CNP compound

Abstract

This application relates to novel C-type natriuretic peptide (CNP) compounds, pharmaceutical compositions comprising these compounds, and these compounds for use as medicaments.

Description

Technical Field

[0001] The present invention relates to novel C-type natriuretic peptide (CNP) compounds, pharmaceutical compositions containing these compounds, and these compounds for use as medicaments.

Background Art

[0002] Natriuretic peptides are a family of three structurally related hormones that play unique roles within the cardiovascular system. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are expressed in the heart and are released in response to volume-induced stretch of the atria and ventricles, respectively. Their physiological effects include the regulation of intracardiac structures, blood pressure, and blood volume.

[0003] C-type natriuretic peptide (CNP) is highly expressed in endothelial cells, where it is constitutively released. Other cells within the cardiovascular system, including cardiomyocytes and fibroblasts, also produce CNP, but to a lesser extent. CNP has direct effects on inflammation, fibrosis, cardiac contractility, endothelial function, angiogenesis, and blood pressure.

[0004] There are three known receptors for natriuretic peptides. Natriuretic peptide receptor-1 (NPR1) is a particulate guanylyl cyclase that catalyzes the synthesis of cGMP upon binding by ANP or BNP. NPR1 is expressed in the kidney, lung, adipose tissue, adrenal gland, brain, heart, testis, and vascular smooth muscle tissue. NPR2, which is homologous to NPR1 and is selectively activated by CNP, is expressed in bone, brain, fibroblasts, heart, kidney, liver, lung, uterus, and vascular smooth muscle tissue. In contrast, NPR3 contains only a 37-residue intracellular domain and lacks guanylyl cyclase activity. This controls local natriuretic peptide concentrations via receptor-mediated internalization and degradation, although evidence for the signaling function of NPR3 continues to accumulate. All three natriuretic peptides bind to NPR3 with high affinity, and this receptor is the most widely and abundantly expressed of the three receptors. Also, the degree of conservation of all three receptors is very high, and NPR2 is the most highly conserved, in line with CNP.

[0005] Clinical and preclinical data have shown the important roles of CNP and its two receptors in cardio-renal metabolic functions, and data continue to accumulate supporting that targeting this system retains therapeutic potential in a wide range of cardiovascular, renal, and metabolic diseases (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). Similarly, human genetics, like preclinical and clinical data, supports NPR2 and CNP as regulators of bone growth with therapeutic potential in a wide range of short stature phenotypes related to NPR2 and CNP (Non-Patent Document 4; Non-Patent Document 5) as well as FGFR3-related skeletal dysplasias (Non-Patent Document 6; Non-Patent Document 7, Non-Patent Document 8) and RASopathies (Non-Patent Document 9). Currently, two CNP compounds are in clinical development for achondroplasia, a form of dwarfism (BMN111 (Vosoritide) and TransCon CNP). Mayo Clinic has previously studied a CNP compound designed to activate both NPR2 and NPR1 in heart failure (Non-Patent Document 10).

[0006] The clearance of CNP in human plasma is very rapid, and the calculated half-life is several minutes. Considering this short half-life, it would be beneficial to develop new long-acting CNP compounds that can be administered at lower frequencies while maintaining an acceptable clinical profile.

[0007] To provide a longer action profile, various different approaches have been used to modify the structure of CNP. In Patent Document 1, a fusion peptide of the CNP domain and the Fc domain is disclosed. In Patent Document 2, a fatty acid-modified NPR1 agonist is disclosed.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

[0010] The present invention provides a CNP compound having improved pharmaceutical properties.

[0011] In one aspect, the present invention is a CNP compound comprising a CNP peptide and a modifying group, wherein the net charge (effective charge) of the compound at physiological pH is 0 or negative, and the CNP peptide has the formula I: AA 01 -AA 02 -AA 03 -AA 04 -AA 05 -AA 06 -AA 07 -AA 08 -AA 09 -AA 10 -AA 11 -AA 12 -AA 13 -AA 14 -AA 15 -AA 16 -AA 17 -AA 18 -AA 19 -AA 20 -AA 21 -AA 22 -AA 23 -AA 24 -AA 25 -AA 26 -AA 27 -AA 28 -AA 29 -AA30 -AA 31 -AA 32 -AA 33 -AA 34 -AA 35 -AA 36 -AA 37 (wherein AA 01 is Gln or absent, AA 02 is Glu or absent, AA 03 is His or absent, AA 04 is Pro or absent, AA 05 is Asn or Gln or Glu or absent, AA 06 is Ala or absent, AA 07 is Arg or His or Ala or absent, AA 08 is Lys or Ser or His or absent, AA 09 is Tyr or Glu or absent, AA 10 is Lys or Glu or Gln or His or absent, AA 11 is Gly, AA 12 is Ala, AA 13 is Gln or Asn or Glu, AA 14 is Lys or His or Glu, AA 15 is Lys, Ser or Glu or Thr or His, AA 16 is Gly, AA 17is Leu or Gly or Ser or Val, AA 18 is Ser or His, AA 19 is Gln or Ser or Lys or His, AA 20 is Gly, AA 21 is Cys, AA 22 is Phe, AA 23 is Gly, AA 24 is Leu, AA 25 is Pro or Lys, AA 26 is Leu, AA 27 is Asp or Glu, AA 28 is Arg, AA 29 is Ile, AA 30 is Gly, AA 31 is Ser, AA 32 is Leu or Nle or Met, AA 33 is Ser, AA 34 is Gly, AA 35 is Leu, AA 36 is Gly, AA 37 contains an amino acid sequence according to (wherein is Cys), The modifying group includes Chem.A, Chem.B, and Chem.C, Chem.A is,

Chemical formula

Chem.

Chem.

[0012] In one embodiment, the present invention provides a CNP compound, and the CNP peptide has any one of the following amino acid sequences. GAQKKGSSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 136), ARKYKGAQKKGLSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 77), YKGAQKKGGSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 88), YKGAQKKGLSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 103), QEHPQARKYKGAQKKGLSSGCFGLPLDRIGSLSGLGC (SEQ ID NO: 67), and QEHPQARKYKGAQKKGLSSGCFGLPLERIGSLSGLGC (SEQ ID NO: 104)

[0013] In another aspect, the present invention provides a pharmaceutical composition comprising a compound according to the present invention and one or more pharmaceutically acceptable excipients.

[0014] The present invention also provides, in one aspect, the use of a compound of the present invention as a medicament for treating or preventing cardio-renal metabolic diseases including heart failure and achondroplasia.

[0015] Due to the short half-life of wt CNP of approximately 2 minutes, CNP is not suitable for pharmaceutical use. In the present invention, the half-life of CNP is prolonged by fatty acylation that promotes albumin binding. However, the combination of the net positively charged CNP peptide (at physiological pH) and fatty acid albumin binder modification (when the CNP compound is overall positively charged) exacerbates injection site reactions upon subcutaneous injection and reduces bioavailability. This is surprising because the net positively charged CNP peptide without the bound fatty acid albumin binder does not show observable injection site reactions. To solve these problems, amino acid substitutions are introduced into the CNP peptide and negative charges are introduced into the modifying group (including fatty acids). The combination of these modifications results in a compound with an overall net negative charge at physiological pH that has good subcutaneous bioavailability and no to mild subcutaneous injection site reactions.

[0016] Surprisingly, adding negative charges also reduces the in vitro and in vivo efficacy of the CNP compound in a manner where a higher net negative charge generally correlates with lower in vivo efficacy. To retain sufficient efficacy of the CNP compound, only a small number of net negative charges are introduced when introduced. However, a small number of net negative charges reduces the solubility and biophysical stability of the CNP compound when formulated for subcutaneous administration at physiologically relevant pH. The present disclosure balances these parameters and surprisingly provides a CNP compound that also has sufficient efficacy and sufficient solubility and biophysical stability for liquid formulations.

[0017] Furthermore, wt CNP does not exhibit the desired chemical stability in liquid formulations required for convenient drug administration. In the present invention, selected amino acid substitutions of the wt CNP sequence are introduced to promote chemical stability in the formulation.

[0018] The present disclosure provides CNP compounds having a prolonged half-life and good tolerance for subcutaneous administration, while maintaining in vivo efficacy sufficient for therapeutic use and high chemical and biophysical stability in liquid formulations.

[0019] The present invention can also solve further problems that become apparent from the disclosure of the exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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[0021] The present invention provides CNP compounds having improved pharmaceutical properties. The present invention also provides pharmaceutical compositions, as well as the use of the present compounds and the present compositions as medicaments for treating diseases.

[0022] C-type natriuretic peptide C-type natriuretic peptide (CNP) (GenBank accession number NP_077720) is a small single-chain peptide in the family of peptides (ANP, BNP, CNP) that has a 17-amino acid residue disulfide ring structure and plays an important role in multiple biological processes. CNP interacts with natriuretic peptide receptor 2 (NPR2, NPR-B, GC-B) to stimulate the production of cyclic guanosine monophosphate (cGMP). CNP is more widely expressed, including in the central nervous system, reproductive tract, bone, and vascular endothelium. The clearance of CNP in human plasma is rapid, and the half-life is several minutes.

[0023] Native CNP genes and polypeptides have been previously described. U.S. Patent No. 5,352,770 discloses CNP-22 isolated and purified from porcine brain that has the same sequence as human CNP (human wild-type CNP-22: GLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 01)). U.S. Patent No. 6,034,231 discloses the human gene and polypeptide of preproCNP (126 amino acids), as well as the human CNP-53 gene and peptide. Human wild-type CNP-37 has the sequence QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 02).

[0024] CNP compounds In one aspect, the present invention provides a CNP compound comprising a CNP peptide and a modifying group, wherein the net charge of the compound at physiological pH is 0 or negative.

[0025] The CNP compounds described herein have been engineered to achieve compounds that are overall neutral or have a net negative charge. This has been achieved by introducing amino acid substitutions in the CNP peptide and introducing a negative charge into the modifying group. It is not easy to reduce the charge of this CNP compound while still retaining the biological activity of this CNP compound towards the NPR2 receptor.

[0026] As used herein, the term "net charge" refers to the sum of all negative and positive charges on the present CNP compound at a given pH, and the charge is defined only by the acid dissociation constants of each ionizable group in the present CNP compound.

[0027] In one embodiment, the net charge of the present CNP compound at physiological pH is negative as determined as described in Example 4.

[0028] In one embodiment, the net charge of the present CNP compound at physiological pH is from 0 to -4 as determined as described in Example 4.

[0029] As used herein, the term "physiological pH" refers to a pH in the range of 7.35 to 7.45, more typically an average pH of 7.4.

[0030] In one aspect, the CNP compounds described herein have an intramolecular disulfide bridge (a disulfide bond between two cysteine residues) that results in a cyclic structure.

[0031] CNP peptide As used herein, the term "CNP peptide" refers to a peptide 37 to 22 amino acids in length that includes the CNP-22 amino acid sequence (SEQ ID NO: 01), and its amino acid sequence including one or more amino acid modifications (e.g., one or more amino acid substitutions, additions, and / or deletions). The term "CNP peptide" as used herein also encompasses CNP variants.

[0032] As used herein, the term "CNP variant" refers to a CNP peptide that is an amino acid variant of the CNP-37 sequence (SEQ ID NO: 02). In other words, a CNP variant is a CNP peptide that includes one or more amino acid modifications, i.e., at least one amino acid is changed compared to SEQ ID NO: 02. These modifications may independently represent one or more amino acid substitutions, additions, and / or deletions.

[0033] CNP variants may be described by reference to the number of amino acid residues that have changed (i.e., the corresponding positions in the CNP-37 sequence (SEQ ID NO: 02)), and the changes (e.g., the identity of the amino acid residues in the variant at a given position). That is, a reference to a particular amino acid residue of the CNP peptide, in the absence of any further stipulation, refers to the CNP-37 sequence (SEQ ID NO: 2) (i.e., residue 1 is glutamine (Q1) and residue 37 is cysteine (C37)). The following are non-limiting examples of suitable variant nomenclature: des1-4, 5Q, 13Q, 32Nle CNP variants designate CNP peptide sequences having the following amino acid changes when compared to CNP-37: deletion of amino acid residues at positions 1-4, substitution of asparagine (N) at position 5 with glutamine (Q), substitution of asparagine (N) at position 13 with glutamine (Q), substitution of methionine (M) at position 32 with norleucine.

[0034] The term "amino acid" refers to any amino acid that is either naturally occurring (including the 20 standard amino acids encoded by the human standard genetic code) or non-naturally occurring. Amino acid residues can be specified by their full names, their one-letter codes, and / or their three-letter codes. These three methods are completely equivalent. The term "non-coded amino acid" refers to all amino acids that do not fall within the 20 standard amino acids encoded by the human standard genetic code. Non-coded amino acids may be naturally occurring or purely synthetic. Non-limiting examples of non-coded amino acids are D-isomers of coded amino acids and glycine residues having side chains attached to a nitrogen atom rather than an alpha-carbon atom. D-isomers of coded amino acids may be referred to as (i) following the full name of the amino acid, the one-letter code, or the three-letter code by "D", or (ii) the lower-case one-letter code of the amino acid. Further abbreviations for non-coded amino acids used in this application are shown below.

Table 1

[0035] In one aspect of the present invention, the present CNP peptide has the formula I: AA 01 -AA 02 -AA 03 -AA 04 -AA 05 -AA 06 -AA 07 -AA 08 -AA 09 -AA 10 -AA 11 -AA 12 -AA 13 -AA 14 -AA 15 -AA 16 -AA 17 -AA 18 -AA 19 -AA 20 -AA 21 -AA 22 -AA 23 -AA 24 -AA 25 -AA 26 -AA 27 -AA 28 -AA 29 -AA 30 -AA 31 -AA 32 -AA 33 -AA 34 -AA 35 -AA 36 -AA 37 (wherein, AA 01 is Gln or absent, AA 02 is Glu or absent, AA 03 is His or absent, AA 04 is Pro or absent, AA 05 is Asn or Gln or Glu or absent, AA 06 is Ala or absent, AA 07 is Arg or His or Ala, or absent, AA 08 is Lys or Ser or His, or absent, AA 09 is Tyr or Glu, or absent, AA 10 is Lys or Glu or Gln or His, or absent, AA 11 is Gly, AA 12 is Ala, AA 13 is Gln or Asn or Glu, AA 14 is Lys or His or Glu, AA 15 is Lys, Ser or Glu or Thr or His, AA 16 is Gly, AA 17 is Leu or Gly or Ser or Val, AA 18 is Ser or His, AA 19 is Gln or Ser or Lys or His, AA 20 is Gly, AA 21 is Cys, AA 22 is Phe, AA 23 is Gly, AA 24 is Leu, AA 25 is Pro or Lys, AA 26 is Leu, AA 27 is Asp or Glu, AA 28 is Arg, AA 29 is Ile, AA 30 is Gly, AA 31 is Ser, AA 32 is Leu or Nle or Met, AA 33 is Ser, AA 34 is Gly, AA 35 is Leu, AA 36 is Gly, AA 37 is Cys) and contains an amino acid sequence according to.

[0036] In certain embodiments of the present invention, amino acid substitutions are introduced into the CNP peptide to reduce the positive charge of the CNP peptide. For example, to remove the positive charge, Lys25 (AA 25 ) is substituted with Pro, and Lys19 (AA19) is substituted with Gln or Ser.

[0037] In certain embodiments of the present invention, the AA of the CNP peptide 13 is Gln, AA 19 is Gln or Ser, AA 25 is Pro, AA 32 is Leu.

[0038] In certain embodiments of the present invention, AA5 of the CNP peptide is Gln, AA 13 is Gln, AA 19 is Gln or Ser, AA 25 is Pro, AA 32 is Leu.

[0039] In certain embodiments of the present invention, the CNP peptide has the following amino acid sequence: GAQKKGSSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 136), ARKYKGAQKKGLSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 77), YKGAQKKGGSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 88), YKGAQKKGLSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 103), QEHPQARKYKGAQKKGLSSGCFGLPLDRIGSLSGLGC (SEQ ID NO: 67), and has any one of QEHPQARKYKGAQKKGLSSGCFGLPLERIGSLSGLGC (SEQ ID NO: 104).

[0040] Modifying group In one aspect of the present invention, the CNP compound contains a modifying group covalently bonded to an amino acid residue of the CNP peptide.

[0041] In one aspect of the present invention, the modifying group can form a non-covalent association with albumin, thereby promoting the circulation of the CNP compound in the bloodstream, which, compared with the plasma half-lives of CNP-22 (SEQ ID NO: 01) and CNP-37 (SEQ ID NO: 02), has the effect of extending the plasma exposure of the CNP compound and prolonging the plasma half-life. Therefore, the modifying group that has the effect of prolonging and extending the action time of the CNP compound may sometimes be also referred to as an extending group.

[0042] In some embodiments of the present invention, the modifying group is covalently bonded to the N-terminal amino acid of the CNP peptide.

[0043] In some embodiments of the present invention, the modifying group is covalently bonded to the N-terminal alpha-amine of the CNP peptide.

[0044] In one aspect of the present invention, the modifying group includes Chem.A, Chem.B, and Chem.C, where Chem.A is

Chemical formula

[0045] Abbreviations for modifying group elements used in this application are shown below. [Table 2]

[0046] In some embodiments of the present invention, the modifying group is the following non-limiting examples: Chem.E, Chem.F, Chem.G, Chem.H, Chem.I, and Chem.J [Chemical formula] [Chemical formula] (wherein the dotted line defines a bond via an amide bond to the CNP peptide) is selected from

[0047] The modifying groups of the present invention may exist in different stereoisomeric forms that have the same molecular formula and sequence of bonding atoms but differ only in the three-dimensional orientation of those atoms in space. The stereoisomers of the exemplified modifying groups of the present invention are shown in the experimental section by name as well as structure using standard nomenclature. Unless otherwise specified, the present invention relates to all stereoisomeric forms of the claimed modifying groups.

[0048] Functional characteristics In some aspects of the present invention, the CNP compound has desirable biophysical properties. The CNP compound exhibits an extended in vivo half-life. Also, the CNP compound of the present invention has biological activity. Furthermore, the compounds of the present invention have desirable stability. Additionally, the compounds of the present invention have desirable solubility. Moreover, the compounds of the present invention can be administered subcutaneously without eliciting an undesirable level of local tissue reaction such as necrosis.

[0049] Biological activity In one aspect of the present invention, the compound has biological activity in vitro, and this biological activity may be determined as the ability to activate the NPR2 receptor in vitro in a cell line that expresses the NPR2 receptor, and this ability may be determined as the ability to increase the production and concentration of cGMP, which represents receptor activation downstream of NPR2 in the cell. The biological activity in vitro may be determined, for example, as described in Example 2.

[0050] In one aspect of the present invention, the compound has biological activity in vivo, and this biological activity may be determined as the ability to increase the concentration of cGMP in plasma after intravenous or subcutaneous administration in any of rats or Göttingen minipigs or domestic LYD pigs.

[0051] Rats are an example of a suitable animal model, and the activity may be determined in vivo in such rats as described in Example 11.

[0052] The Göttingen minipig or domestic LYD pig is another example of a suitable animal model, and the activity may be determined in vivo in such pigs as described in Example 12.

[0053] Plasma half-life In one aspect of the invention, the present CNP compound has an extended plasma exposure and a prolonged in vivo plasma half-life compared to native CNP, which can be determined in a suitable in vivo pharmacokinetic study. The prolonged plasma exposure may be determined as the plasma half-life (T1 / 2) after intravenous or subcutaneous administration to animals such as rats, Göttingen minipigs, or domestic LYD pigs.

[0054] In some embodiments of the invention, the present CNP compound has a plasma half-life of at least 4 hours, more preferably at least 8 hours, or most preferably at least 10 hours, as determined as described in Example 11 after intravenous administration to rats.

[0055] In some embodiments, the present CNP compound has an in vivo plasma half-life of at least 10 hours, more preferably at least 40 hours, or most preferably at least 60 hours, as determined as described in Example 12 after intravenous or subcutaneous administration to Göttingen minipigs.

[0056] Bioavailability In one aspect of the invention, the present compound has suitable bioavailability after subcutaneous injection. The bioavailability (F%) may be determined as known in the art in any suitable animal model.

[0057] The Göttingen minipig or domestic LYD pig is an example of a suitable animal model, and the bioavailability may be determined in vivo in such Göttingen minipigs or domestic LYD pigs as described in Example 12.

[0058] In some embodiments of the present invention, the bioavailability of the present CNP compound after subcutaneous administration to guinea pigs is at least 40%, 50%, 60%, or 70%.

[0059] Stability In one aspect of the present invention, the compound has suitable physical and chemical stability. The lack of physical or chemical stability can lead to changes in the structure of the compound, resulting in the formation of chemical degradation products that may have reduced biological activity, decreased solubility, and / or increased immunogenic effects compared to the intact compound.

[0060] Physical stability can be evaluated, for example, by measuring the tendency of fibril formation using the ThT assay described in Example 6.

[0061] Chemical stability can be evaluated, for example, by measuring the amount of chemical degradation products (such as isomers, isoAsp, and hydrolysis products) at various time points after exposure to different conditions such as elevated temperature, as described in Example 8.

[0062] Stability can also be evaluated, for example, by examining the molecular ability to form covalent dimers and multimers called HMWP, as described in Example 7.

[0063] Solubility In accordance with the functional aspects of the present invention, the present CNP compound has a desired solubility. The low solubility of CNP significantly hinders the properties and therapeutic uses of its pharmaceutical formulations, so developing CNP compounds with high solubility in relevant formulations would improve the therapeutic utility.

[0064] As described herein, solubility is measured as described in Example 5. In certain embodiments, the CNP compound of the present invention has a solubility of at least 2000 μM, 3000 μM, or 4000 μM at either pH 4 or pH 6.5.

[0065] Pharmaceutical composition In one aspect, the present invention relates to a pharmaceutical composition comprising a compound according to the present invention and a pharmaceutically acceptable excipient. This composition is suitable for parenteral administration.

[0066] In one embodiment, the compound is present in the formulation at a concentration of about 0.1 mg / ml to about 50 mg / ml. In another aspect, the compound is present in the formulation at a concentration of about 10 mg / ml to about 30 mg / ml. In another embodiment, the formulation has a pH of 3.0 to 8.0. In another embodiment, the formulation has a pH of 3.5 to 5.5. In a further embodiment, the formulation has a pH of 6.0 to 7.0. A pharmaceutical composition comprising a compound according to the present invention can be prepared by conventional techniques, for example, as described in Remington’s Pharmaceutical Sciences, 1985 or Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.

[0067] The formulation may further comprise a buffer system, a preservative, an isotonic agent, a chelating agent, a stabilizer, and / or a surfactant. The use of such excipients in pharmaceutical compositions is known to those skilled in the art. For convenience, see Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.

[0068] In one embodiment, the pharmaceutical formulation is a liquid formulation, i.e., a formulation containing water. Such formulations are typically solutions or suspensions. In a further embodiment of the present invention, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0069] In another embodiment, the pharmaceutical formulation is a lyophilized formulation to which a solvent and / or diluent is added by a physician or patient before use. In another embodiment, the pharmaceutical formulation is a dry (e.g., lyophilized or spray-dried) formulation that is ready for use without any prior dissolution. The “dry form” is intended to dry a liquid pharmaceutical composition or formulation by any of freeze drying (i.e., lyophilization, see, for example, Williams and Polli (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (Masters (1991) in Spray-Drying Handbook (5 th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491-676, Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206, and Mumenthaler et al. (1994) Pharm. Res. 11:12-20), or air drying (see Carpenter and Crowe (1988) Cryobiology 25:459-470, and Roser (1991) Biopharm. 4:47-53).

[0070] In a further embodiment of the invention, the buffer is selected from the group consisting of acetate, carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, dihydrogen phosphate, hydrogen phosphate, phosphate, and tris(hydroxymethyl)aminomethane, bicine, tricine, malic acid, lactic acid, succinate, maleic acid, fumaric acid, tartaric acid, and aspartic acid, or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the invention.

[0071] In another embodiment of the present invention, the formulation further comprises a pharmaceutically acceptable preservative. In a further embodiment of the present invention, the formulation further comprises an isotonic agent, such as propylene glycol, mannitol, or glycerol. In a further embodiment of the present invention, the formulation further comprises a chelating agent.

[0072] In another embodiment of the present invention, the formulation further comprises a stabilizer. The use of stabilizers in pharmaceutical compositions is known to those skilled in the art. For convenience, see Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.

[0073] The pharmaceutical composition of the present invention may further comprise an amino acid base in an amount sufficient to reduce aggregate formation by the peptide during storage of the composition.

[0074] The term "amino acid base" is intended to mean an amino acid or a combination of amino acids in which any given amino acid is present either in its free base form or in the form of its salt. When a combination of amino acids is used, all the amino acids may be present in their free base form, all may be present in the form of their salts, or some may be present in their free base form and others in the form of salts. In one embodiment, the amino acids used in the preparation of the compositions of the present invention are those having charged side chains such as arginine, lysine, aspartic acid and glutamic acid. Any stereoisomer (i.e., L, D, or a mixture thereof) of a particular amino acid (e.g., methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine, and mixtures thereof), or a combination of these stereoisomers, may be present in the pharmaceutical compositions of the present invention as long as the particular amino acid is present either in its free base form or in the form of its salt. In one embodiment, the L-stereoisomer is used. The compositions of the present invention may also be formulated using derivatives of these amino acids. Suitable arginine derivatives include, for example, aminoguanidine, ornithine, and N-monoethyl-L-arginine, suitable methionine derivatives include ethionine and butionine, and suitable cysteine derivatives include S-methyl-L-cysteine. As with other amino acids, the amino acid derivatives are incorporated into the present compositions either in their free base form or in the form of their salts. In another embodiment of the present invention, the amino acid or its amino acid derivative is used at a concentration sufficient to prevent or delay protein aggregation.

[0075] In another embodiment of the present invention, the formulation further comprises a surfactant. In another embodiment of the present invention, the formulation further comprises a protease inhibitor. The use of protease inhibitors is particularly useful in pharmaceutical compositions containing enzyme precursors of proteases in order to inhibit autocatalysis.

[0076] Other components may be present in the pharmaceutical formulations of the present invention. Such additional components may include wetting agents, emulsifying agents, antioxidants, fillers, tonicity modifiers, chelating agents, metal ions, oily excipients, proteins (e.g., human serum albumin, gelatin, or protein) and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine, or histidine). Such additional components should, of course, not adversely affect the overall stability of the pharmaceutical formulations of the present invention.

[0077] The pharmaceutical composition according to the present invention may be administered to a patient in need of such treatment at several sites, such as local sites (e.g., skin and mucosal sites), sites bypassing absorption (e.g., intra-arterial, intravenous, intracardiac administration), and sites involved in absorption (e.g., intradermal, subcutaneous, intramuscular, or intra-abdominal administration).

[0078] Administration of the pharmaceutical composition according to the present invention can be obtained by several routes of administration to a patient in need of such treatment, such as by tongue, sublingual, buccal, intraoral, oral, enteral, nasal, pulmonary, e.g., bronchial and alveolar or combinations thereof, epidermal, dermal, transdermal, vaginal, rectal, oculus, e.g., by conjunctiva, and parenterally.

[0079] The compositions of the present invention can be administered in several dosage forms, such as solutions, suspensions, emulsions, microemulsions, multiple emulsions, foams, ointments, pastes, plasters, cataplasms, tablets, coated tablets, rinses, capsules, e.g., hard gelatin capsules or soft gelatin capsules, suppositories, rectal capsules, drops, gels, sprays, powders, aerosols, inhalants, eye drops, eye ointments, eye washes, pessaries, vaginal rings, vaginal ointments, injection solutions, in situ conversion solutions, e.g., in situ gelling, in situ coagulation, in situ precipitation, or in situ crystallization, infusions, and implants.

[0080] The compositions of the present invention can be further formulated or conjugated to drug carriers, drug delivery systems, and advanced drug delivery systems, for example, via covalent, hydrophobic, or electrostatic interactions, to enhance the stability of the compounds, increase bioavailability, increase solubility, achieve chronotherapy known to those skilled in the art, enhance patient compliance with medication, or any combination thereof.

[0081] The compositions of the present invention are useful for the preparation of solid, semi-solid, powder, and solution formulations for pulmonary administration of the CNP compounds of the present invention using, for example, metered-dose inhalers, dry powder inhalers, and nebulizers, which are all devices known to those skilled in the art.

[0082] The compositions of the present invention are useful for the preparation of controlled, sustained, extended, delayed, and sustained-release drug delivery systems.

[0083] Parenteral administration can be carried out by subcutaneous, intramuscular, intraperitoneal, or intravenous injection using a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be carried out using an infusion pump. A further option is a composition which may also be a solution or suspension for the administration of the CNP compounds of the present invention in the form of a nasal spray or a lung spray. Still further options include that the pharmaceutical composition containing the peptides of the present invention can also be adapted for transdermal administration, for example, by needle-free injection or from a patch, optionally an iontophoresis patch, or transmucosal administration, for example, oral administration.

[0084] The CNP compounds of the present invention can be administered via the pulmonary route in a vehicle as a solution, suspension, or dry powder using any of the known types of devices suitable for pulmonary drug delivery. Examples of these include, but are not limited to, three general types of aerosol generation for pulmonary drug delivery, which include jet or ultrasonic nebulizers, metered-dose inhalers, or dry powder inhalers (see Yu J, Chien YW. Pulmonary drug delivery: Physiologic and mechanistic aspects. Crit Rev Ther Drug Carr Sys 14(4)(1997)395-453).

[0085] In one embodiment of the present invention, a pharmaceutical formulation comprising a compound of the present invention is stable during use for more than 6 weeks and storage for more than 3 years. In another embodiment of the present invention, a pharmaceutical formulation comprising a compound of the present invention is stable during use for more than 2 weeks and storage for more than 2 years.

[0086] In one aspect, a process for preparing a pharmaceutical composition comprising a compound according to the present invention comprises mixing a compound according to the present invention with at least one pharmaceutically acceptable excipient.

[0087] Indications The present invention also relates to compounds for use as medicaments.

[0088] As used herein, the term "treatment" refers to the medical treatment of any human subject in need thereof. The timing and purpose of such treatment may vary from individual to individual according to the health status of the subject. Such treatment may be prophylactic, palliative, symptomatic, and / or curative.

[0089] In one aspect, the compounds of the present invention may be used for the treatment of cardio-renal metabolic diseases. Cardio-renal metabolic diseases include, but are not limited to, hypertension, arteriosclerosis, atherosclerosis, restenosis, acute myocardial infarction, pulmonary hypertension, acute decompensated heart failure, congestive heart failure, cardiac edema, renal edema, hepatic edema*, acute renal failure, chronic renal failure, insulin resistance, and type 2 diabetes, which are selected from the group consisting of.

[0090] In one embodiment, the compounds of the present invention are used for the treatment of congestive heart failure.

[0091] In one embodiment, the compounds of the present invention are used for the treatment of acute decompensated heart failure.

[0092] In one aspect, the compounds of the present invention may be used for the treatment of growth disorders including short stature, which may be associated with FGFR3-related skeletal dysplasias and related co-existing diseases.

[0093] In some embodiments, the compounds of the present invention are achondroplasia, chondrodysplasia, type 1 and type 2 lethal dysplasia, SHOX deficiency, Noonan syndrome, Costello, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutations, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome with melanosis), Lacrimo-Auriculo-Dento-Digital syndrome (LADD), osteomyelitis deformans, and SADDAN (severe achondroplasia dysplasia delay with melanosis), which are selected from the group consisting of diseases that may be used for the treatment of.

[0094] In some embodiments, the compounds of the present invention may be used for the treatment of diseases selected from the group consisting of osteogenesis imperfecta, achondrogenesis, stippled epiphyseal dysplasia, homozygous achondroplasia, rhizomelic form of stippled epiphyseal dysplasia, congenital spondyloepiphyseal dysplasia, congenital femoral shortening, Langer mesomelic dysplasia, neurofibromatosis, Rothmund syndrome, and neurofibromatosis type 1.

[0095] In some embodiments, the compounds of the present invention may be used for the treatment of phenotypes associated with growth disorders, including short stature that may be associated with FGFR3-related skeletal dysplasia, selected from the group consisting of growth delay, skull deformation, corrective defects, cervical spinal cord compression, spinal stenosis, pain associated with skeletal dysplasia, hydrocephalus, hearing loss due to chronic otitis media, cardiovascular disease, neurological disease, and obesity.

[0096] Unless otherwise indicated herein, terms presented in the singular may also include the plural.

[0097] List of Embodiments The present invention is further illustrated by the following non-limiting embodiments.

[0098] Embodiment 1: A CNP compound comprising a CNP peptide and a modifying group, wherein the net charge of the compound at physiological pH is 0 or negative, and the CNP peptide has the formula I: AA 01 -AA 02 -AA 03 -AA 04 -AA 05 -AA 06 -AA 07 -AA 08 -AA 09 -AA 10 -AA 11 -AA 12 -AA 13 -AA 14 -AA 15 -AA 16 -AA 17 -AA 18 -AA 19 -AA 20 -AA 21 -AA 22 -AA23 -AA 24 -AA 25 -AA 26 -AA 27 -AA 28 -AA 29 -AA 30 -AA 31 -AA 32 -AA 33 -AA 34 -AA 35 -AA 36 -AA 37 (wherein AA 01 is Gln or absent, AA 02 is Glu or absent, AA 03 is His or absent, AA 04 is Pro or absent, AA 05 is Asn or Gln or Glu or absent, AA 06 is Ala or absent, AA 07 is Arg or His or Ala or absent, AA 08 is Lys or Ser or His or absent, AA 09 is Tyr or Glu or absent, AA 10 is Lys or Glu or Gln or His or absent, AA 11 is Gly, AA 12 is Ala, AA 13 is Gln or Asn or Glu, AA 14 is Lys or His or Glu, AA 15is Lys, Ser, Glu, Thr, or His, AA 16 is Gly, AA 17 is Leu, Gly, Ser, or Val, AA 18 is Ser or His, AA 19 is Gln, Ser, Lys, or His, AA 20 is Gly, AA 21 is Cys, AA 22 is Phe, AA 23 is Gly, AA 24 is Leu, AA 25 is Pro or Lys, AA 26 is Leu, AA 27 is Asp or Glu, AA 28 is Arg, AA 29 is Ile, AA 30 is Gly, AA 31 is Ser, AA 32 is Leu, Nle, or Met, AA 33 is Ser, AA 34 is Gly, AA 35 is Leu, AA 36 is Gly, AA 37 contains an amino acid sequence following (wherein it is Cys), the modifying group contains Chem.A, Chem.B, and Chem.C, Chem.A is [Chemical formula] (wherein p is an integer in the range of 14 to 20, * represents an amide bond connecting Chem.A and Chem.B) and is selected from the group consisting of Chem.B is Chem.B1 and Chem.B2 [Chemical formula] (wherein q is an integer in the range of 1 to 8, * represents an amide bond connecting Chem.A- and Chem.B-, ** represents an amide bond connecting Chem.B- and Chem.C-) and is selected from the group consisting of Chem.C is [Chemical formula] (wherein r is an integer in the range of 0 to 4, s is an integer in the range of 0 to 3, t is an integer in the range of 0 to 1, ** represents an amide bond connecting Chem.B- and Chem.C-, *** represents an amide bond connecting Chem.C- and the N-terminal alpha-amine on the CNP peptide), and is a CNP compound selected from the group consisting of Embodiment 2: AA of the CNP peptide 13 is Gln, AA 19 is Gln or Ser, AA 25 is Pro, AA 32 is Leu, and is the CNP compound according to Embodiment 1. Embodiment 3: AA5 of the CNP peptide is Gln, AA 13 is Gln, AA 19 is Gln or Ser, AA 25 is Pro, AA 32 is Leu, and is the CNP compound according to Embodiment 1. Embodiment 4: The CNP compound according to Embodiment 1, wherein the CNP peptide contains one of the following amino acid sequences. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] Embodiment 5: The CNP compound according to Embodiment 1, wherein the CNP peptide has any one of the following amino acid sequences. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] Embodiment 6: The CNP peptide has the following amino acid sequence: GAQKKGSSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 136), ARKYKGAQKKGLSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 77), YKGAQKKGGSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 88), YKGAQKKGLSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 103), QEHPQARKYKGAQKKGLSSGCFGLPLDRIGSLSGLGC (SEQ ID NO: 67), and a CNP compound according to Embodiment 1, having any one of Embodiment 7: The CNP peptide has the following amino acid sequence: QEHPQARKYKGAQKKGLSSGCFGLPLDRIGSLSGLGC (SEQ ID NO: 67), the CNP compound according to Embodiment 1. Embodiment 8: The CNP compound according to any one of the preceding embodiments, wherein the modifying group is covalently bonded to the N-terminal alpha-amine of the CNP peptide. Embodiment 9: The CNP compound according to any one of the preceding embodiments, wherein Chem.A of the modifying group has 16 p. Embodiment 10: Chem.A of the modifying group is Chem.A2

Chemical formula

Chemical formula

Chemical formula

Table 5

Chemical formula

Chemical formula

Chemical formula

Examples

[0099] Materials and Methods List of Abbreviations 2-ClTrt 2-chlorotrityl resin Aldrithiol-4 4,4’-dipyridyldisulfide AUC Area Under the Curve BEH Ethylene Bridged Hybrid Boc tert-butyloxycarbonyl C18d Octadecanoic acid C20d Eicosandioic acid CAD Charged Aerosol Detector cGMP Cyclic Guanosine Monophosphate CL Clearance CSH Charged Surface Hybrid DCM Dichloromethane DgGlu D-configuration gamma-glutamoyl DIC N,N-Diisopropylcarbodiimide DMEM Dulbecco's Modified Eagle Medium DMF Dimethylformamide DMSO Dimethyl Sulfoxide DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid ESI Electrospray Ionization F% Bioavailability Fd Faraday Fmoc Fluorenylmethyloxycarbonyl FPLC High Performance Protein Liquid Chromatography gGlu gamma-glutamoyl HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid HFIP Hexafluoro-2-propanol HMWP High Molecular Weight Protein HSA Human Serum Albumin i.v. Intravenous IPA Isopropanol LCMS Liquid Chromatography Mass Spectrometry LLOQ Lower Limit of Quantification LYD Landrace, Yorkshire, and Duroc MeCN Acetonitrile MMPX 8-Methoxymethyl-3-isobutyl 1-methylxanthine MS Mass Spectrometry NAD No Abnormality Detected NCA Non-Compartmental Analysis n.d. Not Detected Nle(X) Norleucine NMP N-Methyl-2-pyrrolidone OEG Oligoethylene Glycol Oxyma Pure Ethyl Cyano Hydroxyiminoacetate Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl PBS Phosphate Buffered Saline PD Pharmacodynamics PDA Photodiode Array Detection PK Pharmacokinetics QC Quality Control RP Reverse Phase RPM Revolutions Per Minute s.c. Subcutaneous SEC Size Exclusion Chromatography SIL Stable Isotope Labeling SPPS Solid Phase Peptide Synthesis T1 / 2 Half-Life tBu tert-Butyl Tetrazole-C18 17-(2H-tetrazol-5-yl)heptadecanoic acid TF time-of-flight type TFA trifluoroacetic acid ThT Thioflavin T TIPS triisopropylsilane Tris 2-amino-2-(hydroxymethyl)propane-1,3-diol Trt trityl TUV adjustable UV UPLC ultra performance liquid chromatography UV ultraviolet ray

[0100] General preparation method Method A - solid-phase peptide synthesis Peptides can be synthesized by solid-phase peptide synthesis known in the art, and the methods used to synthesize exemplary compounds of the present invention are described below.

[0101] The synthesis of the compound was carried out on a Symphony X solid-phase peptide synthesizer (GYROS Protein Technologies AB, Tucson, AZ).

[0102] Typically, the synthesis was carried out using 450 μmol of resin, although syntheses using 150 or 300 μmol of resin were also used. In a typical synthesis, the resin was washed with DMF. After washing with DMF, the resin was incubated for 30 minutes with Fmoc-protected amino acid (5 equivalents, 0.3 M in 0.3 M Oxyma Pure in DMF, 7.5 mL), DIC (5 equivalents, 0.75 M in DMF, 3.0 mL), and collidine (5 equivalents, 0.75 M in DMF, 3.0 mL). Then, additional DIC (5 equivalents, 0.75 M in DMF, 3.0 mL) was added and the resin was incubated for 90 minutes. After each coupling, the resin was capped by treating it with acetic anhydride (1 M in DMF, 7.5 mL) and collidine (0.75 M, 3.0 mL) for 20 minutes. The fatty acid was coupled for 6 hours using fatty acid mono-tert-butyl ester (5 equivalents, 0.3 M in 0.3 M Oxyma Pure, 7.5 mL), DIC (5 equivalents, 0.75 M in DMF, 3.0 mL), and collidine (5 equivalents, 0.75 M in DMF, 3.0 mL).

[0103] The Fmoc-protected amino acids used were, for example, recommended standards supplied by Anaspec, Bachem, Iris Biotech, or NovabioChem: They were Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, and Fmoc-Nle-OH. Unless otherwise specified, the naturally occurring L-form of the amino acid was used. When serine or threonine is present in the peptide, pseudoproline dipeptides may be used (see also W.R. Sampson et al., J. Pept. Sci. 1999, 5, 403-409).

[0104] For peptides acylated at any position during SPPS, the following suitably protected building blocks such as, but not limited to, Fmoc-8-amino-3,6-dioxaoctanoic acid and Boc-Glu(Fmoc)-OH were supplied, for example, by Anaspec, Bachem, Iris Biotech, or Novabiochem.

[0105] Octadecanedioic acid mono-tert-butyl-ester and eicosanedioic acid mono-tert-butyl ester were prepared as known in the art, for example, as described in WO2010 / 102886(A1).

[0106] (S)-4-(17-(1H-tetrazol-5-yl)heptadecanamido)-5-(tert-butoxy)-5-oxopentanoic acid, a protected building block incorporating 17-(2H-tetrazol-5-yl)heptadecanoic acid (tetrazole-C18), was synthesized according to the following procedure. 5-Chloro-1-((dimethylamino)(dimethyliminio)methyl)-1H-benzo[d][1,2,3]triazol-3-oxide tetrafluoroborate (TCTU, 5.87 g, 16.5 mmol) and N-methylmorpholine (NMM, 8.00 mL, 72.8 mmol) were sequentially added to a stirred suspension of 17-(1H-tetrazol-5-yl)heptadecanoic acid (5.10 g, 15.0 mmol) in dry DMF (70 mL). The mixture was stirred at room temperature for 30 h. H-L-Glu-OtBu (L-glutamic acid 1-tert-butyl ester, 4.58 g, 22.5 mmol) was added in one portion. The suspension turned into a yellow solution within 5 min. After standing at room temperature for 4 h, it was poured into an ice-cooled, stirred solution of hydrochloric acid (35%, 20 mL) in water (1 L). The crude product was filtered off, washed with water (3 × 100 mL), dried briefly by suction on a sintered glass frit, and redissolved in DMF (70 mL). This solution was poured into water (1 L), the resulting suspension was stirred for 5 min, the solid was filtered off, washed with water (3 × 100 mL), and dried in vacuo. The crude product was dissolved in ethyl acetate (400 mL), filtered through a silica gel (Silicagel 60, 0.040–0.063 mm) column (100 g), and the column was washed with ethyl acetate (600 mL). Ethyl acetate was removed in vacuo. The solid residue was boiled in 1,2-dichloroethane (200 mL) with vigorous stirring for 5 min, then the emulsion was cooled to room temperature in 5 min and crystallized overnight at the same temperature. The waxy crystals were filtered off, washed with 1,2-dichloromethane (50 mL) and n-heptane (100 mL), and air-dried. This purification step was repeated once more. Finally, the compound was dissolved in 1,4-dioxane (50 mL) and lyophilized to give (S)-4-(17-(1H-tetrazol-5-yl)heptadecanamido)-5-(tert-butoxy)-5-oxopentanoic acid as an off-white powder.

[0107] For peptides featuring a C-terminal cysteine, the resin was pre-packed H-Cys(Trt)-2-ClTrt (e.g., from Bachem or Novabiochem).

[0108] Method B - Peptide cleavage from resin. After synthesis, the resin was washed with DCM, and the peptide was cleaved by treatment with TFA / TIPS / water / DTT (e.g., in a ratio of 90:5:2.5:2.5 or 92.5:2.5:2.5:5) for 2 - 3 hours, followed by precipitation with diethyl ether. The precipitate was centrifuged and washed several times with diethyl ether.

[0109] Method C - Disulfide bridge formation Native CNP contains a disulfide bridge, which, when reduced, abolishes binding to the NPR2 receptor. Methods for disulfide bridge formation for synthetic peptides are known in the art, and three methods used to prepare exemplary compounds of the present invention are described below.

[0110] Folding using aldithiol - 4 The crude precipitated peptide from 150 μmol synthesis was dissolved in 10 ml of DMSO, followed by addition of water to make 700 mL and an additional 100 mL of MeCN. Finally, 2 mL of 0.5 M NaOAc buffer pH 5.0 was added to the peptide solution. The pH was adjusted to pH 5 - 6.5 with 1 M NaOH until the solution became clear. The amount of peptide in the solution was quantified using CAD, and a fresh stock solution of aldithiol - 4 (2 mg / ml in MeOH) was prepared. 1.0 equivalent of aldithiol - 4 in MeOH was added over 5 - 10 minutes with vigorous stirring, compared to the peptide content. The solution was stirred for 10 minutes, and an additional 0.5 or 1.0 equivalent of the aldithiol - 4 stock solution was added. Once determined to be complete by LCMS, the reaction was quenched with 2 mL of TFA. The peptide was purified by RP - HPLC as described below.

[0111] Folding Using DMSO The crude peptide precipitate from 450 μmol synthesis was dissolved in 1:1 MeCN / water (50 mL), added to 500 mM Tris-HCl pH 8.0 buffer (1 L), and diluted with water (1.4 L) and DMSO (600 mL). The solution was left to stand at room temperature with gentle stirring for 16 - 24 hours. The reaction was quenched by adding 5.0 equivalents of iodoacetamide (50 mM in water), acidified to pH 2 with TFA after 10 minutes, and diluted to 5 L with water. The peptide was purified by RP-HPLC as described below.

[0112] Folding Using Redox Buffer of Cysteine / Cystine or Cysteamine / Cystamine The crude peptide precipitate from 150 μmol synthesis was dissolved in DMSO (20 mL) and added to a buffer of 50 mM Tris-HCl, 3 mM cysteine, 0.3 mM cystine pH 8.2 (800 mL), or a buffer of 50 mM Tris-HCl, 3 mM cysteamine, 0.3 mM cystamine pH 8.2. The solution was left to stand at room temperature with gentle stirring for 16 - 24 hours. The reaction was quenched by adding 3 mL of TFA, diluted with 100 mL of MeCN, and purified by RP-HPLC as described below.

[0113] Method D - Purification Methods for peptide purification are known in the art, and two methods used to purify exemplary compounds of the present invention are described below.

[0114] Reverse Phase Preparative HPLC Using Acidic Eluent The crude folded peptide was purified by preparative reverse-phase HPLC on a C18 column, such as a Waters Xbridge Prep C18 OBD, 5 μm, 250×50 mm or a Phenomenex Gemini Axia NX C18, 5 μm, 250×30 mm, using a Waters Delta Prep 4000 or Gilson Model 322 H2 system. The peptide was eluted from the column using a linear gradient of eluents A (0.1% TFA in water) and B (0.1% TFA in acetonitrile), for example, 27–42% eluent B over 30 min at a constant flow rate of 25 mL / min

Number

Number

[0115] Reverse-phase preparative HPLC using a near-neutral eluent If further purification was required, the lyophilized peptide TFA salt was purified on a similar preparative HPLC system using a neutral or near-neutral pH eluent, for example, eluent A: 100 mM NaOAc pH 6.5, 5% MeCN, eluent B: MeCN. The peptide was eluted from the column using a linear gradient of eluents A and B, for example, 25–55% eluent B over 30 min at a constant flow rate of 60 mL / min

Number

[0116] Method E - Resalting and Desalting In some cases, it was desirable to change the counterion to sodium to facilitate formulation at pH 6.5. Methods for exchanging peptide counterions are known in the art, The methods used to change the counterion for some of the exemplary compounds of the present invention are described below.

[0117] Size exclusion desalting The counterion was exchanged by solubilizing the peptide TFA salt at 2 - 3 mg / mL in 400 mM NaOAc pH 6.5 and 20% MeCN. The dissolved peptide was then desalted on an Akta Purifer FPLC system using a HiPrep 26 / 10 desalting column. The column was pre - equilibrated with 20% MeCN, the peptide was injected, and eluted at 6 mL / min with a uniform concentration of 20% MeCN for 1.5 CV. The peptide was collected, quantified by CAD analysis, and lyophilized to obtain the peptide sodium salt as a colorless powder.

[0118] General methods of detection and characterization To confirm identity, measure purity, and quantify the amount of the synthesized peptide prepared, the peptide was characterized using methods such as LCMS and reverse - phase UPLC known in the art. The methods used to characterize the exemplary compounds of the present invention are described below.

[0119] Method F - Analytical chromatography method The prepared CNP compound was characterized by RP - UPLC and LCMS, and the amount prepared was quantified by RP - UPLC / CAD.

[0120] LCMS34 LCMS was performed on a setup consisting of an ACQUITY UPLC system and an Xevo G2 - XS QTOF mass spectrometer. The system was equipped with a Waters ACQUITY UPLC BEH C18 column, 1.7 μm, 2.1 mm×50 mm, and a column temperature of 60 °C. UV detection was at 214 nm. The MS ionization mode was ESI+.

[0121] Eluent A: 0.1% formic acid in water, Eluent B: 0.1% formic acid in acetonitrile. The analysis was carried out by injecting an appropriate volume of the sample (preferably 1 - 10 μL) into the column and eluting it with a linear gradient of Eluent A and B from 5 - 95% of Eluent B at a constant flow rate of 0.4 mL / min over 4.0 minutes.

[0122] RP-UPLC107: Alternatively, LCMS was carried out on a setup consisting of a Waters ACQUITY UPLC system and a Waters QDA mass detector. The system was equipped with a Waters ACQUITY UPLC BEH C18 column, 1.7 μm, 2.1 mm × 50 mm, and a column temperature of 40 °C. UV detection was at 214 nm.

[0123] Eluent A: 0.05% TFA in water, Eluent B: 0.05% TFA in acetonitrile. The analysis was carried out by injecting an appropriate volume of the sample (preferably 1 - 10 μL) into the column and eluting it with a linear gradient of Eluent A and B from 5 - 60% of Eluent B at a constant flow rate of 0.9 mL / min over 1.6 minutes.

[0124] RP-UPLC02: RP-UPLC02 was carried out on a Waters ACQUITY UPLC system with a TUV or PDA detector. The system was equipped with a Waters ACQUITY UPLC BEH C18 column, 1.7 μm, 2.1 mm × 150 mm, and a column temperature of 40 °C. UV detection was at 214 nm. Eluent A: 0.05% TFA in water, Eluent B: 0.05% TFA in acetonitrile.

[0125] The analysis was carried out by injecting an appropriate volume of the sample (preferably 1 - 10 μL) into the column and eluting it with a linear gradient of Eluent A and B from 5 - 95% of Eluent B at a constant flow rate of 0.4 mL / min over 16.0 minutes.

[0126] RP-UPLC61: RP-UPLC61 was performed on a Waters ACQUITY UPLC system equipped with a TUV or PDA detector. The system was equipped with a Waters ACQUITY UPLC BEH Shield C18 column, 1.7 μm, 2.1 mm × 150 mm, and a column temperature of 60 °C. UV detection was at 214 nm. Eluent A: 20 mM Na2SO4, 2 mM Na2HPO4, 2 mM NaH2PO4 in 9:1 water / MeCN at pH 7.2, Eluent B: 7:3 MeCN / water. The analysis was carried out by injecting an appropriate volume of sample (preferably 1 - 10 μL) onto the column and eluting it with a linear gradient of Eluents A and B of 5 - 20% B over 3 minutes at a constant flow rate of 0.4 mL / min, then 20 - 80% B over 17 minutes, and then 80 - 90% B over 1 minute.

[0127] CAD02 peptide quantification: CAD was performed on a Thermo Scientific Vanquish UPLC system equipped with UV-DAD and CAD (charged aerosol detector). The system was equipped with a Waters ACQUITY UPLC CSH C18 column, 1.7 μm, 2.1 mm × 50 mm, and a column temperature of 40 °C. UV detection was at 214 nm. Eluent A: 0.1% v / v TFA in water, Eluent B: 0.1% v / v TFA in MeCN. The analysis was carried out by injecting an appropriate volume of sample onto the column and eluting it with a linear gradient of Eluents A and B of 0 - 95% B over 4 minutes at a constant flow rate of 0.45 mL / min. Calibration was performed using a standard curve generated by injecting an external standard of Insulin Aspart, 3.55 mg / mL.

[0128] Method G - Preparation of CNP compound formulations for in vivo experiments Formulations for in vivo studies were prepared according to the principle outlined below.

[0129] Procedure: The formulation for in vivo study was prepared by dissolving the lyophilized peptide in a formulation buffer having the same composition as the final formulation (the final formulation composition is specified in Examples 9 to 15). The following final formulation compositions were used: · pH 4.0: 5 mM sodium acetate, 250 mM glycerol, pH 4.0 · pH 4.0: 5 mM sodium acetate, 240 mM propylene glycol, pH 4.0 · pH 4.0: 5 mM sodium acetate, 250 mM glycerol, 0.007% polysorbate 20, pH 4.0 · pH 6.0: 20 mM sodium phosphate, 223 mM propylene glycol, pH 6.0 · pH 6.5: 8 mM sodium phosphate, 250 mM glycerol, pH 6.5 · pH 7.4: 8 mM sodium phosphate, 250 mM glycerol, pH 7.4 · pH 7.4: 8 mM sodium phosphate, 250 mM glycerol, 0.007% polysorbate 20, pH 7.4 · pH 7.5: 8 mM sodium phosphate, 250 mM glycerol, pH 7.5 · pH 5.5: 1.33 mM citric acid monohydrate, 3.67 mM citrate, trisodium, 52 mg / ml trehalose, 15 mg / ml D - mannose, 0.73 mg / ml L - methionine, 0.05 mg / ml polysorbate 80, pH 5.5

[0130] The formulation procedure was slightly different for the pH 6.5 formulation compared to the formulations for other pH targets for practical reasons.

[0131] Formulation at pH 6.5: The lyophilized peptide was dissolved in a buffer solution at pH 7.4 (8 mM sodium phosphate, 250 mM glycerol, pH 7.4) to a nominal concentration exceeding the target peptide concentration by 20 - 50%, and the pH was adjusted to pH 6.5 using 0.2 N NaOH or 0.2 N HCl. The actual peptide concentration was determined using CAD (Method F, CAD02), and a volume of buffer solution at pH 6.5 (8 mM sodium phosphate, 250 mM glycerol, pH 6.5) corresponding to the nominal concentration corresponding to the target peptide concentration was added, and the pH was adjusted to pH 6.5 using 0.2 N NaOH or 0.2 N HCl. The actual peptide concentration was determined using CAD (Method F, CAD02). The formulation was sterile filtered (0.22 μm), the final peptide concentration was determined using CAD (Method F, CAD02), and the formulation was filled into sterile containers.

[0132] Formulation at pH 4.0, pH 6.0, pH 7.4, and pH 7.5: The lyophilized peptide was dissolved in a formulation buffer having a composition corresponding to the composition of the final formulation (specified in Examples 9 - 15) to a nominal concentration exceeding the target peptide concentration by 20 - 50%, and the pH was adjusted to the target using 0.2 N NaOH or 0.2 N HCl. The actual peptide concentration was determined using CAD (Method F, CAD02), and a volume of the same formulation buffer corresponding to the nominal concentration corresponding to the target peptide concentration was added, and the pH was adjusted to the target using 0.2 N NaOH or 0.2 N HCl. The actual peptide concentration was determined using CAD (Method F, CAD02). The formulation was sterile filtered (0.22 μm), the final peptide concentration was determined using CAD (Method F, CAD02), and the formulation was filled into sterile containers.

[0133] [Example 1] Synthesis of CNP Compounds CNP compounds were synthesized and characterized according to the general preparation method described above. Exemplary CNP compounds and their components are summarized in Table 1. The protractor element and linker element in Table 1 together form the modifying group of the CNP compound.

Table 6 - 1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

[0134] Chem.1; Compound ID 0065; Sequence No. 1 hCNP22

Chem.

[0135] Chem.2; Compound ID 1510; Sequence No.2 hCNP37

Chem.

[0136] Chem.3; Compound ID 0312; Sequence No.3 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-4,5Q,13Q,32Nle]-hCNP37

Chem.

[0137] Chem.4; Compound ID 0776; SEQ ID NO: 4 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,13Q,27E,32L]-hCNP37

Chem.

[0138] Chem.5; Compound ID 1235; SEQ ID NO: 5 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,9E,10H,13Q,32L]-hCNP37

Chem.

[0139] Chem.6; Compound ID 0262; SEQ ID NO: 6 [N-terminal([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), des1-8, 32Nle]-hCNP37

Chem.

[0140] Chem.7; Compound ID 0296; Sequence number 7 [N-terminal([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), des1-4, 15S, 19S, 32Nle]-hCNP37

Chem.

[0141] Chem.8; Compound ID 0313; Sequence number 8 [N-terminal([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), des1-10,32Nle]-hCNP37

Chem.

[0142] Chem.9; Compound ID 0334; Sequence number 9 [N-terminal([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), des1-10,13Q,32Nle]-hCNP37

Chem.

[0143] Chem.10; Compound ID 1221; Sequence number 10 [N-terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,32L]-hCNP37

Chem.

[0144] Chem.11; Compound ID 1222; SEQ ID NO: 11 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,9E,10E,13Q,32L]-hCNP37

Chem.

[0145] Chem.12; Compound ID 1223; SEQ ID NO: 12 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,10E,13Q,14E,32L]-hCNP37

Chem.

[0146] Chem.13; Compound ID 1224; SEQ ID NO: 13 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,10E,13Q,19S,32L]-hCNP37

Chem.

[0147] Chem.14; Compound ID 1225; Sequence number 14 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,25P,32L]-hCNP37

Chem.

[0148] Chem.15; Compound ID 1226; Sequence number 15 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,14E,25P,32L]-hCNP37

Chem.

[0149] Chem.16; Compound ID 1227; Sequence number 16 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10E, 13Q, 19S, 25P, 32L]-hCNP37

Chem.

[0150] Chem.17; Compound ID 1228; SEQ ID NO: 17 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10E, 13Q, 14E, 19S, 25P, 32L]-hCNP37

Chem.

[0151] Chem.18; Compound ID 1229; SEQ ID NO: 18 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 9E, 13Q, 14E, 32L]-hCNP37

Chem.

[0152] Chem.19; Compound ID 1230; SEQ ID NO: 19 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,9E,13Q,14E,19S,32L]-hCNP37

Chem.

[0153] Chem.20; Compound ID 1231; SEQ ID NO: 20 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,9E,10Q,13Q,14E,32L]-hCNP37

Chem.

[0154] Chem.21; Compound ID 1232; SEQ ID NO: 21 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,13Q,14E,32L]-hCNP37

Chem.

[0155] Chem.22; Compound ID 1233; SEQ ID NO: 22 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,19S,25P,32L]-hCNP37

Chem.

[0156] Chem.23; Compound ID 1236; SEQ ID NO: 23 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,9E,10H,13Q,14E,32L]-hCNP37

Chem.

[0157] Chem.24; Compound ID 1237; SEQ ID NO: 24 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10H,13Q,14E,32L]-hCNP37

Chem.

[0158] Chem.25; Compound ID 1240; Sequence number 25 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8, 10H, 13Q, 14E, 25P, 32L]-hCNP37

Chem.

[0159] Chem.26; Compound ID 1241; Sequence number 26 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8, 10H, 13Q, 19S, 25P, 32L]-hCNP37

Chem.

[0160] Chem.27; Compound ID 1242; Sequence number 27 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,10H,13Q,14E,19S,25P,32L]-hCNP37

Chem.

[0161] Chem.28; Compound ID 1274; SEQ ID NO: 28 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,10E,13E,19Q,25P,32L]-hCNP37

Chem.

[0162] Chem.29; Compound ID 1287; SEQ ID NO: 29 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-9,10E,13Q,14E,19S,25P,32L]-hCNP37

Chem.

[0163] Chem.30; Compound ID 1288; Sequence number 30 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-9,10E,13Q,15E,19S,25P,32L]-hCNP37

Chem.

[0164] Chem.31; Compound ID 1289; Sequence number 31 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,15E,19S,25P,32L]-hCNP37

Chem.

[0165] Chem.32; Compound ID 1290; Sequence number 16 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl)amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10E, 13Q, 19S, 25P, 32L]-hCNP37

Chem.

[0166] Chem.33; Compound ID 1302; Sequence number 32 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl)amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10H, 13Q, 15T, 19S, 25P, 32L]-hCNP37

Chem.

[0167] Chem.34; Compound ID 1303; Sequence number 33 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl)amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10H, 13Q, 15T, 17V, 19S, 25P, 32L]-hCNP37

Chem.

[0168] Chem.35; Compound ID 1304; Sequence number 34 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10H,13Q,14H,15T,19S,25P,32L]-hCNP37

Chem.

[0169] Chem.36; Compound ID 1305; Sequence number 35 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,14H,15T,19S,25P,32L]-hCNP37

Chem.

[0170] Chem.37; Compound ID 1309; Sequence number 36 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,9E,10H,13Q,25P,32L]-hCNP37

Chem.

[0171] Chem.38; Compound ID 1310; SEQ ID NO: 37 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,9E,10H,13Q,19S,25P,32L]-hCNP37

Chem.

[0172] Chem.39; Compound ID 1311; SEQ ID NO: 38 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,9E,10H,13Q,17V,19S,25P,32L]-hCNP37

Chem.

[0173] Chem.40; Compound ID 1312; SEQ ID NO: 39 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,9E,10H,13Q,15T,17V,19S,25P,32L]-hCNP37

Chem.

[0174] Chem.41; Compound ID 1322; SEQ ID NO: 40 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10H,13Q,14H,15H,27E,32L]-hCNP37

Chem.

[0175] Chem.42; Compound ID 1323; SEQ ID NO: 40 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10H, 13Q, 14H, 15H, 27E, 32L]-hCNP37

Chem.

[0176] Chem.43; Compound ID 1324; Sequence number 41 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 13Q, 14H, 15H, 19Q, 27E, 32L]-hCNP37

Chem.

[0177] Chem.44; Compound ID 1338; Sequence number 42 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10E, 13Q, 19S, 25P, 27E, 32L]-hCNP37

Chem.

[0178] Chem.45; Compound ID 1339; SEQ ID NO: 43 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-9,10E,13Q,19S,25P,32L]-hCNP37

Chem.

[0179] Chem.46; Compound ID 1340; SEQ ID NO: 44 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,14H,15H,19Q,32L]-hCNP37

Chem.

[0180] Chem.47; Compound ID 1341; SEQ ID NO: 45 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,13Q,14H,15H,19Q,25P,32L]-hCNP37

Chem.

[0181] Chem.48; Compound ID 1345; Sequence number 46 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,10H,13Q,14H,15H,32L]-hCNP37

Chem.

[0182] Chem.49; Compound ID 1346; Sequence number 47 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,10H,13Q,14H,15H,25P,32L]-hCNP37

Chem.

[0183] Chem.50; Compound ID 1347; SEQ ID NO: 48 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-10,13Q,19H,25P,32L]-hCNP37

Chem.

[0184] Chem.51; Compound ID 1348; SEQ ID NO: 49 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-10,13Q,18H,19S,25P,32L]-hCNP37

Chem.

[0185] Chem.52; Compound ID 1350; SEQ ID NO: 50 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 10H, 13Q, 14H, 15T, 19S, 25P, 32L]-hCNP37

Chem.

[0186] Chem.53; Compound ID 1351; Sequence number 51 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 10H, 13Q, 14H, 15T, 17V, 19S, 25P, 32L]-hCNP37

Chem.

[0187] Chem.54; Compound ID 1352; Sequence number 33 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10H, 13Q, 15T, 17V, 19S, 25P, 32L]-hCNP37

Chem.

[0188] Chem.55; Compound ID 1353; SEQ ID NO: 36 [N-terminal ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-8,9E,10H,13Q,25P,32L]-hCNP37

Chem.

[0189] Chem.56; Compound ID 1354; SEQ ID NO: 52 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-5,10H,13Q,15T,17V,19S,25P,32L]-hCNP37

Chem.

[0190] Chem.57; Compound ID 1355; SEQ ID NO: 22 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,19S,25P,32L]-hCNP37

Chem.

[0191] Chem.58; Compound ID 1356; SEQ ID NO: 22 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,19S,25P,32L]-hCNP37

Chem.

[0192] Chem.59; Compound ID 1357; SEQ ID NO: 5 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl)amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,9E,10H,13Q,32L]-hCNP37

Chem.

[0193] Chem.60; Compound ID 1359; SEQ ID NO: 53 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl)amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,9E,10H,13Q,15T,17V,19S,25P,32L]-hCNP37

Chem.

[0194] Chem.61; Compound ID 1360; SEQ ID NO: 54 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl)amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,9E,10H,13Q,14H,15T,17V,19S,25P,32L]-hCNP37

Chem.

[0195] Chem.62; Compound ID 1375; Sequence number 14 [N-terminus ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,25P,32L]-hCNP37

Chem.

[0196] Chem.63; Compound ID 1376; Sequence number 14 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,25P,32L]-hCNP37

Chem.

[0197] Chem.64; Compound ID 1377; Sequence number 55 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,17G,25P,32L]-hCNP37

Chem.

[0198] Chem.65; Compound ID 1378; SEQ ID NO: 56 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,6G,7G,8G,10E,13Q,19S,25P,32L]-hCNP37

Chem.

[0199] Chem.66; Compound ID 1379; SEQ ID NO: 57 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), 5Q,8S,10E,13Q,19S,25P,32L]-hCNP37

Chem.

[0200] Chem.67; Compound ID 1380; SEQ ID NO: 58 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), 5E, 8S, 10E, 13Q, 19S, 25P, 32L]-hCNP37

Chem.

[0201] Chem.68; Compound ID 1381; SEQ ID NO: 59 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), 5Q, 7H, 10H, 13Q, 14H, 15H, 17S, 19Q, 25P, 32L]-hCNP37

Chem.

[0202] Chem.69; Compound ID 1382; SEQ ID NO: 60 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,7H,13Q,19Q,25P,32L]-hCNP37

Chem.

[0203] Chem.70; Compound ID 1383; SEQ ID NO: 61 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,7H,10H,13Q,15H,17S,19Q,25P,32L]-hCNP37

Chem.

[0204] Chem.71; Compound ID 9384; SEQ ID NO: 62 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-6,7G,8Q,9A,10P,13Q,17S,19Q,25P,32L]-hCNP37 [Chem.] Molecular weight: 3704.2302. LCMS34: Calculated m / z 1235.7434; Measured m / z 1235.5400; Calculated m / z 927.0576; Measured m / z 926.9010

[0205] Chem.72; Compound ID 1385; SEQ ID NO: 63 [N-terminus([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), des1-5, 10E, 13Q, 25P, 32L]-hCNP37 [Chem.] Molecular weight: 4121.7322. LCMS34: Calculated m / z 1374.9107; Measured m / z 1374.6120; Calculated m / z 1031.4331; Measured m / z 1031.2130

[0206] Chem.73; Compound ID 1386; SEQ ID NO: 64 [N-terminus([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), des1-5, 13Q, 19S, 25P, 32L]-hCNP37 [Chem.] Molecular weight: 4079.6955. LCMS34: Calculated m / z 1360.8985; Measured m / z 1360.7680; Calculated m / z 1020.9239; Measured m / z 1020.8160

[0207] Chem.74; Compound ID 1387; SEQ ID NO: 65 [N-terminal([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), des1-5,6G,7G,8G,13Q,19S,25P,32L]-hCNP37 [Chem.] Molecular weight: 3895.4136. LCMS34: Calculated m / z 1299.4712; Measured m / z 1299.3300; Calculated m / z 974.8534; Measured m / z 974.7500

[0208] Chem.75; Compound ID 1388; SEQ ID NO: 66 [N-terminal([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), des1-5,6G,7G,8G,13Q,25P,32L]-hCNP37 [Chem.] Molecular weight: 3936.5086. LCMS34: Calculated m / z 1313.1695; Measured m / z 1313.0250; Calculated m / z 985.1272; Measured m / z 985.2870

[0209] Chem.76; Compound ID 1389; SEQ ID NO: 67 [N-terminal([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), 5Q,13Q,19S,25P,32L]-hCNP37 [Chem.] Molecular weight: 4699.3224. LCMS34: Calculated m / z: 1567.4408; Measured m / z: 1567.4835; Calculated m / z: 1175.8306; Measured m / z: 1176.1075

[0210] Chem.77; Compound ID 1390; SEQ ID NO: 68 [N-terminal ([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), 5Q, 10E, 13Q, 19S, 25P, 32L]-hCNP37

Chem.

[0211] Chem.78; Compound ID 1391; SEQ ID NO: 55 [N-terminal ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8, 10E, 13Q, 17G, 25P, 32L]-hCNP37

Chem.

[0212] Chem.79; Compound ID 1392; SEQ ID NO:16 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,19S,25P,32L]-hCNP37

Chem.

[0213] Chem.80; Compound ID 1393; SEQ ID NO:69 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,19Q,25P,32L]-hCNP37

Chem.

[0214] Chem.81; Compound ID 1394; SEQ ID NO:70 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,10E,13Q,19Q,25P,32L]-hCNP37

Chem.

[0215] Chem.82; Compound ID 1395; SEQ ID NO: 71 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,7A,10E,13Q,19Q,25P,32L]-hCNP37

Chem.

[0216] Chem.83; Compound ID 1396; SEQ ID NO: 70 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,10E,13Q,19Q,25P,32L]-hCNP37

Chem.

[0217] Chem.84; Compound ID 1398; SEQ ID NO: 72 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), 5Q, 8H, 13Q, 17S, 19Q, 25P, 32L]-hCNP37

Chem.

[0218] Chem.85; Compound ID 1399; SEQ ID NO: 72 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(19-carboxynonadecanoyl amino) butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), 5Q, 8H, 13Q, 17S, 19Q, 25P, 32L]-hCNP37

Chem.

[0219] Chem.86; Compound ID 1400; SEQ ID NO: 73 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), 5Q, 7H, 13Q, 17G, 19Q, 25P, 32L]-hCNP37

Chem.

[0220] Chem.87; Compound ID 1401; SEQ ID NO: 74 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), 5Q, 13Q, 32L]-hCNP37

Chem.

[0221] Chem.88; Compound ID 1402; SEQ ID NO: 75 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,13Q,19Q,25P,32L]-hCNP37

Chem.

[0222] Chem.89; Compound ID 1403; SEQ ID NO: 76 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,13Q,17G,19Q,25P,32L]-hCNP37

Chem.

[0223] Chem.90; Compound ID 1404; SEQ ID NO: 75 [N-terminus ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), 5Q, 13Q, 19Q, 25P, 32L]-hCNP37

Chemical Structure

[0224] Chem.91; Compound ID 1405; SEQ ID NO: 75 [N-terminus ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(19-carboxynonadecanoyl amino) butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), 5Q, 13Q, 19Q, 25P, 32L]-hCNP37 [Chem.] Molecular weight: 5316.9683. LCMS34: Calculated m / z: 1773.3228; Measured m / z: 1773.2192; Calculated m / z: 1330.2421; Measured m / z: 1330.1785

[0225] Chem.92; Compound ID 1406; SEQ ID NO: 75 [N-terminus ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), 5Q, 13Q, 19Q, 25P, 32L]-hCNP37 [Chem.] Molecular weight: 5450.114. LCMS34: Calculated m / z: 1817.7047; Measured m / z: 1817.5808; Calculated m / z: 1363.5285; Measured m / z: 1363.4484

[0226] Chem.93; Compound ID 9407; SEQ ID NO: 77 [N-terminal ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 13Q, 19Q, 25P, 32L]-hCNP37

Chem.

[0227] Chem.94; Compound ID 1419; SEQ ID NO: 51 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 10H, 13Q, 14H, 15T, 17V, 19S, 25P, 32L]-hCNP37

Chem.

[0228] Chem.95; Compound ID 1420; SEQ ID NO: 78 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 10H, 13Q, 14H, 15T, 17V, 25P, 32L]-hCNP37

Chem.

[0229] Chem.96; Compound ID 1421; SEQ ID NO: 79 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 13Q, 15T, 17V, 19S, 25P, 32L]-hCNP37

Chem.

[0230] Chem.97; Compound ID 1422; SEQ ID NO: 80 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 10H, 13Q, 17V, 19S, 25P, 32L]-hCNP37

Chem.

[0231] Chem.98; Compound ID 1423; SEQ ID NO: 81 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 10H, 13Q, 15T, 17V, 25P, 32L]-hCNP37

Chem.

[0232] Chem.99; Compound ID 1424; SEQ ID NO: 82 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,13Q,15T,17V,25P,32L]-hCNP37

Chem.

[0233] Chem.100; Compound ID 1425; Sequence No. 83 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-8,10H,13Q,15T,17V,25P,32L]-hCNP37

Chem.

[0234] Chem.101; Compound ID 1426; Sequence No. 84 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[17-(1H-tetrazol-5-yl)heptadecanoyl amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,10H,13Q,15S,19Q,25P,32L]-hCNP37

Chem.

[0235] Chem.102; Compound ID 1431; Sequence number 85 [N-Terminal([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10E,13Q,17G,19H,25P,32L]-hCNP37

Chem.

[0236] Chem.103; Compound ID 1432; Sequence number 86 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,10E,13Q,14H,17G,19Q,25P,32L]-hCNP37

Chem.

[0237] Chem.104; Compound ID 1434; SEQ ID NO: 87 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10H,13Q,17G,19Q,25P,32L]-hCNP37

Chem.

[0238] Chem.105; Compound ID 9435; SEQ ID NO: 88 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,17G,19Q,25P,32L]-hCNP37

Chem.

[0239] Chem.106; Compound ID 1436; SEQ ID NO: 89 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,17V,19Q,25P,32L]-hCNP37

Chem.

[0240] Chem.107; Compound ID 1437; SEQ ID NO: 90 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,10H,13Q,17V,19Q,25P,32L]-hCNP37

Chem.

[0241] Chem.108; Compound ID 1448; SEQ ID NO: 91 [N-terminus([(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]), 5Q,10E,13Q,25P,32L]-hCNP37

Chem.

[0242] Chem.109; Compound ID 1449; SEQ ID NO: 51 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,10H,13Q,14H,15T,17V,19S,25P,32L]-hCNP37

Chem.

[0243] Chem.110; Compound ID 1450; SEQ ID NO: 52 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,10H,13Q,15T,17V,19S,25P,32L]-hCNP37

Chem.

[0244] Chem.111; Compound ID 1451; SEQ ID NO: 92 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 10H, 13Q, 17V, 19S, 25P, 27E, 32L]-hCNP37

Chem.

[0245] Chem.112; Compound ID 1452; SEQ ID NO: 77 [N-terminal ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5, 13Q, 19Q, 25P, 32L]-hCNP37

Chem.

[0246] Chem.113; Compound ID 1453; SEQ ID NO: 77 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,13Q,19Q,25P,32L]-hCNP37

Chem.

[0247] Chem.114; Compound ID 1454; SEQ ID NO: 77 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,13Q,19Q,25P,32L]-hCNP37

Chem.

[0248] Chem.115; Compound ID 1455; SEQ ID NO: 77 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,13Q,19Q,25P,32L]-hCNP37

Chem.

[0249] Chem.116; Compound ID 1456; SEQ ID NO: 77 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,13Q,19Q,25P,32L]-hCNP37

Chem.

[0250] Chem.117; Compound ID 1457; SEQ ID NO: 93 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),-2G,-1Q,2P,3G,4Q,5A,6P,7G,8Q,9A,10P,13Q,19Q,25P,32L]-hCNP37

Chem.

[0251] Chem.118; Compound ID 1458; SEQ ID NO: 94 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-2, 3G, 4Q, 5A, 6P, 7G, 8Q, 9A, 10P, 13Q, 17S, 19Q, 25P, 32L]-hCNP37

Chem.

[0252] Chem.119; Compound ID 1459; SEQ ID NO: 95 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1,2G,3Q,4A,5P,13Q,17S,19Q,25P,32L]-hCNP37

Chem.

[0253] Chem.120; Compound ID 1460; Sequence number 96 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,13Q,25P,32L]-hCNP37

Chem.

[0254] Chem.121; Compound ID 1461; Sequence number 97 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-8,10H,13Q,19Q,25P,32L]-hCNP37

Chem.

[0255] Chem.122; Compound ID 1462; SEQ ID NO: 96 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,13Q,25P,32L]-hCNP37

Chem.

[0256] Chem.123; Compound ID 1463; SEQ ID NO: 98 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[(17-carboxyheptadecanoyl)amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,13Q,19Q,32L]-hCNP37

Chem.

[0257] Chem.124; Compound ID 1464; SEQ ID NO: 99 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl)amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),des1-5,19Q,25P,32L]-hCNP37

Chem.

[0258] Chem.125; Compound ID 1465; Sequence No. 100 [N-terminal ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-5, 13Q, 19Q, 25P]-hCNP37

Chem.

[0259] Chem.126; Compound ID 1470; Sequence No. 96 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino) butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] butanoyl] amino] ethoxy] ethoxy] acetyl] amino] ethoxy] ethoxy] acetyl]), des1-5, 13Q, 25P, 32L]-hCNP37

Chem.

[0260] Chem.127; Compound ID 1471; Sequence No. 75 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), 5Q, 13Q, 19Q, 25P, 32L]-hCNP37

Chem.

[0261] Chem.128; Compound ID 1472; Sequence No. 75 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), 5Q, 13Q, 19Q, 25P, 32L]-hCNP37

Chem.

[0262] Chem.129; Compound ID 1473; Sequence No. 67 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,13Q,19S,25P,32L]-hCNP37

Chem.

[0263] Chem.130; Compound ID 1474; Sequence number 101 [N-terminus([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),5Q,13Q,25P,32L]-hCNP37

Chem.

[0264] Chem.131; Compound ID 1475; Sequence number 77 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(2S)-4-carboxy-2-[[(2S)-4-carboxy-2-[[(2S)-4-carboxy-2-[[(2S)-4-carboxy-2-[[(2S)-4-carboxy-2-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,13Q,19Q,25P,32L]-hCNP37

Chem.

[0265] Chem.132; Compound ID 1476; SEQ ID NO: 77 [N-terminus([2-[2-[2-[[2-[2-[2-[[(2S)-4-carboxy-2-[[2-[[(2S)-4-carboxy-2-[[2-[[(2S)-4-carboxy-2-[[2-[[(2S)-4-carboxy-2-[[2-[[(2S)-4-carboxy-2-(17-carboxyheptadecanoylamino)butanoyl]amino]acetyl]amino]butanoyl]amino]acetyl]amino]butanoyl]amino]acetyl]amino]butanoyl]amino]acetyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,13Q,19Q,25P,32L]-hCNP37

Chem.

[0266] Chem.133; Compound ID 1477; Sequence No. 102 [N-terminal([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-5,13Q,19Q,25P,27E,32L]-hCNP37

Chem.

[0267] Chem.134; Compound ID 1478; Sequence No. 103 [N-terminal([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,19Q,25P,32L]-hCNP37

Chem.

[0268] Chem.135; Compound ID 9480; SEQ ID NO: 103 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,19Q,25P,32L]-hCNP37

Chem.

[0269] Chem.136; Compound ID 1481; SEQ ID NO: 103 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(2S)-4-carboxy-2-[[(2S)-4-carboxy-2-[[(2S)-4-carboxy-2-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,19Q,25P,32L]-hCNP37

Chem.

[0270] Chem.137; Compound ID 9482; SEQ ID NO: 67 [N - terminal([(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4-(17 - carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]),5Q,13Q,19S,25P,32L]-hCNP37

Chem.

[0271] Chem.138; Compound ID 9483; SEQ ID NO: 104 [N - terminal([(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4-(17 - carboxyheptadecanoyl amino)butanoyl]amino]butanoyl]amino]butanoyl]amino]butanoyl]),5Q,13Q,19S,25P,27E,32L]-hCNP37

Chem.

[0272] Chem.139; Compound ID 1484; SEQ ID NO: 105 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-6,7G,8Q,9A,10P,13Q,19Q,25P,32L]-hCNP37

Chem.

[0273] Chem.140; Compound ID 1486; SEQ ID NO: 106 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-6,7G,8Q,9A,10P,13Q,17S,19Q,25P,27E,32L]-hCNP37

Chem.

[0274] Chem.141; Compound ID 1487; SEQ ID NO: 107 [N-Terminal([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), -2G, -1Q, 2P, 3G, 4Q, 5A, 6P, 7G, 8Q, 9A, 10P, 13Q, 19Q, 25P, 32L]-hCNP37

Chem.

[0275] Chem.142; Compound ID 1488; SEQ ID NO: 107 [N - terminus([(4S)-4 - carboxy - 4-(17 - carboxyheptadecanoylamino)butanoyl]), - 2G, - 1Q, 2P, 3G, 4Q, 5A, 6P, 7G, 8Q, 9A, 10P, 13Q, 19Q, 25P, 32L] - hCNP37

Chem.

[0276] Chem.143; Compound ID 1489; SEQ ID NO: 108 [N - terminus([2 - [2 - [2 - [[2 - [2 - [2 - [[2 - [2 - [2 - [[2 - [2 - [2 - [[(4S)-4 - carboxy - 4 - [[(4S)-4 - carboxy - 4-(17 - carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1 - 8, 13Q, 17G, 19Q, 25P, 27E, 32L] - hCNP37

Chem.

[0277] Chem.144; Compound ID 1493; SEQ ID NO: 109 [N-terminal ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,19Q,25P,27E,32L]-hCNP37

Chem.

[0278] Chem.145; Compound ID 1511; Sequence number 62 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(2S)-4-carboxy-2-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-6,7G,8Q,9A,10P,13Q,17S,19Q,25P,32L]-hCNP37

Chem.

[0279] Chem.146; Compound ID 1512; Sequence number 88 [N-terminus([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(2S)-4-carboxy-2-[[(2S)-4-carboxy-2-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1-8,13Q,17G,19Q,25P,32L]-hCNP37

Chem.

[0280] Chem.147; Compound ID 1513; Sequence number 110 [N-terminus(17-carboxyheptadecanoyl), -5E, -4E, -3E, -2E, -1E, 5Q, 13Q, 19S, 25P, 32L]-hCNP37

Chem.

[0281] Chem.148; Compound ID 1514; Sequence number 111 [N-terminus(17-carboxyheptadecanoyl), -4E, -3E, -2E, -1E, 5Q, 13Q, 19S, 25P, 27E, 32L]-hCNP37

Chem.

[0282] Chem.149; Compound ID 1265; Sequence No. 233 [N - terminal ([2 - [2 - [2 - [[2 - [2 - [2 - [[(4R) - 4 - carboxy - 4 - (17 - carboxyheptadecanoyl amino) butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]), des1 - 5,6a,7r,8k,9y,10k,12a,13Q,14k,15k,17l,18S,19k,21c,22f,24l,25k,26l,27e,28r,29i,31s,32l,33s,35l,37c] - hCNP37

Chem.

[0283] Chem.150; Compound ID 0106; Sequence No. 234 [ - 2P, - 1G] - hCNP37

Chem.

[0284] Chem.151; Compound ID 0089; Sequence No. 1 [N - terminal ([2 - [2 - [2 - [[2 - [2 - [2 - [[(4S) - 4 - carboxy - 4 - (17 - carboxyheptadecanoyl amino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl])] - hCNP22

Chem.

[0285] Chem.152; Compound ID 0230; Sequence number 235 [N-terminal ([2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]),-5G,-4Q,-3A,-2P,-1G,2A,3P,4G,5Q,7P,8G,9Q,10A,11P,12G,13Q,14A,15P]-hCNP37

Chem.

[0286] Chem.153; Compound ID 0231; Sequence number 1 [N-terminal ([2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl])]-hCNP22

Chem.

[0287] [Example 2] In vitro hNPR2 activity assay To evaluate the in vitro activity of the CNP22 (0065; Chem.1) and CNP compounds of the present invention, they were tested for their ability to induce the production of cyclic guanosine monophosphate (cGMP) in reporter cells expressing the human natriuretic peptide receptor 2 (hNPR2).

[0288] Cell culture: To generate reporter cell lines, HEK293 (ATCC® CRL-1573™) cells were stably transfected with the pGloSensor-42F reporter plasmid (GloSensor cGMP reporter - luciferase, Promega) and an expression plasmid encoding the hNPR2 receptor. Single cell clones were isolated for the reporter cell lines. When the reporter cells are exposed to an hNPR2 activating compound, hNPR2 causes the production of cGMP. GloSensor luciferase produced from the pGloSensor-42F plasmid binds to cGMP, and this binding induces a conformational shift in GloSensor luciferase, activating an otherwise inactive enzyme. The active GloSensor luciferase can convert luciferin to oxyluciferin, which is the process that produces bioluminescence. Luciferase activity can be quantified by detection of luminescence upon cell lysis and supply of the luciferase substrate. Thus, addition of an hNPR2 activating compound followed by addition of the detection reagent to the reporter cells causes the generation of luminescence in a dose-dependent manner. By testing several different concentrations of the hNPR2 activating compound, the maximum half maximal effective concentration (EC50) can be determined. This measurement is also called in vitro potency and is reported for all compounds tested. In all in vitro activity experiments, CNP22 was used as a positive plate control for hNPR2 activation. To reduce assay-to-assay variability, the potency of each compound relative to the potency of the CNP22 control on each plate is also reported.

[0289] Procedure: HEK293-hNPR2 / GloSensor clone 35 cells were continuously cultured in selection medium (DMEM containing 10% FCS, 1% P / S, 200 μg / mL G418, and 100 μg / mL hygromycin). To perform the assay, cells were detached with TrypLE Express (Gibco #12604013), passed through a 70 μm cell strainer, counted, and seeded overnight at 10,000 cells / well in 30 μL / well of assay medium (DMEM without phenol red and with 1% P / S) in white 384-well plates (Perkin Elmer #6007680). The next day, test compounds were serially diluted in dilution medium (DMEM without phenol red containing 1% OVA (Sigma #A5505 lot 04M7001V), 0.01% Tween20 (Roche #33766700), and 1% P / S), and subsequently 10 μL / well of the diluted test compounds were added to the cells. For each compound, 10 different concentrations in the range of 0.25 pM to 10 μM were tested. After incubation at 37 °C and 5% CO2 for 30 minutes, 40 μL / well of Bright-Glo detection reagent (Promega #E2650) was added. Luminescence was detected immediately or 15 minutes later by an EnVision Multimode Plate Reader (PerkinElmer). To determine the in vitro potency or EC50 of each test compound, four-parameter logistic regression was performed on the raw data of each test compound using the Python package SciPy optimize. The mean EC50 values are listed in Table 2 as "hNPR2 0% HSA.[EC50 (nM)]". Relative potency was calculated by the following formula: EC50 (CNP22 in-plate control) / EC50 (test compound) * 100 and shown in Table 2 as "hNPR2 0% HSA.[EC50 % relative CNP]". At least two replicates were measured for each test compound. The reported values are the average of the replicates and are listed in Table 2.

Table 7-1

Table 7-2

Table 7-3

Table 7-4

[0290] These data show that the tested CNP compounds span a wide range of in vitro NPR2 activation levels and that negatively charged CNP compounds commonly show a reduction in in vitro NPR2 potency compared to positively charged CNP compounds.

[0291] [Example 3] In Vitro Neprilysin Stability of CNP Compounds It has been reported that native CNP can be metabolized by neprilysin (neutral endopeptidase, NEP), a protease widely present in the body. Therefore, an in vitro neprilysin stability assay was set up to test the neprilysin-mediated degradation of the selected CNP compounds.

[0292] Procedure: Briefly, the assay buffer was composed of PBS buffer (pH 7.4, ThermoFisher Scientific) with 0.005% v / v Tween 20 (Sigma-Aldrich). Recombinant human neprilysin (rhNEP; R&D Systems) and the CNP compound were dissolved in the assay buffer. Native CNP22 (0065, Chem.1) was included as a positive control on each day of incubation. First, the CNP compound (final concentration was 1000 nM) was pre-incubated in the assay buffer at 37 °C for 10 minutes. The assay was initiated by adding either rhNEP (final concentration was 2 μg of protein per mL) or the assay buffer (adsorption experiment), and the incubation was carried out at 37 °C. The reaction was terminated at selected time points (0.5 minutes, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes) by adding 1 volume of the incubation mixture to 3 volumes of ethanol (containing 1% v / v formic acid). The mixture was centrifuged at 13000 rpm for 20 minutes at 4 °C. After centrifugation, 1 volume of the supernatant was mixed with 1 volume of Milli-Q water.

[0293] Analysis by LCMS: The mixture was analyzed by LCMS using an Acquity UPLC Peptide CSH C18 analytical column from Waters (130 Å, 1.7 μm, 1 × 50 mm) operating at 60 °C. Gradient elution was performed using a Nexera UHPLC system (Shimadzu) with mobile phase A (consisting of Milli-Q water with 0.1% formic acid) and mobile phase B (consisting of acetonitrile with 0.1% formic acid). The flow rate was 0.3 ml / min. A zenoTOF 7600 mass spectrometer (Sciex) was used as the detector and operated in positive electrospray ionization mode. The data was recorded in full scan mode (m / z 300 - 1700).

[0294] Calibration curves were prepared in assay buffer and treated as test samples. Using these samples, the concentration of the relevant CNP compounds in the in vitro samples was calculated, including quality control samples. For at least 75% of the standards and QCs, the deviation between the nominal concentration and the calculated concentration was less than 20%.

[0295] All incubations were performed in duplicate. Data were reported as the percentage remaining at 90 minutes (based on the calculated concentration) compared to the average concentration of the 0.5-minute time point samples. Examples of seven compounds and CNP22 (0065, Chem.1) are shown in Table 3.

Table 8

[0296] These data show that the CNP compounds of the present invention are substantially stable in the presence of recombinant human neprylisin compared to CNP22, which is rapidly degraded.

[0297] [Example 4] Peptide Net Charge Calculation The charge of ionizable groups in a peptide at a given pH can be calculated using the Henderson-Hasselbalch equation, for example, by using empirically determined acid dissociation constants (pKa) for each amino acid residue or modified group known in the art, as described by B. Skoog and A. Wichman (Trends in Analytical Chemistry, 1986, vol. 5, pp. 82-83.) and L. Kozlowski (Biology Direct, 2016, 11:55). This is shown for negatively charged amino acids or modified groups in Equation 1 and for positively charged amino acids and modified groups in Equation 2, where pKn is the pKa of the negatively charged group, pKp is the pKa of the positively charged group, and pH in the equation is the pH of interest. The net charge of a peptide at a given pH is the sum of the negative and positive charges of all ionizable groups in the peptide.

Equation

[0298] For natural amino acid residues, tabular pKa constants are used for the free termini and ionizable sites in the side chains. The pKa values of modified amino acids and modifying groups were estimated by the ACD / Labs ver. 12 package and are also shown in Table 4.

[0299] As an example, when using the pKa values shown in Table 4 to calculate the net charge of compound 9482 at pH 7.4, the sum of the negative charges carried by Cys37 (C-terminal acid), Asp27, Glu2, gGlu(1), gGlu(2), gGlu(3), gGlu(4), gGlu(5), and the position of the C18d fatty acid is minus 8.995 Fd. The sum of the positive charges carried by the positions of Arg28, Lys15, Lys14, Lys10, Lys8, Arg7, and His3 is 6.162 Fd. Thus, the net charge is the sum of the above, namely minus 2.833.

[0300] The calculated charge values of the selected pH (pH 7.4 and pH 6.5) for the CNP compounds of the present invention using the above principle are shown in Table 5. [Table 9] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4]

[0301] [Example 5] Solubility of CNP compounds To evaluate the compatibility of the CNP compound with the soluble liquid formulation, the solubility of the CNP compound in pharmaceutically relevant buffers was measured. Since pH was expected to affect solubility, the solubility at two different pH levels (pH 4.0 and pH 6.5) was examined.

[0302] Preparation of the pH 4.0 sample: The pH 4.0 sample of the CNP compound was prepared by mixing the lyophilized peptide with a 5 mM sodium acetate, 250 mM glycerol buffer at pH 4.0 to a nominal concentration of at least 4000 - 5000 nmol / mL at room temperature. The pH of the sample was measured and adjusted to pH 4.0, and if not completely dissolved, the sample was equilibrated at room temperature until the next day.

[0303] Preparation of the pH 6.5 sample: The pH 6.5 sample of the CNP compound was prepared by dissolving the lyophilized peptide in water. While stirring, a portion of the peptide solution was transferred to a buffer composed of 41 mM sodium phosphate and 118 mg / mL glycerol, pH 7.5 - 8.0, followed by the addition of diluted sodium hydroxide, then another portion of the peptide solution was added, and further diluted sodium hydroxide was added. This procedure was repeated until all of the peptide solution was added, reaching a nominal concentration of at least 4000 - 5000 nmol / mL and the buffer composition became 8 mM sodium phosphate, 23 mg / mL glycerol. The pH of the sample was measured and adjusted to pH 6.5, and if not completely dissolved, the sample was equilibrated at room temperature until the next day.

[0304] Procedure: The sample at pH 4.0 was prepared as described above, and the compound concentration in the supernatant was determined using CAD (Method F, CAD02). The compound concentration at pH 4.0 represents the solubility at pH 4.0, and the measured values are shown in Table 6. [Table 11]

[0305] Samples at pH 6.5 were prepared as described above, and the peptide concentration in the supernatant was determined by CAD (Method F, CAD02). The peptide concentration at pH 6.5 represents the solubility at pH 6.5, and the measured values are shown in Table 7. [Table 12]

[0306] Roughly, only samples containing more than 4000 μM were subjected to further tests as described below.

[0307] These results indicate that the CNP compounds of the present invention can be formulated in pharmaceutically relevant buffers at concentrations suitable for subcutaneous administration.

[0308] Example 6 - Thioflavin T (ThT) Assay The ThT assay was used to evaluate the physical stability of the CNP compounds and their compatibility with liquid formulations. The low physical stability of peptides can lead to amyloid fibril formation, which is observed in samples as an ordered thread-like macromolecular structure that can eventually lead to gel formation. This has traditionally been examined by visual inspection of the samples. However, such measurements are highly subjective and vary among observers. Therefore, the application of small molecule indicator probes is much more advantageous. Thioflavin T (ThT) is such a probe and has a distinct fluorescence signature when bound to fibrils [Naiki et al. (1989) Anal. Biochem. 177, 244 - 249, LeVine (1999) Methods. Enzymol. 09, 274 - 284].

[0309] Procedure: Thioflavin T was added to the supernatant samples (pH 4.0 and 6.5) of Example 5 using an aqueous ThT stock solution to a final concentration of approximately 1 μM in the sample. 150 μL aliquots of the sample were placed into a 96-well microtiter plate (Packard OptiPlate™-96, white polystyrene). Typically, two to three replicates of each sample were applied to the plate. The plate was sealed and placed into a fluorescence plate reader (Fluoroskan Ascent FL) and incubated at 37 °C using circular shaking (960 rpm, amplitude 1 mm). Fluorescence measurements were performed every 20 minutes using excitation through a 444 nm filter and emission measurement through a 485 nm filter. Between each measurement, the plate was shaken and heated as described above, and the assay was terminated after 45 hours. The fluorescence measurements for each microtiter plate well were plotted against time and the lag time (the time until an increase in ThT fluorescence was observed) was estimated.

[0310] To evaluate the loss of the dissolved peptide after 45 hours of incubation, peptide recovery was measured as the ratio of the total peak areas after and before incubation using the RP-UPLC method described below.

[0311] Measurement of lag time and peptide recovery % using RP-UPLC: RP-UPLC was performed using an Acquity UPLC BEH C18 1.7 μm (2.1 × 30 mm) Waters column (eluent A: 0.1% v / v% TFA in water, eluent B: 4:1 acetonitrile / water with 0.1% v / v TFA) with gradient elution (0 min: 95% A, 2 min: 20% A, 2.3 min: 20% A, 2.4 min: 95% A, 9 min: 95% A) at a flow rate of 0.9 mL / min and a column temperature of 30 °C. The total peptide area was evaluated using UV detection at 215 nm.

[0312] Samples at pH 4.0 were prepared as described in Example 5 and subjected to the ThT assay described above. The lag time and peptide recovery values are shown in Table 8.

Table 13

[0313] The sample at pH 6.5 was prepared as described in Example 5 and subjected to the above ThT assay. The lag time and peptide recovery values are shown in Table 9. [Table 14]

[0314] As a conclusion, this experiment shows that most of the CNP compounds of the present invention can be formulated in pharmaceutically relevant buffers with a low tendency to form fibrils.

[0315] Example 7 - Measurement of High Molecular Weight Protein (HMWP) To evaluate the tendency of CNP compounds to form covalent dimers or oligomers when formulated, the high molecular weight protein content of CNP compound formulations at pH 4.0 and 6.5 was determined.

[0316] Procedure: The relative amount of covalent HMWP was evaluated using SEC-UPLC. SEC-UPLC was performed using a Waters UPLC Protein BEH SEC column, 125 Å, 4.6 × 150 mm, 1.7 μm, with isocratic elution (0.3 M NaCl, 10 mM NaH2PO4, and 5 mM H3PO4, 50% (v / v) isopropanol, pH 2.4), a flow rate of 0.3 mL / min, a column temperature of 50 °C, and UV detection at 215 nm. The total area of the peaks eluting before the monomer main peak was designated as HMWP and expressed on a percentage scale relative to the total peptide peak area.

[0317] Results: HMWP formation after 2-week incubation at 37°C: Samples at pH 4.0 were prepared as described in Example 5, and the amount of HMWP was determined as described above. The samples were incubated at 37°C for 2 weeks, and the amount of HMWP was determined again. The increase in HMWP over the 2-week incubation at 37°C is reported in Table 10 (when the incubation period deviated from 2 weeks, the increase in HMWP was normalized to 2 weeks assuming a constant formation rate).

Table 15

[0318] Samples at pH 6.5 were prepared as described in Example 5, and the amount of HMWP was determined as described above. The samples were incubated at 37°C for 2 weeks, and the amount of HMWP was determined again. The increase in HMWP over the 2-week incubation at 37°C is reported in Table 11 (when the incubation period deviated from 2 weeks, the increase in HMWP was normalized to 2 weeks assuming a constant formation rate).

Table 16

[0319] As a conclusion, this experiment shows that the CNP compound of the present invention can be formulated in a pharmaceutically relevant buffer with a low tendency to form HMWP.

[0320] Example 8 - Accelerated Chemical Stability of the CNP Compound To determine the chemical stability of the CNP compound in the formulation, samples of the CNP compound in buffer were exposed to heat-accelerated chemical decomposition. The stability of a given peptide was evaluated by measuring the recovery of the intact peptide by quantification using RP-UPLC. The samples were also analyzed by LC-MS to evaluate the chemical decomposition pattern.

[0321] Sample preparation: Samples for the 2-week study were prepared according to the principle described in Example 5.

[0322] Samples for the 6-week study were prepared by dissolving the lyophilized peptide in buffer (8 mM phosphate, 250 mM glycerol, pH 7.4) to a nominal peptide concentration of 5000 nmol / ml and then adjusting the pH to 6.5 with 0.1 N HCl.

[0323] Incubation: For the 2-week study, time zero (TZ) samples were removed, stored at -18 °C, and the remaining volume was incubated at 37 °C. Samples were removed after 2 weeks of incubation and stored at -18 °C. The frozen samples were thawed, diluted to approximately 200 nmol / mL, and the extent of chemical degradation was determined using LCMS as described.

[0324] For the 6-week study, time zero (TZ) samples were removed, stored at -18 °C, and the remaining volume was incubated at 37 °C. Samples were removed weekly and stored at -18 °C until the final sample collection at 6 weeks. The frozen samples were thawed, diluted to approximately 200 nmol / mL, and the extent of chemical degradation was determined using RP-UPLC and LCMS as described.

[0325] Analysis of peptide recovery by RP-UPLC: The RP-UPLC method was performed on a Waters ACQUITY H-CLASS UPLC system equipped with a PDA detector. Separation was carried out on an ACQUITY UPLC BEH C8 Column (130 Å, 1.7 μm, 2.1 mm × 150 mm) operating at a column temperature of 50 °C using eluent A (19.59 mM NH4H2PO4, 41.71 mM (NH4)2HPO4, pH 6.5, and MeCN, 9:1 v:v) and eluent B (80% MeCN in water). UV detection was performed at 214 nm. 3 μL of sample was injected onto the column, which was eluted with a one-step linear gradient of eluent A and B from 27 - 30% B over 20 minutes, followed by washing with 95% B for 2 minutes, and then a re-equilibration step at a constant flow rate of 0.3 mL / min for 7 minutes, thereby obtaining an analysis time of 30 minutes per sample. The area of the peak corresponding to the intact CNP compound was expressed as a percentage scale relative to the total peptide peak area.

[0326] Analysis of Chemical Degradation Patterns by LCMS: Samples were analyzed on a Thermo Scientific VANQUISH UPLC using a Waters Acquity C18 reverse-phase column, 300 Å, 1.7 μM particles, 1 mm × 150 mm. Compounds were separated over 48 minutes with a linear gradient of 5% to 55% solvent B using solvent A: water, 0.1% formic acid containing 0.02% TFA, and solvent B: acetonitrile, 0.1% formic acid containing 0.02% TFA. Mass spectra were acquired on a Q Exactive Plus hybrid Quadrupole-Orbitrap mass spectrometer interfaced with an H-ESI II ion source, BioPharma option (Thermo Scientific). Data acquisition was performed at a scan range of m / z 500 - 2000 and a resolution of 30000.

[0327] The data list was aggregated into a compound summary table. The table headers were defined as follows.

[0328] "Recovery": The compound peak (i.e., the major peak intensity at time zero with the compound monoisotopic mass) recovery is normalized against the compound peak at a given time point. "Isomers": As the total intensity of peaks with isobaric mass relative to the major compound, isolated in the RT dimension. "18 amu Loss": That is, the total intensity of peaks with a mass 18 amu lower than the CNP compound peak, isolated in the RT dimension. Fragment: Peaks isolated in the RT dimension, occurring over time and assumed to result from hydrolysis peptide backbone cleavage, with a mass 50 amu lower than the compound peak and a mass 18 amu higher than the compound mass. Finally, "Others": Includes the sum of the total intensities of peaks with a 32 amu addition or dimer of the major peak, occurring over time.

[0329] Results: The results of the LCMS analysis of the 2-week accelerated degradation study for some CNP compounds of the present invention are summarized in Table 12. The results of the RP-UPLC and LCMS analysis of the 6-week accelerated degradation study for CNP compound 9482 (Chem.137) are summarized in Table 13 and Table 14, respectively.

Table 17

Table 18

Table 19

[0330] These data indicate that the CNP compounds according to the present invention exhibit substantial chemical stability in the formulation.

[0331] Example 9 - Local Tolerance of CNP Compounds in Rats The objective of this study was to evaluate the local tissue reaction to subcutaneous injection of CNP compounds in rats.

[0332] Procedure: The CNP compounds were formulated according to the principle described in the general preparation method - Method G. Each Sprague Dawley rat (male, 10 - 11 weeks old at arrival, Janvier, France) was subcutaneously dosed with 250 or 350 uL of the same compound (formulation concentration of 1000 nmol / mL) 120 hours before tissue collection, and a total of 2 injections were performed for each animal (Table 15). The injection sites (flank or neck) were alternated among animals within each group and time point.

Table 20

[0333] The animals had free access to tap water and diet (Altromin 1324). The group sizes were n = 3 for the vehicle and positive control, and n = 4 for the test compound. The vehicle and compound were formulated in either 5 mM sodium acetate, 250 mM glycerol, pH 4.0, or 8 mM sodium phosphate, 250 mM glycerol, pH 7.4, as shown in Table 16.

[0334] The animals were anesthetized with isoflurane. Using an electric clipper, a square fur area (2 cm × 2 cm) was removed from either the neck area or the flank area of the rats. For dosing, a new injection needle (25G cannula, 1 ml syringe) was placed in the middle of the square boundary closest to the animal's head, and the needle was inserted into the skin towards the center of the square from the boundary. As a result, the needle tip was located at the center of the square, which was marked with an oil-based pen, and the formulation was inserted into the center of the square. On the day of tissue collection, the rats were deeply anesthetized with isoflurane and euthanized by bleeding. The injection sites were examined and scored for lesions. The injection sites were evaluated, and any changes (position, color, shape, and size) were noted and sampled for histological preparation.

[0335] The injection sites were removed and fixed in 4% phosphate-buffered neutral formaldehyde in a sealed plastic container.

[0336] Subsequently, each injection site was cut into three sections at 0.8 cm, 1.0 cm, and 1.2 cm from the boundary of the square where the injection needle was placed. These sections represented the injection site and the adjacent tissue, respectively. The tissues were processed, embedded in paraffin, cut into thin sections of 2 - 4 μm, stained with hematoxylin and eosin (H&E), and microscopically evaluated using an optical microscope. The histopathological changes were graded on a 5-level scale (Min (minimum), Mil (mild), Mod (moderate), Mar (marked), and Sev (severe)). The study is summarized in Table 16.

Table 21

[0337] Results: The results of the local tolerance study are summarized in Table 17. [Table 22]

[0338] Conclusion: The net positively charged control 0776 showed moderate to marked local subcutaneous necrosis 5 days after subcutaneous injection in rats. In comparison, Compounds 1351, 9384, 9407, and 1420 showed no or mild local subcutaneous necrosis 5 days after subcutaneous injection in rats and were all evaluated as acceptable for human subcutaneous administration.

[0339] Example 10 - Local Tolerance Test of CNP in Pigs The purpose of this study was to evaluate the local tissue reaction response to subcutaneous injection of CNP compounds in pigs.

[0340] Procedure: In domestic pigs, the injection sites were subjected to histopathological examination after subcutaneous dosing. The injections were placed on the mid - back between the shoulders and hips on both sides of the dorsal midline. A 2×2 cm area was shaved and marked with oily ink under light anesthesia 1 or 2 days before injection.

[0341] Each injection consisted of 100 or 200 μl of a formulation at an approximate concentration of 4400 μM. The CNP compound formulations were prepared according to the principles of a general preparation method - Method G and were either A) 8 mM sodium phosphate, 250 mM glycerol, pH 6.5, B) 5 mM sodium acetate, 250 mM glycerol, pH 4.0, or C) 5 mM sodium acetate, 240 mM propylene glycol, pH 4.0, and physiological saline was included as a negative control.

[0342] On Day 1, the pigs were lightly anesthetized. An insulin pen (NovoPen 4) and an insulin needle (NovoTwist 32G / 5mm) were used, and the needle was held perpendicular to the skin to accurately deposit the CNP formulation into the subcutaneous adipose tissue. On the 5th day (4 days after injection) or the 6th day (5 days after injection), the pigs were euthanized, the injection sites were removed, and fixed in 4% phosphate-buffered neutral formaldehyde in a sealed plastic container. The tissue blocks were pre-fixed in 10% buffered formalin for 2 - 4 hours, cut into 2-mm thick slabs with a multi-purpose knife, examined for gross changes, and finally fixed overnight in cassettes. Two tissue pieces where the tissue reaction was maximal or in the central part of the injection site were selected for evaluation by light microscopy. Three to four 2 - 4-μm sections were cut from three levels at 100-μm intervals, stained with hematoxylin and eosin (H&E), and evaluated by light microscopy. Histopathological changes were graded on a 5-level scale (Min (minimal), Mil (mild), Mod (moderate), Mar (marked), and Sev (severe)). The study is summarized in Table 18.

Table 23

[0343] Results: The results are summarized in Table 19. *Necrosis was used as a marker for local subcutaneous reaction; **NAD: no abnormality detected

Table 24

[0344] In conclusion, the net positively charged control compound 0776 showed moderate to marked local subcutaneous necrosis in pigs 5 days after subcutaneous injection, while the net positively charged CNP peptide 0106 without bound fatty acid albumin binder showed no observable injection site reaction.

[0345] In comparison, compounds 9480 and 9482 did not show local subcutaneous necrosis in pigs 5 days after subcutaneous injection. Compounds 9384 and compound 9407 did not show local subcutaneous necrosis or showed minimal local subcutaneous necrosis in pigs 5 days after subcutaneous injection, compounds 9435 and 1235 showed minimal to mild local subcutaneous necrosis in pigs 5 days after subcutaneous injection, and compound 9483 did not show local subcutaneous necrosis or showed moderate local subcutaneous necrosis in pigs 5 days after subcutaneous injection. Compounds 9384, 9407, 9435, 9480, and 9482 were all evaluated as acceptable for human subcutaneous administration.

[0346] Example 11 - PK / PD of CNP Compounds after Intravenous and Subcutaneous Administration to Rats To evaluate the intravenous and subcutaneous PK / PD profiles of the CNP compounds of the present invention, formulations of the CNP compounds were administered to rats, blood samples were collected, and exposure and cGMP responses were measured.

[0347] cGMP biomarker assay: The in vivo biological activity of the CNP compounds was evaluated by measuring cGMP in plasma samples using plasma protein precipitation followed by quantification by LC-MS / MS.

[0348] To evaluate the cyclic guanosine monophosphate (cGMP) biomarker response in biological samples, after administering the CNP compounds to test animals, a plasma protein precipitation procedure followed by liquid chromatography using tandem mass spectrometry (LC-MS / MS) analysis was applied. As a deceleration, the concentration of cGMP in the test plasma samples was compared to a stable isotope-labeled (SIL) surrogate analyte as a reference ( 13 C 10 15Determined using a standard curve prepared with N5cGMP. Surrogate analytes were spiked in pooled authentic blank matrix to cover an analytical range of 5 nM to 2000 nM. 8-Methoxymethyl-3-isobutyl 1-methylxanthine (MMPX) was used as an internal standard. A plasma protein precipitation procedure was performed using an organic solvent to precipitate plasma proteins, leaving the supernatant containing cGMP molecules, which was then analyzed on an LC-MS / MS instrument.

[0349] To prepare the standard samples, the frozen authentic blank plasma matrix was thawed and then centrifuged at 4°C and 4000 RPM for 5 minutes. Using the stock solution of SIL-cGMP, a 2000 nM standard in blank plasma was prepared and then transferred to a 1 mL Eppendorf 96-well plate (Patent No. 8,636,965). This was placed in a liquid handler (TECAN Fluent 78) and standards and QCs (5, 10, 20, 50, 100, 200, 500, 1000, 2000 nM) were prepared by stepwise dilution with blank plasma in a 96-well microplate (Chimney Well 651201). Blank plasma samples were prepared on the same plate. The study samples were thawed by ventilating at ambient temperature for 10 minutes, shaken on a tabletop shaker at ambient temperature for 5 minutes, and then centrifuged at 4°C and 4000 RPM for 5 minutes. The samples were then placed in the liquid handler and the plasma protein precipitation procedure was carried out. A certain amount of standard samples, QC samples, zero samples, and study samples were precipitated using 4 volumes of acetonitrile (OPTIMA® LC / MS grade) containing 30 nM of MMPX (Sigma #M2547). The samples were then shaken on a tabletop shaker at ambient temperature for 4 minutes and then centrifuged at 4°C and 4000 RPM for 30 minutes, after which the supernatant was transferred to a new 96-well microplate. Subsequently, the plate was evaporated using an Eppendorf concentrator plus™ at 30°C for 60 minutes. The residue was reconstituted with a certain amount of 5% acetonitrile and 0.1% formic acid (RATHBURN LC / MS Grade), shaken on a tabletop shaker at ambient temperature for 2 minutes, and centrifuged at 4°C for 10 minutes. The prepared samples were then analyzed using an LC-MS / MS instrument (Sciex ExionLC® coupled to a Sciex 6500+ Triple quadrupole). Gradient elution was applied to reverse-phase LC using 0.1% formic acid as mobile phase A and 95% acetonitrile and 0.1% formic acid as mobile phase B. A Waters Acquity UPLC® CSH® Fluoro-Phenyl 1.7 μm, 2.1×100 mm column was used as the stationary phase and thermostated at 60°C.SIL-cGMP, endogenous cGMP, and MMPX were detected by selected reaction monitoring in positive ion mode MS / MS.

[0350] Acceptance criteria for analysis run: An SIL-cGMP standard curve was established with a 1 / ×2 weighting and evaluated from the perspectives of linearity, precision, and accuracy. At least 75% of the calibration standards had to meet the following criteria upon back-calculation. The calibration standards, excluding the LLOQ which had to fall within ±20% of the nominal concentration of the LLOQ standard, had to fall within ±15% of the nominal concentration of each calibration standard. Values outside these limits had to be discarded unless the established model was changed. If discarding one calibration standard caused another calibration standard to be out of bounds, this calibration standard was also discarded.

[0351] At least two-thirds of the QC samples had to be within 15% of their respective nominal values. One-third of the QC samples could be outside 15% of their respective nominal values but must always be at the same nominal concentration and less than 50% of the QC samples.

[0352] Quantitative Plasma Analysis of CNP Compounds: The plasma concentration of CNP compounds was assayed by plasma protein precipitation and analyzed by TurboFlow liquid chromatography-mass spectrometry (TF-LC-MS). Standard substances were prepared by spiking plasma from either blank rats or minipigs with relevant CNP compounds in the range of 0.5 - 2000 nM. For TF-LC-MS by protein precipitation, standard substances, plasma blanks, or study samples were prepared by adding 3 or 4 volumes of either ethanol or methanol containing 20 nM internal standard to 1 volume of the sample. For some compounds, formic acid was added to the precipitation reagent (final concentration was 1% v / v). After adding the precipitation reagent, the mixture was centrifuged at 6200 rpm at 4 °C for 30 minutes. After centrifugation, 1 volume of the supernatant was mixed with 2 volumes of water (containing 1% formic acid). The mixture was analyzed by TF-LC-MS using either a Cyclone TurboFlow column (0.5 × 50 mm, ThermoFisher Scientific) and one of the XBridge Protein BEH C4 3.5 μm 300 Å or XBridge Protein BEH C18 3.5 μm 130 Å analytical columns (both 50 × 2.1 mm, obtained from Waters). Gradient elution was performed using mobile phase A (consisting of milli-Q water containing 1% formic acid and 5% methanol / acetonitrile (50 / 50)) and mobile phase B (consisting of methanol / acetonitrile (50 / 50) containing 1% formic acid and 5% milli-Q water). The mass spectrometer was operated in positive ionization mode. A TSQ Altis mass spectrometer (ThermoFisher Scientific) was used as the detector in selected reaction monitoring mode (specific transitions were optimized for each CNP compound). A linear calibration curve (weighting of 1 / x2) was used to calculate the concentration in plasma samples. Quality control samples were included. The deviation between the nominal and calculated concentrations in the standard substances and quality control samples was less than 15%.

[0353] Animals and Dosing: Groups of 4 - 6 rats (male, Sprague Dawley, 350 g upon arrival, Janvier, France) per compound and route of administration were dosed with the CNP compound formulated either in A) 8 mM sodium phosphate, 250 mM glycerol, 0.007% polysorbate 20, pH 7.4 or B) 5 mM sodium acetate, 250 mM glycerol, 0.007% polysorbate 20, pH 4.0. The subcutaneous dosing amount was 300 nmol / kg at a concentration of 1000 nmol / L, resulting in a dosing volume of 0.3 mL / kg, while the intravenous dosing amount was 100 nmol / kg at a concentration of 100 nmol / L, resulting in a dosing volume of 1 mL / kg.

[0354] Sampling: 250 μL of sublingual blood samples were collected from non - anesthetized rats into EDTA - coated vials (Microvette 600 K3E reference #15.1673.100 Sarstedt) at baseline ( - 1 hour before dosing) and at 5 minutes (intravenous dosing group only), 2 hours, 6 hours, 24 hours, 48 hours, and 72 hours (exposure analysis only) after dosing. Blood samples were stored rapidly on ice and centrifuged within 10 minutes of collection (4°C, 8000 RPM, 5 minutes). 50 μL of plasma samples were transferred to each microtube for either exposure or cGMP analysis, held on dry ice, and stored in a freezer ( - 20°C). Rats had free access to food (Altromin) and tap water.

[0355] Analysis: The PK parameters were calculated for each animal by non-compartmental analysis (NCA) using the software Phoenix WinNonlin (version 8.1 or higher, Certara). Clearance (Cl) was calculated as follows: Dose / AUCinf (linear up, log down). The half-life (t1 / 2) was calculated as ln(2) / λ, where λ was estimated by linear regression of time vs. log concentration typically in the time range of 2 - 48 hours (e.g., 6 - 48 hours). Bioavailability was calculated as dose-normalized AUC: (AUC(sc) / dose(sc)) / (AUC(iv) / dose(iv)). The values in the table are the averages from individual animals (n = 4 - 6 individual animals). Subsequent plots of cGMP plasma concentration vs. time were (Y-axis) the mean cGMP concentration after IV dosing (n = 4 - 6 individual animals), and (X-axis) the nominal plasma sampling times.

[0356] Results: The PK data with clearance (CL), half-life T1 / 2, and bioavailability (F%) are summarized in Table 20. Plasma cGMP concentrations after intravenous administration of the CNP compound are shown in Figures 1 - 27, and the doses are shown in Table 20. Plasma cGMP is reported as the mean.

Table 25

[0357] In conclusion, this experiment shows that the CNP compound of the present invention exhibits an extended half-life and reduced clearance in an in vivo rat model. Furthermore, this experiment shows that the CNP compound is biologically active based on their ability to elicit a cGMP response in the dosed animals (Figures 1 - 27).

[0358] Example 12 - PK / PD of CNP Compound after Intravenous and Subcutaneous Administration to Miniature Swine and LYD Swine The objective of this study was to investigate the pharmacokinetic and pharmacodynamic properties after intravenous (i.v.) and subcutaneous (s.c.) administration to Göttingen minipigs and domestic LYD pigs, and to estimate the bioavailability after subcutaneous administration. Quantitative plasma analysis of CNP and cGMP levels was performed as described in Example 11.

[0359] Animals and dosing: Pharmacokinetic and pharmacodynamic studies in Göttingen minipigs or domestic LYD pigs were conducted by CRO Minerva Imaging or Novo Nordisk A / S, respectively. The CNP compound was dosed to normal female 1) Göttingen minipigs at 6 - 12 months of age with an average body weight of 22 kg, or 2) domestic LYD pigs at 4 - 5 months of age with an average body weight of 80 kg. At least one week before dosing, permanent central venous catheters were implanted for blood sampling and intravenous (i.v.) dosing. For subcutaneous (s.c.) dosing, the pigs were ultrasonically scanned and the optimal injection sites on the lateral side above the neck were marked with indelible ink. Insulin Pens (NovoPen Echo or NovoPen 4) and insulin needles (NovoFine 32G / 4mm or NovoTwist 32G / 5mm) were used, with the needle perpendicular to the skin, to accurately deposit the CNP formulation into the subcutaneous adipose tissue.

[0360] The CNP compound was formulated according to the principles of a general preparation method - Method G and was either A) 8 mM sodium phosphate, 250 mM glycerol, pH 6.5, B) 5 mM sodium acetate, 250 mM glycerol, pH 4.0, C) 5 mM sodium acetate, 240 mM propylene glycol, pH = 4.0, D) 8 mM sodium phosphate, 250 mM glycerol, pH 7.5, or E) 20 mM sodium phosphate, 223 mM propylene glycol, pH 6.0.

[0361] In the case of intravenous administration, the present CNP compound was dosed to minipigs at 30 - 60 nmol / kg and to domestic pigs at 15 - 43 nmol / kg using Formulations A - E. In the case of subcutaneous administration, the present CNP compound was dosed to minipigs at 55 - 62 nmol / kg and to domestic pigs at 20 - 30 nmol / kg using Formulations A - D.

[0362] Sampling: For cGMP and CNP compound analysis by LC - MS, approximately 1 mL of blood samples were collected into EDTA - coated tubes (8 mM) before dosing and up to 14 days after dosing. In a few cases, cGMP was analyzed only up to 48 hours after intravenous dosing in domestic LYD pigs. Blood samples were held on wet ice for up to 30 minutes at 4°C and centrifuged at a minimum of 1500 G for 10 minutes, and plasma samples were stored at - 20°C until analysis.

[0363] Plasma concentration - time profiles were analyzed by non - compartmental PK analysis using Phoenix WinNonlin 8.1, Pharsight Inc., Mountain View, CA, USA. The calculation of the area under the plasma concentration - time curve (AUC) was based on the "linear up log down" method, and a uniform weighting was used for the estimation of the terminal rate constant (λz). Subcutaneous bioavailability (F) was calculated as the ratio of the dose - normalized AUC (AUC / dose) after subcutaneous administration to the AUC / dose after intravenous administration.

[0364] Results: PK data with clearance (CL), half - life T1 / 2, and bioavailability (F%) in minipigs are summarized in Table 21. PK data with clearance (CL), half - life T1 / 2, and bioavailability (F%) in LYD domestic pigs are summarized in Table 22. Plasma cGMP concentrations after intravenous or subcutaneous dosing of the CNP compound in minipigs and domestic pigs are shown in Figures 28 - 48, and the doses are shown in Tables 21 and 22. Plasma cGMP is reported as the mean of n = 2 - 4 for 14 days in minipigs and as the mean of n = 2 - 4 for 2 days in domestic LYD pigs.

Table 26

Table 27

[0365] Conclusion: This experiment shows that the CNP compound of the present invention exhibits an extended half-life and reduced clearance in both minipigs and domestic LYD pigs. Furthermore, this experiment shows that the CNP compound is biologically active based on its ability to elicit a cGMP response in the dosed animals (Figs. 28 - 48).

[0366] Furthermore, this experiment shows that the subcutaneous bioavailability (F) of more than 40% of the compounds of the present invention (Tables 21 and 22). On the other hand, non-functional positively charged examples (Compound IDs 0776 and 0312) show low subcutaneous bioavailability (7 - 13%). A non-functional example with a nearly neutral charge (Compound ID 1235) shows a moderate subcutaneous bioavailability of 25%.

[0367] Example 13 - Telemetric Measurement of Heart Rate and Blood Pressure The purpose of this study was to investigate the effect of an acute dose of the CNP compound 9482 (Chem. 137) on mean blood pressure (MAP) and heart rate (HR) in awake rats.

[0368] Procedure: The transducer was implanted into Sprague Dawley rats (Charles River), and the rats were allowed to recover for a period of 2 - 3 weeks. Before surgery, the device was placed on the receiver plate and registered with the software by turning on the power. Before the start of the surgery, physiological saline was added to the transducer catheter, and then the transmitter was turned on. The transmitter was placed on the abdomen, and the sensor was placed on the abdominal aorta. Isoflurane was used as an anesthetic, and during and after the surgery, the rats were given Temgesic (0.05 mg / kg, subcutaneous), Norodyl (5 mg / kg, subcutaneous), and Baytril (10 mg / kg, subcutaneous) for pain relief and to prevent bacterial infection.

[0369] Study design: Rats were housed at 2 rats / cage (1 rat with the device and 1 rat as a social partner). All animals were to have free access to water and diet (Altromin) throughout the study. Rats were housed on a light cycle of light from 6:00 am to 6:00 pm and dark from 6:00 pm to 6:00 am. Eight rats were divided into two groups according to a crossover study design.

[0370] Telemetry: Each rat was equipped with a transducer (HD-S10 manufactured by DSI) that could measure blood pressure, heart rate, and activity using Ponemah software with a frequency of 100 Hz. During the measurement, the room where the rats were housed was not disturbed so as to minimize the variability of the data. A 4-hour baseline recording was performed before the start of the test to capture the mean arterial pressure (MAP) and heart rate (HR) patterns of untreated and undisturbed rats. The recording period after drug administration was 24 hours.

[0371] Dosing scheme: Rats are dosed in a crossover study design using 4 rats per group. Each rat is given a 1-week washout period prior to the following dosing events. Dosing is performed intravenously (IV) via the tail vein. Dosing of 9482 was tested in separate experiments with intravenous administrations of 30, 100, and 300 nmol / kg. The vehicle is composed of 8 mM phosphate, 250 mM glycerol, pH 7.4.

[0372] Data analysis and statistics were performed with Ponemah software and Graphpad Prism. For statistics, a paired t-test was applied with a 95% confidence level and P < 0.05 indicating statistical significance.

[0373] Results: Within the first 30 minutes after dosing, no differences in MAP and HR were observed with the CNP compound 9482 at 30, 100, or 300 nmol / kg compared to the vehicle (Tables 23 and 25). The mean over the first 12 hours did not produce a difference in MAP between the vehicle and the three tested doses of 9482 (Table 24). Increases in HR of 8.8 ± 10.1% and 6.4 ± 6.6% (P < 0.05) were detected at 100 and 300 nmol / kg for the same period (Table 26). No change in HR was observed at the 30 nmol / kg dose (Table 26).

Table 28

Table 29

Table 30

Table 31

[0374] Conclusion: Acute IV injection of CNP compound 9482 was not associated with any major hemodynamic changes over 12 hours, as indicated by the lack of change in MAP and only a slight increase in HR at the two highest doses.

[0375] Example 14 - Mouse Midfoot Osteotomy and Culture: The aim of this study was to evaluate bone growth after administration of CNP compounds in an ex vivo mouse model.

[0376] Procedure: One-day-old mouse pups (NMRI, Javier Labs France) were euthanized prior to excision. All animal care and use guidelines applied internationally and within Novo Nordisk were followed. Three midfoot bones were excised from each hindlimb. The excised bones were placed in 24-well cell culture plates and cultured at 37 °C and 5% CO2 in 400 μl of α-minimum essential medium supplemented with 0.2% BSA and 1% Pen / Strep. Bones from all animals were randomized into a control group and six CNP compound treatment groups (100 nM, n = 8). The medium was changed every 2 - 3 days. All compounds were formulated according to the general preparation method - the principle described in Method G. Compounds 9435 and 9483 were formulated in A) 5 mM sodium acetate, 250 mM glycerol, pH 4.0, while compounds 9384, 9407, 9480, and 9482 were formulated in B) 8 mM sodium phosphate, 250 mM glycerol, pH 7.4.

[0377] Vehicle alone had no effect on bone growth compared to medium alone.

[0378] Digital photographs were taken on days 0, 4, 7, 11, and 14 using a digital camera attached to a Nikon microscope. The length of each midfoot bone was measured using ImageJ software and the increase in bone length was expressed as the rate of change (mean (SD)) from the length measured on the excision day.

[0379] Results: With all CNP compounds, bone length increased significantly 11 days after treatment compared to the control group (p < 0.05, two-way ANOVA). The overall increase in bone length was 28 - 48% higher after treatment with CNP compounds compared to the control group 14 days after culture. The results of the study are summarized in Table 27.

Table 32

[0380] In Table 27, the midfoot bones were isolated from mice and cultured for up to 14 days. The length was measured every 3 - 4 days throughout the study and presented as the rate of change from day 0 (mean (SD)). Statistically significant differences are shown compared to the control group (two-way ANOVA).

[0381] Conclusion: This experiment shows that the CNP compounds of the present invention can significantly increase bone length in an ex vivo mouse model.

[0382] Example 15 - Mouse Growth Study The objective of this study was to evaluate growth in mice after treatment with two doses of CNP compounds.

[0383] Procedure: Mice (C57BL / 6J, male, 4 weeks old, n = 10) were given daily subcutaneous injections of compound 9482 or vehicle for 35 days. The formulations were prepared according to the principles described in the general preparation method - Method G (30 and 70 nmol / kg, 10 mL / kg; formulation buffer 8 mM sodium phosphate, 250 mM glycerol, 0.007% polysorbate 20, pH 7.4). Body weight, as well as tail length and body length, were measured weekly throughout the study. The increase in growth was presented as the rate of change from day 0 (mean (SD)).

[0384] Results: Administration of both 30 nmol / kg and 70 nmol / kg of the CNP compound 9482 resulted in a significant increase in tail length and body length 7 and 18 days after treatment, respectively, compared to the control (p < 0.05, two-way ANOVA). After 35 days, the change in tail length (from day 0) increased by 45% and 83%, respectively, after treatment with 30 nmol / kg and 70 nmol / kg of the CNP compound compared to control animals. Body length was 30 and 67% higher, respectively, compared to the control group after 35 days of treatment with 30 nmol / kg and 70 nmol / kg of the CNP compound. The results of the study are summarized in Tables 28 and 29.

Table 33

[0385] In Table 28, the increase in tail length is represented as the rate of change (mean (SD)) from day 0. Statistically significant differences are shown compared to the control group (two-way ANOVA).

Table 34

[0386] In Table 29, the increase in body length is represented as the rate of change (mean (SD)) from day 0. Statistically significant differences are shown compared to the control group (two-way ANOVA).

[0387] Conclusion: This experiment shows that subcutaneous administration of the CNP compound of the present invention in a pharmaceutically relevant formulation results in a significant increase in the tail length and body length of mice. Incorporation by reference of the sequence listing This application is filed together with a sequence listing in electronic format. The entire contents of the sequence listing are incorporated herein by reference.

Claims

1. A CNP compound comprising a CNP peptide and a modifying group, wherein the net charge of the compound at physiological pH is 0 or negative, and the CNP peptide has the formula I: AA ０１ -AA ０２ -AA ０３ -AA ０４ -AA ０５ -AA ０６ -AA ０７ -AA ０８ -AA ０９ -AA １０ -AA １１ -AA １２ -AA １３ -AA １４ -AA １５ -AA １６ -AA １７ -AA １８ -AA １９ -AA ２０ -AA ２１ -AA ２２ -AA ２３ -AA ２４ -AA ２５ -AA ２６ -AA ２７ -AA ２８ -AA ２９ -AA ３０ -AA ３１ -AA ３２ -AA ３３ -AA ３４ -AA ３５ -AA ３６ -AA ３７ (wherein, AA ０１ is Gln or absent, AA ０２ is Glu or absent, AA ０３ is His or absent, AA ０４ is Pro or absent, AA ０５ is Asn or Gln or Glu, or absent, AA ０６ is Ala or absent, AA ０７ is Arg or His or Ala, or absent, AA ０８ is Lys or Ser or His, or absent, AA ０９ is Tyr or Glu, or absent, AA １０ is Lys or Glu or Gln or His, or absent, AA １１ is Gly, AA １２ is Ala, and AA １３ is Gln or Asn or Glu, AA １４ is Lys or His or Glu, AA １５ is Lys or Ser or Glu or Thr or His, AA １６ is Gly, and AA １７ is Leu or Gly or Ser or Val, AA １８ is Ser or His, AA １９ is Gln or Ser or Lys or His, AA ２０ is Gly, AA ２１ is Cys, and AA ２２ is Phe, and AA ２３ is Gly, AA ２４ is Leu, AA ２５ is Pro or Lys, AA ２６ is Leu, AA ２７ is Asp or Glu, AA ２８ is Arg, and AA ２９ is ile, AA ３０ is Gly, and AA ３１ is Ser, and AA ３２ is Leu or Nle or Met, AA ３３ is Ser, AA ３４ is Gly, and AA ３５ is Leu, AA ３６ is Gly, AA ３７ comprises an amino acid sequence according to (wherein AA is Cys), the modifying group includes Chem. A, Chem. B, and Chem. C, Chem. A is 【Chemical 1】 (wherein p is an integer in the range of 14 to 20, * represents an amide bond connecting Chem. A and Chem. B) and is selected from the group consisting of, Chem. B is 【Chemical Formula 2】 (wherein q is an integer in the range of 1 to 8, * represents an amide bond connecting Chem. A- and Chem. B-, ** represents an amide bond connecting Chem. B- and Chem. C-) and is selected from the group consisting of, Chem. C is [Chemical Formula 3] (wherein r is an integer in the range of 0 to 4, s is an integer in the range of 0 to 3, t is an integer in the range of 0 to 1, u is an integer in the range of 0 to 3, ** represents an amide bond connecting Chem. B- and Chem. C-, *** represents an amide bond connecting Chem. C- and the N-terminal alpha-amine on the CNP peptide) and is selected from the group consisting of, a CNP compound.

2. The AA of the CNP peptide １３ is Gln, and the AA １９ is Gln or Ser, and the AA ２５ is Pro, and the AA ３２ is Leu. The CNP compound according to claim 1

3. The AA of the CNP peptide ５ is Gln, and the AA １３ is Gln, and the AA １９ is Gln or Ser, and the AA ２５ is Pro, and the AA ３２ is Leu. The CNP compound according to claim 1

4. The CNP compound according to claim 1, wherein the CNP peptide comprises one of the following amino acid sequences. 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】

5. The CNP compound according to claim 1, wherein the CNP peptide has any one of the following amino acid sequences. 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】 【Table 2-5】 【Table 2-6】

6. The CNP peptide has the following amino acid sequence: GAQKKGSSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 136), ARKYKGAQKKGLSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 77), YKGAQKKGGSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 88), YKGAQKKGLSQGCFGLPLDRIGSLSGLGC (SEQ ID NO: 103), QEHPQARKYKGAQKKGLSSGCFGLPLDRIGSLSGLGC (SEQ ID NO: 67), and The CNP compound according to claim 1, which has any one of QEHPQARKYKGAQKKGLSSGCFGLPLERIGSLSGLGC (SEQ ID NO: 104).

7. The CNP peptide has the following amino acid sequence: The CNP compound according to claim 1, which has QEHPQARKYKGAQKKGLSSGCFGLPLDRIGSLSGLGC (SEQ ID NO: 67).

8. The Chem. B of the modifying group has a q of 1 to 5, and the CNP compound according to any one of claims 1 to 7.

9. The modifying group is as follows: Chem. E, Chem. F, Chem. G, Chem. H, Chem. I, and Chem. J 【Chemical Formula 4-1】 【Chemical Formula 4-2】 (wherein the dotted line defines a bond via an amide bond to the N-terminal alpha-amine of the CNP peptide), and the CNP compound according to any one of claims 1 to 8.

10. CNP compound according to formula II: [Chemical Formula 5]

11. CNP compound according to any one of formula II, III, IV, V, VI, and VII: 【Chemical Formula 6-1】 【Chemical Formula 6-2】

12. A CNP compound comprising a CNP peptide and a modifying group, wherein the compound is any one of the compounds disclosed in Table 1. 【Table 3-1】 【Table 3-2】 【Table 3-3】 【Table 3-4】 【Table 3-5】 【Table 3-6】

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 and one or more pharmaceutically acceptable excipients.

14. A compound according to any one of claims 1 to 12 or a composition according to claim 13 for use in the treatment or prevention of cardio-renal metabolic diseases including heart failure and growth disorders including achondroplasia.

15. A method for treating or preventing heart failure, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 to 12, optionally in combination with one or more additional therapeutically active compounds.

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