on different amino acid sequences and taken into consideration in concentration calculations. Representative solubility data is provided in Table 10 below: [Table 10-1] [Table 10-2]

[0154] Example 14: Evaluation of physical stability using thioflavin T assay The physical stability of the compound was evaluated using the thioflavin T (ThT) assay. The low physical stability of peptide solutions can lead to amyloid fibril formation, which is observed in the sample as well-ordered, thread-like polymeric structures and can ultimately result in gel formation. ThT is widely used to visualize and quantify the presence of misfolded protein aggregates (see Biancalana et al., Biochim. Biophys. Acta, 2010, 1804(7), 1405). When it binds to fibrils, such as those in amyloid aggregates, the dye exhibits a distinct fluorescent signature (see Naiki et al., Anal. Biochem., 1989, 177, 244; and LeVine et al., Methods. Enzymol., 1999, 309, 274). The time course of fibril formation often follows a characteristic shape of a sigmoid curve, which can be divided into three regions: the lag phase, the rapid growth phase, and the plateau phase. A typical fibril formation process begins with the lag phase, where a portion of the partially folded peptide is converted into fibrils, but this is not yet detectable. The lag time corresponds to the time it takes for the nuclear critical mass to be constructed. This is followed by a dramatic elongation phase, during which the fibril concentration increases rapidly. Therefore, by measuring the increase in fluorescence intensity due to ThT and the delay time of this increase, the fibrilization tendency of the peptide can be determined, thereby providing a measure of the peptide's physical stability.

[0155] In this study, the test compound was diluted in buffer to a final concentration of 3 mg / ml. Subsequently, 20 μL of 10.1 mM ThT aqueous solution was added to 2 mL of peptide solution to obtain a final concentration of 100 μM ThT. The experiment was performed using two buffers: 100 mM acetate buffer, pH 4.5 (Buffer A); and 100 mM acetate buffer, pH 4.5 and 2.7 mg / mL m-cresol (Buffer B).

[0156] The fibrillation tendency of peptides was determined under stress using a Fluoroskan Ascent FL or Fluoroskan Ascent fluorometer. 200 μL of sample was placed in a 96-well microtiter plate, PS, flat-bottomed, Greiner Fluotrac No. 655076. The plate was sealed with Scotch tape (Quiagen). The sample was stressed by a continuous cycle of shaking at 960 rpm for 10 seconds, followed by a rest at 37°C for 50 seconds. Fibril development was monitored by measuring fluorescence intensity every 20 minutes. Eight replicates were tested for each sample. Representative stability data obtained with buffer A are shown in Table 11 below, where "FI" refers to fluorescence intensity: [Table 11] Representative stability data obtained with buffer B is provided in Table 12 below: [Table 12]

[0157] Example 15: Evaluation of physical stability using a dynamic light scattering assay The physical stability of the compounds was also evaluated using a dynamic light scattering assay. Dynamic light scattering (DLS) measures light scattered from particles undergoing Brownian motion (1 nm ≤ radius ≤ 1 μm). This motion is induced by collisions between the particles and solvent molecules, which themselves move due to their thermal energy. The diffusive motion of the particles results in temporal variations in the scattered light (Pecora, "Dynamic Light Scattering: Applications of Photon Correlation Spectroscopy", Plenum Press, 1985). The variations in scattered light intensity were recorded and converted into an autocorrelation function. By fitting the autocorrelation curve to an exponential function, the diffusion coefficient D of the particles in solution can be derived. Next, the diffusion coefficient is used to calculate the hydrodynamic radius Rh (or apparent Stokes radius) through the Stokes-Einstein equation assuming spherical particles. This calculation is defined in the international standard ISO 13321, Methods for Measuring Particle Size Distribution, Part 8: Photon Correlation Spectroscopy, International Organization for Standardization (ISO) 1996; and the international standard ISO 22412, Dynamic Light Scattering for Particle Size Analysis, International Organization for Standardization (ISO) 2008.

[0158] DLS interaction parameter (k D k is a measure for describing interparticle interactions, where particles are folded proteins or peptides (Yadav et al., J. Pharm. Sc. 2010, 99(3), 1152; and Connolly et al., Biophys. J. 2012, 103, 69). High values ​​indicate strong net repulsive interactions, while low values ​​indicate net attractive interactions. Therefore, k D It can be used for the purpose of relative, qualitative comparison.

[0159] In this study, the physical stability of the test compound was evaluated by its apparent hydrodynamic radius (R) after synthesis (0 weeks) and after storage at 40°C for 4 weeks. h The scattering intensity (I) and mass contribution (M) were determined and evaluated.

[0160] The stability of the compound was evaluated in five different buffer systems: 100 mM acetate buffer, pH 4.5 (Buffer A); 100 mM acetate buffer, pH 4.5, 2.7 mg / mL m-cresol (Buffer B); 20 mM phosphate buffer, pH 6.2 (Buffer C); 20 mM phosphate buffer, pH 7.4 (Buffer D); and 20 mM acetate buffer, pH 4.5 (Buffer E).

[0161] For each solution of the test compound, the hydrodynamic radius Rh and diffusion constant D (related via the Stokes-Einstein equations) were determined as the average over three measurements. Both parameters were determined for different compound concentrations in the same buffer system (e.g., Rh). h1 And D1: 1 mg / ml and R h5 And D5: 5 mg / ml, R h10 and D 10 The concentration was determined at 10 mg / ml. The difference between these parameters between low and high peptide concentrations is the DLS interaction parameter k D It is a substitute for the increase in the value of D or R with increasing peptide concentration. h The decrease in the value of k D This corresponds to >0, and therefore to repulsive inter-particle interactions that result in improved physical (or colloidal) stability. In addition, the hydrodynamic radius R h The corresponding scattering intensity (%) was determined as the average for overlap. The target concentration was 300 μM. Therefore, a solution from the solid sample was prepared in a buffer system with a compound concentration of 300 μM based on the previously determined % purity.

[0162] DLS measurements were performed using a DynaPro Plate Reader I (Wyatt Technology, Santa Barbara, CA, US) with an 837nm laser source at a scattering angle of 150°. Data was collected and processed using Dynamics V 7.8.1.3 or 7.8.2.18 software provided by Wyatt Technology.

[0163] The hydrodynamic radius was determined using the non-negatively constrained least squares (NNLS) method with the DYNALS algorithm with regularization fitting. For comparison, the refractive index of water, n=1.330 and η=0.89 cP, were used for all samples. Samples were tempered at +25°C for 1 hour and visually inspected before analysis.

[0164] The samples were mixed using a pipette tip. Measurements were performed in five replicates. Therefore, five 15 μL aliquots were pipetteed onto a polystyrene 384 assay plate with a clear bottom (Greiner Bio-One, Germany) and sealed. The plate was centrifuged at 600 rcf for 2 minutes. After removing the seal, the samples were measured at +25°C. Representative stability data is provided in Table 13 below: [Table 13]

[0165] Example 16: Evaluation of pharmacokinetic properties The pharmacokinetic properties of exemplary peptides, SEQ ID NO: 35 and SEQ ID NO: 24, were evaluated in mice and rats. The test compound was administered at concentrations of 0.05, 0.1, 0.5, or 1 mg / ml, depending on the dose, species, and administration volume, in an appropriate buffer system (PBS buffer or DPBS solution at pH 7.4). Female C57Bl / 6 mice were administered 0.1 mg / kg or 0.3 mg / kg intravenously or subcutaneously. The animals were sacrificed, and blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 32, and 48 hours after intravenous administration, and at 0.25, 0.5, 1, 2, 4, 8, 24, 32, and 48 hours after subcutaneous administration, respectively. Plasma samples were analyzed after protein precipitation via liquid chromatography-mass spectrometry (LC / MS). Pharmacokinetic parameters and half-lives were calculated using Phoenix-WinNonlin 8.1 with a non-compartmental model and linear trapezoidal interpolation. The results of this test for these peptides are shown in Table 14 below: [Table 14]

[0166] Example 17: Evaluation of the effect on blood pressure The effect of the compound on blood pressure was determined in telemetry studies using a dose of 0.1 mg / kg SC in Sprague Dawley rats. Rats were pre-implanted with telemetry devices (DSI, Saint-Paul, USA, HD-S10, HD-S11, or HD-S21) and allowed to recover for at least two weeks before being treated with a vehicle or different peptides. Blood pressure (BP) was recorded via a catheter inserted into the abdominal aorta. The device itself was placed in the abdomen. At the end of the procedure, the animals were placed in individual cages until the end of the study.

[0167] Pressure signals were recorded one hour before treatment (basal period). Subcutaneous administration of the test compound or its vehicle was then performed under continuous signal recording for 48 hours. Data acquisition was performed using Hem 4.3 acquisition software (Notocord) connected to a telemetry device. (登録商標) The procedure was performed using Le Pecq (France). Hemodynamic parameters were recorded during the 2-hour period before treatment and every 4 hours after subcutaneous administration over a 48-hour period.

[0168] The parameters examined (calculated from the BP signal) were heart rate (HR) / minute (bpm), diastolic and systolic blood pressure (DBP) (mmHg), and mean arterial pressure (MBP) (mmHg).

[0169] Data from Hem is in Microsoft Excel (登録商標)The parameters were manipulated using the following method. For each parameter, the baseline value was determined as the average calculated over a 1-hour period between pretreatment procedures, and the posttreatment value was calculated over 4-hour periods ([0-4], [4-8], [8-12], [12-16], [16-20], [20-24], [24-28], [32-36], [36-40], [40-44], and [44-48] hours) over a 48-hour period after administration.

[0170] Representative data is shown in Table 15 below, where "n" represents the number of animals for which individual values ​​were obtained, and "Duration" indicates the last point in time when a significant effect on BP was still observed: Table 15 [Table 15]

[0171] Example 18: Evaluation of the effect on body weight and body fat content The peptide of Sequence ID No. 35 was evaluated for its ability to reduce body weight and body fat content in mice. Its potency was compared to that of a reference compound, namely the compound of Example 4 of WO2018 / 013803 (Alsina-Fernandez; Eli Lilly and Company) (hereinafter referred to as "Compound A").

[0172] Female C57BL / 6N Crl mice were housed in a vivarium with a 12-hour light / dark cycle and room temperature of 23±1°C. All animals were given free access to water and food (Ssniff-modified high-fat diet: TD.97366, Soest, Germany) for 18 weeks prior to pharmacological intervention (medication phase). After the pre-feeding period, mice were housed individually and randomized according to body weight into n=8 treatment groups, ensuring that each group had a similar mean body weight. At the start of the study, the mice were 25-26 weeks old and weighed 42-50 g. Mice were treated every two days in the late afternoon before lights out with subcutaneous injection of 8 nmol / kg of the test compound or their vehicle (phosphate-buffered saline: PBS). Body weight and food intake were measured daily throughout the administration phase, while body fat content was measured on day 0 and day 15 before the start of treatment. The study was completed on day 15.

[0173] For statistical analysis, a one-way analysis of variance (ANOVA) was performed using SigmaStat 3.5. This test was conducted with a risk alpha of 0.050, and comparisons with the high-fat diet vehicle group were performed using Dunnett's test.

[0174] The results of this study are shown in Figures 1 and 2. From Figure 1, it can be seen that the initial administration of SEQ ID NO: 35 and compound A induced a significant decrease in food intake that normalized after the second treatment, in parallel with the mice treated with the vehicle. Body weight was significantly reduced in mice treated with SEQ ID NO: 35 or compound A after a 15-day treatment period compared to the vehicle control (P<0.001). Mice treated with the vehicle maintained a nearly constant body weight over 15 days (-1.9±1.6% over 2 weeks), while mice treated with SEQ ID NO: 35 lost -10.8±1.4% of their body weight, and mice treated with compound A lost -7.1±1.3% (see Figure 2). The decrease in body fat content was -34.2±2.9% for SEQ ID NO: 35 and -27.5±1.9% for compound A. Furthermore, in the same study, compared to subcutaneous injection of 10 nmol / kg of semaglutide every two days, SEQ ID NO. 35 enlarged similarly, but food intake decreased (cumulative food intake over 15 days: 43.7±1.3g for vehicle, compared to 39.3±2.5g for SEQ ID NO: 35 and 29.6±0.9g for semaglutide). Also, muscle preservation was better: -8.6±1.1% for vehicle, compared to -6.3±1.7% for SEQ ID NO: 35 and -14.8±0.7% for semaglutide).

[0175] Example 19: Fatty acid modification The effect of fatty acid modification is on EC2 at hCRF2 < 0.2 nM. 50 The selectivity profiles of hCRF1 / hCRF2 > 500 and the absence of known metabolic and chemical disadvantages were evaluated for the peptides listed in Table 2. Preferred sequence features include D-valine at position 7 and the presence of a bulky amino acid preferentially adjacent to N31.

[0176] Ten optimized sequences with the aforementioned profiles were selected from the list in Table 2 and are shown in Figure 3. The following additional peptides (Table 16) were synthesized from these peptides. These compounds, as shown in Figure 3, share the same amino acid sequence as the compounds previously disclosed in the patent application, but differ in their linker / fatty acid derivatization. Table 16 below also shows the original sequence numbers from the previous patent application for each of the newly proposed compounds, from which they share the same amino acid sequence.

[0177] Peptide synthesis: Materials and methods The following starting materials and methods were used in the synthesis procedures described in the Examples. Rink Amide AM resin LL 0.29 mmol / g (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin, Novabiochem), 100-200 mesh was used for the synthesis of all peptide amides. Fmoc-protected natural amino acids were purchased from Novabiochem, Iris Biotech, Bachem, or Chem-Impex International. The following standard amino acids were used in the synthesis: Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OMpe)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH. Furthermore, the following amino acids were purchased from the same supplier as above: Fmoc-L-Lys(Dde)-OH, Fmoc-Aib-OH, Fmoc-D-Val-OH. [Table 16-1] [Table 16-2]

[0178] Peptide synthesis: Materials and methods - continued The following side chain components were obtained from Iris Biotech, TCI, Merck: Fmoc-AEEA-OH, Fmoc-L-Glu-OtBu, palmitic acid (HO-C(O)(CH2) 14 CH3), Hexadecanedioic acid (HO-C(O)(CH2) 14 COOH). Crude peptides were purified using a preparative HPLC system with a C4 column. Specifically, the following column was used: Reprosil Gold C4 Prep (Dr. Maisch), 250 x 40 cm, 120 Å, 5 μm. Acetonitrile + 0.1% TFA and water + 0.1% TFA were used as eluents. The product-containing fractions were collected and lyophilized to obtain purified products, typically as TFA salts.

[0179] Alternatively, the compound was isolated as an acetate by the following procedure. Ion exchange was performed using HiTrap. TM The procedure was performed using a Q HP column (GE Healthcare). Pure peptides were dissolved in a 2 mg / ml 0.16 M acetic acid solution, slowly packed into the column, and eluted with a 0.16 M acetic acid solution. The recovered solution was lyophilized.

[0180] Crude and purified peptides were analyzed by ultra-high-performance liquid chromatography using UV and mass spectrometry detection (UPLC-UV-MS). Analytical ULC was performed according to one of the following methods:

[0181] Method A: Detection at 214nm Column: Acquity Waters BEH130 C4, 1.7μm (2.1 x 100mm) 45℃ Solvent: H2O + 0.1% TFA : ACN + 0.1% TFA (flow rate 0.4 ml / min) Gradient: 70:30 (0 min) ~ 70:30 (1 min) ~ 50:50 (5 min) ~ 10:90 (5.2 min) 10:90 (5.5 minutes) ~ 70:30 (5.7 minutes) ~ 70:30 (6 minutes) Mass spectrometer: Waters SQ detector with electrospray ionization in cation detection mode.

[0182] Method B: Detection at 214nm Column: Acquity Waters BEH130 C4, 1.7μm (2.1x100mm) 45 °C Solvent: H2O + 0.1% TFA : ACN + 0.1% TFA (flow rate 0.4 ml / min) Gradient: 65:35 (0 min) ~ 65:35 (1 min) ~ 45:55 (5 min) ~ 10:90 (5.2 min) 10:90 (5.5 minutes) ~ 65:35 (5.7 minutes) ~ 65:35 (6 minutes) Mass spectrometer: Waters SQ detector with electrospray ionization in cation detection mode

[0183] Example 19.1: General Synthesis Procedure Sequence IDs 187-222 All peptides (SEQ ID NOs. 187-222) were synthesized using standard Fmoc stepwise solid-phase synthesis (SPPS) on a Liberty Blue microwave synthesizer (CEM). The process involved using 0.1 mmol scale Rink amide AM Resin LL 0.29 mmol / g with DIC / Oxyma activation. DMF was used as the solvent. For the modified lysine side chain, Fmoc-L-Lys(Dde)-OH was used at position 12.

[0184] The following conditions were adopted: Standard deprotection: 20% piperidine in DMF, 2 x 120 seconds, 90°C Cleaning: 4x DMF. Standard single coupling: 5 equivalents AA 0.4M / 5 equivalents DIC 1M / 5 equivalents Oxyma 1M, 120 seconds, 90°C. Cleaning: 4x DMF.

[0185] At the end of assembly, the N-terminus of the peptide was protected with tert-butoxycarbonyl tert-butyl carbonate in DMF [10 equivalents excess relative to the resin loading, FluoroChem]; the mixture was shaken at room temperature for 30 minutes and the reaction was monitored by Kaiser test. Removal of the Dde group on Lys12 was achieved by adding 2% hydrazine monohydrate dropwise and washing the resin with DMF / DCM / DMF (6 / 6 / 6 times each).

[0186] After the removal of the Dde group, the resin was treated according to the peptide sequence side chain derivatization as described in Examples 2 to 7.

[0187] Peptide cleavage from the resin was performed using the following cleavage cocktail: 87.5% TFA, 5% phenol, 5% water, 2.5% TIPS, for 2–2.5 hours. The resin used in the synthesis was such that the C-terminus was cleaved from the resin as a primary amide.

[0188] The cleavage mixture was recovered by filtration, the crude peptide was precipitated in methyl tert-butyl ether, centrifuged, the supernatant was removed, fresh diethyl ether was added to the peptide, and the process was repeated twice; the crude peptide was then lyophilized. The peptide was analyzed by analytical UPLC and validated by ESI + mass spectrometry. The crude peptide was purified by conventional preparative RP-HPLC purification procedures.

[0189] Example 19.2: Synthesis of the peptide of SEQ ID NO: 187 The compound of Sequence ID No. 186 was prepared according to the procedure described in Example 19.1. Novabiochem Rink amide AM Resin LL 0.29 mmol / g (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh was used. An automated Fmoc synthesis method was applied using DIC / oxima activation. At position 12, Fmoc-L-Lys(Dde)-OH was used in a solid-phase synthesis protocol. At the end of assembly, the N-terminus of the peptide was protected and the Dde protecting group on Lys12 was removed as reported in Example 19.1.

[0190] The γ-carboxyl terminus of glutamic acid was bonded to the ε-amino group of Lys using Fmoc-L-Glu-OtBu in DMF using the DIC / HOAt method (4 equivalents excess relative to the resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on glutamic acid was removed by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes each (25 ml each). The resin was washed with NMP / DCM / NMP (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at the completion of Fmoc deprotection was positive.

[0191] The albumin-binding site is hexadecanedioic acid (HO-C(O)(CH2) 14 The reaction was carried out in NMP using the DIC / HOAt method (5 equivalents in excess of resin loading) with COOH. The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with NMP / DCM / NMP (6 / 6 / 6 times each).

[0192] The peptide was cleaved from the resin as described in Example 19.1. The crude product was purified by preparative RP-HPLC using an acetonitrile / water gradient (containing 0.1% TFA) on Reprosil Gold C4 Prep (Dr. Maisch), 250 × 40 cm, 120 Å, 5 μm. The purified peptide was analyzed by LC / MS (Method A). (M / 4+H)+ The mass signal was found under a peak at a retention time of 3.84 minutes, revealing that the peptide mass of 1213.97 was consistent with the expected value of 1213.47.

[0193] Example 19.3: Synthesis of the peptide of SEQ ID NO: 198 The compound of Sequence ID No. 197 was prepared according to the procedure described in Example 19.1. Novabiochem Rink amide AM Resin LL 0.29 mmol / g (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh was used. An automated Fmoc synthesis method was applied using DIC / oxima activation. At position 12, Fmoc-L-Lys(Dde)-OH was used in a solid-phase synthesis protocol. At the end of assembly, the N-terminus of the peptide was protected and the Dde protecting group on Lys12 was removed as reported in Example 19.1.

[0194] Fmoc-AEEA-OH was bonded to the ε-amino group of Lys in DMF using the DIC / HOAt method (4 equivalents in excess of resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on AEEA was removed by treating the resin with 20% (v / v) piperidine / DMF solution for 5 minutes (25 ml each) twice. The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at the completion of Fmoc deprotection was positive.

[0195] The second Fmoc-AEEA-OH was bonded to the deprotected amino group in DMF using the DIC / HOAt method, and then deprotected from Fmoc as reported above.

[0196] The γ-carboxyl terminus of glutamic acid was bonded to the deprotected amino group using Fmoc-L-Glu-OtBu in DMF via the DIC / HOAt method (4 equivalents in excess of resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on glutamic acid was removed by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes each (25 ml each). The resin was washed with NMP / DCM / NMP (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at completion of Fmoc deprotection was positive.

[0197] The albumin-binding site is hexadecanedioic acid (HO-C(O)(CH2) 14 The reaction was carried out in NMP using the DIC / HOAt method (5 equivalents in excess of resin loading) with COOH. The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with NMP / DCM / NMP (6 / 6 / 6 times each).

[0198] The peptide was cleaved from the resin as described in Example 19.1. The crude product was purified by preparative RP-HPLC using an acetonitrile / water gradient (containing 0.1% TFA) on Reprosil Gold C4 Prep (Dr. Maisch), 250 × 40 cm, 120 Å, 5 μm. The purified peptide was analyzed by LC / MS (Method A). (M / 4+H) + The mass signal was found under a peak at a retention time of 3.66 minutes, revealing that the peptide mass of 1286.7 was consistent with the expected value of 1286.05.

[0199] Example 19.4: Synthesis of the peptide of SEQ ID NO: 199 The compound of Sequence ID No. 198 was prepared according to the procedure described in Example 19.1. Novabiochem Rink amide AM Resin LL 0.29 mmol / g (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh was used. An automated Fmoc synthesis method was applied using DIC / oxima activation. At position 12, Fmoc-L-Lys(Dde)-OH was used in a solid-phase synthesis protocol. At the end of assembly, the N-terminus of the peptide was protected and the Dde protecting group on Lys12 was removed as reported in Example 19.1.

[0200] Fmoc-AEEA-OH was bonded to the ε-amino group of Lys in DMF using the DIC / HOAt method (4 equivalents in excess of resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on AEEA was removed by treating the resin with 20% (v / v) piperidine / DMF solution for 5 minutes (25 ml each) twice. The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at the completion of Fmoc deprotection was positive.

[0201] The second Fmoc-AEEA-OH was bonded to the deprotected amino group in DMF using the DIC / HOAt method, and then deprotected from Fmoc as reported above.

[0202] The γ-carboxyl terminus of glutamic acid was bonded to the deprotected amino group using Fmoc-L-Glu-OtBu in DMF via the DIC / HOAt method (4 equivalents in excess of resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on glutamic acid was removed by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes each (25 ml each). The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at completion of Fmoc deprotection was positive.

[0203] The second Fmoc-L-Glu-OtBu was bonded to the deprotected amino group in DMF using the DIC / HOAt method, and then deprotected from Fmoc as described above. The resin was washed with NMP / DCM / NMP (6 / 6 / 6 times each).

[0204] The albumin-binding site is hexadecanedioic acid (HO-C(O)(CH2) 14 The reaction was carried out in NMP using the DIC / HOAt method (5 equivalents in excess of resin loading) with COOH. The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with NMP / DCM / NMP (6 / 6 / 6 times each).

[0205] The peptide was cleaved from the resin as described in Example 19.1. The crude product was purified by preparative RP-HPLC using an acetonitrile / water gradient (containing 0.1% TFA) on Reprosil Gold C4 Prep (Dr. Maisch), 250 × 40 cm, 120 Å, 5 μm. The purified peptide was analyzed by LC / MS (Method A). (M / 4+H) + The mass signal was found under a peak at a retention time of 3.50 minutes, revealing that the peptide mass of 1318.70 was consistent with the expected value of 1318.33.

[0206] Example 19.5: Synthesis of the peptide of SEQ ID NO: 204 The compound of SEQ ID NO: 203 was prepared according to the procedure described in Example 19.1. Novabiochem Rink amide AM Resin LL 0.29 mmol / g (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh was used. An automated Fmoc synthesis method was applied using DIC / oxima activation. At position 12, Fmoc-L-Lys(Dde)-OH was used in a solid-phase synthesis protocol. At the end of assembly, the N-terminus of the peptide was protected and the Dde protecting group on Lys12 was removed as reported in Example 19.1.

[0207] The γ-carboxyl terminus of glutamic acid was bonded to the ε-amino group of Lys using Fmoc-L-Glu-OtBu in DMF using the DIC / HOAt method (4 equivalents excess relative to the resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on glutamic acid was removed by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes each (25 ml each). The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at the completion of Fmoc deprotection was positive.

[0208] The albumin-binding site is connected to palmitic acid (HO-C(O)(CH2) 14 CH 3) The reaction was carried out in DMF using the DIC / HOAt method (5 equivalents in excess of resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each).

[0209] The peptide was cleaved from the resin as described in Example 19.1. The crude product was purified by preparative RP-HPLC using an acetonitrile / water gradient (containing 0.1% TFA) on Reprosil Gold C4 Prep (Dr. Maisch), 250 × 40 cm, 120 Å, 5 μm. The purified peptide was analyzed by LC / MS (Method B). (M / 4+H) + The mass signal was found under a peak at a retention time of 3.84 minutes, revealing that the peptide mass of 1206.50 was consistent with the expected value of 1205.98.

[0210] Example 19.6: Synthesis of the peptide of SEQ ID NO: 205 The compound of Sequence ID No. 204 was prepared according to the procedure described in Example 1. Novabiochem Rink amide AM Resin LL 0.29 mmol / g (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh was used. An automated Fmoc synthesis method was applied using DIC / oxima activation. At position 12, Fmoc-L-Lys(Dde)-OH was used in a solid-phase synthesis protocol. At the end of assembly, the N-terminus of the peptide was protected and the Dde protecting group on Lys12 was removed as reported in Example 19.1.

[0211] Fmoc-AEEA-OH was bonded to the ε-amino group of Lys in DMF using the DIC / HOAt method (4 equivalents in excess of resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on AEEA was removed by treating the resin with 20% (v / v) piperidine / DMF solution for 5 minutes (25 ml each) twice. The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at the completion of Fmoc deprotection was positive.

[0212] The second Fmoc-AEEA-OH was bonded to the deprotected amino group in DMF using the DIC / HOAt method, and then deprotected from Fmoc as reported above.

[0213] The γ-carboxyl terminus of glutamic acid was bonded to the ε-amino group of Lys using Fmoc-L-Glu-OtBu in DMF using the DIC / HOAt method (4 equivalents excess relative to the resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on glutamic acid was removed by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes each (25 ml each). The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at the completion of Fmoc deprotection was positive.

[0214] The albumin-binding site is connected to palmitic acid (HO-C(O)(CH2) 14 The reaction was carried out in DMF using the DIC / HOAt method (5 equivalents in excess of resin loading) with CH3). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each).

[0215] The peptide was cleaved from the resin as described in Example 19.1. The crude product was purified by preparative RP-HPLC using an acetonitrile / water gradient (containing 0.1% TFA) on Reprosil Gold C4 Prep (Dr. Maisch), 250 × 40 cm, 120 Å, 5 μm. The purified peptide was analyzed by LC / MS (Method B). (M / 4+H) + The mass signal was found under a peak at a retention time of 3.45 minutes, revealing that the peptide mass of 1279.0 was consistent with the expected value of 1278.56.

[0216] Example 19.7: Synthesis of the peptide of SEQ ID NO: 206 The compound of Sequence ID No. 205 was prepared according to the procedure described in Example 19.1. Novabiochem Rink amide AM Resin LL 0.29 mmol / g (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamide-norleucylaminomethyl resin), 100-200 mesh was used. An automated Fmoc synthesis method was applied using DIC / oxima activation. At position 12, Fmoc-L-Lys(Dde)-OH was used in a solid-phase synthesis protocol.

[0217] At the end of the assembly, the N-terminus of the peptide was protected, and the Dde protecting group on Lys12 was removed, as reported in Example 19.1.

[0218] Fmoc-AEEA-OH was bonded to the ε-amino group of Lys in DMF using the DIC / HOAt method (4 equivalents in excess of resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on AEEA was removed by treating the resin with 20% (v / v) piperidine / DMF solution for 5 minutes (25 ml each) twice. The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at the completion of Fmoc deprotection was positive.

[0219] The second Fmoc-AEEA-OH was bonded to the deprotected amino group in DMF using the DIC / HOAt method, and then deprotected from Fmoc as reported above.

[0220] The γ-carboxyl terminus of glutamic acid was bonded to the deprotected amino group using Fmoc-L-Glu-OtBu in DMF via the DIC / HOAt method (4 equivalents in excess of resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Fmoc group on glutamic acid was removed by treating it twice with 20% (v / v) piperidine / DMF solution for 5 minutes each (25 ml each). The resin was washed with DMF / DCM / DMF (6 / 6 / 6 times each). The Kaiser test on the peptide resin aliquot at completion of Fmoc deprotection was positive.

[0221] The second Fmoc-L-Glu-OtBu was bonded to the deprotected amino group in DMF using the DIC / HOAt method, and then deprotected from Fmoc as reported above.

[0222] The albumin-binding site is connected to palmitic acid (HO-C(O)(CH2) 14 The reaction was carried out in DMF using the DIC / HOAt method (5 equivalents in excess of resin loading) with CH3). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by the Kaiser test. The resin was filtered and washed with DMF / DCM / DMF (6 / 6 / 6 times each).

[0223] The peptide was cleaved from the resin as described in Example 19.1. The crude product was purified by preparative RP-HPLC using an acetonitrile / water gradient (containing 0.1% TFA) on Reprosil Gold C4 Prep (Dr. Maisch), 250 × 40 cm, 120 Å, 5 μm. The purified peptide was analyzed by LC / MS (Method B). (M / 4+H) + The mass signal was found under a peak at a retention time of 3.30 minutes, revealing that the peptide mass of 1311.35 was consistent with the expected value of 1310.83.

[0224] Example 19.8: Further peptide synthesis The following peptides were synthesized according to the procedures described in Examples 19.1-7. The calculated and found masses and retention times of these peptides, along with those of the compounds in Examples 19.2-7, are shown in Table 3 below: [Table 17-1] [Table 17-2]

[0225] Abbreviation Certain abbreviations are used in the examples and elsewhere in this specification: "AA" refers to amino acids; "AEEA" refers to [2-(2-aminoethoxy)ethoxy]acetyl; "Aib" refers to 2-aminoisobutyric acid; "Boc" refers to tert-butyloxycarbonyl; "tBu" refers to tertiary butyl; "DCM" refers to dichloromethane; "Dde" refers to 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl; "DIC" refers to N,N'-diisopropylcarbodiimide; "DMF" refers to dimethylformamide; "Fmoc" refers to fluorenylmethyloxycarbonyl; "gGlu" refers to gamma-glutamic acid (γE); "HOAt" refers to 1-hydroxy-7-azabenzotriazole; "HPLC" refers to high-performance liquid chromatography. "LC / MS" refers to liquid chromatography / mass spectrometry; "MS" refers to mass spectrometry; "NMP" refers to N-methyl-2-pyrrolidone; "OtBu" refers to o-tert-butyl; "Oxyma" refers to ethyl cyanohydroxyiminoacetate; "Pbf" refers to 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; "RP-HPLC" refers to reverse-phase high-performance liquid chromatography. "TIPS" refers to triisopropylsilane; "TFA" refers to trifluoroacetic acid; "Trt" refers to the tritille.

[0226] Example 19.9: Evaluation of the activity of these FA-modified peptides in the human CRF2α receptor. The agonism of compounds against human corticotropin-releasing factor 2α (CRF2α receptor) was determined using a functional assay that measured cAMP regulation during treatment, with TeloHEAC cells stably expressing the human CRF2α receptor.

[0227] The compound was dissolved in 100% DMSO at a concentration of 0.5 mM and then sequentially diluted 16 times in 100% DMSO (1:2). Each 20 nL dilution was then transferred to a 384-well assay plate using an acoustic droplet dispenser. Next, 5 μL of compound buffer (1x HBSS; 20 mM HEPES, 2 mM IBMX, 0% or 0.2% HSA) was added to each well.

[0228] Before use, frozen cells were rapidly thawed at 37°C and washed with 20 mL of cell buffer (1x HBSS; 20 mM HEPES) for 5 minutes at 900 rpm. The cells were resuspended in cell buffer and adjusted to a cell density of 800,000 cells / ml. 5 μL of cells (final delta density: 4000 cells / well) were dispensed into a compound-containing 384-well assay plate and incubated at 37°C for 30 minutes.

[0229] Using the Cisbio 62AM4PEC kit, cAMP levels in treated cells were determined according to the manufacturer's instructions. Finally, after incubating the plates at room temperature for 1 hour, the fluorescence ratio between 665 / 620 nm was measured.

[0230] Percent activity values ​​(E%) were calculated by setting 100 nM urocortin 2 (UCN2) as 100%. The in vitro potency of the compound was quantified by determining the concentration that elicits 50% activation of the maximum response (EC50) compared to urocortin 2. Representative EC50 values ​​are provided in Table 18 below: [Table 18]

[0231] Example 19.10: Evaluation of the activity of the compound according to the present invention at the anti-target CFR1R receptor. We purchased CRF1R-overexpressing CHO-K1 cell line clone 2 from PerkinElmer. Cells were grown in a 10 cm dish in culture medium [F12 (Hams) / 10% FBS / 400 μg / ml G418] at 37°C / 5% CO2 until near confluence. At this stage, the cells were harvested and resuspended at 10 million / mL in culture medium containing 10% DMSO but without G418. 1 mL vials were slowly frozen at -80°C in isopropanol and then transferred to liquid nitrogen for storage. Using these vials, experiments were performed according to the procedure described above, except for a compound used in a control well to define 100% activation (sauvagine at 100 nM). Typical EC50 and Emax values ​​are shown in Table 19 below: [Table 19-1] [Table 19-2]

[0232] Of note in this table is the clear effect of the presence of free carboxylic acids in the FA modification of K12. For example, as albumin-binding moieties, -gGlu-C(O)(CH2) 14 COOH and {AEEA}2-gGlu-C(O)(CH2) 18Peptides 223, 225, and 226, which contain COOH, are compared, for example, with peptides 222 and 224, to see how albumin-binding moiety-gGlu-C(O)(CH2) 14 See also whether CH3 is present. This is even clearer in the comparative analysis of peptides that differ only in the presence of the free carboxylic acid (Table 20). [Table 20-1] [Table 20-2]

[0233] It was also observed that extending the linkers such as -{AEEA}2-gGlu- and {AEEA}2-(gGlu)2- in the albumin-binding moiety containing free carboxylic acid further improved the CRF2 selectivity of the peptide (data not shown).

[0234] Example 19.11: DMPK protocol and results of these FA-modified peptides In vitro plasma stability In vitro plasma metabolic stability was investigated using male Sprague Dawley rats and humans. The test compounds were incubated at 37°C for 2 hours at 3 mM. At each time point (0, 0.25, 1, and 2 hours), the samples were prepared for analysis by a one-step protein precipitation technique, by adding 150 μL of ethanol and 0.1% formic acid to a fixed volume of 50 μL of plasma. The samples were homogeneously mixed using a vortex mixer and then centrifuged at 14000 rpm for 15 minutes. The supernatant (100 μL) was collected, diluted with 100 μL of water and 0.1% formic acid, and analyzed by LC-HRMS (TripleTOF 6600+, AB Sciex). For each test compound, the area ratio at each time point was compared to the area ratio at 0 hours and converted to the remaining proportion. Representative plasma stability data is provided in Table 21 below: [Table 21]

[0235] Solubility and chemical stability Similar to Example 15 above, the chemical stability of representative examples of these FA-modified peptides was evaluated using LC-UV instead of light scattering. Solubility and chemical stability were examined in 100 mM phosphate buffer (pH 7.4) and 100 mM acetate buffer (pH 4.5). The test compound powder was dissolved in both buffers at a target concentration of 10 mg / mL and incubated at room temperature for 1 hour. After centrifugation at 2500 rcf for 15 minutes, 10 mL of the supernatant was diluted in 190 mL of incubation buffer and analyzed by LC-UV (Acquity UPLC-DAD, Waters). Solubility was calculated by comparing the peak area of ​​the test compound in the buffer sample with the peak area of ​​the same compound dissolved at 0.5 mg / mL in water:acetonitrile 1:1 and 0.1% formic acid.

[0236] Two additional 80 mL aliquots were collected from the supernatant after centrifugation and diluted in 160 mL of incubation buffer. One was stored at 5°C and the other at 40°C. After 28 days, the samples were analyzed by LC-UV. Chemical stability was calculated as a percentage loss using the following formula: * Chemical stability (as % loss) = [(Purity after 28 days at 5°C) - (Purity after 28 days at 40°C)] × 100 / (Purity after 28 days at 5°C) * % Purity = [(Peak Area Compound) × 100 / (Total Peak Area)] Typical buffer solubility data is provided in Table 22 below: [Table 22]

[0237] Representative chemical stability data is provided in Table 23 below: [Table 23]

[0238] All sequences disclosed herein are listed in the attached sequence listing, the entire contents of which form part of this specification.

[0239] All publications, patent applications, patents, and other references mentioned herein are incorporated as references. In addition, the materials, methods, and examples given are illustrative and not intended to limit the scope. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. This disclosure is provided together with a detailed description, but it should be understood that the above description is intended to illustrate and not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are found in the claims.

Claims

1. Compounds that are peptides containing the amino acid sequence of the following formula (I): X1-X2-X3-X4-X5-X6-X7-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X 21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38 Formula (I) In the formula, each element has the following meaning: X1 is isoleucine (I) or phenylalanine (F); X2 is either valine (V) or threonine (T); X3 is leucine (L); X4 is serine(S); X5 is leucine (L); X6 is aspartic acid (D); X7 is valine(V) or D-valine(v); X8 is proline (P); X9 is isoleucine (I) or threonine (T); X10 is lysine (K), glutamic acid (E), histidine (H), or glycine (G); X11 is isoleucine(I) or leucine(L); X12 is lysine (K), where the epsilon (ε)-amino group of the lysine side chain is covalently bonded to the albumin-binding site; X13 is glutamine (Q) or lysine (K); X14 is isoleucine (I), lysine (K), or 2-aminoisobutyric acid (Aib); X15 is leucine (L); X16 is either leucine (L) or phenylalanine (F); X17 is glutamic acid (E) or lysine (K); X18 is glutamine (Q); X19 is alanine (A), glutamic acid (E), or glutamine (Q); X20 is either lysine (K) or arginine (R); X21 is glutamine (Q) or lysine (K); X22 is lysine (K), arginine (R), or glutamic acid (E); X23 is lysine (K) or 2-aminoisobutyric acid (Aib); X24 is glutamine (Q), 2-aminoisobutyric acid (Aib), leucine (L), or glutamic acid (E); X25 is arginine (R), lysine (K), or 2-aminoisobutyric acid (Aib); X26 is alanine (A), glutamic acid (E), 2-aminoisobutyric acid (Aib), or glutamine (Q); X27 is glutamine (Q), 2-aminoisobutyric acid (Aib), or lysine (K); X28 is alanine (A); X29 is glutamic acid (E) or lysine (K); X30 is either lysine (K) or threonine (T); X31 is either asparagine (N) or alanine (A); X32 is lysine (K), alanine (A), valine (V), threonine (T), glutamic acid (E), or 2-aminoisobutyric acid (Aib); X33 is arginine (R), lysine (K), or glutamine (Q); X34 is isoleucine(I) or leucine(L); X35 is leucine (L); X36 is either alanine (A) or glutamic acid (E); X37 is glutamine (Q) or arginine (R); and X38 is isoleucine(I) or valine(V); or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1; where X1 is isoleucine(I); X2 is valine (V); X7 is valine(V) or D-valine(v); X9 is isoleucine (I) or threonine (T); X10 is lysine (K) or glycine (G); X11 is leucine (L); X13 is either lysine (K) or glutamine (Q); X14 is lysine (K) or isoleucine (I); X16 is leucine (L); X19 is either alanine (A) or glutamic acid (E); X21 is glutamine (Q); X22 is glutamic acid (E), arginine (R), or lysine (K); X23 is lysine (K) or 2-aminoisobutyric acid (Aib); in particular, X24 is lysine (K), glutamic acid (E) or glutamine (Q) or 2-aminoisobutyric acid (Aib); X25 is either lysine (K) or arginine (R); X26 is alanine (A), glutamic acid (E), or glutamine (Q); in particular, glutamic acid (E) or glutamine (Q); X27 is 2-aminoisobutyric acid (Aib), lysine (K), or glutamine (Q); particularly lysine (K) or glutamine (Q); more preferably glutamine (Q); X29 is glutamic acid (E); X30 is lysine (K) or threonine (T); especially lysine (K); X31 is asparagine(N); X32 is lysine (K) or alanine (A); X33 is arginine(R) or glutamine(Q); in particular, arginine(R); X34 is isoleucine(I); X35 is leucine (L); X36 is alanine (A) or glutamic acid (E); in particular, glutamic acid (E); X37 is arginine (R) or glutamine (Q); in particular glutamine (Q); and X38 is a Valine (V) tube.

3. A compound according to claim 1 or 2; where X7 is D-valine(v); X10 is lysine (K) or glycine (G); especially lysine (K); X19 is alanine (A) or glutamic acid (E); in particular, glutamic acid (E); X34 (23?) is lysine (K) or 2-aminoisobutyric acid (Aib); in particular, lysine (K); X24 is glutamine (Q) or 2-aminoisobutyric acid (Aib); in particular, glutamine (Q); X25 is arginine(R); X26 is alanine (A), glutamic acid (E), or glutamine (Q); particularly glutamic acid (E) or glutamine (Q); more preferably glutamic acid (E); X27 is lysine (K) or glutamine (Q); in particular glutamine (Q); X30 is lysine (K) or threonine (T); especially lysine (K); X33 is arginine (R) or glutamine (Q); in particular glutamine (Q); X36 is alanine (A) or glutamic acid (E); in particular alanine (A); and X37 is arginine (R) or glutamine (Q); in particular glutamine (Q).

4. A compound according to any one of claims 1 to 3; here X10 is lysine (K); X19 is glutamic acid (E); X24 is glutamine (Q); X25 is arginine(R); X26 is glutamic acid (E); X27 is glutamine (Q); X30 is lysine (K); X33 is glutamine (Q); X36 is alanine (A); and X37 is glutamine (Q).

5. A compound according to any one of claims 1 to 4, wherein the compound is a peptide having the amino acid sequence of formula (I), wherein the amino acid residue X1 is acetylated and the amino acid residue X38 is amidated; or a pharmaceutically acceptable salt thereof.

6. A compound according to claim 5, wherein the amino acid residue X38 is amidated as a C-terminal primary amide.

7. A compound according to any one of claims 1 to 4, wherein the albumin-binding portion is a group of the following formula (II): -YZC(O)R 1 (II) [Here, Y is AEEA, {AEEA} 2 , {AEEA} 3 Gly, {Gly} 2 , {Gly} 3 , N-MeGly, {N-MeGly} 2 {N-MeGly} 3 Or lack thereof, where AEEA means [2-(2-aminoethoxy)ethoxy]-acetyl; Z is gGlu, {gGlu} 2 or lack; and R 1 is -(CH 2 ) x COOH or -(CH 2 ) x CH 3 , especially -(CH 2 ) x COOH, where x is an integer from 12 to 22 )

8. A compound according to claim 7, wherein the albumin-binding portion is selected from the following groups: -{AEEA} 2 -gGlu-C(O)(CH). 2 ) 16 COOH; -{AEEA} 2 -gGlu-C(O)(CH). 2 ) 18 COOH; -{AEEA} 2 -{gGlu} 2 -C(O)(CH). 2 ) 16 COOH; -{AEEA} 2 -{gGlu} 2 -C(O)(CH). 2 ) 18 COOH; -C(O)(CH 2 ) 16 COOH; -gGlu-C(O)(CH 2 ) 16 COOH; -gGlu-C(O)(CH 2 ) 14 COOH; -{AEEA} 2 -gGlu-C(O)(CH 2 ) 14 COOH; and -{AEEA} 2 -{gGlu} 2 -C(O)(CH). 2 ) 14 COOH。

9. A compound according to claim 7, wherein the albumin-binding portion is as follows: -{AEEA} 2 -gGlu-C(O)(CH). 2 ) 14 COOH; or -{AEEA} 2 -gGlu-C(O)(CH). 2 ) 16 COOH.

10. A compound according to claim 1, wherein the compound is a peptide of any one of SEQ ID NOs: 1 to 227, or a pharmaceutically acceptable salt thereof.

11. A compound according to claim 1, wherein the compound is a peptide of any one of SEQ ID NOs: 3, 7, 35, 83, 130, 135, 136, 137, 138, 139, 140, 141, 142, 147, 149, 151, 152, 171, 172, 173, and 174, or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient, diluent, or carrier.

13. A compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12, for therapeutic purposes.

14. A compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12, for use in the treatment or prevention of cardiovascular disease, obesity, and diabetes in patients.

15. A compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12, which is used as an agonist of corticotropin-releasing factor receptor 2 (CRF2).

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