CNP prodrug

Abstract

To provide prodrugs of C-type natriuretic peptide (CNP), pharmaceutical compositions comprising such CNP prodrugs, and uses thereof.SOLUTION: In an embodiment, the CNP prodrug is a conjugate of a CNP polypeptide and poly (ethylene glycol) with a reversible linker.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to CNP prodrugs, pharmaceutically acceptable salts thereof, pharmaceutical compositions containing such CNP prodrugs or pharmaceutically acceptable salts thereof, and uses thereof. [Background technology]

[0002] Gain-of-function mutations in FGFR3 lead to achondroplasia (ACH), hypochondroplasia (HCH), and thanatophoric dysplasia (TD). These conditions, all caused by increased signaling from fibroblast growth factor receptor 3 (FGFR3), are characterized by disproportionate proximal short-limb dwarfism, with varying degrees of severity, ranging from mild (HCH) to severe (ACH) to lethal (TD). FGFR3 is a key regulator of endochondral bone growth and transduces signals through several intracellular pathways, including those of signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase (MAPK). Constitutive activation of FGFR3 impairs growth plate chondrocyte proliferation and terminal differentiation as well as extracellular matrix synthesis. FGFR3 activation is associated with increased phosphorylation of the STAT and MAPK pathways. The MAPK signaling pathway is regulated by C-type natriuretic peptide (CNP). Binding of CNP to its receptor, the natriuretic peptide receptor-B (NPR-B), inhibits FGFR3 downstream signaling, thus inducing endochondral growth and skeletal overgrowth, as observed in both CNP-overexpressing mice and humans. Overproduction of CNP in cartilage or sustained delivery of CNP by intravenous (iv) infusion normalizes the short stature of achondroplasia mice, suggesting that administration of supraphysiological levels of CNP may be a therapeutic strategy for ACH.

[0003] However, given its short half-life (2 minutes after intravenous (iv) administration), CNP as a therapeutic agent is challenging in the pediatric population, as it would require continuous infusion. Furthermore, CNP is severely inactivated in subcutaneous tissue, necessitating intravenous administration.

[0004] Potter (FEBS Journal 278 (2011) 1808-1817) describes the clearance of CNP by two pathways: receptor-mediated degradation and degradation by extracellular proteases. CNP is degraded by the action of neutral endopeptidase 24.11 (NEP) and removed from the systemic circulation by the natriuretic peptide clearance receptor, NPR-C, which binds CNP and deposits it in lysosomes where it is degraded.

[0005] The ability of an individual's organs to remove a molecule from the circulation is described by the removal rate, which is calculated by subtracting the venous concentration from the arterial concentration and dividing this value by the arterial blood concentration of the molecule. This so-called A / V difference quantifies how efficiently an organ removes or degrades the molecule in question. In humans, the CNP A / V slope is negative for kidney, liver, and lung tissues, consistent with CNP degradation occurring in these tissues.

[0006] Reducing degradation by one or both of these clearance pathways would help extend the half-life of CNP.

[0007] Due to the limited size of its active site cavity, NEP preferentially recognizes substrates smaller than approximately 3 kDa. U.S. Patent No. 8,377,884 B2 describes variants of CNP, optionally permanently conjugated to PEG polymers to increase resistance to NEP degradation. However, it has been found that the addition of PEG to wild-type CNP, even with PEGs as small as 0.6 kDa, reduces CNP activity, and the addition of PEGs larger than approximately 2 or 3 kDa to CNP or its variants reduces the functional activity of CNP in a size-dependent manner. Thus, the attachment of PEG molecules larger than 2-3 kDa to reduce NEP degradation is accompanied by a loss of activity that may reduce the therapeutic potential of such molecules.

[0008] In addition to adversely affecting peptide activity, attachment of PEG or other polymers to CNP can also prevent effective distribution to the growth plate. Farnum et al. (Anat Rec A Discov Mol Cell Evol Biol. 2006 Jan; 288(1):91-103) demonstrated that distribution of molecules from the systemic vasculature to the growth plate is size-dependent, and that small molecules (10 kDa or less) can distribute to the growth plate, but molecular sizes greater than 40 kDa prevent penetration into the growth plate.

[0009] International application WO 2009 / 156481 A1 relates to reversible PEG conjugates of BNP, a term defined to include all members of the natriuretic peptide family. This application focuses solely on the cardiovascular effects of this class of peptides, which are mediated by natriuretic peptide receptor A (NPR-A). WO 2009 / 156481 A1 does not disclose the specific properties of BNP with regard to regulating the growth, proliferation, and differentiation of cartilage growth plate chondrocytes, which are mediated by activation of natriuretic peptide receptor B (NPR-B).

[0010] Another approach to generating a NEP-resistant CNP molecule and enabling subcutaneous administration is described in The American Journal of Human Genetics 91, 1108–1114. BMN-111 is a modified recombinant human C-type natriuretic peptide (CNP) in which 17 amino acids have been added to form a 39-amino acid CNP pharmacological analog. BMN-111 mimics CNP pharmacological activity in the growth plate and has an extended half-life as a result of neutral endopeptidase (NEP) resistance, which allows for once-daily subcutaneous (SC) administration. Because BMN-111 is a non-naturally occurring peptide, the risk of inducing an immune response is higher compared to native peptides. As described by Martz in "sFGFR for achondroplasia" (SciBx, Biocentury, October 2013), immune responses to BMN-111 were observed in animal studies, but the presence of antibodies did not affect the pharmacological activity of the drug. However, BMN-111 has a half-life of only 20 minutes, resulting in a short duration of exposure to effective drug levels when administered daily.

[0011] To increase exposure to effective drug levels, the dose of drugs with CNP activity may be increased. Natriuretic peptides are a family of hormones that can affect blood volume and blood pressure, and increased doses may be associated with cardiovascular side effects. Studies of BMN-11 in animals and humans have demonstrated that increased doses decrease arterial blood pressure and increase heart rate. Doses of BMN-111 below 15 μg / kg were associated with mild hypotension in healthy volunteers. Therefore, increasing the dose of drugs with CNP activity to increase drug exposure may be associated with unacceptable cardiovascular side effects.

[0012] In short, there is a need for more convenient and / or effective treatments for CNP. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Patent No. 8,377,884 B2 [Patent Document 2] WO 2009 / 156481 A1 [Non-patent literature]

[0014] [Non-Patent Document 1] Potter, FEBS Journal 278(2011)1808-1817 [Non-patent document 2] Farnum et al., Anat Rec A Discov Mol Cell Evol Biol. January 2006, 288(1):91-103 [Non-patent document 3] The American Journal of Human Genetics 91, 1108-1114 [Non-patent document 4] Martz, “sFGFR for achondroplasia” (SciBx, Biocentury October 2013) Summary of the Invention [Problem to be solved by the invention]

[0015] SUMMARY OF THE INVENTION It is therefore an object of the present invention to at least partially overcome the above-mentioned drawbacks. [Means for solving the problem]

[0016] The object is to provide a compound of formula (Ia) or (Ib): [ka] (In the formula, -D is a CNP substructure, -L 1 - is a reversible prodrug linker moiety, -L 2 - is a single chemical bond or spacer moiety; -Z is a water-soluble carrier moiety; x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; y is an integer selected from the group consisting of 1, 2, 3, 4 and 5. or a pharmaceutically acceptable salt thereof.

[0017] In another embodiment, the present invention provides a conjugate DL: (In the formula, -D is a CNP substructure, -L is a reversible prodrug linker moiety -L 1 - includes -L 1 -L 2 -Z', optionally further substituted; -L 2 - is a single chemical bond or spacer moiety; -Z' is a water-insoluble carrier moiety. or a pharmaceutically acceptable salt thereof.

[0018] Many substructures -L 2 -L 1 It is understood that -D is linked to the water-insoluble carrier -Z'. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the structure of CNP according to SEQ ID NO:1. DETAILED DESCRIPTION OF THE INVENTION

[0020] Surprisingly, it has been found that the CNP prodrugs and pharmaceutically acceptable salts thereof of the present invention result in an extended circulation time of CNP in the bloodstream, leading to more convenient and patient-friendly administration methods, such as SC injection once a week or once a month or less. At the same time, unmodified CNP is released, thereby ensuring distribution of the active substance to the growth plate. Because the CNP prodrugs of the present invention have low residual activity, i.e., low binding affinity to NPR-B, the risk of neovascular side effects such as hypotension is significantly reduced.

[0021] More surprisingly, it has been found that the compounds of the present invention achieve more stable blood levels than those observed after daily bolus injection, which more closely mimics physiological exposure to endogenous CNP. These more stable blood levels also apply to various dosing regimens, such as daily administration, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every other week, and once a month.

[0022] Even more surprisingly, sustained release of CNP, such as from a sustained release system, for example from a prodrug of the present invention, has been found to be more effective than a once-daily bolus injection.

[0023] In the present invention, terms having the following meanings are used.

[0024] As used herein, the term "CNP" refers to all CNP polypeptides characterized by regulating the growth, proliferation, and differentiation of cartilage growth plate chondrocytes, preferably from mammalian species, more preferably from human and mammalian species, more preferably from human and murine species, as well as variants, analogs, orthologs, homologs, and derivatives and fragments thereof. Preferably, the term "CNP" refers to the CNP polypeptide of SEQ ID NO: 1, as well as variants, homologs, and derivatives thereof that exhibit essentially the same biological activity, i.e., regulating the growth, proliferation, and differentiation of cartilage growth plate chondrocytes. More preferably, the term "CNP" refers to the polypeptide of SEQ ID NO: 1. It is also preferred that the term "CNP" refers to SEQ ID NO: 24, i.e., the 38-amino acid CNP substructure, as well as variants, homologs, and derivatives thereof that exhibit essentially the same biological activity, i.e., regulating the growth, proliferation, and differentiation of cartilage growth plate chondrocytes.

[0025] SEQ ID NO: 1 is the following sequence: GLSKGCFGLKLDRIGSMSGLGC The cysteines at positions 6 and 22 in this sequence are linked by a disulfide bridge, as shown in FIG.

[0026] SEQ ID NO: 24 is the following sequence: LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC in which the cysteines at positions 22 and 38 are linked by a disulfide bridge.

[0027] The term "CNP" also includes all CNP variants, analogs, orthologs, homologs, and derivatives and fragments thereof as disclosed in WO 2009 / 067639 A2 and WO 2010 / 135541 A2, which are incorporated herein by reference.

[0028] Thus, the term "CNP" preferably also refers to the following peptide sequence: SEQ ID NO:2 (CNP-53): DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 3 (G-CNP-53): GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 4 (M-CNP-53): MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 5 (P-CNP-53): PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 6 (CNP-53 M48N): DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC; SEQ ID NO: 7 (CNP-53 Δ15-31): DLRVDTKSRAAWARGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO:8 (CNP-52): LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 9 (CNP-51): RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 10 (CNP-50): VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 11 (CNP-49): DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 12 (CNP-48): TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 13 (CNP-47): KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 14 (CNP-46): SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 15 (CNP-45): RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 16 (CNP-44): AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 17 (CNP-44 Δ14-22): AAWARLLQEHPNAGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 18 (CNP-44 Δ15-22): AAWARLLQEHPNARGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 19 (CNP-43): AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 20 (CNP-42): WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 21 (CNP-41): ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 22 (CNP-40): RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 23 (CNP-39): LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 24 (CNP-38): LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 25 (CNP-37): QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 26 (CNP-37 Q1pQ, where pQ = pyroglutamate): pQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 27 (G-CNP-37): GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 28 (P-CNP-37): PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 29 (M-CNP-37): MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 30 (PG-CNP-37): PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 31 (MG-CNP-37): MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 32 (CNP-37 M32N): QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC; SEQ ID NO: 33 (G-CNP-37 M32N): GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC; SEQ ID NO: 34 (G-CNP-37 K14Q): GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 35 (G-CNP-37 K14P): GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 36 (G-CNP-37 K14Q, Δ15): GQEHPNARKYKGANQGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 37 (G-CNP-37 K14Q, K15Q): GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 38 (CNP-36): EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 39 (CNP-35): HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 40 (CNP-34): PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 41 (CNP-33): NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 42 (CNP-32): ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 43 (CNP-31): RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 44 (CNP-30): KYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 45 (CNP-29): YKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 46 (CNP-28): KGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 47 (GHKSEVAHRF-CNP-28): GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 48 (CNP-27): GANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 49 (CNP-27 K4Q, K5Q): GANQQGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 50 (CNP-27 K4R, K5R): GANRRGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 51 (CNP-27 K4P, K5R): GANPRGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 52 (CNP-27 K4S, K5S): GANSSGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 53 (CNP-27 K4P, K5R): GANGANPRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 54 (CNP-27 K4R, K5R, K9R): GANRRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 55 (CNP-27 K4R, K5R, K9R, M22N): GANRRGLSRGCFGLKLDRIGSNSGLGC; SEQ ID NO: 56 (P-CNP-27 K4R, K5R, K9R): PGANRRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 57 (M-CNP-27 K4R, K5R, K9R): MGANRRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 58 (HSA fragment-CNP-27): GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLG; SEQ ID NO: 59 (HSA fragment-CNP-27 M22N): GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSNSGLGC; SEQ ID NO: 60 (M-HSA fragment-CNP-27): MGHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 61 (P-HSA fragment-CNP-27): PGHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 62 (CNP-26): ANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 63 (CNP-25): NKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 64 (CNP-24): KKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 65 (CNP-23): KGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 66 (R-CNP-22): RGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 67 (ER-CNP-22): ERGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 68 (R-CNP-22 K4R): RGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 69 (ER-CNP-22 4KR): ERGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 70 (RR-CNP-22): RRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 71 (HRGP fragment-CNP-22): GHHSHEQHPHGANQQGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 72 (HRGP fragment-CNP-22): GAHHPHEHDTHGANQQGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 73 (HRGP fragment-CNP-22): GHHSHEQHPHGANPRGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 74 (IgG1(F c ) fragment-CNP-22): GQPREPQVYTLPSGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 75 (HSA fragment-CNP-22): GQHKDDNPNLPRGANPRGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 76 (HSA fragment-CNP-22): GERAFKAWAVARLSQGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 77 (Osteocrine NPR C inhibitor fragment - CNP22): FGIPMDRIGRNPRGLSKGCFGLKLDRIGSMSGLGC, SEQ ID NO: 78 (FGF2 heparin binding domain fragment-CNP22): GKRTGQYKLGSKTGPGPKGLSKGCFGLKLDRIGSMSGLGC, SEQ ID NO: 79 (IgG1(F c )Fragment-CNP-22 K4R): GQPREPQVYTGANQQGLSRGCFGLKLDRIGSMSGLGC, SEQ ID NO: 80 (HSA fragment-CNP-22 K4R): GVPQVSTSTGANQQGLSRGCFGLKLDRIGSMSGLGC, SEQ ID NO: 81 (Fibronectin fragment-CNP-22 K4R): GQPSSSSQSTGANQQGLSRGCFGLKLDRIGSMSGLGC, SEQ ID NO: 82 (Fibronectin fragment-CNP-22 K4R): GQTHSSGTQSGANQQGLSRGCFGLKLDRIGSMSGLGC, SEQ ID NO: 83 (Fibronectin fragment-CNP-22 K4R): GSTGQWHSESGANQQGLSRGCFGLKLDRIGSMSGLGC, SEQ ID NO: 84 (Zinc finger fragment - CNP-22 K4R): GSSSSSSSSSGANQQGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 85 (CNP-21): LSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 86 (CNP-20): SKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 87 (CNP-19): KGCFGLKLDRIGSMSGLGC; SEQ ID NO: 88 (CNP-18): GCFGLKLDRIGSMSGLGC; SEQ ID NO: 89 (CNP-17): CFGLKLDRIGSMSGLGC; SEQ ID NO: 90 (BNP fragment-CNP-17-BNP fragment): SPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH; SEQ ID NO: 91 (CNP-38 L1G): GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 92 (Ac-CNP-37, where Ac = acetyl): Ac-QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC.

[0029] It is understood that the cysteines at positions 6 and 22 of SEQ ID NO: 1 are linked by disulfide bridges in SEQ ID NOs: 2 to 92.

[0030] More preferably, the term "CNP" refers to the sequences of SEQ ID NOs: 2, 19, 20, 21, 22, 23, 24, 25, 26, 30, 32, 38, 39, 40, 41, 42, 43, 91, 92. Even more preferably, the term "CNP" refers to the sequences of SEQ ID NOs: 23, 24, 25, 26, 38, 39, 91 and 92. In a particularly preferred embodiment, the term "CNP" refers to the sequence of SEQ ID NO: 24.

[0031] In particularly preferred embodiments, the term "CNP" refers to the sequences SEQ ID NOs: 23, 24, 25 and 38, even more preferably the sequences SEQ ID NOs: 24 and 25, and most preferably the sequence SEQ ID NO: 25. In an equally preferred embodiment, the term "CNP" refers to the sequence SEQ ID NO: 24.

[0032] In another preferred embodiment, the term "CNP" refers to the sequence of SEQ ID NO: 93 QEHPNARX1YX2GANX3X4GLSX5GCFGLX6LDRIGSMSGLGC wherein X1, X2, X3, X4, X5 and X6 are each independently selected from the group consisting of K, R, P, S and Q, with the proviso that at least one of X1, X2, X3, X4, X5 and X6 is selected from the group consisting of R, P, S and Q; preferably, X1, X2, X3, X4, X5 and X6 are selected from the group consisting of K and R, with the proviso that at least one of X1, X2, X3, X4, X5 and X6 is R; Even more preferably, the sequence of SEQ ID NO: 94 QEHPNARKYKGANX1X2GLSX3GCFGLX4LDRIGSMSGLGC wherein X1, X2, X3 and X4 are independently selected from the group consisting of K, R, P, S and Q, with the proviso that at least one of X1, X2, X3 and X4 is selected from the group consisting of R, P, S and Q; preferably, X1, X2, X3 and X4 are selected from K and R, with the proviso that at least one of X1, X2, X3 and X4 is R; Most preferably, the sequence of SEQ ID NO: 95 QEHPNARKYKGANX1X2GLSKGCFGLKLDRIGSMSGLGC wherein X1X2 is selected from the group consisting of KR, RK, KP, PK, SS, RS, SR, QK, QR, KQ, RQ, RR, and QQ. Refers to...

[0033] It is understood that for all CNP sequences shown herein, the cysteines at positions 6 and 22 of SEQ ID NO: 1 are linked by disulfide bridges in SEQ ID NOs: 93-95.

[0034] It is understood that the present invention also encompasses CNP variants in which any one or more, up to all, of the residues susceptible to deamidation or deamidation-like reactions (e.g., isomerization) may be converted to other residues by deamidation or deamidation-like reactions to any extent up to 100% for each converted residue. (1) a CNP variant in which any one or more, up to all, asparagine (Asn / N) residues may be converted to aspartic acid or aspartate and / or isoaspartic acid or isoaspartate by deamidation to a conversion rate of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% per converted residue; or (2) a CNP variant in which any one or more, up to all, of the glutamine (Gln / Q) residues may be converted to glutamic acid or glutamate and / or isoglutamic acid or isoglutamate by deamidation to a conversion rate of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% per converted residue; or (3) a CNP variant in which one or more, up to all, of the aspartic acid or aspartate (Asp / D) residues may be converted to isoaspartic acid or isoaspartate by a deamidation-like reaction (also called isomerization) to a conversion rate of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% per converted residue; or (4) a CNP variant in which one or more, up to all, of the glutamic acid or glutamate (Glu / E) residues may be converted to isoglutamic acid or isoglutamate by a deamidation-like reaction (also called isomerization) to a conversion rate of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% per converted residue; or (5) a CNP variant in which the N-terminal glutamine, if present, may be converted to pyroglutamate to a conversion rate of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%; or (5) Combination of the above Includes.

[0035] As used herein, the term "CNP polypeptide variant" refers to a polypeptide from the same species that differs from a reference CNP polypeptide. Preferably, such a reference CNP polypeptide sequence is the sequence of SEQ ID NO: 1. In an equally preferred embodiment, the reference CNP polypeptide sequence is the sequence of SEQ ID NO: 24. Generally, differences are limited, such that the amino acid sequences of the reference and variant are similar throughout and, in many regions, identical. Preferably, a CNP polypeptide variant is at least 70%, 80%, 90%, or 95% identical to a reference CNP polypeptide, preferably the CNP polypeptide of SEQ ID NO: 1. In an equally preferred embodiment, a CNP polypeptide variant is at least 70%, 80%, 90%, or 95% identical to a reference CNP polypeptide, preferably the CNP polypeptide of SEQ ID NO: 24. A polypeptide having an amino acid sequence at least, e.g., 95%, "identical" to a query amino acid sequence means that the amino acid sequence of the subject polypeptide is identical to the query sequence, except that the amino acid sequence of the subject polypeptide may contain no more than 5 amino acid changes per 100 amino acids of the query amino acid sequence. These changes in the reference sequence may occur at the amino-terminal (N-terminal) or carboxy-terminal (C-terminal) positions of the reference amino acid sequence, or anywhere between these terminal positions, interspersed individually among residues in the reference sequence, or interspersed among one or more adjacent groups in the reference sequence. The query sequence may be the entire amino acid sequence of the reference sequence, or any fragment designated as described herein. Preferably, the query sequence is the sequence of SEQ ID NO: 1. In an equally preferred embodiment, the query sequence is the sequence of SEQ ID NO: 24.

[0036] Such CNP peptide variants may be naturally occurring variants, such as naturally occurring allelic variants encoded by one of several alternative forms of CNP occupying a given locus in a chromosome or organism, or may be isoforms encoded by naturally occurring splice variants derived from a single primary transcript. Alternatively, the CNP polypeptide variant may be a variant not known to occur naturally and amenable to mutagenesis methods known in the art.

[0037] It is known in the art that one or more amino acids can be deleted from the N-terminus or C-terminus of a biologically active peptide or protein without substantial loss of biological function. Such N- and / or C-terminal deletions are also encompassed by the term CNP polypeptide variant.

[0038] Those skilled in the art also recognize that some amino acid sequences of CNP polypeptides can be altered without significantly affecting the structure or function of the peptide. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have minimal effect on activity. For example, guidance on how to make phenotypically silent amino acid substitutions is provided in Bowie et al. (1990), Science 247:1306-1310, the entire contents of which are incorporated herein by reference, and the authors of this reference indicate that there are two main approaches to studying the tolerance of amino acid sequences to changes.

[0039] The term CNP polypeptide also encompasses all CNP polypeptides encoded by CNP analogs, orthologs, and / or species homologs. As used herein, the term "CNP analog" refers to CNPs from different, unrelated organisms that perform the same function in each organism, but that do not originate from an ancestral structure shared by the ancestors of those organisms. Instead, similar CNPs arose separately and then evolved to perform the same or similar functions. In other words, similar CNP polypeptides are polypeptides that perform the same biological activity, i.e., regulating the growth, proliferation, and differentiation of cartilage growth plate chondrocytes, but have completely different amino acid sequences.

[0040] As used herein, the term "CNP ortholog" refers to CNPs in two different species that have evolved to be distinct from each other, although their sequences are related by a common homologous CNP in an ancestral species.

[0041] As used herein, the term "CNP homolog" refers to a CNP from a different organism that performs the same function in each organism and originates from an ancestral structure shared by the ancestors of those organisms. In other words, a homologous CNP polypeptide is a polypeptide with an identical amino acid sequence that performs the same biological activity, i.e., regulating the growth, proliferation, and differentiation of cartilage growth plate chondrocytes. Preferably, a CNP polypeptide homolog can be defined as a polypeptide that exhibits at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% identity to a reference CNP polypeptide, preferably the CNP polypeptide of SEQ ID NO: 1. In an equally preferred embodiment, the reference CNP polypeptide is the CNP polypeptide of SEQ ID NO: 24.

[0042] Thus, a CNP polypeptide according to the present invention may be, for example, (i) one in which at least one of the amino acid residues has been substituted with a conserved or non-conserved amino acid residue, preferably a conserved amino acid residue, which may or may not be one encoded by the genetic code, and / or (ii) one in which at least one of the amino acid residues includes a substituent group, and / or (iii) one in which the CNP polypeptide is fused to another compound, for example, a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol), and / or (iv) one in which additional amino acids have been fused to the CNP polypeptide, such as an IgG Fc fusion region peptide or a leader or secretory sequence, or a sequence used in the purification of such forms of the polypeptide, or a protein precursor sequence.

[0043] As used herein, the term "CNP polypeptide fragment" refers to any peptide that comprises a contiguous stretch of a portion of the amino acid sequence of a CNP polypeptide, preferably the polypeptide of SEQ ID NO: 1. In an equally preferred embodiment, the term "CNP polypeptide fragment" refers to any peptide that comprises a contiguous stretch of a portion of the amino acid sequence of the polypeptide of SEQ ID NO: 24.

[0044] More specifically, a CNP polypeptide fragment comprises at least 6, e.g., at least 8, at least 10, or at least 17 contiguous amino acids of a CNP polypeptide, more preferably, the polypeptide of SEQ ID NO: 1. It is also preferred that a CNP polypeptide fragment comprises at least 6, e.g., at least 8, at least 10, or at least 17 contiguous amino acids of the CNP polypeptide of SEQ ID NO: 24. Furthermore, a CNP polypeptide fragment may be described as a subgenus of CNP polypeptides comprising at least 6 amino acids, where "at least 6" is defined as any integer between 6 and the integer representing the C-terminal amino acid of a CNP polypeptide, preferably of SEQ ID NO: 1, or of the equally preferred polypeptide of SEQ ID NO: 24. Further included are species of CNP polypeptide fragments at least 6 amino acids in length, as described above, which are further specified with respect to N- and C-terminal positions. All CNP polypeptide fragments at least 6 amino acids in length, as described above, which may be specifically specified by N- and C-terminal positions, are also encompassed as individual species by the term "CNP polypeptide fragment." That is, any combination of N-terminal and C-terminal positions that can be occupied by a contiguous fragment of at least 6 amino acids in length on any given amino acid sequence of a CNP polypeptide, preferably SEQ ID NO: 1, or the equally preferred CNP polypeptide of SEQ ID NO: 24, is included in the present invention.

[0045] The term "CNP" also includes poly(amino acid) conjugates having the above sequence but with a backbone containing both amide and non-amide bonds, e.g., ester bonds, such as depsipeptides. Depsipeptides are chains of amino acid residues whose backbone contains both amide (peptide) and ester bonds. Thus, as used herein, the term "side chain" refers to the moiety attached to the alpha-carbon of an amino acid moiety when the amino acid moieties are linked by amine bonds, e.g., in the case of a polypeptide, or any carbon atom-containing moiety conjugated to the backbone of a poly(amino acid) conjugate, e.g., in the case of a depsipeptide. Preferably, the term "CNP" refers to a polypeptide having a backbone formed by amide (peptide) conjugates.

[0046] The term CNP includes the above variants, analogs, orthologs, homologs, derivatives and fragments of CNP, and therefore all references to a particular position within a reference sequence also include the equivalent position within variants, analogs, orthologs, homologs, derivatives and fragments of the CNP substructure, even if not specifically stated.

[0047] As used herein, the term "ring substructure" refers to a stretch of consecutive amino acid residues in a CNP drug or substructure that is located between two cysteine ​​residues that form an intermolecular disulfide bridge, or between homologous amino acid residues that are linked via a chemical linker. Preferably, the ring substructure is located between the two cysteine ​​residues that form an intermolecular disulfide bridge. These two cysteines correspond to the cysteines at positions 22 and 38 of the sequence of CNP-38 (SEQ ID NO: 24). Thus, when a CNP drug or substructure has the sequence of CNP-38, amino acids 23-37 are located within the ring substructure.

[0048] Regardless of the length of the CNP moiety, the sequence of the ring moiety of wild-type CNP is FGLKLDRIGSMSGLG (SEQ ID NO: 96).

[0049] As explained above, the term "CNP" refers to CNP drugs or substructures having various numbers of amino acids. Those skilled in the art will understand that the same amino acids occur at different positions in CNP drugs or substructures of different lengths, and will have no difficulty identifying two cysteines that form a disulfide bridge, or two homologous amino acid residues that are linked to each other via a chemical linker, in longer, shorter, and / or otherwise modified versions of CNP.

[0050] The term CNP includes the above variants, analogs, orthologs, homologs, derivatives and fragments of CNP, and therefore the term "ring substructure" also includes the corresponding variants, analogs, orthologs, homologs, derivatives and fragments of the sequence of SEQ ID NO: 96. Thus, all references to a particular position in a reference sequence also include the equivalent position in variants, analogs, orthologs, homologs, derivatives and fragments of the CNP substructure, even if not explicitly stated.

[0051] As used herein, the term "pharmaceutical composition" refers to a composition containing one or more active ingredients, such as drugs or prodrugs, particularly CNP prodrugs of the present invention, and optionally one or more excipients, as well as any product resulting directly or indirectly from the combination, complexation, or aggregation of any two or more of the components of the composition, or from the dissociation of one or more of the components, or from other types of reactions or interactions of one or more of the components. Thus, pharmaceutical compositions of the present invention encompass any composition produced by mixing one or more CNP prodrugs of the present invention and, optionally, a pharmaceutically acceptable excipient.

[0052] As used herein, the term "liquid composition" refers to a mixture comprising a water-soluble CNP prodrug and one or more solvents, such as water.

[0053] The term "suspension composition" refers to a mixture containing a water-insoluble CNP prodrug, for example, where the carrier Z' is a hydrogel, and one or more solvents, for example, water. Due to the water-insoluble polymer, the polymeric prodrug cannot dissolve and remains in a particulate state.

[0054] The term "dry composition" as used herein means that the pharmaceutical composition is provided in a dry form. Suitable drying methods are spray drying and lyophilization, i.e., freeze-drying. Such dry compositions of prodrugs have a maximum of 10%, preferably less than 5%, and more preferably less than 2% residual moisture as determined by Karl Fischer. Preferably, the pharmaceutical composition of the present invention is dried by lyophilization.

[0055] As used herein, the term "drug" refers to a substance used in the treatment, cure, prevention, or diagnosis of disease, or to otherwise improve physical or mental well-being. When a drug is conjugated to another moiety, the moiety of the resulting product that is derived from the drug is referred to as a "biologically active moiety."

[0056] As used herein, the term "prodrug" refers to a biologically active moiety reversibly covalently linked to a special protecting group via a reversible prodrug linker moiety, which is a linker moiety that contains a reversible bond with the biologically active moiety, and the special protecting group alters or eliminates undesirable properties of the parent molecule. This includes enhancing desirable properties and suppressing undesirable properties of the drug. The special non-toxic protecting group is referred to as a "carrier." A prodrug releases the biologically active moiety to which it is reversibly covalently attached in the form of its corresponding drug. In other words, a prodrug is a conjugate that contains a biologically active moiety reversibly covalently conjugated to a carrier moiety via a reversible prodrug linker moiety, where the reversible covalent bond of the carrier to the reversible linker moiety is either direct or via a spacer. Such a conjugate releases the previously conjugated biologically active moiety in the form of a free drug.

[0057] A "biodegradable bond" or "reversible bond" is a bond that can be hydrolytically degraded, i.e., cleaved, in the absence of enzymes under physiological conditions (aqueous buffer solution at pH 7.4, 37°C) with a half-life ranging from 1 hour to 6 months, preferably from 1 hour to 4 months, even more preferably from 1 hour to 3 months, even more preferably from 1 hour to 2 months, and even more preferably from 1 hour to 1 month. Thus, a stable bond is a bond that has a half-life of greater than 6 months under physiological conditions (aqueous buffer solution at pH 7.4, 37°C).

[0058] Thus, a "reversible prodrug linker moiety" is covalently conjugated by a reversible bond to a biologically active moiety, such as CNP, via a moiety that is also covalently conjugated to a carrier moiety, such as -Z or -Z', and the covalent bond to the carrier moiety can be direct or via -L 2 Preferably, -Z or -Z' and -L 2 -The bond between is a stable bond.

[0059] As used herein, the term "traceless prodrug linker" refers to a reversible prodrug linker that, upon degradation, releases the drug in its free form. As used herein, the term "free form" drug refers to the drug in its unmodified, pharmacologically active form.

[0060] As used herein, the term "excipient" refers to a diluent, adjuvant, or vehicle used to administer a therapeutic agent, such as a drug or prodrug. Such pharmaceutical excipients can be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose solutions are preferred excipients when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably used as liquid excipients for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc. Pharmaceutical compositions, if desired, may also contain minor amounts of wetting or emulsifying agents, pH buffering agents, such as acetate, succinate, tris, carbonate, phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), or surfactants such as Tween, poloxamer, poloxamine, CHAPS, Igepal, or amino acids such as glycine, lysine, or histidine. These pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Pharmaceutical compositions may also be formulated as suppositories, using traditional binders and excipients such as triglycerides. Oral formulations can contain standard excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such compositions will contain a therapeutically effective amount of the drug or biologically active moiety, together with a suitable amount of excipients so as to produce the form for proper administration to the patient. The formulation should suit the mode of administration.

[0061] As used herein, the term "reagent" refers to a compound that contains at least one functional group for reaction with a functional group of another compound or drug. It is understood that drugs that contain a functional group (e.g., a primary or secondary amine or hydroxyl functional group) are also reagents.

[0062] As used herein, the term "substructure" refers to a portion of a molecule that is missing one or more atoms compared to the corresponding reagent. For example, when a reagent of the formula "HXH" reacts with another reagent and becomes part of a reaction product, the corresponding substructure of the reaction product has the structure "HX-" or "-X-", where each "-" indicates a bond to another substructure. Thus, the biologically active substructure is released as a drug from the prodrug.

[0063] When a grouping of atoms or a chemical structure is provided in which a grouping of atoms is attached to two substructures or interrupts one substructure, it is understood that the grouping or chemical structure may be attached to the two substructures in either orientation, unless expressly stated otherwise. For example, the substructure "-C(O)N(R 12 )-" is "-C(O)N(R 12 )-" or "-N(R 12 It may be attached to two substructures or may be interrupted by one substructure, such as ")C(O)-". [ka] teeth, [ka] as, or [ka] It may be attached to two substructures or interrupt one substructure as a

[0064] As used herein, the term "functional group" means a group of atoms that can react with another group of atoms. Functional groups include, but are not limited to, the following groups: carboxylic acid (-(C=O)OH), primary or secondary amine (-NH, -NH-), maleimide, thiol (-SH), sulfonic acid (-(O=S=O)OH), carbonate, carbamate (-O(C=O)N<), hydroxyl (-OH), aldehyde (-(C=O)H), ketone (-(C=O)-), hydrazine (>NN<), isocyanate, isothiocyanate, phosphate (-O(P=O)OHOH), phosphonate (-O(P=O)OHH), haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine, disulfide, sulfonamide, sulfonic acid, vinyl sulfone, vinyl ketone, diazoalkane, oxirane, and aziridine.

[0065] When the prodrugs of the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, particularly their pharmaceutically usable salts. Thus, prodrugs of the present invention containing acidic groups can be used in accordance with the present invention, for example, as alkali metal salts, alkaline earth metal salts, or ammonium salts. More specific examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts, or salts with ammonia or organic amines (e.g., ethylamine, ethanolamine, triethanolamine, etc.) or amino acids. Prodrugs of the present invention may also exist that contain one or more basic groups, i.e., groups that can be protonated, and such prodrugs can be used in accordance with the present invention in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenolpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. Further methods for converting basic groups to cations are known to those skilled in the art, such as alkylation of amine groups to provide a positively charged ammonium group and a suitable counterion for its salt. When the prodrug of the present invention contains both an acidic group and a basic group, the present invention also includes internal salts or betaines (zwitterions) in addition to the salt forms mentioned above. The respective salts can be obtained by conventional methods known to those skilled in the art, such as, for example, by contacting these prodrugs with an organic or inorganic acid or base in a solvent or dispersion, or by anion or cation exchange with other salts. The present invention also includes all salts of the prodrugs of the present invention which are not suitable for direct use in pharmaceuticals due to their low physiological compatibility, but which can be used, for example, as intermediates in chemical reactions or in the preparation of pharmaceutically acceptable salts.

[0066] The term "pharmaceutically acceptable" means a substance that is not harmful when administered to a patient, and preferably means approved for use in animals, preferably for use in humans, by a regulatory body, such as the EMA (Europe) and / or the FDA (USA) and / or any other national regulatory body.

[0067] The term "about" used in conjunction with a numerical value herein refers to the numerical value itself, plus or minus a range of up to 10%, more preferably up to 8%, even more preferably up to 5%, and most preferably up to 2% of the numerical value. For example, the phrase "about 200" refers to a range of 200 + / - 10%, i.e., 180 to 220, preferably 200 + / - 8%, i.e., 184 to 216, even more preferably 200 + / - 5%, i.e., 190 to 210, and most preferably 200 + / - 2%, i.e., 196 to 204. It is understood that a percentage indicated as "about 20%" does not mean "20% + / - 10%, i.e., a range of 10 to 30%, but rather "about 20%" refers to 18 to 22%, i.e., a range of plus or minus 10% of the numerical value 20.

[0068] As used herein, the term "polymer" refers to a molecule comprising repeating structural units, i.e., monomers, linked by chemical bonds in a linear, cyclic, branched, cross-linked, dendrimeric, or combination thereof, and may be of synthetic or biological origin, or a combination of both. It is understood that a polymer may also comprise one or more other chemical groups and / or moieties, such as one or more functional groups. Preferably, a soluble polymer has a molecular weight of at least 0.5 kDa, e.g., at least 1 kDa, at least 2 kDa, at least 3 kDa, or at least 5 kDa. If the polymer is soluble, it preferably has a molecular weight of at most 1000 kDa, e.g., at most 750 kDa, e.g., at most 500 kDa, e.g., at most 300 kDa, e.g., at most 200 kDa, e.g., at most 100 kDa. It is understood that for insoluble polymers, such as hydrogels, no meaningful molecular weight range can be provided.

[0069] As used herein, the term "polymeric" refers to a reagent or moiety that includes one or more polymers or polymeric moieties. The polymeric reagent or moiety may also optionally include one or more other moieties, preferably selected from the group consisting of: C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl, and A bond selected from the group comprising: [ka] (In the formula, The dashed line indicates the bond to the remainder of the moiety or reagent; -R and -R aare each independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0070] Those skilled in the art will understand that the polymerization products resulting from a polymerization reaction will not all have the same molecular weight, but rather will exhibit a distribution of molecular weights. Therefore, as used herein, molecular weight range, molecular weight, range of the number of monomers in a polymer, and number of monomers in a polymer refer to number average molecular weight and monomer number average, i.e., the arithmetic mean of the molecular weight of the polymer or polymeric substructure and the arithmetic mean of the number of monomers in the polymer or polymeric substructure.

[0071] Thus, for a polymeric moiety comprising "x" monomer units, any integer assigned to "x" corresponds to the arithmetic average number of monomers. Any range of integers assigned to "x" provides a range of integers within which the arithmetic average number of monomers lies. An integer "x" designated as "about x" means that the arithmetic average number of monomers lies within the integer range of x + / - 10%, preferably x + / - 8%, more preferably x + / - 5%, and most preferably x + / - 2%.

[0072] As used herein, the term "number average molecular weight" refers to the ordinary arithmetic mean of the molecular weights of the individual polymers.

[0073] The term "water-soluble" as used herein with respect to a carrier means that, when such a carrier is part of a CNP prodrug of the present invention, at least 1 g of the CNP prodrug including such a water-soluble carrier can be dissolved in 1 liter of water at 20° C. to form a homogeneous solution. Accordingly, the term "water-insoluble" with respect to a carrier means that, when such a carrier is part of a CNP prodrug of the present invention, less than 1 g of the CNP prodrug including such a water-insoluble carrier can be dissolved in 1 liter of water at 20° C. to form a homogeneous solution.

[0074] As used herein, the term "hydrogel" refers to a hydrophilic or amphiphilic polymeric network composed of homopolymers or copolymers that is insoluble due to the presence of covalent chemical bonds. Crosslinkers provide the structure and physical integrity of the network.

[0075] As used herein, the term "thermogelling" refers to a compound that is a liquid or low viscosity solution having a viscosity of less than 500 csp at 25°C at a shear rate of about 0.1 / sec at low temperatures ranging between about 0°C and about 10°C, but is a higher viscosity compound at higher temperatures ranging between about 30°C and about 40°C, e.g., about 37°C, at a shear rate of about 0.1 / sec and less than 10,000 csp at about 25°C.

[0076] The term "PEG-based," as used herein with respect to a moiety or reagent, means that the moiety or reagent comprises PEG. Preferably, the PEG-based moiety or reagent comprises at least 10% (w / w) PEG, such as at least 20% (w / w) PEG, for example at least 30% (w / w) PEG, such as at least 40% (w / w) PEG, for example at least 50% (w / w), such as at least 60% (w / w) PEG, for example at least 70% (w / w) PEG, such as at least 80% (w / w) PEG, for example at least 90% (w / w), such as at least 95%. The remaining weight percentage of the PEG-based moiety or reagent is other moieties preferably selected from the following moieties and linkages: C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl, and A bond selected from the group comprising: [ka] (In the formula, The dashed line indicates the bond to the remainder of the moiety or reagent; -R and -R a are each independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0077] The term "PEG-based comprising at least X% PEG" as used herein with respect to a moiety or reagent means that the moiety or reagent comprises at least X% (w / w) ethylene glycol units (-CHCHO-), which may be arranged in alternating blocks or may be randomly distributed within the moiety or reagent, preferably all ethylene glycol units of the moiety or reagent are present in one block, with the remaining weight percentage of the PEG-based moiety or reagent being other moieties preferably selected from the following moieties and linkages: C 1-50 Alkyl, C 2-50 Alkenyl, C 2-50 Alkynyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl, and A bond selected from the group comprising: [ka] (In the formula, The dashed line indicates the bond to the remainder of the moiety or reagent; -R and -R a are each independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0078] The term "a hyaluronic acid system containing at least X% hyaluronic acid" is used accordingly.

[0079] As used herein, the term "substituted" means that one or more -H atoms of a molecule or substructure have been replaced with another atom or group of atoms, which are referred to as "substituents."

[0080] Preferably, the one or more further optional substituents are, independently of one another, halogen, —CN, —COOR x1 , -OR x1 , -C(O)R x1 , -C(O)N(R x1 R x1a ), -S(O)2N(R x1 R x1a ), -S(O)N(R x1 R x1a ), -S(O)R x1 , -S(O)R x1 , -N(R x1 )S(O)2N(R x1a R x1b ), -SR x1 , -N(R x1 R x1a ), -NO2, -OC(O)R x1 , -N(R x1 )C(O)R x1a , -N(R x1 )S(O)2R x1a , -N(R x1 )S(O)R x1a , -N(R x1 )C(O)OR x1a , -N(R x1 )C(O)N(R x1a R x1b ), -OC(O)N(R x1 R x1a ), -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, wherein -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R x2 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl has -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3)-, -S(O)2N(R x3 )-, -S(O)N(R x3 )-, -S(O)2-, -S(O)-, -N(R x3 )S(O)2N(R x3a )-, -S-, -N(R x3 )-, -OC(OR x3 )(R x3a )-, -N(R x3 )C(O)N(R x3a )- and -OC(O)N(R x3 )-, optionally interrupted by one or more groups selected from the group consisting of -R x1 , -R x1a , -R x1b are independent of each other, -H, -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, wherein -T 0 , C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R x2 and wherein C is optionally substituted with 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl has -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-, -S(O)2N(R x3 )-, -S(O)N(R x3 )-;-S(O)2-, -S(O)-, -N(R x3 )S(O)2N(R x3a )-, -S-, -N(R x3 )-, -OC(OR x3 )(R x3a )-, -N(R x3 )C(O)N(R x3a )- and -OC(O)N(R x3 )-, optionally interrupted by one or more groups selected from the group consisting of Each T 0are independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; 0 may independently be the same or different one or more -R x2 and optionally substituted with Each R x2 are independently halogen, -CN, oxo(=O), -COOR x4 , -OR x4 , -C(O)R x4 , -C(O)N(R x4 R x4a ), -S(O)2N(R x4 R x4a ), -S(O)N(R x4 R x4a ), -S(O)R x4 , -S(O)R x4 , -N(R x4 )S(O)2N(R x4a R x4b ), -SR x4 , -N(R x4 R x4a ), -NO2, -OC(O)R x4 , -N(R x4 )C(O)R x4a , -N(R x4 )S(O)2R x4a , -N(R x4 )S(O)R x4a , -N(R x4 )C(O)OR x4a , -N(R x4 )C(O)N(R x4a R x4b ), -OC(O)N(R x4 R x4a ), and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R x3 , -R x3a , -R x4 , -R x4a , -R x4b are independently -H and C 1-6alkyl, 1-6 The alkyl is optionally substituted with one or more halogens which may be the same or different.

[0081] More preferably, the one or more further optional substituents are, independently of one another, halogen, —CN, —COOR x1 , -OR x1 , -C(O)R x1 , -C(O)N(R x1 R x1a ), -S(O)2N(R x1 R x1a ), -S(O)N(R x1 R x1a ), -S(O)R x1 , -S(O)R x1 , -N(R x1 )S(O)2N(R x1a R x1b ), -SR x1 , -N(R x1 R x1a ), -NO2, -OC(O)R x1 , -N(R x1 )C(O)R x1a , -N(R x1 )S(O)2R x1a , -N(R x1 )S(O)R x1a , -N(R x1 )C(O)OR x1a , -N(R x1 )C(O)N(R x1a R x1b ), -OC(O)N(R x1 R x1a ), -T 0 , C 1-10 Alkyl, C 2-10 Alkenyl, and C 2-10 alkynyl, wherein -T 0 , C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl may be one or more -R x2 optionally substituted with C 1-10 Alkyl, C 2-10 Alkenyl and C2-10 Alkynyl has -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-, -S(O)2N(R x3 )-, -S(O)N(R x3 )-, -S(O)2-, -S(O)-, -N(R x3 )S(O)2N(R x3a )-, -S-, -N(R x3 )-, -OC(OR x3 )(R x3a )-, -N(R x3 )C(O)N(R x3a )- and -OC(O)N(R x3 )-; Each-R x1 , -R x1a , -R x1b , -R x3 , -R x3a are independently -H, halogen, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl, Each T 0 are independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; 0 may independently be the same or different one or more -R x2 and optionally substituted with Each R x2 are independently halogen, -CN, oxo(=O), -COOR x4 , -OR x4 , -C(O)R x4 , -C(O)N(R x4 R x4a ), -S(O)2N(R x4 R x4a ), -S(O)N(R x4 R x4a ), -S(O)R x4 , -S(O)R x4 , -N(R x4 )S(O)2N(Rx4a R x4b ), -SR x4 , -N(R x4 R x4a ), -NO2, -OC(O)R x4 , -N(R x4 )C(O)R x4a , -N(R x4 )S(O)2R x4a , -N(R x4 )S(O)R x4a , -N(R x4 )C(O)OR x4a , -N(R x4 )C(O)N(R x4a R x4b ), -OC(O)N(R x4 R x4a ), and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R x4 , -R x4a , -R x4b are independently -H, halogen, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl.

[0082] Even more preferably, the one or more further optional substituents are, independently of one another, halogen, —CN, —COOR x1 , -OR x1 , -C(O)R x1 , -C(O)N(R x1 R x1a ), -S(O)2N(R x1 R x1a ), -S(O)N(R x1 R x1a ), -S(O)R x1 , -S(O)R x1 , -N(R x1 )S(O)2N(R x1a R x1b ), -SR x1 , -N(R x1 R x1a ), -NO2, -OC(O)R x1, -N(R x1 )C(O)R x1a , -N(R x1 )S(O)2R x1a , -N(R x1 )S(O)R x1a , -N(R x1 )C(O)OR x1a , -N(R x1 )C(O)N(R x1a R x1b ), -OC(O)N(R x1 R x1a ), -T 0 , C 1-6 Alkyl, C 2-6 Alkenyl, and C 2-6 alkynyl, wherein -T 0 , C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl may be one or more -R x2 and wherein C is optionally substituted with 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl has -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-, -S(O)2N(R x3 )-, -S(O)N(R x3 )-, -S(O)2-, -S(O)-, -N(R x3 )S(O)2N(R x3a )-, -S-, -N(R x3 )-, -OC(OR x3 )(R x3a )-, -N(R x3 )C(O)N(R x3a )- and -OC(O)N(R x3 )-; Each-R x1 , -R x1a , -R x1b , -R x2 , -R x3 , -R x3a are independently -H, halogen, C 1-6 Alkyl, C2-6 Alkenyl and C 2-6 alkynyl, Each T 0 are independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; 0 may independently be the same or different one or more -R x2 is optionally replaced by

[0083] Preferably, up to six -H atoms of an optionally substituted molecule are independently replaced by substituents, e.g., five -H atoms are independently replaced by substituents, four -H atoms are independently replaced by substituents, three -H atoms are independently replaced by substituents, two -H atoms are independently replaced by substituents, or one -H atom is replaced by a substituent.

[0084] The term "interposed" means that the moiety is inserted between two carbon atoms, or, if the insertion is terminal to the moiety, between a carbon or heteroatom and a hydrogen atom, preferably between a carbon and a hydrogen atom.

[0085] As used herein alone or in combination, the term "C 1-4 "Alkyl" means a linear or branched alkyl moiety having 1 to 4 carbon atoms. When present at the terminal end of a molecule, it is a linear or branched alkyl moiety having 1 to 4 carbon atoms. 1-4 Examples of alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. 1-4 When bonded by an alkyl, such C 1-4 Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, and -C(CH3)2-. 1-4Each hydrogen of an alkyl carbon may be optionally replaced by a substituent as defined above. 1-4 The alkyl may be interrupted by one or more moieties as defined below.

[0086] As used herein alone or in combination, the term "C 1-6 "Alkyl" means a straight or branched alkyl moiety having 1 to 6 carbon atoms. When present at the terminal end of a molecule, straight and branched C 1-6 Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl. 1-6 When bonded by an alkyl group, such C 1-6 Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)- and -C(CH3)2-. 1-6 Each hydrogen atom of the carbon may optionally be replaced by a substituent as defined above. 1-6 The alkyl may be interrupted by one or more moieties as defined below.

[0087] Therefore, "C 1-10 Alkyl," "C 1-20 Alkyl" or "C 1-50 "Alkyl" means an alkyl chain having 1 to 10, 1 to 20, or 1 to 50 carbon atoms, respectively; C 1-10 , C 1-20 or C 1-50 Each hydrogen atom of the carbon may optionally be replaced by a substituent as defined above. 1-10 or C 1-50 The alkyl may be interrupted by one or more moieties as defined below.

[0088] As used herein alone or in combination, the term "C 2-6 "Alkenyl" means a straight or branched hydrocarbon moiety containing at least one carbon-carbon double bond, having 2 to 6 carbon atoms. When present at the end of the molecule, examples are -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CHCH2-CH3, and -CH=CH-CH=CH2. When two moieties of the molecule are C 2-6 When bonded by an alkenyl, such C 2-6 An example of an alkenyl is -CH=CH-. 2-6 Each hydrogen atom of the alkenyl moiety may be optionally replaced by a substituent as defined above. 2-6 The alkenyl may be interrupted by one or more moieties as defined below.

[0089] Therefore, the term "C" alone or in combination 2-10 alkenyl," "C 2-20 alkenyl" or "C 2-50 "Alkenyl" means a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon double bond and having 2 to 10, 2 to 20, or 2 to 50 carbon atoms. 2-10 Alkenyl, C 2-20 Alkenyl or C 2-50 Each hydrogen atom of the alkenyl group may be optionally replaced by a substituent as defined above. 2-10 Alkenyl, C 2-20 Alkenyl or C 2-50 The alkenyl may be interrupted by one or more moieties as defined below.

[0090] As used herein alone or in combination, the term "C 2-6"Alkynyl" means a straight or branched hydrocarbon moiety containing at least one carbon-carbon triple bond having 2 to 6 carbon atoms. When present at the end of a molecule, examples are -C≡CH, -CH2-C≡CH, CH2-CH2-C≡CH, and CH2-C≡C-CH3. When two moieties of a molecule are connected by an alkynyl group, an example is -C≡C-. C 2-6 Each hydrogen atom of the alkynyl group may optionally be replaced by a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, C 2-6 The alkynyl may be optionally interrupted by one or more moieties as defined below.

[0091] Thus, the term "C" as used herein alone or in combination 2-10 alkynyl", "C 2-20 alkynyl" or "C 2-50 "Alkynyl" means a linear or branched hydrocarbon moiety containing at least one carbon-carbon triple bond, having 2 to 10, 2 to 20, or 2 to 50 carbon atoms, respectively. 2-10 Alkynyl, C 2-20 Alkynyl or C 2-50 Each hydrogen atom of the alkynyl group may optionally be replaced by a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, C 2-10 Alkynyl, C 2-20 Alkynyl or C 2-50 The alkynyl may be optionally interrupted by one or more moieties as defined below.

[0092] As mentioned above, C 1-4 Alkyl, C 1-6 Alkyl, C 1-10 Alkyl, C 1-20 Alkyl, C 1-50 Alkyl, C 2-6 Alkenyl, C 2-10 Alkenyl, C 2-20 Alkenyl, C 2-50 Alkenyl, C 2-6 Alkynyl, C 2-10Alkynyl, C 2-20 Alkenyl or C 2-50 The alkynyl may be optionally interrupted by one or more moieties, preferably [ka] (In the formula, The dashed line indicates the bond to the remainder of the moiety or reagent; -R and -R a are each independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl. is selected from the group consisting of:

[0093] As used herein, the term "C 3-10 "Cycloalkyl" means a cyclic alkyl chain having 3 to 10 carbon atoms, which may be saturated or unsaturated, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl. 3-10 Each hydrogen atom of a cycloalkyl carbon may be replaced by a substituent as defined above. 3-10 "Cycloalkyl" also includes bridged bicycles such as norbornane or norbornene.

[0094] The term "8- to 30-membered carbopolycyclyl" or "8- to 30-membered carbopolycycle" refers to a cyclic moiety having 8 to 30 ring atoms, two or more rings, two adjacent rings sharing at least one ring atom, and which may contain up to a maximum number of double bonds (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic rings). Preferably, 8- to 30-membered carbopolycyclyl refers to a cyclic moiety having 2, 3, 4, or 5 rings, more preferably 2, 3, or 4 rings.

[0095] As used herein, the term "3- to 10-membered heterocyclyl" or "3- to 10-membered heterocycle" refers to a ring having 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms, in which at least one ring atom and up to four ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and which may contain up to a maximum number of double bonds (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic rings), and which is attached to the remainder of the molecule by a carbon or nitrogen atom. Examples of 3- to 10-membered heterocycles include aziridine, oxirane, thiirane, azirine, oxirene, thiylene, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazol- ine, thiadiazole, thiadiazoline, tetrahydrofuran, and tetrahydrothiophene. , pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine, and homopiperazine. Each hydrogen atom of the 3- to 10-membered heterocyclyl or 3- to 10-membered heterocyclic group may be optionally replaced by a substituent as defined below.

[0096] As used herein, the term "8- to 11-membered heterobicyclyl" or "8- to 11-membered heterobicycle" refers to a two-ring heterocyclic moiety having 8 to 11 ring atoms and which may contain up to a maximum number of double bonds (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic rings), in which at least one ring atom is shared by both rings, and at least one ring atom, up to a maximum of six ring atoms, is replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)-), oxygen, and nitrogen (including =N(O)-), and the rings are bonded to the remainder of the molecule by a carbon or nitrogen atom. Examples of 8- to 11-membered heterobicycles are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine, and pteridine. The term 8- to 11-membered heterobicycle also includes two-ring spiro structures such as 1,4-dioxa-8-azaspiro[4.5]decane, or bridged heterocycles such as 8-aza-bicyclo[3.2.1]octane. Each hydrogen atom of an 8- to 11-membered heterobicyclyl or an 8- to 11-membered heterobicycle carbon may optionally be replaced by a substituent as defined below.

[0097] Similarly, the term "8- to 30-membered heteropolycyclyl" or "8- to 30-membered heteropolycycle" refers to a heterocyclic moiety of more than two rings, preferably 3, 4, or 5 rings, having 8 to 30 ring atoms and which may contain up to a maximum number of double bonds (fully saturated, partially saturated, or unsaturated, aromatic or non-aromatic rings), wherein two adjacent rings share at least one ring atom, and at least one ring atom, up to a maximum of 10 ring atoms, is replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)-), oxygen, and nitrogen (including =N(O)-), and wherein the ring is bonded to the remainder of the molecule by a carbon or nitrogen atom.

[0098] structure: [ka] Regarding the partial structure of x / R y together with the atoms to which they are attached, C 3-10 It is understood that the phrase "form a cycloalkyl or a 3- to 10-membered heterocyclyl" means that Rx and Ry form the following structure: [ka] (Wherein R is C 3-10 cycloalkyl or 3- to 10-membered heterocyclyl).

[0099] structure: [ka] Regarding the partial structure of x / R y together with the atoms to which they are attached form a ring A" is x and R y is also understood to mean forming the structure: [ka]

[0100] As used herein, the term "terminal alkyne" refers to the following moiety: [ka]

[0101] As used herein, "halogen" means fluoro, chloro, bromo, or iodo. It is generally preferred that the halogen is fluoro or chloro.

[0102] In general, the terms "comprise" or "comprising" also encompass "consist" or "consisting of."

[0103] Preferably, -D has the sequence SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:30, even more preferably the sequences SEQ ID NO:24 and SEQ ID NO:25.

[0104] In one embodiment, -D has the sequence of SEQ ID NO:25.

[0105] In another embodiment, -D has the sequence of SEQ ID NO:30.

[0106] In a preferred embodiment, -D has the sequence SEQ ID NO:24.

[0107] Substructure-L 1 - is a reversible prodrug linker that releases the drug, i.e., CNP, in its free form, i.e., it is a traceless prodrug linker. Suitable prodrug linkers are known in the art, such as the reversible prodrug linker moieties disclosed in WO 2005 / 099768 A2, WO 2006 / 136586 A2, WO 2011 / 089216 A1, and WO 2013 / 024053 A1, which are incorporated herein by reference.

[0108] In another embodiment, -L 1 - is a reversible prodrug linker as described in WO 2011 / 012722 A1, WO 2011 / 089214 A1, WO 2011 / 089215 A1, WO 2013 / 024052 A1 and WO 2013 / 160340 A1, which are incorporated herein by reference.

[0109] Substructure-L 1 - may be connected to -D by any type of bond provided that it is reversible. 1 - is linked to -D by a bond selected from the group consisting of amide, ester, carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide and acylguanidine. 1 - is linked to -D by a bond selected from the group consisting of amide, ester, carbamate, and acylguanidine.

[0110] In a preferred embodiment, the moiety -L 1 - is linked to -D by an amide bond. It is noted that amide bonds are generally not reversible, however, in the present invention, -L 1 It is understood that the adjacent group contained in - makes the amide bond reversible.

[0111] Particularly preferred substructure -L 1 - is disclosed in WO 2009 / 095479 A2. Thus, in one preferred embodiment, the moiety -L 1 - is a group represented by the formula (II): [ka] (In the formula, The dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; -X- is -C(R 4 R 4a )-, -N(R 4 )-, -O-, -C(R 4 R4a )-C(R 5 R 5a )-, -C(R 5 R 5a )-C(R 4 R 4a )-, -C(R 4 R 4a )-N(R 6 )-, -N(R 6 )-C(R 4 R 4a )-, -C(R 4 R 4a )-O-, -OC(R 4 R 4a )- or -C(R 7 R 7a )- and X 1 is C or S(O), -X 2 - is -C(R 8 R 8a )- or -C(R 8 R 8a )-C(R 9 R 9a )- and =X 3 is =O, =S, or =N-CN, -R 1 , -R 1a , -R 2 , -R 2a , -R 4 , -R 4a , -R 5 , -R 5a , -R 6 , -R 8 , -R 8a , -R 9 , -R 9a are independently -H and C 1-6 is selected from the group consisting of alkyl, -R 3 , -R 3a are independently -H and C 1-6 alkyl, with the proviso that -R 3 , -R 3a If one or both of are other than -H, they may be attached to the N to which they are attached by SP 3 are linked by hybridized carbon atoms, -R 7 is -N(R 10 R 10a ), or -NR 10 -(C=O)-R 11 and -R 7a , -R 10 , -R 10a , -R 11 are, independently of each other, -H or C 1-6 is alkyl, In some cases, Pair-R 1a / -R 4a , -R 1a / -R 5a , -R 1a / -R 7a , -R 4a / -R 5a , -R 8a / -R 9a one or more of the following forms a chemical bond; In some cases, Pair-R 1 / -R 1a , -R 2 / -R 2a , -R 4 / -R 4a , -R 5 / -R 5a , -R 8 / -R 8a , -R 9 / -R 9a One or more of the following, together with the atoms to which they are attached, form a C 3-10 forming a cycloalkyl or a 3- to 10-membered heterocyclyl; In some cases, Pair-R 1 / -R 4 , -R 1 / -R 5 , -R 1 / -R 6 , -R 1 / -R 7a , -R 4 / -R 5 , -R 4 / -R 6 , -R 8 / -R 9 , -R 2 / -R 3together with the atoms to which they are attached form ring A, In some cases, R 3 / R 3a together with the nitrogen atom to which they are attached form a 3- to 10-membered heterocyclic ring, A is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; -L 1 -ga, -L 2 -Z or -L 2 -Z' and optionally -L 1 - is further substituted, provided that the hydrogen with an asterisk in formula (II) is -L 2 -Z or -L 2 is not replaced by -Z' or a substituent, -L 2 - is a single chemical bond or spacer, -Z is a water-soluble carrier; -Z' is a water-insoluble carrier.

[0112] Preferably, -L of formula (II) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0113] In one embodiment, -L of formula (II) 1 - is not further substituted.

[0114] -R in formula (II) 3 / -R 3a However, when they are combined with the nitrogen atom to form a 3- to 10-membered heterocycle, the atom directly bonded to the nitrogen is SP 3 It is understood that only such 3- to 10-membered heterocyclic rings can be formed that are hybridized carbon atoms. 3 / -R 3aand the nitrogen atom to which they are attached have the following structure: [ka] (In the formula, The dashed line is -L 1 - indicates the bond to the remainder of the ring contains 3 to 10 atoms, including at least one nitrogen atom, and R # and R ## SP 3 represents a hybridized carbon atom).

[0115] It is also understood that the 3- to 10-membered heterocycle may be further substituted.

[0116] -R in formula (II) 3 / -R 3a and the nitrogen atom to which they are attached are: [ka] (In the formula, The dashed line indicates the bond to the rest of the molecule; -R is -H and C 1-6 alkyl).

[0117] Optionally, -L of formula (II) 1 - may be further substituted. In general, any substituent may be used as long as it does not affect the principle of cleavage, i.e., the hydrogen with an asterisk in formula (II) is not replaced and the partial structure of formula (II) [ka] The nitrogen in remains part of a primary, secondary, or tertiary amine, i.e., -R 3 and -R 3a are each independently -H or SP 3 It is linked to -N< by a hybridized carbon atom.

[0118] In one embodiment, -R of formula (II) 1 or -R 1a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 2 or -R 2a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 3 or -R 3a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 4 -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 5 or -R 5a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 6 -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 7 or -R 7a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 8 or -R 8a -L 2 -Z or -L 2 In another embodiment, the -R of formula (II) is substituted with -Z'. 9 or -R 9a -L 2 -Z or -L 2 -Z' is substituted.

[0119] Most preferably, -R of formula (II) 4 -L 2 -Z or -L 2 -Z' is substituted.

[0120] Preferably, -X- in formula (II) is -C(R 4 R 4a )- or -N(R 4 )-. Most preferably, -X- in formula (II) is -C(R 4 R 4a )-.

[0121] Preferably, X in formula (II) 1 is C.

[0122] Preferably, =X in formula (II) 3 is =O.

[0123] Preferably, -X in formula (II) 2 - is -C(R 8 R 8a )-.

[0124] Preferably, -R in formula (II) 8 and -R 8a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (II) 8 and -R 8a At least one of -R in formula (II) is -H. 8 and -R 8a Both are -H.

[0125] Preferably, -R in formula (II) 1 and -R 1a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (II) 1 and -R 1a At least one of -R in formula (II) is -H. 1 and -R 1a Both are -H.

[0126] Preferably, -R in formula (II) 2 and -R 2ais independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (II) 2 and -R 2a At least one of -R in formula (II) is -H. 2 and -R 2a Both are H.

[0127] Preferably, -R in formula (II) 3 and -R 3a is independently selected from the group consisting of -H, methyl, ethyl, propyl, and butyl. Even more preferably, -R in formula (II) 3 and -R 3a In a similarly preferred embodiment, at least one of -R in formula (II) is methyl. 3 and -R 3a In another equally preferred embodiment, -R in formula (II) 3 and -R 3a are both methyl.

[0128] Preferably, R in formula (II) 3 is -H, and -R in formula (II) 3a is methyl.

[0129] Preferably, -R in formula (II) 4 and -R 4a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (II) 4 and -R 4a At least one of -R in formula (II) is -H. 4 and -R 4a Both are -H.

[0130] Preferably, the substructure -L 1 - is a group represented by the formula (IIa): [ka] (In the formula, The dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; -R 1 , -R 1a , -R 2 , -R 2a , -R 3 , -R 3a , -R 4 , -R 4a and -X 2 - is used as defined in relation to formula (II) and Said-L 1 -L 2 -Z or -L 2 -Z', and optionally -L 1 - is further substituted, provided that the hydrogen marked with an asterisk in formula (IIa) is -L 2 -Z or -L 2 It is not replaced by -Z' or a substituent.

[0131] Preferably, -L of formula (IIa) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0132] Preferably, the moiety -L of formula (IIa) 1 - is not further substituted.

[0133] Preferably, -R of formula (IIa) 1 and -R 1a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (IIa) 1 and -R 1a At least one of -R in formula (IIa) is -H. Even more preferably, 1 and -R 1a Both are -H.

[0134] Preferably, -R of formula (IIa) 4 and -R 4ais independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (IIa) 4 and -R 4a At least one of -R in formula (IIa) is -H. Even more preferably, 4 and -R 4a Both are -H.

[0135] Preferably, -X in formula (IIa) 2 - is -C(R 8 R 8a )-.

[0136] Preferably, -R of formula (IIa) 8 and -R 8a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (IIa) 8 and -R 8a At least one of -R in formula (IIa) is -H. Even more preferably, 8 and -R 8a Both are -H.

[0137] Preferably, -R of formula (IIa) 2 and -R 2a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (IIa) 2 and -R 2a At least one of -R in formula (IIa) is -H. Even more preferably, 2 and -R 2a Both are H.

[0138] Preferably, -R of formula (IIa) 3 and -R 3a is independently selected from the group consisting of -H, methyl, ethyl, propyl, and butyl. Even more preferably, -R in formula (IIa) 3 and -R 3a In a similarly preferred embodiment, at least one of -R in formula (IIa) is methyl. 3 and -R 3aare both -H. In another equally preferred embodiment, -R in formula (IIa) 3 and -R 3a are both methyl.

[0139] Preferably, R in formula (IIa) 3 is -H, and -R in formula (IIa) 3a is methyl.

[0140] Preferably, the substructure -L 1 - is a group represented by the formula (IIb): [ka] (In the formula, The dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; -R 2 , -R 2a , -R 3 , -R 3a and -X 2 - is used as defined in relation to formula (II) and Said-L 1 -L 2 -Z or -L 2 -Z', and optionally -L 1 - is further substituted, provided that the hydrogen marked with an asterisk in formula (IIb) is -L 2 -Z or -L 2 It is not replaced by -Z' or a substituent.

[0141] Preferably, -L of formula (IIb) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0142] Preferably, the moiety -L of formula (IIb) 1 - is not further substituted.

[0143] Preferably, -X in formula (IIb) 2- is -C(R 8 R 8a )-.

[0144] Preferably, -R of formula (IIb) 8 and -R 8a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (IIb) 8 and -R 8a At least one of -R in formula (IIb) is -H. 8 and -R 8a Both are -H.

[0145] Preferably, -R of formula (IIb) 2 and -R 2a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (IIb) 2 and -R 2a At least one of -R in formula (IIb) is -H. 2 and -R 2a Both are H.

[0146] Preferably, -R of formula (IIb) 3 and -R 3a is independently selected from the group consisting of -H, methyl, ethyl, propyl, and butyl. Even more preferably, -R in formula (IIb) 3 and -R 3a In a similarly preferred embodiment, at least one of -R in formula (IIb) is methyl. 3 and -R 3a In another equally preferred embodiment, -R in formula (IIb) 3 and -R 3a are both methyl.

[0147] Most preferably, R of formula (IIb) 3 is -H, and -R in formula (IIb) 3a is methyl.

[0148] Even more preferably, the substructure -L1 - is a group represented by the formula (IIb'): [ka] (In the formula, The dashed line indicates the attachment of the CNP moiety, D, to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates -L 2 - indicates a bond to -R 2 , -R 2a , -R 3 , -R 3a , and -X 2 - is used as defined in relation to formula (II) and Optionally, said -L 1 - is further substituted, provided that the hydrogen marked with an asterisk in formula (IIb') is not replaced by a substituent.

[0149] Preferably, the partial structure -L of formula (IIb') 1 - is not further substituted.

[0150] Preferably, -X in formula (IIb') 2 - is -C(R 8 R 8a )-.

[0151] Preferably, -R in formula (IIb') 8 and -R 8a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (IIb') 8 and -R 8a At least one of -R in formula (IIb') is -H. 8 and -R 8a Both are -H.

[0152] Preferably, -R in formula (IIb') 2 and -R 2a is independently selected from the group consisting of -H, methyl and ethyl. More preferably, -R in formula (IIb')2 and -R 2a At least one of -R in formula (IIb') is -H. 2 and -R 2a Both are H.

[0153] Preferably, -R in formula (IIb') 3 and -R 3a is independently selected from the group consisting of -H, methyl, ethyl, propyl and butyl. Even more preferably, -R in formula (IIb') 3 and -R 3a In a similarly preferred embodiment, at least one of -R in formula (IIb') is methyl. 3 and -R 3a In another equally preferred embodiment, -R in formula (IIb') 3 and -R 3a are both methyl.

[0154] Most preferably, R of formula (IIb') 3 is -H, and -R in formula (IIb') 3a is methyl.

[0155] Preferably, the substructure -L 1 - is a group represented by the formula (IIc): [ka] where the dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond. and Said-L 1 -L 2 -Z or -L 2 -Z' is substituted, Said-L 1 - is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIc) is -L 2 -Z or -L 2 It is not replaced by -Z' or a substituent.

[0156] Preferably, -L of formula (IIc) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0157] Preferably, the moiety -L of formula (IIc) 1 - is not further substituted.

[0158] In another preferred embodiment, the moiety -L 1 - is a group represented by the formula (IIc-a): [ka] where the dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond. and Said-L 1 -L 2 -Z or -L 2 -Z' is substituted, Said-L 1 - is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIc-a) is -L 2 -Z or -L 2 It is not replaced by -Z' or a substituent.

[0159] Preferably, -L in formula (IIc-a) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0160] Preferably, the partial structure -L of formula (IIc-a) 1 - is not further substituted.

[0161] In another preferred embodiment, the moiety -L 1 - is a group represented by the formula (IIc-b): [ka] where the dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond. and Said-L 1 -L 2 -Z or -L 2 -Z' is substituted, Optionally, said -L 1 - is further substituted, provided that the hydrogen marked with an asterisk in formula (IIc) is -L 2 -Z or -L 2 It is not replaced by -Z' or a substituent.

[0162] Preferably, -L in formula (IIc-b) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0163] Preferably, the partial structure -L of formula (IIc-b) 1 - is not further substituted.

[0164] Even more preferably, the substructure -L 1 represents the formulae (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv) and (IIc-v): [ka] TIFF2026012776000026.tif5479(in the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates -L 2 -Z or -L 2 -Z') is selected from the group consisting of Said-L 1 - is optionally further substituted, provided that the hydrogen marked with an asterisk in formulas (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv) and (IIc-v) is not replaced by a substituent.

[0165] Preferably, the partial structure -L of the formula (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv) and (IIc-v) 1 - is not further substituted.

[0166] In a particularly preferred embodiment, the moiety -L 1 -teeth, [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates -L 2 -Z or -L 2 -Z') is.

[0167] Preferably, -L in formula (IIc-ii) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0168] In an equally preferred embodiment, the moiety -L 1 - represents the groups of the formulae (IIc-i'), (IIc-ii'), (IIc-iii'), (IIc-iv') and (IIc-v'): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates -L 2 -Z or -L 2 -Z') is selected from the group consisting of Optionally, said -L 1- is further substituted, and the hydrogen marked with an asterisk in formulas (IIc-i'), (IIc-ii'), (IIc-iii'), (IIc-iv') and (IIc-v') is not replaced by a substituent.

[0169] Preferably, the partial structure -L of the formula (IIc-i'), (IIc-ii'), (IIc-iii'), (IIc-iv') and (IIc-v') 1 - is not further substituted.

[0170] In another particularly preferred embodiment, the moiety -L 1 -teeth, [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates -L 2 -Z or -L 2 -Z') is.

[0171] Preferably, -L in formula (IIc-ii') 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0172] In an equally preferred embodiment, the moiety -L 1 - is a group of the formulae (IIc-i″), (IIc-ii″), (IIc-iii″) and (IIc-iv″): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates -L 2 -Z or -L 2 -Z') is selected from the group consisting of Optionally, said -L 1 - is further substituted, with the proviso that the hydrogen marked with an asterisk in formulae (IIc-i''), (IIc-ii''), (IIc-iii'') and (IIc-iv'') is not replaced by a substituent.

[0173] Preferably, the partial structure -L of formula (IIc-i″), (IIc-ii″), (IIc-iii″) and (IIc-iv″) 1 - is not further substituted.

[0174] In another particularly preferred embodiment, the moiety -L 1 -teeth, [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates -L 2 -Z or -L 2 -Z') is.

[0175] Preferably, -L in formula (IIc-ii″) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0176] Formula (II), (IIa), (IIb), (IIb'), (IIc), (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv), (IIc-v), (IIc-i'), -L of (IIc-ii'), (IIc-iii'), (IIc-iv'), (IIc-v'), (IIc-i''), (IIc-ii''), (IIc-iii) and (IIc-iv'') 1 The optional further substituents of - are preferably as described above.

[0177] Another particularly preferred substructure -L 1 - is disclosed in unpublished European Patent No. 14180004, which corresponds to the international application having application number PCT / EP2015 / 067929. Thus, in another preferred embodiment, the moiety -L 1 - is a group represented by the formula (III): [ka] (In the formula, The dashed line indicates attachment of D to a primary or secondary amine or hydroxyl by forming an amide or ester bond, respectively; -R 1 , -R 1a , -R 2 , -R 2a , -R 3 and -R 3a are, independently of each other, -H, -C(R 8 R 8a R 8b ), -C(=O)R 8 , -C≡N, -C(=NR 8 )R 8a , -CR 8 (=CR 8a R 8b ), -C≡CR 8 and -T, -R 4 , -R 5 and -R 5a are, independently of each other, -H, -C(R 9 R 9a R 9b ) and -T; a1 and a2 are independently 0 or 1; Each-R 6 , -R 6a , -R 7 , -R 7a , -R 8 , -R 8a , -R 8b , -R 9 , -R 9a , -R 9b are, independently of each other, -H, halogen, -CN, -COOR 10 , -OR10 , -C(O)R 10 , -C(O)N(R 10 R 10a ), -S(O)2N(R 10 R 10a ), -S(O)N(R 10 R 10a ), -S(O)R 10 , -S(O)R 10 , -N(R 10 )S(O)2N(R 10a R 10b ), -SR 10 , -N(R 10 R 10a ), -NO2, -OC(O)R 10 , -N(R 10 )C(O)R 10a , -N(R 10 )S(O)2R 10a , -N(R 10 )S(O)R 10a , -N(R 10 )C(O)OR 10a , -N(R 10 )C(O)N(R 10a R 10b ), -OC(O)N(R 10 R 10a ), -T, C 1-20 Alkyl, C 2-20 Alkenyl, and C 2-20 alkynyl, wherein said -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl may be one or more -R 11 optionally substituted with C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 12 )-, -S(O)2N(R 12 )-, -S(O)N(R 12 )-, -S(O)2-, -S(O)-, -N(R 12 )S(O)2N(R 12a )-, -S-, -N(R 12 )-, -OC(OR 12)(R 12a )-, -N(R 12 )C(O)N(R 12a )- and -OC(O)N(R 12 )-, optionally interrupted by one or more groups selected from the group consisting of Each-R 10 , -R 10a , -R 10b are independently -H, -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 alkynyl, wherein -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl may be one or more -R 11 and wherein C is optionally substituted with 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 12 )-, -S(O)2N(R 12 )-, -S(O)N(R 12 )-, -S(O)2-, -S(O)-, -N(R 12 )S(O)2N(R 12a )-, -S-, -N(R 12 )-, -OC(OR 12 )(R 12a )-, -N(R 12 )C(O)N(R 12a )- and -OC(O)N(R 12 )-, optionally interrupted by one or more groups selected from the group consisting of Each T is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl, wherein each T is independently selected from the group consisting of one or more —R 11 and optionally substituted with Each-R 11 are each independently a halogen, -CN, oxo (=O), -COOR 13 , -OR13 , -C(O)R 13 , -C(O)N(R 13 R 13a ), -S(O)2N(R 13 R 13a ), -S(O)N(R 13 R 13a ), -S(O)R 13 , -S(O)R 13 , -N(R 13 )S(O)2N(R 13a R 13b ), -SR 13 , -N(R 13 R 13a ), -NO2, -OC(O)R 13 , -N(R 13 )C(O)R 13a , -N(R 13 )S(O)2R 13a , -N(R 13 )S(O)R 13a , -N(R 13 )C(O)OR 13a , -N(R 13 )C(O)N(R 13a R 13b ), -OC(O)N(R 13 R 13a ), and C 1-6 alkyl, wherein C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 12 , -R 12a , -R 13 , -R 13a , -R 13b are independently -H and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; In some cases, Pair-R 1 / -R 1a , -R 2 / -R 2a , -R 3 / -R 3a , -R 6 / -R 6a , -R 7 / -R 7aOne or more of the following, together with the atoms to which they are attached, form a C 3-10 forming a cycloalkyl or a 3- to 10-membered heterocyclyl; In some cases, Pair-R 1 / -R 2 , -R 1 / -R 3 , -R 1 / -R 4 , -R 1 / -R 5 , -R 1 / -R 6 , -R 1 / -R 7 , -R 2 / -R 3 , -R 2 / -R 4 , -R 2 / -R 5 , -R 2 / -R 6 , -R 2 / -R 7 , -R 3 / -R 4 , -R 3 / -R 5 , -R 3 / -R 6 , -R 3 / -R 7 , -R 4 / -R 5 , -R 4 / -R 6 , -R 4 / -R 7 , -R 5 / -R 6 , -R 5 / -R 7 , -R 6 / -R 7 together with the atoms to which they are attached form ring A, A is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl and Said-L 1 -L 2 -Z or -L 2-Z', and optionally -L 1 - is further substituted, -L 2 - is a single chemical bond or spacer, -Z is a water-soluble carrier; -Z' is a water-insoluble carrier.

[0178] -L in formula (III) 1 The optional further substituents of - are preferably as described above.

[0179] Preferably, -L of formula (III) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0180] In one embodiment, -L of formula (III) 1 - is not further substituted.

[0181] -L 1 Further preferred embodiments for - are disclosed in EP 1536334 B1, WO 2009 / 009712 A1, WO 2008 / 034122 A1, WO 2009 / 143412 A2, WO 2011 / 082368 A2, and U.S. Pat. No. 8,618,124 B2, which are incorporated herein by reference in their entirety.

[0182] -L 1 Further preferred embodiments of -L are disclosed in U.S. Pat. No. 8,946,405 B2 and U.S. Pat. No. 8,754,190 B2, which are incorporated herein by reference in their entirety. Accordingly, preferred moieties -L 1 - is a group represented by the formula (IV): [ka] (In the formula, The dashed line indicates a bond to the CNP moiety, -D, said bond being through a functional group of -D selected from the group consisting of -OH, -SH, and -NH; m is 0 or 1; -R 1 and -R 2 At least one or both of the following are, independently of each other, -CN, -NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -C(O)R 3 , -S(O)R 3 , -S(O)2R 3 , and -SR 4 is selected from the group consisting of -R 1 and -R 2 is selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl; -R 3 is -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 9 and -N(R 9 )2, -R 4 is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; Each-R 5 is independently selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted alkenylalkyl, optionally substituted alkynylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; -R9 is selected from the group consisting of —H and optionally substituted alkyl; -Y- is absent and -X- is -O- or -S-; or -Y- is -N(Q)CH2- and -X- is -O-; Q is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; In some cases, -R 1 and R 2 may combine to form a 3- to 8-membered ring, and In some cases, both -R 9 together with the nitrogen to which they are attached form a heterocyclic ring) and Said-L 1 -L 2 -Z or -L 2 -Z', and optionally -L 1 - is further substituted, -L 2 - is a single chemical bond or spacer, -Z is a water-soluble carrier; -Z' is a water-insoluble carrier.

[0183] The terms used exclusively in connection with formula (IV) have the following meanings: As used herein, the term "alkyl" includes straight-chain, branched, or cyclic saturated hydrocarbon groups of 1 to 8 carbons, or in some embodiments, 1 to 6 or 1 to 4 carbon atoms.

[0184] The term "alkoxy" includes an alkyl group attached to an oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.

[0185] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons containing a carbon-carbon double bond.

[0186] The term "alkynyl" includes non-aromatic unsaturated hydrocarbons containing a carbon-carbon triple bond.

[0187] The term "aryl" includes aromatic hydrocarbon groups of 6 to 18 carbon atoms, preferably 6 to 10 carbon atoms, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" includes aromatic rings of 3 to 15 carbon atoms and containing at least one N, O, or S atom, preferably 3 to 7 carbon atoms and containing at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.

[0188] In some instances, an alkenyl, alkynyl, aryl, or heteroaryl moiety may be coupled to the remainder of the molecule by an alkylene bond. In these situations, the substituent will be referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl, or heteroaryl is coupled.

[0189] The term "halogen" includes bromo, fluoro, chloro and iodo.

[0190] The term "heterocyclic ring" refers to a 4- to 8-membered aromatic or non-aromatic ring containing 3 to 7 carbon atoms and at least one N, O, or S atom. Examples are piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided above for the term "heteroaryl."

[0191] When the ring system is optionally substituted, suitable substituents are selected from the group consisting of alkyl, alkenyl, alkynyl, or additional rings, each of which is optionally further substituted. Optional substituents for any of the groups, including those described above, include halo, nitro, cyano, -OR, -SR, -NR, -OCOR, -NRCOR, -COOR, -CONR, -SOR, -SOR, -SONR, -SONR, where each R is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, or two R groups, together with the atoms to which they are attached, form a ring.

[0192] Preferably, -L in formula (IV) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0193] -L 1 Further preferred embodiments of - are disclosed in WO2013 / 036857A1, which is incorporated herein by reference in its entirety. Accordingly, preferred moieties -L 1 - is a compound of formula (V): [ka] (In the formula, The dashed line indicates the bond to the CNP moiety -D, said bond being through the amine functional group of -D; -R 1 is an optionally substituted C1-C6 linear, branched, or cyclic alkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an alkoxy, and -NR 5 2, -R 2 is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; -R 3is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; -R 4 is selected from the group consisting of -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl; Each-R 5 are each independently selected from the group consisting of: -H, optionally substituted C1-C6 alkyl, optionally substituted aryl, and optionally substituted heteroaryl, or when taken together, two -R 5 can be cycloalkyl or cycloheteroalkyl) and Said-L 1 -L 2 -Z or -L 2 -Z', and optionally -L 1 - is further substituted, -L 2 - is a single chemical bond or spacer, -Z is a water-soluble carrier; -Z' is a water-insoluble carrier.

[0194] The terms used exclusively in connection with formula (V) have the following meanings: "Alkyl," "alkenyl," and "alkynyl" include straight-chain, branched, or cyclic hydrocarbon groups of 1 to 8, or 1 to 6, or 1 to 4 carbons, where alkyl is a saturated hydrocarbon, alkenyl contains one or more carbon-carbon double bonds, and alkynyl contains one or more carbon-carbon triple bonds. Unless otherwise specified, they contain 1 to 6 C.

[0195] "Aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. "Heteroaryl" includes aromatic rings of 3 to 15 carbons and at least one N, O, or S atom, preferably 3 to 7 carbons and at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.

[0196] The term "substituted" refers to an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group that contains one or more substituents in place of one or more hydrogen atoms. Substituents generally include halogen (including F, Cl, Br, and I), lower alkyl (including straight-chain, branched, and cyclic), lower haloalkyl (including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl), OH, lower alkoxy (including straight-chain, branched, and cyclic), SH, lower alkylthio (including straight-chain, branched, and cyclic), amino, alkylamino, dialkylamino, silyl (including alkylsilyl, alkoxysilyl, and arylsilyl), nitro, cyano, carbonyl, carboxylic acid, carboxylic acid ester, carboxylic acid amide, aminocarbonyl, aminoacyl, carbamate, urea, thio may be selected from carbamates, thioureas, ketenes, sulfones, sulfonamides, aryls (including phenyl, naphthyl and anthracenyl), heteroaryls (including 5-membered heteroaryls such as pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole and tetrazole, 6-membered heteroaryls such as pyridine, pyrimidine and pyrazine, and fused heteroaryls such as benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole and benzisothiazole).

[0197] Preferably, -L in formula (V) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0198] -L 1 Further preferred embodiments of -L are disclosed in U.S. Patent No. 7,585,837 B2, which is incorporated herein by reference in its entirety. Accordingly, preferred moieties -L 1 - is a group represented by the formula (VI): [ka] (In the formula, The dashed line indicates the bond to the CNP moiety -D, said bond being through the amine functional group of -D; R 1 and R 2 are independently hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, -SO3H, -SO2NHR 5 , amino, ammonium, carboxyl, PO3H2, and OPO3H2; R 3 , R 4 and R 5 are independently selected from the group consisting of hydrogen, alkyl, and aryl; Said-L 1 -L 2 -Z or -L 2 -Z', and optionally -L 1 - is further substituted, -L 2 - is a single chemical bond or spacer, -Z is a water-soluble carrier; -Z' is a water-insoluble carrier.

[0199] Suitable substituents of formula (VI) are alkyl (e.g., C 1-6 alkyl), alkenyl (e.g., C 2-6alkenyl), alkynyl (e.g., C 2-6 alkynyl), aryl (e.g., phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (e.g., aromatic 4- to 7-membered heterocycle), or halogen moieties.

[0200] The terms used exclusively in connection with formula (VI) have the following meanings: The terms "alkyl," "alkoxy," "alkoxyalkyl," "aryl," "alkaryl," and "aralkyl" refer to alkyl radicals of 1 to 8, preferably 1 to 4, carbon atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, and aryl radicals of 6 to 10 carbon atoms, such as phenyl and naphthyl. The term "halogen" includes bromo, fluoro, chloro, and iodo.

[0201] Preferably, -L of formula (VI) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0202] -L 1 Further preferred embodiments of - are disclosed in WO2002 / 089789A1, which is incorporated herein by reference in its entirety. Accordingly, the preferred moiety -L 1 - is a group represented by the formula (VII): [ka] (In the formula, The dashed line indicates the bond to the CNP moiety -D, said bond being through the amine functional group of -D; L1 is a bifunctional linking group, Y1 and Y2 are independently O, S, or NR 7 and R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are independently hydrogen, C1-6 Alkyl, C 3-12 Branched alkyl, C 3-8 Cycloalkyl, C 1-6 Substituted alkyl, C 3-8 Substituted cycloalkyl, aryl, substituted aryl, aralkyl, C 1-6 Heteroalkyl, substituted C 1-6 Heteroalkyl, C 1-6 Alkoxy, phenoxy and C 1-6 heteroalkoxy; When Ar is contained in formula (VII), it is a partial structure forming a polysubstituted aromatic hydrocarbon or a polysubstituted heterocyclic group, X is a chemical bond, a moiety that is actively transported into a target cell, a hydrophobic moiety, or a combination thereof; Y is 0 or 1. and Said-L 1 -L 2 -Z or -L 2 -Z', and optionally -L 1 - is further substituted, -L 2 - is a single chemical bond or a spacer moiety; -Z is a water-soluble carrier; -Z' is a water-insoluble carrier.

[0203] The terms used exclusively in connection with formula (VII) have the following meanings: The term "alkyl" includes, for example, alkoxy, C 3-8 Linear, branched, substituted C, including cycloalkyl or substituted cycloalkyl 1-12 It shall be understood to include alkyl.

[0204] The term "substituted" shall be understood to include the addition and replacement of one or more atoms contained in a functional group or compound with one or more different atoms.

[0205] Substituted alkyl includes carboxyalkyl, aminoalkyl, dialkylamino, hydroxyalkyl, and mercaptoalkyl, substituted cycloalkyl includes moieties such as 4-chlorocyclohexyl, aryl includes moieties such as naphthyl, substituted aryl includes moieties such as 3-bromo-phenyl, aralkyl includes moieties such as toluyl, heteroalkyl includes moieties such as ethylthiophene, substituted heteroalkyl includes moieties such as 3-methoxythiophene, alkoxy includes moieties such as methoxy, and phenoxy includes moieties such as 3-nitrophenoxy. Halo- shall be understood to include fluoro, chloro, iodo, and bromo.

[0206] Preferably, -L in formula (VII) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0207] In another preferred embodiment, -L 1 - is a substructure of formula (VIII) [ka] (In the formula, The dashed line with an asterisk indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; Unmarked dashed lines are -L 1 - indicates the bond to the rest of Including, Said-L 1 -L 2 -Z or -L 2 -Z', and optionally -L 1 - is further substituted, where -L 2 - is a single chemical bond or spacer, -Z is a water-soluble carrier; -Z' is a water-insoluble carrier.

[0208] Preferably, -L in formula (VIII) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0209] In one embodiment, -L of formula (VIII) 1 - is not further substituted.

[0210] In another preferred embodiment, -L 1 - is a substructure of formula (IX) [ka] (In the formula, The dashed line with an asterisk indicates attachment by forming a carbamate bond to the nitrogen of the CNP substructure, -D; Unmarked dashed lines are -L 1 - indicates the bond to the rest of Including, Said-L 1 -L 2 -Z or -L 2 -Z', and optionally -L 1 - is further substituted, -L 2 - is a single chemical bond or spacer, -Z is a water-soluble carrier; -Z' is a water-insoluble carrier.

[0211] Preferably, -L in formula (IX) 1 - is one substructure -L 2 -Z or -L 2 -Z' is substituted.

[0212] In one embodiment, -L of formula (IX) 1 - is not further substituted.

[0213] Substructure-L 1- may be linked to -D through any functional group of -D, preferably through the amine functional group of -D, which may be an N-terminal functional group or an amine functional group provided by a lysine side chain, i.e., by the lysine at position 4 or 10 when CNP has the sequence of SEQ ID NO: 1, by the lysine at positions 7, 9, 13, 14, 18, and 24 when CNP has the sequence of SEQ ID NO: 38, by the lysine at positions 8, 10, 14, 15, 19, or 25 when CNP has the sequence of SEQ ID NO: 25, by the lysine at positions 9, 11, 15, 16, 20, or 26 when CNP has the sequence of SEQ ID NO: 24, and by the lysine at positions 10, 12, 16, 17, 21, or 27 when the CNP moiety has the sequence of SEQ ID NO: 23.

[0214] In one embodiment, the CNP moiety is linked to -L by the N-terminal amine functional group of the CNP moiety. 1 - and is linked.

[0215] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 4, where the CNP moiety has the sequence of SEQ ID NO: 1. 1 - and is linked.

[0216] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of the lysine at position 10, where the CNP moiety has the sequence of SEQ ID NO: 1. 1 - and is linked.

[0217] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of the lysine at position 8, where the CNP moiety has the sequence of SEQ ID NO: 25. 1 - and is linked.

[0218] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of the lysine at position 10, where the CNP moiety has the sequence of SEQ ID NO: 25. 1 - and is linked.

[0219] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 14, where the CNP moiety has the sequence of SEQ ID NO: 25. 1 - and is linked.

[0220] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 15, where the CNP moiety has the sequence of SEQ ID NO: 25. 1 - and is linked.

[0221] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 19, where the CNP moiety has the sequence of SEQ ID NO:25. 1 - and is linked.

[0222] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 25, where the CNP moiety has the sequence of SEQ ID NO: 25. 1 - and is linked.

[0223] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of the lysine at position 9, where the CNP moiety has the sequence of SEQ ID NO:24. 1 - and is linked.

[0224] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 11, where the CNP moiety has the sequence of SEQ ID NO:24. 1 - and is linked.

[0225] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 15, where the CNP moiety has the sequence of SEQ ID NO:24. 1 - and is linked.

[0226] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 16, where the CNP moiety has the sequence of SEQ ID NO:24. 1 - and is linked.

[0227] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 20, where the CNP moiety has the sequence of SEQ ID NO:24. 1 - and is linked.

[0228] In another embodiment, the CNP moiety is linked to -L by the amine functionality provided by the side chain of lysine at position 26, where the CNP moiety has the sequence of SEQ ID NO:24. 1 - and is linked.

[0229] Most preferably, the CNP moiety has the sequence of SEQ ID NO: 24, and is linked to -L by the amine functionality provided by the side chain of lysine at position 26. 1 - and is linked.

[0230] -L to CNP ring 1 It has surprisingly been found that conjugation of - significantly reduces the affinity of CNP prodrugs for NPR-B compared to conjugation at the N-terminus or to non-cyclic portions of CNP, which reduces the affinity for NPR-B and also reduces the risk of cardiovascular side effects such as hypotension.

[0231] Therefore, -L 1 - is preferably conjugated to the side chain of an amino acid residue of said ring substructure of -D or to the backbone of said ring substructure of -D. 1 Even more preferably, - is reversibly covalently conjugated to the side chain of an amino acid residue of said ring moiety of -D.

[0232] The amino acid residue located in the ring substructure of -D is preferably any amino acid bearing a functional group.

[0233] Preferably, -L 1 The amino acid residue of the ring substructure of -D to which - is conjugated contains a functional group selected from the group consisting of carboxylic acid, primary and secondary amine, maleimide, thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate, phosphate, phosphonate, haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine, sulfate, disulfide, vinyl sulfone, vinyl ketone, diazoalkane, oxirane, guanidine and aziridine. 1 The amino acid residue of the ring substructure of -D to which - is conjugated contains a functional group selected from the group consisting of hydroxyl, primary and secondary amines, and guanidine.

[0234] Substructure-L 1 - may be connected to -D by any type of bond provided that it is reversible. 1 - is linked to -D by a bond selected from the group consisting of amide, ester, carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide and acylguanidine. 1 - is linked to -D by a bond selected from the group consisting of amide, ester, carbamate, and acylguanidine.

[0235] In one embodiment, -L 1 - is linked to -D by an ester bond.

[0236] In another embodiment, -L 1 - is linked to -D by a carbamate bond.

[0237] In another embodiment, -L 1 - is linked to -D by an acylguanidine.

[0238] In a preferred embodiment, -L 1- is linked to -D by an amide bond.

[0239] -L 1 The amino acid residue of the ring moiety of -D to which - is conjugated is selected from the group consisting of proteinogenic and non-proteinogenic amino acid residues.

[0240] In one embodiment, -L 1 The amino acid residue of the ring moiety of -D to which - is conjugated is a non-proteinogenic amino acid.

[0241] In a preferred embodiment, -L 1 The amino acid residue of the ring substructure of -D to which - is conjugated is a proteinogenic amino acid. Even more preferably, said amino acid is selected from the group consisting of histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, and arginine. Even more preferably, said amino acid is selected from the group consisting of lysine, aspartic acid, arginine, and serine. Even more preferably, said amino acid is selected from the group consisting of lysine, arginine, and serine.

[0242] In one embodiment, -L 1 The amino acid residue of the ring substructure of -D to which - is conjugated is a histidine, it being understood that such a histidine is not present in the sequence of SEQ ID NO: 96, but only in its variants, analogs, orthologs, homologs and derivatives.

[0243] In one embodiment, -L 1 The amino acid residue of the ring substructure of -D to which - is conjugated is tryptophan, which is absent from the sequence of SEQ ID NO: 96 and is present only in its variants, analogs, orthologs, homologs and derivatives.

[0244] In one embodiment, -L 1The amino acid residue of the ring substructure of -D to which - is conjugated is threonine, it being understood that such threonine is absent in the sequence of SEQ ID NO: 96, but only in its variants, analogs, orthologs, homologs and derivatives.

[0245] In one embodiment, -L 1 The amino acid residue of the ring substructure of -D to which - is conjugated is a tyrosine, it being understood that such a tyrosine is absent in the sequence of SEQ ID NO: 96, but only in its variants, analogs, orthologs, homologs and derivatives.

[0246] In one embodiment, -L 1 The amino acid residue of the ring substructure of -D to which - is conjugated is glutamic acid, it being understood that such glutamic acid is not present in the sequence of SEQ ID NO: 96, but only in its variants, analogs, orthologs, homologs and derivatives.

[0247] In one embodiment, -L 1 The amino acid residue of the ring substructure of -D to which - is conjugated is lysine. Preferably, said amino acid is lysine at position 4 of SEQ ID NO: 96, which corresponds to lysine at position 26 of SEQ ID NO: 24.

[0248] In another embodiment, -L 1 The amino acid residue of the ring moiety of -D to which - is conjugated is aspartic acid. Preferably, said amino acid is the aspartic acid at position 6 of SEQ ID NO: 96, which corresponds to the aspartic acid at position 28 of SEQ ID NO: 24.

[0249] In another embodiment, -L 1 The amino acid residue of the ring substructure of -D to which - is conjugated is arginine. Preferably, said amino acid is arginine at position 7 of SEQ ID NO: 96, which corresponds to arginine at position 29 of SEQ ID NO: 24.

[0250] In another embodiment, -L 1The amino acid residue of the ring substructure of -D to which - is conjugated is serine. Preferably, said amino acid is serine at position 10 or 12 of SEQ ID NO: 96. In one embodiment, said amino acid is serine at position 10 of SEQ ID NO: 96, which corresponds to serine at position 32 of SEQ ID NO: 24. In another embodiment, said amino acid is serine at position 12 of SEQ ID NO: 96, which corresponds to serine at position 34 of SEQ ID NO: 24.

[0251] In a preferred embodiment, -L 1 The amino acid residue of the ring moiety of -D to which - is conjugated is lysine. Most preferably, -D has the sequence of SEQ ID NO: 24 and -L 1 - is conjugated to the lysine at position 26.

[0252] It was also surprisingly found that increasing the length of the CNP sequence was beneficial with respect to NEP stability: CNP-22 was more susceptible to NEP degradation than CNP-34, which in turn was more susceptible than CNP-38.

[0253] In the prodrugs of the present invention, -L 2 - is a chemical bond or a spacer moiety.

[0254] In one embodiment, -L 2 - is a chemical bond.

[0255] In another embodiment, -L 2 - is a spacer moiety.

[0256] -L 2 If - is other than a single chemical bond, -L 2 - is preferably -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-, -S(O)2N(R y1 )-, -S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(Ry1 )-, -OC(OR y1 )(R y1a )-, -N(R y1 )C(O)N(R y1a )-, -OC(O)N(R y1 )-, C 1-50 Alkyl, C 2~50 Alkenyl, and C 2~50 alkynyl, wherein -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R y2 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-, -S(O)2N(R y3 )-, -S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-, -N(R y3 )C(O)N(R y3a )- and -OC(O)N(R y3 )-, optionally interrupted by one or more groups selected from the group consisting of -R y1 and -R y1a are, independently of each other, -H, -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, wherein -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl may be one or more of the same or different -Ry 2 and wherein C is optionally substituted with 1-50 Alkyl, C 2-50 Alkenyl and C 2-50Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y4 )-, -S(O)2N(R y4 )-, -S(O)N(R y4 )-, -S(O)2-, -S(O)-, -N(R y4 )S(O)2N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-, -N(R y4 )C(O)N(R y4a )- and -OC(O)N(R y4 )-, optionally interrupted by one or more groups selected from the group consisting of Each T is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, wherein each T is independently selected from the group consisting of one or more of the same or different -R y2 and optionally substituted with Each R y2 are independently halogen, -CN, oxo(=O), -COOR y5 , -OR y5 , -C(O)R y5 , -C(O)N(R y5 R y5a ), -S(O)2N(R y5 R y5a ), -S(O)N(R y5 R y5a ), -S(O)R y5 , -S(O)R y5 , -N(R y5 )S(O)2N(R y5a R y5b ), -SR y5 , -N(R y5 R y5a ), -NO2, -OC(O)R y5 , -N(R y5 )C(O)R y5a , -N(R y5 )S(O)2R y5a , -N(R y5)S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5a R y5b ), -OC(O)N(R y5 R y5a ), and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b are independently -H and C 1-6 alkyl, 1-6 The alkyl is optionally substituted with one or more halogens which may be the same or different.

[0257] -L 2 If - is other than a single chemical bond, -L 2 - is even more preferably -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-, -S(O)2N(R y1 )-, -S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-, -N(R y1 )C(O)N(R y1a )-, -OC(O)N(R y1 )-, C 1-50 Alkyl, C 2~50 Alkenyl, and C 2~50 alkynyl, wherein -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl may be one or more -R y2optionally substituted with C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-, -S(O)2N(R y3 )-, -S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-, -N(R y3 )C(O)N(R y3a )- and -OC(O)N(R y3 )-, optionally interrupted by one or more groups selected from the group consisting of -R y1 and -R y1a are, independently of each other, -H, -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 alkynyl, wherein -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl may be one or more -R y2 and wherein C is optionally substituted with 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y4 )-, -S(O)2N(R y4 )-, -S(O)N(R y4 )-, -S(O)2-, -S(O)-, -N(R y4 )S(O)2N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-, -N(R y4 )C(O)N(R y4a )- and -OC(O)N(R y4)-, optionally interrupted by one or more groups selected from the group consisting of Each T is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, wherein each T is independently selected from the group consisting of one or more of the same or different -R y2 and optionally substituted with -R y2 is halogen, -CN, oxo(=O), -COOR y5 , -OR y5 , -C(O)R y5 , -C(O)N(R y5 R y5a ), -S(O)2N(R y5 R y5a ), -S(O)N(R y5 R y5a ), -S(O)R y5 , -S(O)R y5 , -N(R y5 )S(O)2N(R y5a R y5b ), -SR y5 , -N(R y5 R y5a ), -NO2, -OC(O)R y5 , -N(R y5 )C(O)R y5a , -N(R y5 )S(O)2R y5a , -N(R y5 )S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5a R y5b ), -OC(O)N(R y5 R y5a ), and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R y3 , -R y3a , -R y4, -R y4a , -R y5 , -R y5a and -R y5b are independently -H and C 1-6 alkyl, 1-6 The alkyl is optionally substituted with one or more halogens which may be the same or different.

[0258] -L 2 If - is other than a single chemical bond, -L 2 - is even more preferably -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-, -S(O)2N(R y1 )-, -S(O)N(R y1 )-, -S(O)2-, -S(O)-, -N(R y1 )S(O)2N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-, -N(R y1 )C(O)N(R y1a )-, -OC(O)N(R y1 )-, C 1-50 Alkyl, C 2~50 Alkenyl, and C 2~50 alkynyl, wherein -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R y2 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-, -S(O)2N(R y3 )-, -S(O)N(R y3 )-, -S(O)2-, -S(O)-, -N(R y3 )S(O)2N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(Ry3a )-, -N(R y3 )C(O)N(R y3a )- and -OC(O)N(R y3 )-, optionally interrupted by one or more groups selected from the group consisting of -R y1 and -R y1a are independently -H, -T, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 alkynyl, Each T is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 selected from the group consisting of cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; -R y2 are independently halogen and C 1-6 is selected from the group consisting of alkyl, Each-R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b are, independently of each other, -H and C 1-6 alkyl, 1-6 The alkyl is optionally substituted with one or more halogens which may be the same or different.

[0259] Even more preferably, -L 2 - is -O-, -T- and -C(O)N(R y1 C optionally interrupted by one or more groups independently selected from 1-20 is an alkyl chain, 1-20 The alkyl chains are -OH, -T and -C(O)N(R y6 R y6a ) optionally substituted with one or more groups independently selected from the group consisting of -R y1 , -R y6 , -R y6a are independently H and C 1-4alkyl, wherein T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 It is selected from the group consisting of cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl.

[0260] Preferably, -L 2 - has a molecular weight in the range of 14 g / mol to 750 g / mol.

[0261] Preferably, -L 2 - includes a substructure selected from the following: [ka] (In the formula, The dashed line is -L 2 -, -L 1 -, -Z and / or -Z' each represent a bond to the remainder; -R and -R a are each independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0262] In one preferred embodiment, -L 2 - has a chain length of 1 to 20 atoms.

[0263] Substructure-L 2 As used herein, the term "chain length" refers to -L 1 -L at the shortest connection between - and -Z 2 -Refers to the number of atoms.

[0264] Preferably, -L 2 - is a group represented by the formula (i) [ka] (In the formula, A dashed line with an asterisk indicates -L 1 - indicates a bond to An unmarked dashed line indicates a bond to -Z or -Z'; -R 1 -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl, n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. and optionally, this moiety of formula (i) is further substituted.

[0265] Preferably, -R in formula (i) 1 is selected from the group consisting of -H, methyl, ethyl, propyl and butyl. Even more preferably, -R in formula (i) 1 is selected from the group consisting of -H, methyl, ethyl and propyl. Even more preferably, -R in formula (i) 1 is selected from the group consisting of -H and methyl. Most preferably, -R in formula (i) 1 is methyl.

[0266] Preferably, n in formula (i) is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Even more preferably, n in formula (i) is selected from the group consisting of 0, 1, 2, 3, 4, and 5. Even more preferably, n in formula (i) is selected from the group consisting of 0, 1, 2, and 3. Even more preferably, n in formula (i) is selected from the group consisting of 0 and 1. Most preferably, n in formula (i) is 0.

[0267] In one preferred embodiment, -L 2 -teeth, [ka] (In the formula, A dashed line with an asterisk indicates -L 1 - indicates a bond to An unmarked dashed line indicates a bond to -Z or -Z'. and wherein the substructures (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi), (xii), (xiii), (xiv), (xv), (xvi) and (xvii) are optionally further substituted.

[0268] In a preferred embodiment, -L 2 is selected from the group consisting of: [ka] (In the formula, A dashed line with an asterisk indicates -L 1 - indicates a bond to An unmarked dashed line indicates a bond to -Z or -Z').

[0269] In an even more preferred embodiment, -L 2 is selected from the group consisting of: [ka] (In the formula, A dashed line with an asterisk indicates -L 1 - indicates a bond to An unmarked dashed line indicates a bond to -Z or -Z').

[0270] Even more preferably, -L 2 - is: [ka] (In the formula, A dashed line with an asterisk indicates -L 1 - indicates a bond to An unmarked dashed line indicates a bond to -Z or -Z').

[0271] In one preferred embodiment, the moiety -L 1 -L 2 is selected from the group consisting of: [ka] TIFF2026012776000046.tif42144 (in the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0272] In an even more preferred embodiment, the moiety -L 1 -L 2 - is: [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0273] In the most preferred embodiment, the moiety -L 1 -L 2 has the formula (IId-ii′): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0274] In another preferred embodiment, the moiety -L 1 -L 2 is selected from the group consisting of: [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0275] In an even more preferred embodiment, the moiety -L 1 -L 2 - is: [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0276] In the most preferred embodiment, the moiety -L 1 -L 2 has the formula (IId-iia'): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0277] In another preferred embodiment, the moiety -L 1 -L 2 is selected from the group consisting of: [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0278] In an even more preferred embodiment, the moiety -L 1 -L 2 - is: [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0279] In the most preferred embodiment, the moiety -L 1 -L 2 has the formula (IId-iib'): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; A dashed line with an asterisk indicates a bond to -Z or -Z').

[0280] Substructure -L by replacing any -H present 2 -A-L 1 - may be bonded to

[0281] Preferably, -R in formula (II) 1 , -R 1a , -R 2 , -R 2a , -R 3 , -R 3a , -R 4 , -R 4a , -R 5 , -R 5a , -R 6 , -R 7 , -R 7a , -R 8 , -R 8a , -R 9 , -R 9a , -R 10 , -R 10aand / or -R 11 1 to 5, preferably 1, of the hydrogen atoms given by -L 2 Preferably, -R in formula (III) 1 , -R 1a , -R 2 , -R 2a , -R 3 , -R 3a , -R 4 , -R 5 , -R 5a , -R 6 , -R 6a , -R 7 , -R 7a , -R 8 , -R 8a , -R 8b , -R 9 , -R 9a , -R 9b , -R 10 , -R 10a , -R 10b , -R 11 , -R 12 , -R 12a , -R 13 , -R 13a and / or -R 13b 1 to 5, preferably 1, of the hydrogen atoms given by -L 2 - is replaced by

[0282] Preferably, -Z has a molecular weight in the range of 5 to 200 kDa. Even more preferably, -Z has a molecular weight in the range of 8 to 100 kDa, even more preferably 10 to 80 kDa, even more preferably 12 to 60 kDa, even more preferably 15 to 40 kDa, and most preferably, -Z has a molecular weight of about 20 kDa. In another equally preferred embodiment, -Z has a molecular weight of about 40 kDa.

[0283] Carrier-Z is C 8-24Preferably, -Z is a polymer, preferably 2-methacryloyl-oxyethyl phosphorylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyloxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl-oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylate ... and copolymers thereof. The polymers include polymers selected from the group consisting of poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate-based polymers, xylan, and copolymers thereof.

[0284] In one embodiment, such water-soluble carrier-Z comprises a protein. Preferred proteins are selected from the group consisting of a carboxyl terminal peptide of chorionic gonadotropin as described in U.S. Patent Application Publication No. 2012 / 0035101 A1, which is incorporated herein by reference, albumin, an XTEN sequence as described in WO 2011123813 A2, which is incorporated herein by reference, a proline / alanine random coil sequence as described in WO 2011 / 144756 A1, which is incorporated herein by reference, a proline / alanine / serine random coil sequence as described in WO 2008 / 155134 A1 and WO 2013 / 024049 A1, which are incorporated herein by reference, and an Fc fusion protein.

[0285] In another preferred embodiment, -Z comprises a fatty acid derivative. Preferred fatty acid derivatives are those disclosed in WO 2005 / 027978 A2 and WO 2014 / 060512 A1, which are incorporated herein by reference.

[0286] In another preferred embodiment, -Z is a hyaluronic acid based polymer.

[0287] In one embodiment, -Z is a carrier as disclosed in WO 2012 / 02047 A1, which is incorporated herein by reference.

[0288] In another embodiment, -Z is a carrier as described in WO 2013 / 024048 A1, which is incorporated herein by reference.

[0289] In another preferred embodiment, -Z is a PEG-based polymer. Even more preferably, -Z is a branched or multi-arm PEG-based polymer. Most preferably, -Z is a multi-arm PEG-based polymer. Even more preferably, -Z is a multi-arm PEG-based polymer having at least four PEG-based arms.

[0290] Preferably, such branched or multi-arm PEG-based polymer-Z, preferably multi-arm PEG-based polymer-Z, comprises a number of substructures -L 2 -L 1 -D, and preferably each of the substructures -L 2 -L 1 -D is linked to the end of a branch or arm, preferably to the end of an arm. Preferably, such a branched or multi-arm PEG-based polymer-Z, preferably a multi-arm PEG-based polymer-Z, comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 moieties -L 2 -L 1 Even more preferably, such a branched or multi-arm PEG-based polymer-Z, preferably a multi-arm PEG-based polymer-Z, is linked to 2, 3, 4, 6 or 8 moieties -L 2 -L 1 Even more preferably, such a branched or multi-arm PEG-based polymer-Z, preferably a multi-arm PEG-based polymer-Z, is linked to two, four or six moieties -L 2 -L 1 -D, and even more preferably, such branched or multi-arm PEG-based polymer-Z, preferably multi-arm PEG-based polymer-Z, is linked to 4 or 6 moieties -L 2 -L 1 -D, and most preferably, such branched or multi-arm PEG-based polymer-Z, preferably multi-arm PEG-based polymer-Z, is linked to four moieties -L 2 -L 1 -It is connected with D.

[0291] More than one substructure -L 2 -L 1 Linking -D with one moiety -Z is advantageous because it ensures a sufficiently high drug loading that allows for the provision of a small, pharmaceutically effective dose of CNP, and also increases patient convenience.

[0292] Preferred water-soluble PEG-based carrier-Z is a multi-arm PEG derivative, such as a 4-arm PEG derivative, particularly a 4-arm PEG containing a pentaerythritol core, an 8-arm PEG containing a hexaglycerin core, and an 8-arm PEG containing a tripentaerythritol core, as listed in, for example, the product list of JenKem Technology, USA (downloaded from http: / / www.jenkemusa.com / Pages / PEGProducts.aspx on December 18, 2014). More preferably, the water-soluble PEG-based carrier-Z is 4-arm PEG amine containing a pentaerythritol core: [ka] (n is in the range of 20 to 500), 8-arm PEG amine with a hexaglycerin core: [ka] (n is in the range of 20 to 500, R = hexaglycerol or tripentaerythritol core structure), and 6-arm PEG amines containing a sorbitol or dipentaerythritol core: [ka] (n is in the range of 20 to 500, R = contains a sorbitol or dipentaerythritol core The dashed line in these formulas indicates the bond to the remainder of the CNP prodrug.

[0293] In formula (Ia), x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. Preferably, x is an integer selected from the group consisting of 2, 3, 4, 6, and 8. More preferably, x is an integer selected from the group consisting of 2, 4, and 6. Even more preferably, x is an integer selected from the group consisting of 4 and 6, and most preferably, x is 4.

[0294] In formula (Ib), y is an integer selected from the group consisting of 1, 2, 3, 4, or 5. Preferably, y is an integer selected from the group consisting of 1, 2, or 3. In one preferred embodiment, y is 1. In an equally preferred embodiment, y is 2.

[0295] In another preferred embodiment, one moiety -L 2 -L 1 -D is linked to one substructure -Z.

[0296] In a particularly preferred embodiment, -Z is a branched polymer. In one embodiment, -Z is a branched polymer having 1, 2, 3, 4, 5, or 6 branch points. Preferably, -Z is a branched polymer having 1, 2, or 3 branch points. In one embodiment, -Z is a branched polymer having 1 branch point. In another embodiment, -Z is a branched polymer having 2 branch points. In another embodiment, -Z is a branched polymer having 3 branch points.

[0297] The branch points are preferably selected from the group consisting of -N<, -CH< and >C<.

[0298] Preferably, such branched moieties -Z are PEG-based.

[0299] Preferably, such branched substructure-Z has a molecular weight of at least 10 kDa.

[0300] In one embodiment, such branched moiety-Z has a molecular weight in the range of 10 kDa to 500 kDa, more preferably in the range of 10 kDa to 250 kDa, even more preferably in the range of 10 kDa to 150 kDa, even more preferably in the range of 12 kDa to 100 kDa, and most preferably in the range of 15 kDa to 80 kDa.

[0301] Preferably, such branched substructure-Z has a molecular weight in the range of 10 kDa to 80 kDa. In one embodiment, the molecular weight is about 10 kDa. In another embodiment, the molecular weight of such branched substructure-Z is about 20 kDa. In another embodiment, the molecular weight of such branched substructure-Z is about 30 kDa. In another embodiment, the molecular weight of such branched substructure-Z is about 40 kDa. In another embodiment, the molecular weight of such branched substructure-Z is about 50 kDa. In another embodiment, the molecular weight of such branched substructure-Z is about 60 kDa. In another embodiment, the molecular weight of such branched substructure-Z is about 70 kDa. In another embodiment, the molecular weight of such branched substructure-Z is about 80 kDa. Most preferably, such branched substructure-Z has a molecular weight of about 40 kDa.

[0302] The applicant has identified the partial structure -L 1 -L 2It has been surprisingly discovered that N-terminal attachment of -Z is significantly more efficient than attachment at an internal site in increasing NEP stability, and that attachment at the ring portion of the CNP moiety is the least efficient attachment in increasing NEP stability. However, the applicant has surprisingly discovered that this drawback of ring attachment in increasing NEP stability can be compensated for by using a branched moiety -Z having a molecular weight of at least 10 kDa, such as at least 12 kDa, for example, at least 15 kDa, for example, at least 18 kDa, for example, at least 20 kDa, for example, at least 24 kDa, for example, at least 25 kDa, for example, at least 27 kDa, for example, at least 30 kDa. Preferably, such a branched moiety -Z has a molecular weight of 500 kDa or less, preferably 250 kDa or less, preferably 200 kDa or less, preferably 150 kDa or less, and most preferably 100 kDa or less. Most preferably, such a branched moiety -Z has a molecular weight of about 40 kDa. Thus, the use of such a branched substructure -Z in the ring portion of the CNP substructure not only results in increased NEP stability, but also combines increased NEP stability with reduced NPR-B binding associated with binding to the ring.

[0303] Preferably, -Z or -Z' comprises the following moiety: [ka]

[0304] In one embodiment, -Z comprises a moiety of formula (a): [ka] (In the formula, The dashed line is -L 2 represents the bond to - or to the remainder of -Z, BP a is a branch point selected from the group consisting of -N<, -CR< and >C<; -R is -H and C 1-6 is selected from the group consisting of alkyl, a is BPa is 0 when -N< or -CR<, and n is BP a is 1 if >C<, -S a -, -S a' -, -S a'' -and-S a''' - are, independently of each other, a chemical bond or C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R 1 and wherein C is optionally substituted with 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 2 )-, -S(O)2N(R 2 )-, -S(O)N(R 2 )-, -S(O)2-, -S(O)-, -N(R 2 )S(O)2N(R 2a )-, -S-, -N(R 2 )-, -OC(OR 2 )(R 2a )-, -N(R 2 )C(O)N(R 2a )- and -OC(O)N(R 2 )-, optionally interrupted by one or more groups selected from the group consisting of Each -T- is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, wherein each -T- is independently selected from the group consisting of one or more -R 1 and optionally substituted with Each-R 1 are independently halogen, -CN, oxo(=O), -COOR 3 , -OR 3, -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O)2N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O)R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 2 , -R 2a , -R 3 , -R 3a and -R 3b are independently -H and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -P a' , -P a'' and -P a''' are independently polymeric substructures).

[0305] In one embodiment, BP of formula (a) a is -N<.

[0306] In another embodiment, BP of formula (a) a is -CR<. Preferably, -R is -H. Therefore, a in formula (a) is preferably 0.

[0307] In another embodiment, BP of formula (a) a is >C<.

[0308] In one embodiment, the -S a - is a chemical bond.

[0309] In another embodiment, the -S a -C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 alkynyl, wherein C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl includes -C(O)O-, -O-, -C(O)-, -C(O)N(R 4 )-, -S(O)2N(R 4 )-, -S(O)N(R 4 )-, -S(O)2-, -S(O)-, -N(R 4 )S(O)2N(R 4a )-, -S-, -N(R 4 )-, -OC(OR 4 )(R 4a )-, -N(R 4 )C(O)N(R 4a )- and -OC(O)N(R 4 )-, wherein said -R 4 and -R 4a is independently selected from the group consisting of -H, methyl, ethyl, propyl and butyl. Preferably, -S in formula (a) a - is selected from the group consisting of methyl, ethyl, propyl, and butyl, including -O-, -C(O)-, and -C(O)N(R 4 )-.

[0310] In one embodiment, the -S a' - is a chemical bond.

[0311] In another embodiment, the -S a' -C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 alkynyl, wherein C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl includes -C(O)O-, -O-, -C(O)-, -C(O)N(R 4 )-, -S(O)2N(R 4 )-, -S(O)N(R 4 )-, -S(O)2-, -S(O)-, -N(R 4 )S(O)2N(R 4a )-, -S-, -N(R 4 )-, -OC(OR 4 )(R 4a )-, -N(R 4 )C(O)N(R 4a )- and -OC(O)N(R 4 )-, wherein said -R 4 and -R 4a is independently selected from the group consisting of -H, methyl, ethyl, propyl and butyl. Preferably, -S in formula (a) a' - is selected from the group consisting of methyl, ethyl, propyl, and butyl, including -O-, -C(O)-, and -C(O)N(R 4 )-.

[0312] In one embodiment, the -S a'' - is a chemical bond.

[0313] In another embodiment, the -S a'' -C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10alkynyl, wherein C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl includes -C(O)O-, -O-, -C(O)-, -C(O)N(R 4 )-, -S(O)2N(R 4 )-, -S(O)N(R 4 )-, -S(O)2-, -S(O)-, -N(R 4 )S(O)2N(R 4a )-, -S-, -N(R 4 )-, -OC(OR 4 )(R 4a )-, -N(R 4 )C(O)N(R 4a )- and -OC(O)N(R 4 )-, wherein said -R 4 and -R 4a is independently selected from the group consisting of -H, methyl, ethyl, propyl and butyl. Preferably, -S in formula (a) a'' - is selected from the group consisting of methyl, ethyl, propyl, and butyl, including -O-, -C(O)-, and -C(O)N(R 4 )-.

[0314] In one embodiment, the -S a''' - is a chemical bond.

[0315] In another embodiment, the -S a''' -C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 alkynyl, wherein C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl includes -C(O)O-, -O-, -C(O)-, -C(O)N(R 4 )-, -S(O)2N(R 4 )-, -S(O)N(R 4 )-, -S(O)2-, -S(O)-, -N(R4 )S(O)2N(R 4a )-, -S-, -N(R 4 )-, -OC(OR 4 )(R 4a )-, -N(R 4 )C(O)N(R 4a )- and -OC(O)N(R 4 )-, wherein said -R 4 and -R 4a is independently selected from the group consisting of -H, methyl, ethyl, propyl and butyl. Preferably, -S in formula (a) a''' - is selected from the group consisting of methyl, ethyl, propyl, and butyl, including -O-, -C(O)-, and -C(O)N(R 4 )-.

[0316] Preferably, -P of formula (a) a' , -P a'' and -P a'''are, independently, 2-methacryloyl-oxyethyl phosphorylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl-oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylate ... and copolymers thereof. The polymers include polymers selected from the group consisting of poly(propylene glycol), poly(propyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyl oxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate-based polymers, xylan, and copolymers thereof.

[0317] Preferably, -P of formula (a) a' , -P a'' and -P a''' independently comprises a PEG-based moiety. Even more preferably, -P of formula (a) a' , -P a'' and -P a'''independently comprise a PEG-based moiety comprising at least 20% PEG, more preferably at least 30%, even more preferably at least 40% PEG, even more preferably at least 50% PEG, even more preferably at least 60% PEG, even more preferably at least 70% PEG, even more preferably at least 80% PEG, and most preferably at least 90% PEG.

[0318] Preferably, P of formula (a) a' , -P a'' and -P a''' independently have a molecular weight in the range of 5 kDa to 50 kDa, more preferably in the range of 5 kDa to 40 kDa, even more preferably in the range of 7.5 kDa to 30 kDa, and even more preferably in the range of 10 kDa to 30 kDa.

[0319] In one embodiment, P of formula (a) a' , -P a'' and -P a''' has a molecular weight of approximately 5 kDa.

[0320] In another embodiment, P of formula (a) a' , -P a'' and -P a''' has a molecular weight of approximately 7.5 kDa.

[0321] In another embodiment, P of formula (a) a' , -P a'' and -P a''' has a molecular weight of approximately 10 kDa.

[0322] In another embodiment, P of formula (a) a' , -P a'' and -P a''' has a molecular weight of approximately 12.5 kDa.

[0323] In another embodiment, P of formula (a) a' , -P a'' and -P a''' has a molecular weight of approximately 15 kDa.

[0324] In another embodiment, P of formula (a) a' , -P a'' and -P a''' has a molecular weight of approximately 20 kDa.

[0325] In one embodiment, -Z comprises one moiety of formula (a).

[0326] In another embodiment, -Z comprises two moieties of formula (a).

[0327] In another embodiment, -Z comprises three moieties of formula (a).

[0328] In another embodiment, -Z comprises four moieties of formula (a).

[0329] In another embodiment, -Z comprises five moieties of formula (a).

[0330] In another embodiment, -Z comprises six moieties of formula (a).

[0331] In a preferred embodiment, -Z comprises two moieties of formula (a).

[0332] In a preferred embodiment, -Z comprises a moiety of formula (b): [ka] (In the formula, The dashed line is -L 2 represents the bond to - or to the remainder of -Z, b1 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7 and 8; b2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8; b3 is an integer ranging from 150 to 1000, preferably ranging from 150 to 500, and most preferably ranging from 200 to 460; b4 is an integer in the range of 150 to 1000, preferably in the range of 150 to 500, and most preferably in the range of 200 to 460).

[0333] Preferably, b3 and b4 in formula (b) are the same integer.

[0334] In one preferred embodiment, b3 and b4 are both integers in the range of 200-250, and most preferably, b3 and b4 in formula (b) are about 225.

[0335] In another preferred embodiment, b3 and b4 are both integers in the range of 400-500, and most preferably, b3 and b4 in formula (b) are about 450.

[0336] Preferably, b1 in formula (b) is selected from the group consisting of 0, 1, 2, 3 and 4. More preferably, b1 in formula (b) is selected from the group consisting of 1, 2 and 3. Most preferably, b1 in formula (b) is 2.

[0337] Preferably, b2 in formula (b) is selected from the group consisting of 1, 2, 3, 4 and 5. More preferably, b2 in formula (b) is selected from the group consisting of 2, 3 and 4. Most preferably, b2 in formula (b) is 3.

[0338] In one particularly preferred embodiment, b1 of formula (b) is 2, b2 of formula (b) is 3, and b3 and b4 are both about 450.

[0339] In another particularly preferred embodiment, b1 of formula (b) is 2, b2 of formula (b) is 3, and b3 and b4 are both about 225.

[0340] In one embodiment, -Z comprises one moiety of formula (b).

[0341] In another embodiment, -Z comprises two moieties of formula (b).

[0342] In another embodiment, -Z comprises three moieties of formula (b).

[0343] In another embodiment, -Z comprises four moieties of formula (b).

[0344] In another embodiment, -Z comprises five moieties of formula (b).

[0345] In another embodiment, -Z comprises six moieties of formula (b).

[0346] In a preferred embodiment, -Z comprises two moieties of formula (b).

[0347] In an even more preferred embodiment, -Z comprises a moiety of formula (c): [ka] (In the formula, The dashed line is -L 2 represents the bond to - or to the remainder of -Z, c1 and c2 are independently integers in the range of 150 to 500, preferably in the range of 200 to 460).

[0348] Preferably, c1 and c2 in formula (c) are the same integer.

[0349] In one preferred embodiment, c1 and c2 in formula (c) are in the range of 200 to 250, and most preferably about 225. In one preferred embodiment, c1 and c2 in formula (c) are in the range of 400 to 500, and most preferably about 450.

[0350] In a preferred embodiment, the substructure -Z is a branched PEG-based polymer containing at least 10% PEG, has one branch point, two PEG-based polymer arms, and has a molecular weight of about 40 kDa. Thus, each of the two PEG-based polymer arms has a molecular weight of about 20 kDa. Preferably, the branch point is -CH<.

[0351] In one embodiment, -Z comprises one moiety of formula (c).

[0352] In another embodiment, -Z comprises two moieties of formula (c).

[0353] In another embodiment, -Z comprises three moieties of formula (c).

[0354] In another embodiment, -Z comprises four moieties of formula (c).

[0355] In another embodiment, -Z comprises five moieties of formula (c).

[0356] In another embodiment, -Z comprises six moieties of formula (c).

[0357] In a preferred embodiment, -Z comprises two moieties of formula (c).

[0358] In one preferred embodiment, the moiety -Z has the formula (d): [ka] (In the formula, The dashed line is -L 2 - indicates a bond to -Z b -C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R 1 and wherein C is optionally substituted with 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 2 )-, -S(O)2N(R 2 )-, -S(O)N(R 2 )-, -S(O)2-, -S(O)-, -N(R 2 )S(O)2N(R2a )-, -S-, -N(R 2 )-, -OC(OR 2 )(R 2a )-, -N(R 2 )C(O)N(R 2a )- and -OC(O)N(R 2 )-, optionally interrupted by one or more groups selected from the group consisting of Each -T- is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, wherein each -T- is independently selected from the group consisting of one or more -R 1 and optionally substituted with Each-R 1 are independently halogen, -CN, oxo(=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O)2N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O)R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R3a ), and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 2 , -R 2a , -R 3 , -R 3a and -R 3b are independently -H and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -Z a teeth, [ka] In this formula, BP a , -S a -, -S a' -, -S a'' -, -S a''' -, -P a' , -P a'' , -P a''' and a are used as defined for formula (a).

[0359] BP in equation (d) a , -S a -, -S a' -, -S a'' -, -S a''' -, -P a' , -P a'' , -P a''' Preferred embodiments of are as defined above for formula (a).

[0360] In an even more preferred embodiment, the moiety -Z has the formula (e): [ka] (In the formula, The dashed line is -L 2 - indicates a bond to e is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; -Z a teeth, [ka] and in this formula: b1 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7 and 8; b2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8; b3 is an integer ranging from 150 to 1000, preferably ranging from 150 to 500, and most preferably ranging from 200 to 460; b4 is an integer in the range of 150 to 1000, preferably in the range of 150 to 500, and most preferably in the range of 200 to 460).

[0361] Preferred embodiments of b1, b2, b3 and b4 in formula (e) are as defined above for formula (b).

[0362] In one embodiment, e in Formula (e) is 1. In another embodiment, e in Formula (e) is 2. In another embodiment, e in Formula (e) is 3. In another embodiment, e in Formula (e) is 4. In another embodiment, e in Formula (e) is 5. In another embodiment, e in Formula (e) is 6. In another embodiment, e in Formula (e) is 7. In another embodiment, e in Formula (e) is 8. In another embodiment, e in Formula (e) is 9. In another embodiment, e in Formula (e) is 10. In another embodiment, e in Formula (e) is 11. In another embodiment, e in Formula (e) is 12. In another embodiment, e in Formula (e) is 13. In another embodiment, e in Formula (e) is 14. In another embodiment, e in Formula (e) is 15.

[0363] Preferably, e in formula (e) is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, and 9. Even more preferably, e in formula (e) is selected from 3, 4, 5, and 6. Most preferably, e in formula (e) is 5.

[0364] Preferably, e in formula (e) is 5, b1 in formula (e) is 2, b2 in formula (e) is 3, and b3 and b4 in formula (e) are both about 450.

[0365] In another preferred embodiment, the substructure -Z is a branched PEG-based polymer containing at least 10% PEG, has three branch points and four PEG-based polymer arms, and has a molecular weight of about 40 kDa. Thus, each of the four PEG-based polymer arms has a molecular weight of about 10 kDa. Preferably, each of the three branch points is -CH<.

[0366] In one preferred embodiment, the moiety -Z has the formula (f): [ka] (In the formula, The dashed line is -L 2 - indicates a bond to BP f is a branch point selected from the group consisting of -N<, -CR< and >C<; -R is -H and C 1-6 is selected from the group consisting of alkyl, f is BP f is 0 when -N< or -CR<, and f is BP f is 1 if >C<, -S f -, -S f' -, -S f'' -and-S f''' - is a chemical bond or, independently, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, 1-50 Alkyl, C 2-50Alkenyl and C 2-50 Alkynyl may be one or more -R 1 and wherein C is optionally substituted with 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 2 )-, -S(O)2N(R 2 )-, -S(O)N(R 2 )-, -S(O)2-, -S(O)-, -N(R 2 )S(O)2N(R 2a )-, -S-, -N(R 2 )-, -OC(OR 2 )(R 2a )-, -N(R 2 )C(O)N(R 2a )- and -OC(O)N(R 2 )-, optionally interrupted by one or more groups selected from the group consisting of Each -T- is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, wherein each -T- is independently selected from the group consisting of one or more -R 1 and optionally substituted with Each R 1 are independently halogen, -CN, oxo(=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O)2N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O)R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a R 3b ), -SR 3 , -N(R 3 R3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 2 , -R 2a , -R 3 , -R 3a and -R 3b are independently -H and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -Z a' , -Z a'' and -Z a''' is, independently, [ka] In this formula, BP a , -S a -, -S a' -, -S a'' -, -S a''' -, -P a' , -P a'' , -P a''' and a are used as defined for formula (a).

[0367] BP in equation (f) a , -S a -, -S a' -, -S a'' -, -S a''' -, -P a' , -Pa'' , -P a''' Preferred embodiments of are as defined above for formula (a).

[0368] Preferably, the BP of formula (f) 2 is -CR< and r is 0. Preferably, -R is -H.

[0369] Preferably, -S in formula (f) f - is a chemical bond.

[0370] Preferably, -Z in formula (f) a' , -Z a'' and -Z a''' have the same structure. Preferably, -Z in formula (f) a' , -Z a'' and -Z a''' has the formula (b).

[0371] Preferably, -S in formula (f) f - is a chemical bond, and BP in formula (f) a is -CR< (wherein -R is -H). Even more preferably, -S of formula (f) f - is a chemical bond, and BP in formula (f) a is -CR< (wherein -R is -H), and -Z of formula (f) a' , -Z a'' and -Z a''' has the formula (b).

[0372] Even more preferably, -Z has the formula (g): [ka] (In the formula, The dashed line is -L 2 - indicates a bond to -S g -, -S g' -and-S g'' independently, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl,1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R 1 and wherein C is optionally substituted with 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 2 )-, -S(O)2N(R 2 )-, -S(O)N(R 2 )-, -S(O)2-, -S(O)-, -N(R 2 )S(O)2N(R 2a )-, -S-, -N(R 2 )-, -OC(OR 2 )(R 2a )-, -N(R 2 )C(O)N(R 2a )- and -OC(O)N(R 2 )-, optionally interrupted by one or more groups selected from the group consisting of Each -T- is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl, wherein each -T- is independently selected from the group consisting of one or more -R 1 and optionally substituted with Each R 1 are independently halogen, -CN, oxo(=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O)2N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O)R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a R 3b ), -SR3 , -N(R 3 R 3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 2 , -R 2a , -R 3 , -R 3a and -R 3b are independently -H and C 1-6 alkyl, 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -Z a and -Z a' is, independently, [ka] In this formula, BP a , -S a -, -S a' -, -S a'' -, -S a''' -, -P a' , -P a'' , -P a''' and a are used as defined for formula (a).

[0373] BP in formula (g) a , -S a -, -S a' -, -S a'' -, -S a''' -, -Pa' , -P a'' and -P a''' Preferred embodiments of are as defined above for formula (a).

[0374] Preferably, -S in formula (g) g - is one or more of the same or different -R 1 optionally substituted with C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl, -R 1 is halogen, oxo (=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O)2N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O)R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, wherein C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -R 3 , -R3a and -R 3b is independently selected from —H, methyl, ethyl, propyl, and butyl.

[0375] Even more preferably, -S of formula (g) g -C 1-6 alkyl.

[0376] Preferably, -S in formula (g) g' - is one or more of the same or different -R 1 optionally substituted with C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl, -R 1 is halogen, oxo (=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O)2N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O)R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6alkyl, wherein C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -R 3 , -R 3a and -R 3b is independently selected from —H, methyl, ethyl, propyl, and butyl.

[0377] Even more preferably, -S of formula (g) g' -C 1-6 alkyl.

[0378] Preferably, -S in formula (g) g'' - is one or more of the same or different -R 1 optionally substituted with C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl, -R 1 is halogen, oxo (=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O)2N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O)R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3)C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, wherein C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -R 3 , -R 3a and -R 3b is independently selected from —H, methyl, ethyl, propyl, and butyl.

[0379] Even more preferably, -S of formula (g) g'' -C 1-6 alkyl.

[0380] Preferably, -Z in formula (g) a and -Z a' have the same structure. Preferably, -Z in formula (g) a and -Z a' has the formula (b).

[0381] Even more preferably, -Z has the formula (h): [ka] (In the formula, The dashed line is -L 2 - indicates a bond to Each-Z c is a substructure [ka] where each c1 is independently an integer ranging from about 200 to 250.

[0382] Preferably, both c1's in formula (h) are the same.

[0383] Preferably, both c1's in formula (h) are about 225.

[0384] In an even more preferred embodiment, the moiety -Z has the formula (hi): [ka] (In the formula, The dashed line is -L 2 - indicates a bond to Each-Z c is the following partial structure [ka] (each c1 is independently an integer in the range of about 200 to 250)).

[0385] Preferably, both c1's in formula (hi) are the same.

[0386] Preferably, both c1's in formula (hi) are about 225.

[0387] Preferably, the CNP prodrugs of the present invention have the formula (Ia):

[0388] Preferably, the CNP prodrugs of the present invention have the formula (Ia) where x=1.

[0389] In a preferred embodiment, the CNP prodrug of the present invention has the formula (IIe): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; Dashed lines with asterisks represent substructures: [ka] indicates a bond to Each c1 is independently an integer in the range of about 400 to 500).

[0390] Preferably, c1 in formula (IIe) is about 450.

[0391] In an equally preferred embodiment, the CNP prodrug of the present invention has the formula (IIe-i): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; Dashed lines with asterisks represent substructures: [ka] indicates a bond to Each c1 is independently an integer in the range of about 400 to 500).

[0392] Preferably, c1 in formula (IIe-i) is about 450.

[0393] In another equally preferred embodiment, the CNP prodrug of the present invention has the formula (IIe-ii): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; Dashed lines with asterisks represent substructures: [ka] indicates a bond to Each c1 is independently an integer in the range of about 400 to 500).

[0394] Preferably, c1 in formula (IIe-ii) is about 450.

[0395] In one embodiment, the CNP moiety of the CNP prodrugs of formulae (IIe), (IIe-i) and (IIe-ii) has the sequence of SEQ ID NO:25.

[0396] In another embodiment, the CNP moiety of the CNP prodrugs of formulae (IIe), (IIe-i) and (IIe-ii) has the sequence of SEQ ID NO:30.

[0397] In a preferred embodiment, the CNP moiety of the CNP prodrugs of formulae (IIe), (IIe-i) and (IIe-ii) has the sequence of SEQ ID NO:24.

[0398] In one embodiment, the CNP moiety is connected to the nitrogen of the N-terminal amine functional group of CNP by -L in the CNP prodrugs of formula (IIe), (IIe-i), and (IIe-ii). 1 - is bonded to

[0399] In a preferred embodiment, the CNP moiety is linked to -L in the CNP prodrugs of formulas (IIe), (IIe-i), and (IIe-ii) by a nitrogen provided by an amine functionality of the lysine side chain of CNP. 1 - is bonded to

[0400] In one embodiment, this lysine side chain is not part of the ring formed by the disulfide bridge between the cysteine ​​residues at positions 22 and 38 when the CNP substructure has SEQ ID NO:24.

[0401] Thus, in one embodiment, the CNP moiety is connected to -L in the CNP prodrugs of formulas (IIe), (IIe-i), and (IIe-ii) by the amine functionality provided by the side chain of the lysine at position 9, where CNP has the sequence of SEQ ID NO:24. 1 - and is linked.

[0402] In another embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIe), (IIe-i), and (IIe-ii) by virtue of the amine functionality provided by the side chain of lysine at position 11, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0403] In another embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIe), (IIe-i), and (IIe-ii) by virtue of the amine functionality provided by the side chain of lysine at position 15, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0404] In another embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIe), (IIe-i), and (IIe-ii) by virtue of the amine functionality provided by the side chain of lysine at position 16, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0405] In another embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIe), (IIe-i), and (IIe-ii) by virtue of the amine functionality provided by the side chain of the lysine at position 20, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0406] In a preferred embodiment, the lysine side chain for attachment to the remainder of the CNP prodrug of formula (IIe), (IIe-i), and (IIe-ii) is part of the ring formed by the disulfide bridge between the cysteine ​​residues at positions 22 and 38 when the CNP moiety has SEQ ID NO:24.

[0407] Thus, in a preferred embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIe), (IIe-i), and (IIe-ii) by virtue of the amine functionality provided by the side chain of the lysine at position 26, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0408] The positions of the cysteines and lysines described above vary depending on the length of the CNP substructure, and those skilled in the art will understand that it will not be difficult to identify corresponding cysteines and lysines in longer or shorter versions of the CNP substructure. Those skilled in the art will also understand that, for example, some lysines may not be present in shorter CNP substructures. Furthermore, it will be understood that, for example, as a result of site-directed mutagenesis, more lysine residues may be present in the non-cyclizing and / or cyclizing portions of the CNP substructure.

[0409] In a preferred embodiment, the CNP prodrug of the present invention has a c1 of about 450, the CNP moiety has the sequence of SEQ ID NO: 24, and is linked to -L by the amine functionality provided by the side chain of the lysine at position 26. 1 -, having the formula (IIe):

[0410] In another preferred embodiment, the CNP prodrug of the present invention has a c1 of about 450, the CNP moiety has the sequence of SEQ ID NO: 24, and is linked to -L by the amine functionality provided by the side chain of the lysine at position 26. 1 -, having the formula (IIe-i):

[0411] In another preferred embodiment, the CNP prodrug of the present invention has a c1 of about 450, the CNP moiety has the sequence of SEQ ID NO: 24, and is linked to -L by the amine functionality provided by the side chain of the lysine at position 26. 1 -, having the formula (IIe-ii):

[0412] Thus, in a preferred embodiment, the CNP prodrug of the present invention has the formula (IIe'): [ka] (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; Dashed lines with asterisks represent substructures: [ka] indicates a bond to Each c1 is independently an integer in the range of about 400 to 500).

[0413] Preferably, each c1 in formula (IIe') is about 450.

[0414] In a preferred embodiment, the CNP prodrug of the present invention has the formula (IIe-i'): [ka] (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; Dashed lines with asterisks represent substructures: [ka] indicates a bond to Each c1 is independently an integer in the range of about 400 to 500).

[0415] Preferably, each c1 in formula (IIe-i') is about 450.

[0416] In another preferred embodiment, the CNP prodrug of the present invention has the formula (IIe-ii'): [ka] (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; Dashed lines with asterisks represent substructures: [ka] indicates a bond to Each c1 is independently an integer in the range of about 400 to 500).

[0417] Preferably, each c1 in formula (IIe-ii') is about 450.

[0418] In another preferred embodiment, the CNP prodrug of the present invention has the formula (IIf): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; The dashed lines with asterisks indicate the structure: [ka] represents a bond to -Z having the formula Each-Z a teeth, [ka] wherein each c1 is independently an integer ranging from about 200 to 250, and preferably each n is about 225.

[0419] Preferably, each c1 in formula (IIf) is about 225.

[0420] In another preferred embodiment, the CNP prodrug of the present invention has the formula (IIf-i): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; The dashed lines with asterisks indicate the structure: [ka] represents a bond to -Z having the formula Each-Z a teeth, [ka] wherein each c1 is independently an integer ranging from about 200 to 250, and preferably each n is about 225).

[0421] Preferably, each c1 in formula (IIf-i) is about 225.

[0422] In another preferred embodiment, the CNP prodrug of the present invention has the formula (IIf-ii): [ka] (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; The dashed lines with asterisks indicate the structure: [ka] represents a bond to -Z having the formula Each-Z a teeth, [ka] wherein each c1 is independently an integer ranging from about 200 to 250, and preferably each n is about 225).

[0423] Preferably, each c1 in formula (IIf-ii) is about 225.

[0424] In a preferred embodiment, the CNP moiety of the CNP prodrugs of formulae (IIf), (IIf-i) and (IIf-ii) has the sequence of SEQ ID NO:25.

[0425] In a preferred embodiment, the CNP moiety of the CNP prodrugs of formulae (IIf), (IIf-i) and (IIf-ii) has the sequence of SEQ ID NO:24.

[0426] In one embodiment, the CNP moiety is connected by the nitrogen of the N-terminal amine functional group of CNP to -L in the CNP prodrugs of formula (IIf), (IIf-i) and (IIf-ii). 1 - is bonded to

[0427] In a preferred embodiment, the CNP moiety is linked to -L in the CNP prodrugs of formulas (IIf), (IIf-i) and (IIf-ii) by a nitrogen provided by the amine functionality of the lysine side chain of CNP. 1 - is bonded to

[0428] In one embodiment, this lysine side chain is not part of the ring formed by the disulfide bridge between the cysteine ​​residues at positions 22 and 38 when the CNP substructure has SEQ ID NO:24.

[0429] Thus, in one embodiment, the CNP moiety is provided by the amine functionality provided by the side chain of the lysine at position 9, i.e., -L in the CNP prodrugs of formulas (IIf), (IIf-i), and (IIf-ii), where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0430] In another embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIf), (IIf-i), and (IIf-ii) by virtue of the amine functionality provided by the side chain of lysine at position 11, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0431] In another embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIf), (IIf-i), and (IIf-ii) by virtue of the amine functionality provided by the side chain of lysine at position 15, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0432] In another embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIf), (IIf-i), and (IIf-ii) by virtue of the amine functionality provided by the side chain of lysine at position 16, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0433] In another embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIf), (IIf-i) and (IIf-ii) by virtue of the amine functionality provided by the side chain of the lysine at position 20, where CNP has the sequence of SEQ ID NO: 24. 1 - and is linked.

[0434] In a preferred embodiment, the lysine side chain for attachment to the remainder of the CNP prodrug of formula (IIf), (IIf-i) and (IIf-ii) is part of the ring formed by the disulfide bridge between the cysteine ​​residues at positions 22 and 38 when the CNP moiety has SEQ ID NO:24.

[0435] Thus, in a preferred embodiment, the CNP moiety is -L in the CNP prodrugs of formulas (IIf), (IIf-i) and (IIf-ii) by virtue of the amine functionality provided by the side chain of lysine at position 26, where CNP has the sequence of SEQ ID NO:24. 1 - and is linked.

[0436] The positions of the cysteines and lysines described above vary depending on the length of the CNP substructure, and those skilled in the art will understand that it will not be difficult to identify corresponding cysteines and lysines in longer or shorter versions of the CNP substructure. Those skilled in the art will also understand that, for example, some lysines may not be present in shorter CNP substructures. Furthermore, it will be understood that, for example, as a result of site-directed mutagenesis, more lysine residues may be present in the non-cyclizing and / or cyclizing portions of the CNP substructure.

[0437] In a preferred embodiment, the CNP prodrug of the present invention has a c1 of about 225, the CNP moiety has the sequence of SEQ ID NO: 24, and is linked to -L by the amine functionality provided by the side chain of the lysine at position 26. 1 -, having the formula (IIf):

[0438] In another preferred embodiment, the CNP prodrug of the present invention has a c1 of about 225, the CNP moiety has the sequence of SEQ ID NO: 24, and is linked to -L by the amine functionality provided by the side chain of the lysine at position 26. 1 -, having the formula (IIf-i):

[0439] In another preferred embodiment, the CNP prodrug of the present invention has a c1 of about 225, the CNP moiety has the sequence of SEQ ID NO: 24, and is linked to -L by the amine functionality provided by the side chain of the lysine at position 26. 1 -, having the formula (IIf-ii):

[0440] In another preferred embodiment, the CNP prodrug of the present invention has the formula (IIf'): [ka] (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; The dashed lines with asterisks indicate the structure: [ka] represents a bond to -Z having the formula each Z a teeth, [ka] where each c1 is independently an integer ranging from about 200 to 250.

[0441] Preferably, each c1 in formula (IIf') is about 225.

[0442] In another preferred embodiment, the CNP prodrug of the present invention has the formula (IIf-i'): [ka] (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; The dashed lines with asterisks indicate the structure: [ka] represents a bond to -Z having the formula each Z a teeth, [ka] where each c1 is independently an integer ranging from about 200 to 250.

[0443] Preferably, each c1 in formula (IIf-i') is about 225.

[0444] In another preferred embodiment, the CNP prodrug of the present invention has the formula (IIf-ii'): [ka] (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; The dashed lines with asterisks indicate the structure: [ka] represents a bond to -Z having the formula each Z a teeth, [ka] where each c1 is independently an integer ranging from about 200 to 250.

[0445] Preferably, each c1 in formula (IIf-ii') is about 225.

[0446] In short, the -L bond to the CNP substructure via the side chain of the amino acid located in the ring substructure of CNP. 1 We have discovered that by combining reversible binding to - and the use of a branched substructure -Z having a molecular weight of at least 10 kDa with the use of a CNP substructure larger than CNP-22, we can obtain CNP prodrugs with unexpectedly long in vivo half-lives.

[0447] Carrier-Z' is a water-insoluble polymer, more preferably a hydrogel. Preferably, such a hydrogel is selected from the group consisting of 2-methacryloyl-oxyethyl phosphorylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl-oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylate), poly( hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate-based polymers, xylan, and copolymers thereof.

[0448] When Carrier-Z' is a hydrogel, Carrier-Z' is preferably a hydrogel containing PEG or hyaluronic acid. Most preferably, such a hydrogel contains PEG.

[0449] Even more preferably, Carrier-Z' is a hydrogel as described in WO 2006 / 003014 A2, WO 2011 / 012715 A1 or WO 2014 / 056926 A1, which are incorporated herein by reference in their entirety.

[0450] In another embodiment, -Z' is a polymer network formed by physical aggregation of polymer chains, said physical aggregation preferably resulting from hydrogen bonding, crystallization, helix formation or complexation. In one embodiment, such a polymer network is a thermogelling polymer.

[0451] Preferably, the total mass of the CNP prodrug of the present invention is at least 10 kDa, e.g., at least 12 kDa, e.g., at least 15 kDa, e.g., at least 20 kDa, or e.g., at least 30 kDa. The total mass of the CNP prodrug of the present invention is preferably at most 250 kDa, e.g., at most 200 kDa, 180 kDa, 150 kDa, or 100 kDa.

[0452] In preferred embodiments, the residual activity of the CNP prodrugs of the present invention is less than 10%, more preferably less than 1%, even more preferably less than 0.1%, even more preferably less than 0.01%, even more preferably less than 0.001%, and most preferably less than 0.0001%.

[0453] As used herein, the term "residual activity" refers to the activity exhibited by the CNP prodrug of the present invention in which the CNP moiety is bound to a carrier, relative to the activity exhibited by the corresponding free CNP.In this context, "activity" refers to NPR-B binding.It is understood that when measuring the residual activity of the CNP prodrug of the present invention, it takes time for a certain amount of CNP to be released from the CNP prodrug of the present invention, and such released CNP will distort the measurement results of the CNP prodrug.Therefore, it is customary to test the residual activity of a prodrug using a conjugate in which the drug moiety, in this case CNP, is irreversibly, i.e., stably, bound to a carrier, and which is as similar as possible to the structure of the CNP prodrug whose residual activity is to be measured.

[0454] Suitable assays for measuring the CNP activity and residual activity of CNP prodrugs of the present invention, preferably CNP prodrugs in the form of stable analogs, are described in WO 2010 / 135541 A1, Example 4, pages 143 / 144.

[0455] Another aspect of the present invention is a pharmaceutical composition comprising at least one CNP prodrug of the present invention and at least one excipient.

[0456] In one embodiment, pharmaceutical compositions comprising the CNP prodrug molecules of the present invention comprise CNP moieties connected to -L by different functional groups, preferably by amine functional groups provided by the CNP, i.e., by the N-terminal amine functional group, by the amine functional group provided by the side chain of lysine at position 4 and / or by the side chain of lysine at position 10 when CNP has the sequence of SEQ ID NO: 1; by the N-terminal amine functional group, by the amine functional groups provided by the side chain of lysine at positions 8, 10, 14, 15, 19, and / or 25 when CNP has the sequence of SEQ ID NO: 25; or by the N-terminal amine functional group, by the amine functional groups provided by the side chain of lysine at positions 9, 11, 15, 16, 20, and / or 26 when CNP has the sequence of SEQ ID NO: 24. 1-Contains a mixture of CNP prodrugs linked to

[0457] In a preferred embodiment, the CNP moiety of all CNP prodrug molecules included in the pharmaceutical composition is linked to the same amine functional group provided by CNP, i.e., either by the N-terminal amine functional group or by the amine functional group provided by the side chain of lysine at position 4 or by the side chain of lysine at position 10 when CNP has the sequence of SEQ ID NO: 1; by the amine functional groups provided by the side chain of lysine at positions 8, 10, 14, 15, 19, and / or 25 when CNP has the sequence of SEQ ID NO: 25; or by the amine functional groups provided by the side chain of lysine at positions 9, 11, 15, 16, 20, and / or 26 when CNP has the sequence of SEQ ID NO: 24, -L 1 Most preferably, the CNP moiety of all CNP prodrug molecules included in the pharmaceutical composition is linked to -L by the same functional group, which is the amine functionality provided by the side chain of lysine at position 26 when CNP has the sequence of SEQ ID NO:24. 1 - is bonded to

[0458] Preferably, pharmaceutical compositions containing at least one CNP prodrug of the present invention have a pH in the range of pH 3 to pH 8. More preferably, pharmaceutical compositions have a pH in the range of pH 4 to pH 6. Most preferably, pharmaceutical compositions have a pH in the range of pH 4 to pH 5.

[0459] In one embodiment, a pharmaceutical composition comprising at least one CNP prodrug of the present invention and at least one excipient is a liquid or suspension formulation. It is understood that the pharmaceutical composition is a suspension formulation when the CNP prodrug of the present invention comprises a water-insoluble carrier-Z'.

[0460] In another embodiment, a pharmaceutical composition comprising at least one CNP prodrug of the present invention and at least one excipient is a dry formulation.

[0461] Such liquid, suspension or dry pharmaceutical compositions comprise at least one excipient. Excipients used in parenteral formulations can be classified, for example, as buffers, tonicity adjusting agents, preservatives, stabilizers, absorption inhibitors, antioxidants, thickeners / viscosifiers, or other auxiliary agents. However, in some cases, a single excipient may have dual or triple functions. Preferably, the at least one excipient included in the pharmaceutical composition of the present invention is selected from the group consisting of: (i) Buffers: physiologically acceptable buffers for maintaining pH within a desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyruvate. Acid suppressants, such as Mg(OH)2 or ZnCO3, may also be used; (ii) Tonicity adjusting agents: to minimize pain that may result from cell damage due to osmotic pressure differences at the injection depot. Examples include glycerin and sodium chloride. Effective concentrations can be determined by osmometry using an assumed osmolality of 285-315 mOsmol / kg for serum; (iii) Preservatives and / or Antimicrobials: Multi-dose parenteral formulations require the addition of preservatives in sufficient concentrations to minimize the patient's risk of infection upon injection, and corresponding regulatory requirements have been established. Typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosal, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride; (iv) Stabilizers: Stabilization can be achieved by enhancing the protein stabilizing power, destabilizing the denatured state, or directly binding excipients to the protein. Stabilizers can be amino acids such as alanine, arginine, aspartic acid, glycine, histidine, lysine, and proline; sugars such as glucose, sucrose, and trehalose; polyols such as glycerol, mannitol, and sorbitol; salts such as potassium phosphate and sodium sulfate; chelating agents such as EDTA and hexaphosphate; ligands such as divalent metal ions (zinc, calcium, and the like); other salts or organic molecules such as phenolic derivatives. In addition, oligomers or polymers such as cyclodextrin, dextran, dendrimers, PEG, PVP, or protamine or HSA can also be used. (v) Absorption inhibitors: Primarily ionic or nonionic surfactants or other proteins or soluble polymers, such as poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA, and gelatin, are used to coat the inner surface of the formulation container or competitively adsorb to the inner surface. The excipient concentration and type selected depend on the effect to be avoided, but typically a surfactant monolayer is formed at the interface just above the CMC value; (vi) Antioxidant: antioxidants such as ascorbic acid, ectoine, methionine, glutathione, monothioglycerol, morin, polyethyleneimine (PEI), propyl gallate, and vitamin E. Chelating agents such as citric acid, EDTA, hexaphosphate, and thioglycolic acid may also be used; (vii) Thickeners or Viscosity Increasing Agents: These retard particle settling in vials and syringes, aid in particle mixing and resuspension, and make suspensions easier to inject (i.e., require less force on the syringe plunger). Suitable thickeners or viscosity increasing agents include, for example, carbomer thickeners such as Carbopol 940, Carbopol Ultrez 10; cellulose derivatives such as hydroxypropylmethylcellulose (hypromellose, HPMC) or diethylaminoethylcellulose (DEAE or DEAE-C); colloidal magnesium silicate (Veegum) or sodium silicate; hydroxyapatite gel; tricalcium phosphate gel; xanthan; carrageenan such as Satia gum UTC. 30; aliphatic poly(hydroxy acids), such as poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and their copolymers (PLGA), terpolymers of D,L-lactide, glycolide, and caprolactone; poloxamers; hydrophilic poly(oxyethylene) blocks and hydrophobic poly(oxypropylene) blocks to form poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) triblocks (e.g., Pluronic®); polyether ester copolymers, such as polyethylene glycol terephthalate / polybutylene terephthalate copolymers; sucrose acetate isobutyrate (SAIB); dextran or its derivatives Conductors; combinations of dextran and PEG; polydimethylsiloxane; collagen; chitosan; vinyl alcohol (PVA) and derivatives; polyalkylimides; poly(acrylamide-co-diallyldimethylammonium (DADMA)); polyvinylpyrrolidone (PVP); glycosaminoglycans (GAGs), such as dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, and hyaluronan; ABA triblock or AB block copolymers, consisting of a hydrophobic A block, such as polylactide (PLA) or poly(lactide-co-glycolide) (PLGA), and a hydrophilic B block, such as polyethylene glycol (PEG) or polyvinylpyrrolidone.Such block copolymers and the poloxamers mentioned above may exhibit inverse thermogelation behavior (fluid state at room temperature to facilitate administration, and gel state above the sol-gel transition temperature at body temperature after injection); (viii) Spreading or Diffusing Agents: Modulate the permeability of connective tissue by hydrolyzing components of the extracellular matrix within the interstitial space (e.g., but not limited to, hyaluronic acid, a polysaccharide found in the intercellular spaces of connective tissue). Spreading agents, such as, but not limited to, hyaluronidase, reduce the viscosity of the extracellular matrix and facilitate the diffusion of injected drugs; and (ix) Other auxiliary agents: wetting agents, viscosity adjusting agents, antibiotics, hyaluronidase, etc. Hyaluronic acid and acids and bases such as sodium hydroxide are auxiliary agents necessary for adjusting the pH during production.

[0462] Another aspect of the present invention is the use of a CNP prodrug of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one CNP prodrug of the present invention, as a medicament.

[0463] Another aspect of the present invention is a pharmaceutical composition comprising a CNP prodrug of the present invention or a pharmaceutically acceptable salt thereof, or at least one CNP prodrug of the present invention, for use in a method for treating a disease treatable with CNP.

[0464] Preferably, the disease is selected from the group consisting of achondroplasia, hypochondroplasia, short stature, dwarfism, chondro-osteo-dysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, chiropterygous dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, proximal short chondrodysplasia punctata, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, osteogenesis imperfecta, diastrophic dysplasia, congenital femoral brachyosteogenesis imperfecta, Langer metaphyseal dysplasia, Niebergelt metaphyseal dysplasia, Robinow syndrome, Reinhardt syndrome, acroostosis imperfecta, peripheral osteogenesis imperfecta, Kniest dysplasia dysplasia, fibrochondrogenesis, Roberts syndrome, distal intermediolum dysplasia, brachymetaphyseal dysplasia, Morquio syndrome, Kniest syndrome, metamorphic bone dysplasia, spondyloepiphyseal dysplasia, neurofibromatosis, Legius syndrome, Leopard syndrome, Noonan syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Legius syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, cleidocranial dysostosis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzondermoskeletal syndrome) syndrome), dactyly, brachydactyly, camptodactyly, polydactyly, syndactyly, segmental dysplasia, enchondrosis, fibrous dysplasia, hereditary multiple osteochondromas, hypophosphatemic rickets, Jaffe-Liechtenstein syndrome, Marfan syndrome, McCune-Albright syndrome, osteopetrosis and bone poikilosis.

[0465] Preferably, the disease is achondroplasia, hypochondroplasia, short stature, dwarfism, chondro-osteodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, chiropteran dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, proximal short-limbed chondrodysplasia punctata, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, osteogenesis imperfecta, diastrophic dysplasia, congenital femoral short-limbed dysplasia, Langer metaphyseal dysplasia, Niebergelt metaphyseal dysplasia The present invention relates to a genetic disorder characterized by ...

[0466] In another embodiment, the disease is an ophthalmic disease, for example, glaucoma and / or elevated intraocular pressure.

[0467] In another embodiment, the disease is associated with overactivation of FGFR3 in cancer, for example multiple myeloma, myeloproliferative syndrome, leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer, bladder cancer, or breast cancer.

[0468] In another embodiment, the disease is a vascular smooth muscle disorder, preferably selected from the group consisting of hypertension, restenosis, arteriosclerosis, acute decompensated heart failure, congestive heart failure, cardiac edema, renal edema, hepatic edema, acute renal failure, and chronic renal failure.

[0469] Preferably, the disease is an achondroplasia phenotype selected from the group consisting of growth retardation, cranial deformities, orthodontic defects, cervical spinal cord compression, spinal stenosis, hydrocephalus, hearing loss due to chronic otitis media, cardiovascular disease, neurological disease and obesity.

[0470] Most preferably, the disease is achondroplasia.

[0471] In one embodiment, the patient receiving the therapeutic method of the present invention is a mammalian patient, preferably a human patient. In one embodiment, the human patient is an adult. In a preferred embodiment, the human patient is a pediatric patient.

[0472] Another aspect of the present invention is the use of a CNP prodrug of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one CNP prodrug of the present invention, for the manufacture of a medicament for treating a disease treatable with CNP.

[0473] Preferably, the disease is selected from the group consisting of achondroplasia, hypochondroplasia, short stature, dwarfism, chondro-osteodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, chiropteran dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, proximal short-limbed chondrodysplasia punctata, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, osteogenesis imperfecta, diastrophic dysplasia, femoral brachykinesia, Langer metaphyseal dysplasia, Niebergelt metaphyseal dysplasia, Robinow syndrome, Reinhardt syndrome, acroostosis imperfecta, peripheral osteogenesis imperfecta, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, distal metapodial dysplasia, brachymelia, Morquio syndrome, Kniest syndrome, variant osteogenesis imperfecta dysplasia, spondyloepiphyseal dysplasia, neurofibromatosis, Legius syndrome, Leopard syndrome, Noonan syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Legius syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, cleidocranial dysostosis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzon-cutaneous-skeletal syndrome), digital abnormalities, brachydactyly, camptodactyly, polydactyly, syndactyly, segmental dysplasia, enchondrosis, fibrous dysplasia, hereditary multiple osteochondromas, hypophosphatemic rickets, Jaffe-Liechtenstein syndrome, Marfan syndrome, McCune-Albright syndrome, osteopetrosis, and bone poikilosis.

[0474] Preferably, the disease is achondroplasia, hypochondroplasia, short stature, dwarfism, chondro-osteodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, chiropteran dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, proximal short-limbed chondrodysplasia punctata, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, osteogenesis imperfecta, diastrophic dysplasia, congenital femoral short-limbed dysplasia, Langer metaphyseal dysplasia, Niebergelt metaphyseal dysplasia The present invention relates to a genetic disorder characterized by ...

[0475] In another embodiment, the disease is an ophthalmic disease, for example, glaucoma and / or elevated intraocular pressure.

[0476] In another embodiment, the disease is associated with overactivation of FGFR3 in cancer, for example multiple myeloma, myeloproliferative syndrome, leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer, bladder cancer, or breast cancer.

[0477] In another embodiment, the disease is a vascular smooth muscle disorder, preferably selected from the group consisting of hypertension, restenosis, arteriosclerosis, acute decompensated heart failure, congestive heart failure, cardiac edema, renal edema, hepatic edema, acute renal failure, and chronic renal failure.

[0478] Preferably, the disease is an achondroplasia phenotype selected from the group consisting of growth retardation, cranial deformities, orthodontic defects, cervical spinal cord compression, spinal stenosis, hydrocephalus, hearing loss due to chronic otitis media, cardiovascular disease, neurological disease and obesity.

[0479] Most preferably, the disease is achondroplasia.

[0480] In one embodiment, the disease to be treated with a CNP prodrug of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one CNP prodrug of the present invention, occurs in a mammalian patient, preferably a human patient. In one embodiment, the human patient is an adult. In a preferred embodiment, the human patient is a pediatric patient.

[0481] A further aspect of the present invention is a method for treating, managing, delaying, or preventing one or more diseases treatable with CNP in a mammalian patient, preferably a human patient, in need of treatment, comprising administering to said patient in need thereof a therapeutically effective amount of a CNP prodrug of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a CNP prodrug of the present invention. In one embodiment, the human patient is an adult. In a preferred embodiment, the human patient is a pediatric patient.

[0482] Preferably, the one or more diseases that can be treated with CNP include achondroplasia, hypochondroplasia, short stature, dwarfism, chondro-osteodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, kyphotic dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, proximal short-limbed chondrodysplasia punctata, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, osteogenesis imperfecta, diastrophic dysplasia, femoral brachykinesia, Langer metaphyseal dysplasia, Niebergelt metaphyseal dysplasia, Robinow syndrome, Reinhardt syndrome, acroostosis imperfecta, peripheral osteogenesis imperfecta, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, distal metaphyseal dysplasia, brachymelia, Morquio syndrome, Kniest syndrome, In some embodiments, the inflammatory bowel disease is caused by a genetic disorder characterized by a genetic predisposition to osteoarthritis, osteoporosis, osteoporosis, osteoarthritis of the spine, osteoporosis ...

[0483] In another embodiment, the one or more diseases that can be treated with CNP are ophthalmic diseases, such as glaucoma and / or elevated intraocular pressure.

[0484] In another embodiment, the one or more diseases that can be treated with CNP are associated with overactivation of FGFR3 in cancer, e.g., multiple myeloma, myeloproliferative syndrome, leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer, bladder cancer, or breast cancer.

[0485] In another embodiment, the one or more diseases that can be treated with CNP are vascular smooth muscle disorders, preferably selected from the group consisting of hypertension, restenosis, arteriosclerosis, acute decompensated heart failure, congestive heart failure, cardiac edema, renal edema, hepatic edema, acute renal failure, and chronic renal failure.

[0486] Preferably, the one or more disorders that can be treated with CNP are achondroplasia phenotypes selected from the group consisting of growth retardation, cranial deformities, orthodontic defects, cervical spinal cord compression, spinal stenosis, hydrocephalus, hearing loss due to chronic otitis media, cardiovascular disease, neurological disease, and obesity.

[0487] Most preferably, the one or more diseases that can be treated with CNP is achondroplasia.

[0488] A further aspect of the present invention is a method of administering a CNP prodrug of the present invention, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention, comprising the step of administering a CNP prodrug of the present invention, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention by topical, enteral, or parenteral administration, as well as by topical, injection, or infusion methods including intra-articular, peri-articular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular, intraorbital, intravitreal, intratympanic, intravesical, intracardiac, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, intraventricular, intrasternal injection and infusion, direct delivery to the brain by an implantable device (e.g., an Ommaya Reservoir) that allows delivery of the present invention and the like to brain tissue or brain fluid, direct intraventricular injection or infusion, injection or infusion into the brain or brain-related regions, injection into the subchoroidal space, retro-orbital injection, and eye drops, preferably by subcutaneous injection.

[0489] In a preferred embodiment, the present invention relates to a CNP prodrug or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention for use in treating achondroplasia by subcutaneous injection.

[0490] Another aspect of the present invention is the irreversible conjugates of formula (IVa) and (IVb): [ka] (In the formula, -D is a CNP substructure, -L 2 - is a single chemical bond or spacer moiety; -Z is a water-soluble carrier moiety; x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and y is an integer selected from the group consisting of 1, 2, 3, 4, and 5.

[0491] Preferred embodiments fD, -L 2 -, -Z, x and y are as described above. [Example]

[0492] Materials and Methods CNP SEQ ID NO: 1 was obtained from Bachem AG, Bubendorf, Switzerland (CNP-22, human, catalogue number H-1296). CNP-34 SEQ ID NO: 40 and CNP-38 SEQ ID NO: 24 were obtained from CASLO ApS, Kongens Lyngby, Denmark.

[0493] Side-chain protected CNP-38 (synthesized with the Fmoc strategy) on TCP resin with a Boc-protected N-terminus and an ivDde-protected side chain of Lys26 was obtained from CASLO ApS, Kongens Lyngby, Denmark.

[0494] Side-chain protected CNP-34 (synthesized by the Fmoc strategy) on TCP Tentagel resin with a Boc-protected N-terminus and an ivDde-protected side chain at either Lys12, Lys16, or Lys22 was obtained from Peptide Specialty Laboratories GmbH, Heidelberg, Germany. Side-chain protected CNP-38 (synthesized by the Fmoc strategy) on TCP Tentagel resin with a free N-terminus was obtained from Peptide Specialty Laboratories GmbH, Heidelberg, Germany.

[0495] Methoxy PEG amine 5 kDa was obtained from Rapp Rapp Polymere GmbH, Tübingen, Germany. All other PEGs used in this study were obtained from NOF Europe NV, Grobbendonk, Belgium.

[0496] FmocN-Me-Asp(OtBu)-OH was obtained from Bachem AG, Bubendorf, Switzerland. S-Trityl-6-mercaptohexanoic acid was purchased from Polypeptide, Strasbourg, France. HATU was obtained from Merck Biosciences GmbH, Schwalbach / Ts, Germany.

[0497] 2,4-Dimethylbenzyl alcohol was obtained from abcr GmbH, Karlsruhe, Germany.

[0498] Fmoc-N-Me-Asp(OBn)-OH was obtained from Peptide International Inc., Louisville, KY, USA.

[0499] Neutral endopeptidase (NEP) was obtained from Enzo Life Sciences GmbH, Lorrach, Germany.

[0500] All other chemicals and reagents were purchased from Sigma Aldrich GmbH, Taufkirchen, Germany.

[0501] Syringes equipped with polyethylene frits (MultiSynTech GmbH, Witten, Germany) were used as reaction vessels or for washing steps of the peptide resin.

[0502] General procedure for removal of ivDde protecting groups from on-resin side-chain protected CNPs The resin was pre-swollen in DMF for 30 min, and the solvent was discarded. The ivDde group was removed by incubating the resin with DMF / hydrazine hydrate 4 / 1 (v / v, 2.5 mL / g resin) for 8 × 15 min. A fresh DMF / hydrazine hydrate solution was used for each step. Finally, the resin was washed with DMF (10 ×), DCM (10 ×) and dried under vacuum.

[0503] RP-HPLC purification: For preparative RP-HPLC, a Waters 600 controller and a 2487 Dual Absorbance Detector were used, equipped with the following columns: a Waters XBridge™ BEH300 Prep C18 5 μm, 150 × 10 mm, flow rate 6 mL / min, or a Waters XBridge™ BEH300 Prep C18 10 μm, 150 × 30 mm, flow rate 40 mL / min. A linear gradient of solvent system A (water containing 0.1% TFA v / v or 0.01% concentrated HCl v / v) and solvent system B (acetonitrile containing 0.1% TFA v / v or 0.01% concentrated HCl v / v) was used.

[0504] Unless otherwise stated, HPLC fractions containing the product were pooled and lyophilized to dryness.

[0505] Flash chromatography Flash chromatography purification was performed on an Isolera One system from Biotage AB, Sweden using Biotage KP-Sil silica cartridges with n-heptane and ethyl acetate as eluents. Products were detected at 254 nm.

[0506] Analysis method Ultra-performance LC (UPLC)-MS analysis was performed on a Waters Acquity system equipped with a Waters BEH300 C18 column (2.1 × 50 mm, particle size 1.7 μm, flow rate: 0.25 mL / min, solvent A: water containing 0.04% TFA (v / v), solvent B: acetonitrile containing 0.05% TFA (v / v)) coupled to a Thermo Scientific LTQ Orbitrap Discovery mass spectrometer or coupled to a Waters Micromass ZQ.

[0507] Size-exclusion chromatography (SEC) was performed using an Amersham Bioscience AEKTAbasic system equipped with a Superdex 200 5 / 150 GL column (Amersham Bioscience / GE Healthcare) equipped with a 0.45 μm inlet filter unless otherwise stated. 20 mM sodium phosphate, 140 mM NaCl, pH 7.4 was used as the mobile phase.

[0508] -L 1 Due to the reversible nature of the binding of - to -D, measurements of NEP stability and receptor affinity were performed using stable analogs of the CNP prodrugs of the present invention, i.e., structures similar to those of the CNP prodrugs of the present invention that have stable rather than reversible binding of -Z to -D.

[0509] This was necessary because the CNP prodrugs of the present invention would release CNP during the course of the experiment, and the released CNP would affect the results.

[0510] Quantitation of plasma total CNP-38 concentration Total plasma CNP-38 concentrations were determined by quantification of the N-terminal signature peptide (sequence: LQEHPNAR) and C-terminal signature peptide (sequence: IGSMSGLGC) after trypsin digestion.

[0511] LC-MS analysis was performed using an Agilent 1290 UPLC coupled to an Agilent 6550 iFunnel Q-TOF mass spectrometer with an ESI probe. Chromatography was performed using a Waters Acquity BEH300 C18 analytical column (50 × 2.1 mm i.d., 1.7 μm particle size) equipped with a prefilter at a flow rate of 0.25 mL / min (T = 25 °C). Water (UPLC grade) containing 0.2% formic acid (v / v) was used as mobile phase A, and acetonitrile (UPLC grade) containing 0.2% formic acid (v / v) was used as mobile phase B. The gradient system included a short isocratic step with initial parameters of 0.1% B for 3.0 min, followed by a linear increase from 0.1% B to 16% B over 17 min. Mass spectrometry was performed in single ion monitoring (SIM) mode to identify the ion m / z 482.75 [M+2H]. 2+ (N-terminus) and m / z 824.36 [M+H] 1+ (C-terminus) was monitored. Denatured CNP-38 peptide was used as an internal standard.

[0512] Calibration standards for CNP-38 conjugates in blank plasma were prepared as follows: thawed Li-heparin cynomolgus monkey plasma was first homogenized and then centrifuged for 5 minutes. The CNP-38 conjugate formulation was diluted in DMSO to a working solution of 10 μg / mL (equivalent of conjugated CNP-38) and spiked into blank plasma at concentrations between 9.3 ng / 100 μL (equivalent of conjugated CNP-38) and 139.5 ng / 100 μL (equivalent of conjugated CNP-38). These solutions were used to generate a calibration curve. The calibration curve included a 1 / x ratio for both signature peptides (N- and C-termini). 2For quality control, three quality control samples were prepared with contents of 116.2 ng / 100 μL (high QC, conjugated CNP-38 equivalents), 69.75 ng / 100 μL (medium QC, conjugated CNP-38 equivalents), and 23.25 ng / 100 μL (low QC, conjugated CNP-38 equivalents).

[0513] For sample preparation, protein precipitation was performed by adding 300 μL of pre-chilled (0 °C) methanol to 100 μL of plasma sample. 200 μL of the supernatant was transferred to a new well plate and evaporated to dryness (under a gentle nitrogen stream at 35 °C). 100 μL of reconstitution solvent (Thermo Digestion Buffer, order number 60109-101, Thermo Fisher Scientific GmbH, Dreieich, Germany) was used to dissolve the residue. 20 μg of trypsin (order number V5111, Promega GmbH, Mannheim, Germany) was dissolved in 20 μL of 10 mM acetic acid. 2 μL of the trypsin solution was added to each cavity.

[0514] After 4 hours of incubation at 37°C (water bath), 5 μL of 0.5 M TCEP solution was added to each cavity and incubated again for 5 minutes at 96°C. After the samples cooled to room temperature, 3 μL of acetonitrile was added. The eluate was transferred to a vial. 10 μL was injected into the UPLC-MS system.

[0515] [Example 1] Synthesis of linker reagent 1f Linker reagent 1f was synthesized according to the following scheme:

[0516] [ka]

[0517] To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48 mmol) and NaCNBH (819 mg, 12.63 mmol) in MeOH (20 mL) was added 2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) in portions. The mixture was stirred at room temperature for 90 min, acidified with 3 M HCl (4 mL), and stirred for an additional 15 min. The reaction mixture was added to saturated NaHCO solution (200 mL) and extracted five times with CHCl. ​​The combined organic phases were dried over NaSO, and the solvent was evaporated under vacuum. The resulting N-methyl-N-Boc-N'-Tmob-ethylenediamine 1a was dried under high vacuum and used in the next reaction step without further purification. Yield: 3.76 g (11.48 mmol, 89% purity, 1a: double Tmob protected product = 8:1) MS: m / z 355.22 = [M+H] + , (calculated monoisotopic mass = 354.21).

[0518] To a solution of 1a (2 g, 5.65 mmol) in CHCl (24 mL) was added COMU (4.84 g, 11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol), and 2,4,6-collidine (2.65 mL, 20.34 mmol). The reaction mixture was stirred at room temperature for 3 h, diluted with CHCl (250 mL), and washed three times with 0.1 M HSO (100 mL) and three times with saturated brine (100 mL). The aqueous phase was re-extracted with CHCl (100 mL). The combined organic phases were dried over NaSO, filtered, and the residue was concentrated to a volume of 24 mL. 1b was purified using flash chromatography. Yield: 5.31 g (148%, 6.66 mmol) MS: m / z 796.38=[M+H] + , (calculated monoisotopic mass = 795.37).

[0519] To a solution of 1b (5.31 g, 4.52 mmol with respect to N-Fmoc-N-Me-Asp(OBn)-OH) in THF (60 mL) was added DBU (1.8 mL, 3% v / v). The solution was stirred at room temperature for 12 min, diluted with CHCl (400 mL), and washed three times with 0.1 M HSO (150 mL) and three times with saturated brine (150 mL). The aqueous phase was re-extracted with CHCl (100 mL). The combined organic phases were dried over NaSO and filtered. 1c was isolated by evaporation of the solvent and used in the next reaction without further purification. MS: m / z 574.31=[M+H] + , (calculated monoisotopic mass = 573.30).

[0520] 1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile (26 mL), and COMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid (2.12 g, 5.42 mmol), and 2,4,6-collidine (2.35 mL, 18.08 mmol) were added. The reaction mixture was stirred at room temperature for 4 h, diluted with CHCl (400 mL), and washed three times with 0.1 M HSO (100 mL) and three times with saturated brine (100 mL). The aqueous phase was re-extracted with CHCl (100 mL). The combined organic phases were dried over NaSO, filtered, and 1d was isolated by evaporation of the solvent. Product 1d was purified using flash chromatography. Yield: 2.63g (62%, purity 94%) MS: m / z 856.41=[M+H] + , (calculated monoisotopic mass = 855.41).

[0521] To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and HO (11 mL) was added LiOH (267 mg, 11.12 mmol), and the reaction mixture was stirred for 70 min at room temperature. The mixture was diluted with CHCl (200 mL) and washed three times with 0.1 M HSO (50 mL) and three times with saturated brine (50 mL). The aqueous phase was re-extracted with CHCl (100 mL). The combined organic phases were dried over NaSO, filtered, and 1e was isolated by evaporation of the solvent. 1e was purified using flash chromatography. Yield: 2.1g (88%) MS: m / z 878.4 = [M+Na] + , (calculated monoisotopic mass = 837.40).

[0522] To a solution of 1e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) was added DCC (123 mg, 0.59 mmol) and a catalytic amount of DMAP. After 5 min, N-hydroxy-succinimide (114 mg, 0.99 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered, the solvent was removed in vacuo, and the residue was dissolved in 90% acetonitrile + 0.1% TFA (3.4 mL). The crude mixture was purified by RP-HPLC. The product fractions were neutralized with 0.5 M pH 7.4 phosphate buffer and concentrated. The remaining aqueous phase was extracted with DCM, and 1f was isolated by evaporation of the solvent. Yield: 154mg (81%) MS: m / z 953.4 = [M+H] + , (calculated monoisotopic mass = 952.43).

[0523] [Example 2] N εK4 / εK10 -CNP Monolinker Thiol 2,N εK4 -CNP monolinker thiol 2c and N εK10 Synthesis of -CNP monolinker thiol 2d

[0524] [ka] Prepare N by dissolving CNP-22 (5.2 μmol) in 0.6 mL DMSO. εK4 / εK10 To prepare 1f (6.1 mg, 7.1 μmol) in 0.34 mL of DMSO, 60 μL of DIPEA and 0.15 mL of 0.375 M borate buffer adjusted to pH 8.5 with tetrabutylammonium hydroxide hydrate were added, and the mixture was stirred for 30 min at room temperature. The reaction mixture was diluted with 2 mL of acetonitrile / water 1 / 1 (v / v) and 200 μL of AcOH, and the protected N was isolated from the reaction mixture by RP-HPLC. εK4 / εK10 -CNP monolinker conjugates are isolated.

[0525] N εK4 -CNP monolinker thiols 2a and N εK10 For isolation of -CNP monolinker thiol 2b, an optimized RP-HPLC gradient can be used.

[0526] Removal of the protecting groups is achieved by treatment of the frozen product fraction with 0.6 mL of 90 / 10 / 2 / 2 (v / v / v / v) HFIP / TFA / TES / water for 1 hour at room temperature. εK4 / εK10 -CNP monolinker thiol 2 is purified by RP-HPLC. The identity and purity of the product are determined by ESI-LCMS.

[0527] Deprotected N εK4 -CNP monolinker thiol 2c and N εK10 -CNP monolinker thiol 2d can be similarly obtained from 2a and 2b, respectively.

[0528] [Example 3] N αG1 Synthesis of -CNP monolinker thiol 3

[0529] [ka] Prepare N by dissolving CNP-22 (5.2 μmol) in 0.6 mL DMSO. αG1 To prepare the -CNP monolinker thiol 3, 0.25 mL of 0.5 M phosphate buffer pH 7.4 and 1f (6.1 mg, 7.1 μmol) in 0.34 mL of DMSO were added, and the mixture was stirred for several hours at room temperature. The reaction mixture was diluted with 2 mL of acetonitrile / water 1 / 1 (v / v) and 200 μL of AcOH, and the protected N was isolated from the reaction mixture by RP-HPLC. αG1 -CNP monolinker thiol is isolated.

[0530] Removal of the protecting groups is achieved by treatment of the frozen product fraction with 0.6 mL of 90 / 10 / 2 / 2 (v / v / v / v) HFIP / TFA / TES / water for 1 hour at room temperature. αG1 The -CNP monolinker thiol 3 is purified by RP-HPLC. The identity and purity of the product are determined by ESI-LCMS.

[0531] [Example 4] PEGylation of CNP monolinker thiols 2c, 2d, and 3

[0532] [ka] 1 μmol of CNP monolinker thiol 2c was dissolved in 0.5 mL of acetonitrile / 0.2 M succinate buffer, pH 3.8 (1 / 1, v / v), and 1.2 μmol of linear 40 kDa PEG-maleimide was added. The mixture was stirred at room temperature. The reaction was stopped by adding 20 μL of AcOH, and the CNP conjugate 4 was purified by preparative RP-HPLC. CNP conjugates 5 and 6 are similarly prepared from 1 μmol CNP monolinker thiols 2d and 3.

[0533] After complete hydrolysis under acidic conditions, the CNP content is determined by quantitative amino acid analysis.

[0534] [Example 5] In vitro release kinetics CNP conjugates 4, 5, and 6 are dissolved in 60 mM sodium phosphate, 3 mM EDTA, 0.01% Tween-20, pH 7.4 at a concentration of approximately 2 mg / mL and sterile filtered. The mixture is incubated at 37°C, at which point aliquots are withdrawn and analyzed by RP-HPLC and ESI-MS. The UV signal corresponding to free CNP is integrated and plotted against the incubation time.

[0535] Curve fitting software is used to estimate the corresponding release half-life.

[0536] [Example 6] Pharmacokinetics and cGMP production in rats Equimolar doses of CNP-22, CNP conjugates 4, 5, or 6 were injected intravenously and subcutaneously into normal rats. Plasma CNP and cGMP levels were determined over time as described in the literature (U.S. Pat. No. 8,377,884 B2).

[0537] [Example 7] Synthesis of Dmb-protected 6-mercaptohexanoic acid 7 Compound 7 was synthesized according to the following scheme:

[0538] [ka]

[0539] To a solution of 6-mercaptohexanoic acid (7.10 g, 47.90 mmol) in trifluoroacetic acid (20 mL) was added 2,4-dimethylbenzyl alcohol (13.5 g, 95.80 mmol). The mixture was stirred at room temperature for 60 min, after which the trifluoroacetic acid was removed in vacuo. The residue was dissolved in a mixture of 95.8 mL LiOH (3 M) and THF (81 mL) and stirred at room temperature for 60 min. The solvent was removed in vacuo, and the aqueous residue was extracted three times with EtoAc (200 mL). The combined organic phases were dried over MgSO4, and the solvent was removed in vacuo. 7 was purified by RP-HPLC. Yield: 2.27 g (8.52 mmol, 18%) MS: m / z 267.01=[M+H] + , (calculated monoisotopic mass = 266.13).

[0540] [Example 8] Synthesis of linker reagent 8c Linker reagent 8c was synthesized according to the following scheme:

[0541] [ka]

[0542] To a solution of 1c (21.6 g, 27.18 mmol) in isopropanol (401 mL) was added water (130 mL) and LiOH (3.90 g, 163.06 mmol). The reaction mixture was stirred for 3 h at room temperature, then diluted with toluene (300 mL) and washed three times with 0.1 M HCl (200 mL). The combined aqueous phases were washed three times with toluene (100 mL). The aqueous phase was basified to pH 8.5 with 4 M NaOH (4 mL) and extracted eight times with CHCl (200 mL). The combined CHCl phases were washed with saturated brine (50 mL) and dried over NaSO. 8b was isolated by evaporation of the solvent and used in the next reaction without further purification. Yield: 11.89 g (24.59 mmol, 90%) MS: m / z 484.16=[M+H] + , (calculated monoisotopic mass = 483.26).

[0543] To a solution of 7 (293 mg, 1.10 mmol) and PyBOP (572 mg, 1.10 mmol) in THF (10 mL) under a N atmosphere was added DIEA (0.52 mL, 3.00 mmol). The reaction mixture was stirred for 60 min at room temperature. A solution of 8b (484 mg, 1.00 mmol) in THF (2 mL) was added, and the reaction was stirred for an additional 60 min. The reaction was quenched with 2 M citric acid solution (10 mL), and the THF was removed in vacuo. The resulting aqueous phase was then extracted twice with EtOAc (15 mL), and the combined organic layers were washed with water (10 mL) and saturated brine (10 mL), and dried over MgSO. The solvent was removed in vacuo, and 8c was purified by RP HPLC. Yield: 330 mg (0.451 mmol, 45%) MS: m / z 732.34 = [M+H] + , (calculated monoisotopic mass = 731.38).

[0544] [Example 9] Synthesis of linker reagent 9 Linker reagent 9 was synthesized according to the following scheme:

[0545] [ka]

[0546] To a solution of 8b (2.00 g, 4.14 mmol) and Fmoc-Cl (1.07 g, 4.14 mmol) in dioxane (20 mL) was added 1 M Na2CO3 solution (20 mL). The reaction mixture was stirred for 40 min at room temperature. Water (100 mL) and diethyl ether (100 mL) were added, and the aqueous phase was extracted twice with diethyl ether (100 mL). The aqueous phase was acidified with concentrated HCl to pH 1 and again extracted three times with diethyl ether. The combined organic phases were dried over Na2SO4, and the solvent was evaporated in vacuo. 9 was used in the next step without further purification. Yield: 2.63 g (3.73 mmol, 90%) MS: m / z 728.32 = [M+Na] +, (calculated monoisotopic mass = 705.33).

[0547] [Example 10] Synthesis of reversible Lys26 CNP-38 PEG2x20kDa conjugate 10f Conjugate 10f was synthesized according to the following scheme:

[0548] [ka] TIFF2026012776000113.tif159140

[0549] 2.00 g (0.21 mmol) of side-chain-protected CNP-38 on TCP resin bearing a Boc-protected N-terminus and an ivDde-protected side chain of Lys26 was deprotected according to the procedure given in "Materials and Methods" to give 10a. A solution of linker reagent 8c (336 mg, 0.46 mmol), PyBOP (239 mg, 0.46 mmol), and DIEA (182 μL, 1.04 mmol) in DMF (5 mL) was incubated for 10 min at room temperature and then added to resin 10a. The suspension was shaken for 2 h at room temperature. The resin was washed 10 times with DMF (10 mL) and 10 times with DCM (10 mL) and dried under vacuum for 15 min. Cleavage of the peptide from the resin and removal of the protecting groups was achieved by treatment of the resin with 15 mL of pre-chilled (-18 °C) cleavage cocktail 68.5 / 10 / 10 / 5 / 3.5 / 1 (v / w / v / v / v / v) TFA / DTT / thioanisole / phenol / water / TIPS. The mixture was allowed to warm to room temperature and stirred for 60 min. Crude 10C was precipitated into pre-chilled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The combined HPLC fractions were used directly in the next step. MS: m / z 1124.60=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1124.59).

[0550] To the combined HPLC fractions of 10c (250 mL) were added 40 mL of 0.5 M citrate buffer (pH = 5.00) and 7 mL of a 0.01 M solution of 2,2'-dithiobis(pyridine-N-oxide) in 1 / 1 (v / v) acetonitrile / water. After 5 min of incubation at room temperature, the reaction was complete. The mixture was diluted with 500 mL of water containing 0.1% TFA (v / v) and acidified to a pH of approximately 2 with AcOH (20 mL). 10d was purified by RP-HPLC. Yield: 101 mg (17.3 μmol, 9%) CNP-38-Linker-Dmb * 10TFA MS: m / z 1124.10=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1124.09).

[0551] Cleavage of the Dmb protecting group was achieved by adding 30 mL of pre-cooled (-18 °C) cleavage cocktail 100 / 5 / 3 / 2 / 1 (v / v / w / v / v) TFA / MSA / DTT / water / thioanisole to 10d (101 mg, 17.3 μmol) and stirring for 3 h at 0 °C. Crude 10e was precipitated in pre-cooled (-18 °C) diethyl ether. The precipitate was dissolved in water containing 0.1% TFA (v / v) and incubated for 10 min to hydrolyze any TFA esters. 10e was purified by RP-HPLC. Yield: 46 mg (8.34 μmol, 48%) CNP-38-linker-thiol * 10TFA MS: m / z 1094.58=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1094.57).

[0552] To a solution of 10e (46 mg, 8.43 μmol) in 1.15 mL of water containing 0.1% TFA (v / v), a solution of PEG 2x20 kDa maleimide (Sunbright GL2-400MA, 870 mg, 21.75 μmol) in 4.35 mL of water containing 0.1% TFA (v / v) was added, followed by the addition of 0.5 M lactate buffer (1.07 mL, pH = 4.20). The mixture was stirred at room temperature for 4 h. Conjugate 10f was purified by RP-HPLC. Yield: 233 mg (5.21 μmol, 62%) of conjugate 10f * 10HCl

[0553] [Example 11] Synthesis of reversible Lys26 CNP-38 PEG4x10kDa conjugate 11i Conjugate 11i was synthesized according to the following scheme:

[0554] [ka] TIFF2026012776000115.tif215156

[0555] To a solution of 9 (353 mg, 0.50 mmol) and PyBOP (260 mg, 0.50 mmol) in DMF (9 mL) was added DIEA (105 μL, 0.60 mmol). This mixture was drained onto Lys26 side chain protected CNP-38 resin 10a (2.00 g, 0.21 mmol), and the suspension was shaken for 2 h at room temperature to give resin 11a. The resin was washed 10 times with DMF (7 mL). Cleavage of the Fmoc protecting group in 11a was carried out using a solution of HOBt (0.68 g, 5.03 mmol) and piperazine (3.00 g, 34.83 mmol) in DMF (47 mL). To this end, the resin was incubated with 10 mL of the cleavage mixture five times, for 15 min each time at room temperature. The resin was then washed seven times with DMF (7 mL).

[0556] A solution of Fmoc-Lys(Fmoc)-OH (449 mg, 0.76 mmol), COMU (325 mg, 0.76 mmol), and DIEA (165 μL, 0.95 mmol) in DMF (9 mL) was prepared and drained onto the resin. The mixture was shaken for 2 h at room temperature. This procedure was repeated twice, for 1 h each, using freshly prepared coupling mixtures. The resin was washed 10 times with DMF (7 mL), and any remaining free amino groups were capped with 8 mL 1 / 1 / 2 (v / v / v) AcO / pyridine / DMF.

[0557] Cleavage of the Fmoc protecting group in 11c was carried out using a solution of HOBt (0.68 g, 5.03 mmol), piperazine (3.00 g, 34.83 mmol) in DMF (47 mL). To do so, the resin was incubated with 10 mL of the cleavage mixture five times for 15 min each at room temperature. The resin was washed seven times with DMF (7 mL).

[0558] To a solution of 7 (266 mg, 1.00 mmol) and PyBOP (520 mg, 1.00 mmol) in DMF (9 mL) was added DIEA (209 μL, 1.20 mmol). The mixture was drained onto the resin and shaken for 2 h at room temperature. The resin was washed seven times with DMF (7 mL) to give resin 11e. Cleavage of the peptide from the resin and removal of the protecting groups was achieved by treating the resin with 15 mL of pre-cooled (−18 °C) cleavage cocktail 68.5 / 10 / 10 / 5 / 3.5 / 1 (v / w / v / v / v / v) TFA / DTT / thioanisole / phenol / water / TIPS. The mixture was allowed to warm to room temperature and stirred for 3 h. Crude 11f was precipitated into pre-cooled (−18 °C) diethyl ether and purified by RP-HPLC. The combined HPLC fractions were used directly in the next step. MS: m / z 1218.66=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1218.65).

[0559] To the combined HPLC product fractions of 11f (1 L) was added 160 mL of 0.5 M citrate buffer (pH = 5.00) and 100 mL of a 50 mM solution of 2,2'-dithiobis(pyridine-N-oxide) in 9 / 1 (v / v) acetonitrile / water. The mixture was stirred for 4 hours at room temperature and then diluted with 1 L of water containing 0.1% TFA (v / v). 11g was purified by RP-HPLC. Yield: 64.3 mg (10.7 μmol, 6%) CNP-38-Linker-DMB * 10TFA MS: m / z 1218.15=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1218.14).

[0560] Cleavage of the Dmb protecting group was achieved by adding 45 mL of pre-cooled (-18 °C) cleavage cocktail 100 / 5 / 3 / 2 / 1 (v / v / w / v / v) TFA / MSA / DTT / water / thioanisole to 11 g (61.8 mg, 10.3 μmol) and stirring for 4 h at 0 °C. Crude 11h was precipitated in pre-cooled (-18 °C) ether. The precipitate was dissolved in a 1 / 1 (v / v) acetonitrile / water solution containing 0.1% TFA (v / v) and incubated for 4 h at room temperature to hydrolyze any TFA esters. 11h was purified by RP-HPLC. Yield: 38.4 mg (6.65 μmol, 65%) CNP-38-linker-thiol * 10TFA MS: m / z 1159.11=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1159.10).

[0561] To a solution of 11h (34.6 mg, 5.99 μmol) in 1 mL of water containing 0.1% TFA (v / v), a solution of PEG 2x10 kDa maleimide (Sunbright GL2-200MA, 1.12 g, 56.03 μmol) in 6.1 mL of water containing 0.1% TFA (v / v) was added, followed by the addition of 0.5 M lactate buffer (1.46 mL, pH = 4.00). The mixture was stirred for 4 h. Conjugate 11i was purified by RP-HPLC. Yield: 227 mg (4.96 μmol, 83%) of conjugate 11i * 10HCl

[0562] [Example 12] Synthesis of persistent Lys26 CNP-38 PEG4x10kDa conjugate 12g Conjugate 12g was synthesized according to the following scheme:

[0563] [ka] TIFF2026012776000117.tif215147TIFF2026012776000118.tif95109

[0564] To a solution of Fmoc-Lys(Fmoc)-OH (365 mg, 0.62 mmol) and PyBOP (322 mg, 0.62 mmol) in DMF (4.6 mL) was added DIEA (0.11 mL, 0.62 mmol). The mixture was drained onto resin 10a (2.0 g, 0.21 mmol). The suspension was shaken for 2 h at room temperature. The resin was washed ten times with DMF (7 mL). Cleavage of the Fmoc protecting group in 12a was carried out using a solution of HOBt (1.35 g, 9.99 mmol), piperazine (6.00 g, 69.66 mmol) in DMF (94 mL). To this end, the resin was incubated with the cleavage mixture five times for 15 min each time at room temperature to give resin 12b. The resin was then washed seven times with DMF (7 mL).

[0565] To a solution of 7 (283 mg, 1.06 mmol) and PyBOP (552 mg, 1.06 mmol) in DMF (6.5 mL) was added DIEA (185 μL, 1.06 mmol) and drained onto resin 12b (2.07 g, 0.10 mmol / g, 0.21 mmol). The mixture was shaken for 2 h at room temperature. The resin was then washed 10 times with DMF (7 mL) and CHCl (7 mL) and dried under vacuum.

[0566] Cleavage of the peptide from the resin and removal of the protecting groups was achieved by treatment of the resin with 15 mL of pre-chilled (-18 °C) cleavage cocktail 68.5 / 10 / 10 / 5 / 3.5 / 1 (v / w / v / v / v / v) TFA / DTT / thioanisole / phenol / water / TIPS. The mixture was allowed to warm to room temperature and stirred for 2.5 h. Crude 12d was precipitated into pre-chilled diethyl ether (-18 °C) and purified by RP-HPLC. The combined HPLC fractions were used directly in the next step. MS: m / z 1172.37=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1172.37).

[0567] To the combined HPLC product fractions of 12d (390 mL) were added 58.5 mL of 0.5 M citrate buffer (pH = 5.00) and 8.9 mL of a 10 mM solution of 2,2'-dithiobis(pyridine-N-oxide) in 1 / 1 (v / v) acetonitrile / water. The mixture was stirred for 10 min at room temperature and then diluted with 400 mL of water containing 0.1% TFA (v / v). 12e was purified by RP-HPLC. Yield: 100 mg (17.5 μmol, 8% after 6 steps) CNP-38-Linker-Dmb * 9TFA MS: m / z 1171.87=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1171.86).

[0568] Cleavage of the Dmb protecting group was achieved by adding 65 mL of pre-cooled (-18 °C) cleavage cocktail 100 / 5 / 3 / 2 / 1 (v / v / w / v / v) TFA / MSA / DTT / water / thioanisole to 12e (100 mg, 17.5 μmol) and stirring for 3.5 h at 0 °C. Crude 12f was precipitated in pre-cooled (-18 °C) diethyl ether. The precipitate was dissolved in water containing 0.1% TFA (v / v) and incubated for 2 h at room temperature to hydrolyze any TFA esters. 12f was purified by RP-HPLC. Yield: 43.4 mg (7.92 μmol, 45%) CNP-38-linker-thiol * 9TFA MS: m / z 1112.83=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for 1112.82).

[0569] To a solution of 12f (39.6 mg, 7.22 μmol) in 1 mL of water containing 0.1% TFA (v / v) was added a solution of PEG 2x10 kDa maleimide (Sunbright GL2-200MA, 1.12 g, 59.94 μmol) in 6.16 mL of water containing 0.1% TFA (v / v), followed by 0.5 M lactate buffer (1.41 mL, pH = 4.20). The mixture was stirred for 4 h. Conjugate 12g was purified by RP-HPLC. Yield: 204 mg (4.48 μmol, 57%) conjugate 12 g * 9HCl

[0570] [Example 13] Synthesis of PEG 5kDa thiol 13c PEG 5kDa thiol 13c was synthesized according to the following scheme:

[0571] [ka]

[0572] To a solution of 13b (58.6 mg, 0.15 mmol), HOBt (22.9 mg, 0.15 mmol), and EDC hydrochloride (28.8 mg, 0.15 mmol) in DCM (1.00 mL) was added 2,4,6-collidine (121 mg, 1.00 mmol). Then, methoxy PEG amine 5 kDa 13a (500 mg, 0.10 mmol) in DCM (4.00 mL) was added, and the mixture was stirred for 16 h at room temperature. The solvent was evaporated, and the mixture was dissolved in ACN / water and purified by RP-HPLC. The volume of solvent was reduced in vacuo, and the aqueous residue was extracted with DCM (1 × 100 mL, 2 × 50 mL). The combined organic layers were reduced to 20 mL in vacuo. TEA (1.6 mL) and TES (3.5 mL) were added, and the mixture was stirred at room temperature for 4.5 h. 13c was precipitated in diethyl ether, stored overnight at −20° C., filtered, and dried under vacuum. Yield: 372 mg (72 μmol, 72%)

[0573] [Example 14] Synthesis of persistent N-terminal CNP-34 PEG 5 kDa conjugate 14e Conjugate 14e was synthesized according to the following scheme:

[0574] [ka] TIFF2026012776000121.tif77111

[0575] Side-chain protected CNP-34 14a (0.78 g, 70 μmol) on TCP Tentagel resin with a free N-terminus was pre-swollen in DMF for 30 min. A solution of maleimidohexanoic acid (85.3 mg, 0.40 mmol), DIC (50.9 mg, 0.40 mmol), and oxyma (57.4 mL, 0.40 mmol) in DMF (6 mL) was dispensed onto the resin, and the mixture was shaken for 30 min at room temperature. This coupling was then repeated once more using freshly prepared coupling solution. The resin was washed 10 times with DMF and CHCl and dried under vacuum to give 14b.

[0576] Cleavage of the peptide from the resin and removal of the protecting groups was achieved by treatment of the resin with 6 mL of cleavage cocktail 100 / 3 / 2 / 1 (v / v / v / v) TFA / TES / water / thioanisole for 1.5 h at room temperature, and the crude peptide was precipitated into pre-cooled (-18 °C) diethyl ether. MS: m / z 937.77 = [M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 937.74).

[0577] The precipitate was dissolved in 15 mL of TFA. A solution of diphenyl sulfoxide (68.06 mg, 0.34 mmol) and anisole (0.18 mL, 1.68 mmol) in 5 mL of TFA was added. Trichloromethylsilane (0.47 mL, 4.17 mmol) was added, and the mixture was stirred for 15 min at room temperature. Ammonium fluoride (0.38 g, 10.3 mmol) was added, and the solution was stirred for an additional 2 min. The crude material was precipitated into pre-cooled (-18 °C) diethyl ether and purified by RP-HPLC to give 14d. Yield: 8.30 mg (1.78 μmol, 82% purity, 1.4% after 3 steps) CNP-34-Malhx*8TFA MS: m / z 937.26=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 937.23).

[0578] To a solution of 14d (7.34 mg, 1.57 μmol) in 200 μL of 1 / 1 (v / v) acetonitrile / water containing 0.1% TFA (v / v), a solution of 13c (20 mg, 3.90 μmol) in 200 μL of water containing 0.1% TFA (v / v) was added, followed by the addition of 200 μL of 0.5 M lactate buffer (pH = 5.00). The mixture was incubated at room temperature for 30 min. Conjugate 14e was purified by RP-HPLC. Yield: 9.92 mg (1.01 μmol, 57%) conjugate 14e*8TFA

[0579] [Example 15] Synthesis of persistent N-terminal CNP-38 PEG 5 kDa conjugate 15e Conjugate 15e was synthesized according to the following scheme:

[0580] [ka]

[0581] Compound 15d was synthesized as described for 14d, except that side-chain protected CNP-38 15a (1.34 g, 0.12 mmol) on TCP Tentagel resin with a free N-terminus was used as the starting material. Yield: 15.6 mg (2.94 μmol, 6.6%) CNP-38-Malhx*9TFA MS: m / z 1064.05=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1064.04).

[0582] Conjugate 15e was synthesized as described for 14e, except that 15d (8.34 g, 1.58 mmol) was used as the starting material. Yield: 9.47 mg (0.91 μmol, 31%) conjugate 15e*9TFA

[0583] [Example 16] Synthesis of persistent Lys12 CNP-34 PEG 5kDa conjugate 16e Conjugate 16e was synthesized according to the following scheme:

[0584] [ka]

[0585] 1.00 g (0.10 mmol) of side-chain protected CNP-34 on TCP Tentagel resin with a Boc-protected N-terminus and an ivDde-protected side chain of Lys12 was deprotected according to the procedure given in Materials and Methods to give 16a.

[0586] Compound 16d was synthesized as described for 14d, except that resin 16a (1.00 g, 0.10 mmol) was used as the starting material. Yield: 17.0 mg (3.65 μmol, 3.7%) CNP-34-Lys12-Malhx*8TFA MS: m / z 937.25=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 937.23).

[0587] Conjugate 16e was synthesized as described for 14e, except that 16d (17 mg, 3.65 μmol) was used as the starting material. Yield: 12.2 mg (1.25 μmol, 34%) conjugate 16e*8TFA

[0588] [Example 17] Synthesis of persistent Lys16 CNP-34 PEG 5kDa conjugate 17e Conjugate 17e was synthesized according to the following scheme:

[0589] [ka]

[0590] 0.78 g (0.07 mmol) of side-chain protected CNP-34 on TCP Tentagel resin with a Boc-protected N-terminus and an ivDde-protected side chain of Lys16 was deprotected according to the procedure given in Materials and Methods to give 17a.

[0591] Compound 17d was synthesized as described for 14d, except that resin 17a (0.78 g, 0.13 mmol) was used as the starting material. Yield: 5.39 mg (1.16 μmol, 1.7%) CNP-34-Lys16-Malhx*8TFA MS: m / z 937.26=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 937.23).

[0592] Conjugate 17e was synthesized as described for 14e, except that 17d (5.39 mg, 1.16 μmol) was used as the starting material. Yield: 10.7 mg (1.09 μmol, 94%) conjugate 17e*8TFA

[0593] [Example 18] Synthesis of persistent Lys22 CNP-34 PEG 5kDa conjugate 18e Conjugate 18e was synthesized according to the following scheme:

[0594] [ka]

[0595] 1.07 g (0.11 mmol) of side-chain protected CNP-34 on TCP Tentagel resin with a Boc-protected N-terminus and an ivDde-protected side chain of Lys12 was deprotected according to the procedure given in Materials and Methods to give 18a.

[0596] Compound 18d was synthesized as described for 14d, except that resin 18a (1.07 g, 0.11 mmol) was used as the starting material. Yield: 5.20 mg (1.12 μmol, 1.0%) CNP-34-Lys22-Malhx*8TFA MS: m / z 937.26=[M+4H] 4+ , ([M+4H] 4+Calculated monoisotopic mass for α = 937.23).

[0597] Conjugate 18e was synthesized as described for 14e, except that 18d (5.2 mg, 1.12 μmol) was used as the starting material. Yield: 4.20 mg (0.43 μmol, 38%) conjugate 18e*8TFA

[0598] [Example 19] Synthesis of persistent Lys26 CNP-38 PEG 5kDa conjugate 19e Conjugate 19e was synthesized according to the following scheme:

[0599] [ka]

[0600] The ivDde of side-chain protected CNP-38 (0.865 g, 0.10 mmol) on TCP Tentagel resin bearing a Boc-protected N-terminus and an ivDde-protected side chain of Lys26 was deprotected according to the procedure given in Materials and Methods to give 19a.

[0601] Compound 19d was synthesized as described for 14d, except that resin 19a (0.865 g, 0.10 mmol) was used as the starting material. Yield: 10.3 mg (1.95 μmol, 2.0%) CNP-38-Malhx*9TFA MS: m / z 1064.05=[M+4H] 4+ , ([M+4H] 4+ Calculated monoisotopic mass for α = 1064.04).

[0602] Conjugate 19e was synthesized as described for 14e, except that 19d (4.70 mg, 1.10 μmol) was used as the starting material. Yield: 3.20 mg (0.31 μmol, 28%) conjugate 19e*9TFA

[0603] [Example 20] In vitro release kinetics CNP conjugates 10f and 11i were dissolved in PBS buffer (pH 7.4) containing 3 mM EDTA and 10 mM methionine at a concentration of approximately 1 mg conjugate / mL. The solution was sterile filtered and incubated at 37°C. At that time, aliquots were withdrawn and analyzed by RP-HPLC and ESI-MS. The UV signal corresponding to free CNP was integrated and plotted against the incubation time.

[0604] Curve fitting software was used to estimate the corresponding release half-lives.

[0605] result: A half-life time of 8.5 days (±1 day) was obtained for conjugate 10f.

[0606] A half-life of 9.5 days (±1.5 days) was obtained for conjugate 11i.

[0607] [Example 21] Digestion of CNP variants by neutral endopeptidase in vitro To determine the in vitro stability of various CNP variants containing different peptide chain lengths and PEGylation using different PEGylation sites and molecular weights in the presence of neutral endopeptidase (NEP), we established a NEP digestion assay that monitored the decrease of undigested CNP variants over time (normalized to the internal standard PEP) relative to time point t0.

[0608] Specifically, recombinant human NEP (final concentration 2.5 μg / mL) and standard pentafluorophenol (PFP, final concentration 40 μg / mL) were added to CNP variants (100 μg CNP equivalents / mL) in digestion buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaCl). The solution was incubated at 37°C and 500 rpm for up to 4 days. Samples were taken at different time points. The reaction was stopped by combined reduction and heat denaturation via the addition of TCEP (tris(2-carboxyethyl)phosphine, final concentration 25 mM) and incubation of the mixture at 95°C and 500 rpm for 5 min. HPLC-MS was used to identify the resulting reaction products. The half-life of each CNP variant was calculated by the time-dependent change in the ratio of the HPLC-UV peak areas of CNP and PFP. To compensate for variations in protease activity, CNP-38 or CNP-34 digestion was performed as a reference for each batch measurement.

[0609] Table 1 lists the half-lives, based on an in vitro NEP cleavage assay, of various CNP variants of different lengths with various PEG molecules attached to different side chains.

[0610] [Table 1]

[0611] The rank order of resistance to NEP is as follows: the longer CNP variant (CNP-38) is more stable than the shorter CNP variant (CNP-34), which in turn is more stable than the shorter CNP-22. The order of PEG attachment sites is as follows: N-terminus > adjacent ring > ring. Thus, N-terminal PEG attachment confers the highest stability against protein digestion with NEP for the conjugates tested. The stability of CNP-38 PEGylation at Lys26 can be increased with increasing PEG size.

[0612] [Example 22] Functional cGMP stimulation in NIH-3T3 cells with CNP variants The functional activity of CNP variants was determined in a cell-based assay using NIH-3T3 cells (a mouse embryonic fibroblast cell line). These cells express endogenous NPR-B on the cell surface. Stimulation of NPR-B with CNP leads to intracellular production of the second messenger cGMP, which is detected using a commercially available cGMP assay. NIH-3T3 cells were routinely cultured in DMEM F-12 medium containing 5% FBS and 5 mM glutamine at 37°C and 5% CO2. 50,000 cells per assay were resuspended in stimulation buffer (Dulbecco's PBS containing IBMX) and incubated with different concentrations of CNP variants (dilutions were made in PBS containing 0.2% BSA). After 30 minutes of incubation at 37°C and 5% CO2, cells were lysed, and cGMP levels were determined using a commercially available cGMP TR-FRET assay (Cisbio, cGMP Kit, catalog number 62GM2PEB). PEGylated CNP variants were always characterized in comparison with the non-PEGylated version of the same experimental batch. When possible, residual activity was assessed by the EC50 parameter of the resulting dose-response curves (restricted model with a common slope).

[0613] [Table 2]

[0614] Comparing the PEG attachment sites tested, attachment at Lys26 (cyclic lysine) showed the highest reduction in functional activity, while N-terminal attachment showed relatively high residual functional activity values. Increasing the PEG size resulted in better shielding of the CNP molecule and lower residual functional activity.

[0615] [Example 23] Growth studies after 5 weeks of treatment with CNP-38 by daily subcutaneous bolus injection or by continuous subcutaneous infusion in FVB mice This study was conducted to examine the effect of daily subcutaneous bolus injections of CNP-38 versus continuous subcutaneous infusion on animal growth. 21-22-day-old wild-type FVB male mice (n = 9 / group) were administered 50 nmol / kg / day CNP-38 or vehicle (30 mM acetate, pH 4, containing 5% sucrose and 1% benzyl alcohol) by either daily subcutaneous bolus injection or continuous subcutaneous infusion in the scapular region for 35 days. Continuous infusion was administered via an Alzet osmotic pump, Model 1002, for weeks 1-2, followed by a Model 1004 pump for weeks 3-5. The CNP-38 concentration in the pump was adjusted to the average animal weight on study day 7 (Pump Model 1002) and study day 25 (Pump Model 1004). Growth was assessed on day 35 by total body length measurement and radiographic measurements of the right femur and tibia.

[0616] Results for animals treated by daily subcutaneous bolus injection: On day 35, compared with vehicle-treated animals, CNP-38-treated animals had an overall length of 110.2%, right femur length of 105.6%, and right tibia length of 104.0%.

[0617] Results for animals treated by continuous subcutaneous infusion: On day 35, compared with vehicle-treated animals, CNP-38-treated animals had a total length of 121.7%, a right femur length of 107.5%, and a right tibia length of 112.2%.

[0618] We conclude that continuous subcutaneous infusion of CNP-38 or related sustained-release formulations (e.g., sustained-release CNP-38 prodrugs) is more effective than daily subcutaneous bolus injections in inducing limb and axial skeletal growth.

[0619] [Example 24] Pharmacokinetic study of 12g of a permanent Lys26 CNP-38 PEG4x10kDa conjugate in cynomolgus monkeys This study was conducted to demonstrate the suitability of 12g as a model compound for sustained-release CNP-38 prodrugs in cynomolgus monkeys. Male cynomolgus monkeys (2-4 years old, 3.5-4.1 kg) received either a single intravenous dose (n = 3) or a single subcutaneous dose (n = 2) of 12g at a dose of 0.146 mg CNP-38 equivalents / kg. Blood samples were collected up to 168 hours post-dose to generate plasma. Plasma CNP-38 concentrations were determined by quantification of the N-terminal signature peptide (sequence: LQEHPNAR) and C-terminal signature peptide (sequence: IGSMSGLGC) after trypsin digestion as described in Materials and Methods.

[0620] Results: Dose administration was well tolerated, with no signs of discomfort during or after administration. No administration site reactions were observed at any time point throughout the study. After intravenous injection, CNP-38 t max After subcutaneous injection, CNP-38 concentrations were observed to be approximately 0.25% of the 48-hour t max At 168 hours, CNP-38 concentrations were still at c max The bioavailability was approximately 50%.

[0621] Similar PK curves were obtained for the N- and C-terminal signature peptides up to 168 hours after administration, indicating the presence of intact CNP-38 in the conjugate.

[0622] The favorable, long-lasting PK and stability of CNP-38 in the conjugate over several days demonstrates the suitability of the permanent model compound Lys26 CNP-38 PEG 4x10 kDa conjugate 12g as a sustained-release CNP-38 prodrug after subcutaneous injection. It can be concluded that similar conjugates with CNP-38 temporarily linked to Lys26 (e.g., 11i) are suitable CNP-38 prodrugs that provide long-lasting, bioactive CNP-38 release levels over several days.

[0623] [Example 25] Pharmacokinetic study of transient Lys26 CNP-38 PEG4x10kDa conjugate 11i in cynomolgus monkeys This study is conducted to demonstrate the suitability of 11i as a sustained-release CNP-38 prodrug in cynomolgus monkeys. This study is conducted as described in Example 24. The plasma levels of total CNP-38 content (conjugated CNP-38 and released CNP-38) are analyzed as described in Example 24. To analyze the plasma content of free CNP-38, blood samples must be acidified after collection (e.g., by adding 20% ​​by volume of 0.5 M sodium citrate buffer, pH 4) to stop further CNP-38 release from the conjugate. The free CNP-38 level in plasma can be determined, for example, by ELISA using a CNP antibody that binds to the ring region of CNP, as described in the literature (U.S. Patent No. 8,377,884 B2), or by LC-MS / MS.

[0624] [Example 26] Pharmacodynamic Study of Transient Lys26 CNP-38 PEG4x10kDa Conjugate 11i in Cynomolgus Monkeys The effects of weekly treatment with transient Lys26 CNP-38 PEG4x10kDa conjugate 11i on bone growth and bone growth-related biomarker levels are evaluated in cynomolgus monkeys. Eight normal male juvenile cynomolgus monkeys (approximately 2 years old at the start of the study) are subcutaneously injected once weekly with 16 or 56 nmol / kg / week. Four such monkeys receive subcutaneous injections of CNP-38 at a daily dose of 8 nmol / kg / day, resulting in a cumulative weekly dose of 56 nmol / kg / week. Four additional monkeys receive vehicle as controls. The total duration of treatment is 6 months. Various measurements of growth plate elongation and bone growth are performed by digital X-ray and magnetic resonance imaging, as well as by external limb and body length measurements. Blood and urine samples will be collected periodically for clinical pathology examination and measurements. At the end of the study, gross pathology will be performed and tissue samples will be evaluated histologically for efficacy and safety assessments.

[0625] Abbreviation: ACH Achondroplasia ACN Acetonitrile AcOH acetic acid Bn Benzyl Boc tert-butyloxycarbonyl BSA Bovine serum albumin cGMP cyclic guanosine monophosphate CNP C-type natriuretic peptide COMU (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate conc. concentration d day DBU 1,3-diazabicyclo[5.4.0]undecene DCC N,N'-dicyclohexylcarbodiimide DCM dichloromethane DIC N,N'-diisopropylcarbodiimide DIEA N,N-Diisopropylethylamine DIPEA N,N-Diisopropylethylamine DMAP dimethylamino-pyridine DMEM Dulbecco's Modified Eagle's Medium Dmb 2,4-dimethylbenzyl DMEM Dulbecco's Modified Eagle's Medium DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DTT Dithiothreitol EC50 half-maximal effective concentration EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA Ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay eq stoichiometric equivalent ESI-MS Electrospray Ionization Mass Spectrometry Et Ethyl EtOAc ethyl acetate EtOH ethanol FBS Fetal Bovine Serum FGFR3 fibroblast growth factor receptor 3 Fmoc 9-Fluorenylmethyloxycarbonyl h time HATU M(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HCH cartilage hypoplasia HFIP Hexafluoroisopropanol HPLC High Performance Liquid Chromatography HOBt N-hydroxybenzotriazole IBMX 3-isobutyl-1-methylxanthine iPrOH 2-propanol iv intravenous ivDde 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl LC liquid chromatography LTQ Linear Trap Quadrupole Mal 3-Maleimidopropyl Me methyl MeOH Methanol min Mmt Monomethoxytrityl MS mass spectrometry / mass spectrometry MSA methanesulfonic acid MW molecular weight m / z mass-to-charge ratio NEP Neutral endopeptidase NHS N-hydroxysuccinimide NPR natriuretic peptide receptor OtBu tert-butyloxy PBS Phosphate Buffered Saline PEG Poly(ethylene glycol) PFP Pentafluorophenol pH Hydrogen ion potential Pr Propyl PyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Q-TOF quadrupole time-of-flight RP-HPLC Reversed-phase high-performance liquid chromatography rpm Revolutions per minute rt room temperature SIM Single Ion Monitoring SEC size exclusion chromatography sc subcutaneous t 1 / 2 Half-life TCEP Tris(2-carboxyethyl)phosphine TCP Trityl Chloride Polystyrene TD Thanatophoric dysplasia TES Triethylsilane TFA trifluoroacetic acid THF tetrahydrofuran TIPS Triisopropylsilane TMEDA N,N,N',N'-Tetramethylethylenediamine Tmob 2,4,6-trimethoxybenzyl TR-FRET Time-resolved fluorescence energy transfer Trt triphenylmethyl, trityl UPLC Ultra High Performance Liquid Chromatography UV ultraviolet light vs. ZQ Single Quadrupole

Claims

1. Formula (Ia) or (Ib) 【Chemistry 1】 (In the formula, -D is a CNP substructure, -L 1 - is a reversible prodrug linker moiety, -L 2 - is a single chemical bond or spacer moiety; -Z is a water-soluble carrier moiety; x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; y is an integer selected from the group consisting of 1, 2, 3, 4 and 5. or a pharmaceutically acceptable salt thereof.

2. Conjugate DL (In the formula, -D is a CNP substructure, -L is a reversible prodrug linker moiety -L 1 - includes -L 1 -L 2 -Z', optionally further substituted; -L 2 - is a single chemical bond or spacer moiety; -Z' is a water-insoluble carrier moiety. or a pharmaceutically acceptable salt thereof.

3. 3. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 2, wherein -Z' is a hydrogel.

4. 2. The CNP prodrug of claim 1 having the formula (Ia) or a pharmaceutically acceptable salt thereof.

5. 5. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1 or 4, wherein x is 1.

6. 6. The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the CNP partial structure has the sequence of SEQ ID NO:25 or SEQ ID NO:

24.

7. 7. The CNP prodrug of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein the CNP partial structure has the sequence of SEQ ID NO:

24.

8. -L 1 8. The CNP prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein - is conjugated to the side chain of an amino acid residue of the ring substructure of -D or to the backbone of the ring substructure of -D.

9. -L 1 9. The CNP prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein - is conjugated to the side chain of an amino acid residue of the ring substructure of -D selected from the group consisting of histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid, and arginine.

10. -D has the sequence SEQ ID NO: 24, and -L 1 10. The CNP prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein - is conjugated to the lysine at position 26 of -D.

11. Substructure-L 1 - is a group represented by the formula (II) 【Chemistry 2】 (In the formula, The dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; -X- is -C(R 4 R 4a )-, -N(R 4 )-, -O-, -C(R 4 R 4a )-C(R 5 R 5a )-, -C(R 5 R 5a )-C(R 4 R 4a )-, -C(R 4 R 4a )-N(R 6 )-, -N(R 6 )-C(R 4 R 4a )-, -C(R 4 R 4a )-O-, -OC(R 4 R 4a )- or -C(R 7 R 7a )- and X 1 is C or S(O), -X 2 - is -C(R 8 R 8a )- or -C(R 8 R 8a )-C(R 9 R 9a )- and =X 3 is =O, =S, or =N-CN, -R 1 , -R 1a , -R 2 , -R 2a , -R 4 , -R 4a , -R 5 , -R 5a , -R 6 , -R 8 , -R 8a , -R 9 , -R 9a are independently -H and C 1-6 is selected from the group consisting of alkyl, -R 3 , -R 3a are independently -H and C 1-6 alkyl, with the proviso that -R 3 , -R 3a If one or both of are other than -H, they may be attached to the N to which they are attached by SP 3 are linked by hybridized carbon atoms, -R 7 is -N(R 10 R 10a ), or -NR 10 -(C=O)-R 11 and -R 7a , -R 10 , -R 10a , -R 11 are, independently of each other, -H or C 1-6 is alkyl, In some cases, Pair-R 1a / -R 4a , -R 1a / -R 5a , -R 1a / -R 7a , -R 4a / -R 5a , -R 8a / -R 9a one or more of the following forms a chemical bond; In some cases, Pair-R 1 / -R 1a , -R 2 / -R 2a , -R 4 / -R 4a , -R 5 / -R 5a , -R 8 / -R 8a , -R 9 / -R 9a One or more of the following, together with the atoms to which they are attached, form a C 3-10 forming a cycloalkyl or a 3- to 10-membered heterocyclyl, In some cases, Pair-R 1 / -R 4 , -R 1 / -R 5 , -R 1 / -R 6 , -R 1 / -R 7a , -R 4 / -R 5 , -R 4 / -R 6 , -R 8 / -R 9 , -R 2 / -R 3 together with the atoms to which they are attached form ring A, In some cases, R 3 / R 3a together with the nitrogen atom to which they are attached form a 3- to 10-membered heterocyclic ring, A is phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; -L 1 -ga, -L 2 -Z or -L 2 -Z' and optionally -L 1 - is further substituted, provided that the hydrogen with an asterisk in formula (II) is -L 2 -Z or -L 2 is not replaced by -Z' or a substituent, -L 2 - is a single chemical bond or spacer, -Z is a water-soluble carrier; The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein -Z' is a water-insoluble carrier.

12. -X- is -C(R 4 R 4a )- or -N(R 4 12. The CNP prodrug of claim 11, or a pharmaceutically acceptable salt thereof, wherein:

13. -R 4 But -L 2 -Z or -L 2 13. The CNP prodrug of claim 11 or 12, or a pharmaceutically acceptable salt thereof, substituted with -Z'.

14. X 1 The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 13, wherein is C.

15. =X 3 The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 14, wherein is =O.

16. -X 2 -, -C(R 8 R 8a 16. The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 15, wherein:

17. -R 1 and -R 1a The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 16, wherein is -H.

18. -R 2 and -R 2a The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 17, wherein is -H.

19. -R 3 is -H and -R 3a The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 18, wherein is methyl.

20. -R 4 and -R 4a The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 19, wherein is -H.

21. -R 8 and -R 8a The CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 20, wherein is -H.

22. -L 2 - is -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-, -S(O) 2 N(R y1 )-, -S(O)N(R y1 )-, -S(O) 2 -, -S(O)-;-N(R y1 )S(O) 2 N(R y1a )-, -S-, -N(R y1 )-, -OC(OR y1 )(R y1a )-, -N(R y1 )C(O)N(R y1a )-, -OC(O)N(R y1 )-, C 1-50 Alkyl, C 2-50 Alkenyl, and C 2-50 alkynyl, wherein -T-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R y2 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-, -S(O) 2 N(R y3 )-, -S(O)N(R y3 )-, -S(O) 2 -, -S(O)-, -N(R y3 )S(O) 2 N(R y3a )-, -S-, -N(R y3 )-, -OC(OR y3 )(R y3a )-, -N(R y3 )C(O)N(R y3a )- and -OC(O)N(R y3 )-, optionally interrupted by one or more groups selected from the group consisting of -R y1 and -R y1a However, independently of each other, -H, -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, -T, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl may be one or more of the same or different -Ry 2 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y4 )-, -S(O) 2 N(R y4 )-, -S(O)N(R y4 )-, -S(O) 2 -, -S(O)-, -N(R y4 )S(O) 2 N(R y4a )-, -S-, -N(R y4 )-, -OC(OR y4 )(R y4a )-, -N(R y4 )C(O)N(R y4a )- and -OC(O)N(R y4 )-, optionally interrupted by one or more groups selected from the group consisting of Each T is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; each T is independently selected from the group consisting of one or more -R y2 and optionally substituted with Each R y2 are independently halogen, -CN, oxo(=O), -COOR y5 , -OR y5 , -C(O)R y5 , -C(O)N(R y5 R y5a ), -S(O) 2 N(R y5 R y5a ), -S(O)N(R y5 R y5a ), -S(O) 2 R y5 , -S(O)R y5 , -N(R y5 )S(O) 2 N(R y5a R y5b ), -SR y5 , -N(R y5 R y5a ), -NO 2 , -OC(O)R y5 , -N(R y5 )C(O)R y5a , -N(R y5 )S(O) 2 R y5a , -N(R y5 )S(O)R y5a , -N(R y5 )C(O)OR y5a , -N(R y5 )C(O)N(R y5a R y5b ), -OC(O)N(R y5 R y5a ), and C 1-6 alkyl, 1-6 alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R y3 , -R y3a , -R y4 , -R y4a , -R y5 , -R y5a and -R y5b independently, -H and C 1-6 alkyl, C 1-6 22. The CNP prodrug of any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof, wherein the alkyl is optionally substituted with one or more halogens, which may be the same or different.

23. -L 2 - is -O-, -T- and -C(O)N(R y1 C optionally interrupted by one or more groups independently selected from 1-20 is an alkyl chain, 1-20 The alkyl chains are -OH, -T and -C(O)N(R y6 R y6a ) optionally substituted with one or more groups independently selected from -R y1 , -R y6 , -R y6a but independently, H and C 1-4 alkyl, T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 23. The CNP prodrug of any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, selected from the group consisting of cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl.

24. -L 2 - is a formula (i) 【Transformation 3】 (In the formula, A dashed line with an asterisk indicates -L 1 - indicates a bond to An unmarked dashed line indicates a bond to -Z or -Z'; -R 1 -H, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 alkynyl, n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. and 24. The CNP prodrug or pharmaceutically acceptable salt thereof according to any one of claims 1 to 23, wherein the substructure of formula (i) is optionally further substituted.

25. 25. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1 or 4 to 24, wherein -Z has a molecular weight in the range of 5 to 200 kDa.

26. Carrier-Z is C 8-24 26. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1 or 4 to 25, comprising an alkyl, or polymer.

27. -Z is 2-methacryloyl-oxyethyl phosphorylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl-oxazoline), poly(hydroxymethacrylate), poly(hydroxypropyl methacrylamide), poly(hydroxypropyl methacrylate), poly(hydroxypropyl oxazoline), poly(iminocarbonate), 27. The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1 or 4 to 26, comprising a polymer selected from the group consisting of cellulose, poly(lactic acid), poly(lactic-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(propylene glycol), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, cellulose, carbomethylcellulose, hydroxypropylmethylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate-based polymers, xylan, and copolymers thereof.

28. 28. The CNP prodrug or pharmaceutically acceptable salt thereof of any one of claims 1 or 4 to 27, wherein -Z is a branched polymer.

29. 29. The CNP prodrug or pharmaceutically acceptable salt thereof of any one of claims 1 or 4 to 28, wherein -Z has a molecular weight of at least 10 kDa.

30. -Z or -Z' is a partial structure 【Chemistry 4】 30. The CNP prodrug of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, comprising:

31. -Z is a partial structure of formula (a) 【Transformation 5】 (In the formula, The dashed line is -L 2 represents the bond to - or to the remainder of -Z, BP a is a branch point selected from the group consisting of -N<, -CR< and >C<; -R is -H and C 1-6 is selected from the group consisting of alkyl, a is BP a is 0 when -N< or -CR<, and n is BP a is 1 if >C<, -S a -, -S a' -, -S a'' -and-S a''' - are, independently of each other, a chemical bond or C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R 1 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 2 )-, -S(O) 2 N(R 2 )-, -S(O)N(R 2 )-, -S(O) 2 -, -S(O)-, -N(R 2 )S(O) 2 N(R 2a )-, -S-, -N(R 2 )-, -OC(OR 2 )(R 2a )-, -N(R 2 )C(O)N(R 2a )- and -OC(O)N(R 2 )-, optionally interrupted by one or more groups selected from the group consisting of Each -T- is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; each -T- is independently selected from the group consisting of one or more -R 1 and optionally substituted with Each-R 1 are independently halogen, -CN, oxo(=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O) 2 N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O) 2 R 3 , -S(O)R 3 , -N(R 3 )S(O) 2 N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO 2 , -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O) 2 R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 2 , -R 2a , -R 3 , -R 3a and -R 3b are independently -H and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -P a' , -P a'' and -P a''' are independently polymer substructures) 31. The CNP prodrug of any one of claims 1 or 4 to 30, or a pharmaceutically acceptable salt thereof, comprising:

32. -Z is the formula (d) 【Transformation 6】 (In the formula, The dashed line is -L 2 - indicates a bond to -Z b -C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R 1 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 2 )-, -S(O) 2 N(R 2 )-, -S(O)N(R 2 )-, -S(O) 2 -, -S(O)-, -N(R 2 )S(O) 2 N(R 2a )-, -S-, -N(R 2 )-, -OC(OR 2 )(R 2a )-, -N(R 2 )C(O)N(R 2a )- and -OC(O)N(R 2 )-, each -T- is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; each -T- is independently selected from the group consisting of one or more -R 1 and optionally substituted with Each-R 1 are independently halogen, -CN, oxo(=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O) 2 N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O) 2 R 3 , -S(O)R 3 , -N(R 3 )S(O) 2 N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO 2 , -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O) 2 R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 2 , -R 2a , -R 3 , -R 3a and -R 3b are independently -H and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -Z a teeth, 【Transformation 7】 where BP a , -S a -, -S a' -, -S a'' -, -S a''' -, -P a' , -P a'' , -P a''' and a are used as defined in claim 31) 32. The CNP prodrug of claim 1 or any one of claims 4 to 31, or a pharmaceutically acceptable salt thereof, having the formula:

33. -Z is a group represented by the formula (e) 【Transformation 8】 (In the formula, The dashed line is -L 2 - indicates a bond to e is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; -Z a teeth, 【Chemistry 9】 where: b1 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7 and 8; b2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8; b3 is an integer ranging from 150 to 1000; and b4 is an integer between 150 and 1000) 33. The CNP prodrug of claim 1 or any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, having the formula:

34. 34. The CNP prodrug of claim 33, or a pharmaceutically acceptable salt thereof, wherein e is 5, b1 is 2, b2 is 3, and b3 and b4 are both about 450.

35. -Z is a group represented by the formula (f) 【Chemistry 10】 (In the formula, The dashed line is -L 2 - indicates a bond to BP f is a branch point selected from the group consisting of -N<, -CR< and >C<; -R is -H and C 1-6 is selected from the group consisting of alkyl, f is BP f is 0 when -N< or -CR<, and f is BP f is 1 if >C<, -S f -, -S f' -, -S f'' -and-S f''' - is independently a chemical bond or C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R 1 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 2 )-, -S(O) 2 N(R 2 )-, -S(O)N(R 2 )-, -S(O) 2 -, -S(O)-, -N(R 2 )S(O) 2 N(R 2a )-, -S-, -N(R 2 )-, -OC(OR 2 )(R 2a )-, -N(R 2 )C(O)N(R 2a )- and -OC(O)N(R 2 )-, optionally interrupted by one or more groups selected from the group consisting of Each -T- is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; each -T- is independently selected from the group consisting of one or more -R 1 and optionally substituted with Each R 1 are independently halogen, -CN, oxo(=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O) 2 N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O) 2 R 3 , -S(O)R 3 , -N(R 3 )S(O) 2 N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO 2 , -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O) 2 R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 2 , -R 2a , -R 3 , -R 3a and -R 3b are independently -H and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -Z a' , -Z a'' and -Z a''' is, independently, 【Chemistry 11】 where BP a , -S a -, -S a' -, -S a'' -, -S a''' -, -P a' , -P a'' , -P a''' and a are used as defined in claim 31) 32. The CNP prodrug of claim 1 or any one of claims 4 to 31, or a pharmaceutically acceptable salt thereof, having the formula:

36. -Z is a group represented by the formula (g) 【Chemistry 12】 (In the formula, The dashed line is -L 2 - indicates a bond to -S g -, -S g' -and-S g'' independently, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 alkynyl, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl may be one or more -R 1 optionally substituted with C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl includes -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 2 )-, -S(O) 2 N(R 2 )-, -S(O)N(R 2 )-, -S(O) 2 -, -S(O)-, -N(R 2 )S(O) 2 N(R 2a )-, -S-, -N(R 2 )-, -OC(OR 2 )(R 2a )-, -N(R 2 )C(O)N(R 2a )- and -OC(O)N(R 2 )-, optionally interrupted by one or more groups selected from the group consisting of Each -T- is independently phenyl, naphthyl, indenyl, indanyl, tetralinyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; each -T- is independently selected from the group consisting of one or more -R 1 and optionally substituted with Each R 1 are independently halogen, -CN, oxo(=O), -COOR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 R 3a ), -S(O) 2 N(R 3 R 3a ), -S(O)N(R 3 R 3a ), -S(O) 2 R 3 , -S(O)R 3 , -N(R 3 )S(O) 2 N(R 3a R 3b ), -SR 3 , -N(R 3 R 3a ), -NO 2 , -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O) 2 R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a R 3b ), -OC(O)N(R 3 R 3a ), and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; Each-R 2 , -R 2a , -R 3 , -R 3a and -R 3b are independently -H and C 1-6 alkyl, C 1-6 the alkyl is optionally substituted with one or more halogens which may be the same or different; -Z a and -Z a' is, independently, 【Chemistry 13】 where BP a , -S a -, -S a' -, -S a'' -, -S a''' -, -P a' , -P a'' , -P a''' and a are used as defined in claim 31) 36. The CNP prodrug of any one of claims 1, 4-31, or 35, or a pharmaceutically acceptable salt thereof, having the formula:

37. -Z is a group represented by the formula (h) 【Chemistry 14】 (In the formula, The dashed line is -L 2 - indicates a bond to Each-Z c is a substructure 【Chemistry 15】 wherein each c1 is independently an integer ranging from about 200 to 250.

37. The CNP prodrug of any one of claims 1, 4-31, 35 or 36, or a pharmaceutically acceptable salt thereof, having the formula:

38. Formula (IIe) 【Chemistry 16】 (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; Dashed lines with asterisks represent substructures: 【Chemistry 17】 indicates a bond to each c1 is independently an integer ranging from about 400 to 500 35. The CNP prodrug of claim 1 or any one of claims 4 to 34, or a pharmaceutically acceptable salt thereof, having the formula:

39. Formula (IIe') [Chemistry 18] (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; Dashed lines with asterisks represent substructures: 【Chemistry 19】 indicates a bond to each c1 is independently an integer ranging from about 400 to 500 39. The CNP prodrug of any one of claims 1, 4-34, or 38, or a pharmaceutically acceptable salt thereof, having the formula:

40. Formula (IIe-i') 【Chemistry 20】 (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; Dashed lines with asterisks represent substructures: 【Chemistry 21】 indicates a bond to each c1 is independently an integer ranging from about 400 to 500 35. The CNP prodrug of claim 1, or a pharmaceutically acceptable salt thereof, having the formula:

41. Formula (IIe-ii') 【Chemistry 22】 (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; Dashed lines with asterisks represent substructures: 【Chemistry 23】 indicates a bond to each c1 is independently an integer ranging from about 400 to 500 35. The CNP prodrug of claim 1, or a pharmaceutically acceptable salt thereof, having the formula:

42. Expression (IIf) 【Chemistry 24】 (In the formula, The unmarked dashed line indicates the attachment of the CNP moiety -D to the nitrogen by forming an amide bond; The dashed lines with asterisks indicate the structure: 【Chemistry 25】 represents a bond to -Z having the formula Each-Z a teeth, 【Chemistry 26】 wherein each c1 is independently an integer ranging from about 200 to 250.

38. The CNP prodrug of any one of claims 1, 4 to 31, or 35 to 37, or a pharmaceutically acceptable salt thereof, having the formula:

43. Expression (IIf') 【Chemistry 27】 (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; The dashed lines with asterisks indicate the structure: 【Chemistry 28】 represents a bond to -Z having the formula each Z a teeth, 【Chemistry 29】 wherein each c1 is independently an integer ranging from about 200 to 250.

43. The CNP prodrug of any one of claims 1, 4 to 31, 35 to 37, or 42, or a pharmaceutically acceptable salt thereof, having the formula:

44. Formula (IIf-i') 【Transformation 30】 (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; The dashed lines with asterisks indicate the structure: 【Chemistry 31】 represents a bond to -Z having the formula each Z a teeth, 【Chemistry 32】 wherein each c1 is independently an integer ranging from about 200 to 250.

38. The CNP prodrug of any one of claims 1, 4 to 31, or 35 to 37, or a pharmaceutically acceptable salt thereof, having the formula:

45. Formula (IIf-ii') 【Transformation 33】 (In the formula, The unmarked dashed line indicates attachment to the nitrogen provided by the side chain of the lysine at position 26 of the CNP substructure of SEQ ID NO: 24 by forming an amide bond; The dashed lines with asterisks indicate the structure: 【Transformation 34】 represents a bond to -Z, each Z a teeth, 【Chemistry 35】 wherein each c1 is independently an integer ranging from about 200 to 250.

38. The CNP prodrug of any one of claims 1, 4 to 31, or 35 to 37, or a pharmaceutically acceptable salt thereof, having the formula:

46. 46. ​​The prodrug or pharmaceutically acceptable salt thereof of any one of claims 1 to 45, wherein the residual activity of the CNP prodrug is less than 10%.

47. 47. A pharmaceutical composition comprising at least one CNP prodrug of any one of claims 1 to 46, or a pharmaceutically acceptable salt thereof, and at least one excipient.

48. 48. The pharmaceutical composition of claim 47, having a pH in the range of pH 4 to pH 6.

49. 49. Use of the prodrug according to any one of claims 1 to 46 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 47 or 48, as a medicament.

50. Use of the prodrug of any one of claims 1 to 46 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 47 or 48 in a method for treating a disease that can be treated with CNP.

51. The diseases include achondroplasia, hypochondroplasia, short stature, dwarfism, chondro-osseous dysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, kyphotic dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, proximal short-limbed chondrodysplasia punctata, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia , osteogenesis imperfecta, diastrophic dysplasia, congenital femoral brachyosteosis, Langer type metapodial dysplasia, Niebergelt type metapodial dysplasia, Robinow syndrome, Reinhardt syndrome, acroostosis imperfecta, peripheral osteogenesis imperfecta, Kniest dysplasia, fibrochondromatosis, Roberts syndrome, distal metapodial dysplasia, brachymelia, Morquio syndrome, Kniest syndrome, metamorphic dysplasia, vertebral epiphyseal diaphysis 51. The use of claim 50, wherein the condition is selected from the group consisting of acromyosplasia, neurofibromatosis, Legius syndrome, Leopard syndrome, Noonan syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Legius syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, cleidocranial dysostosis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzon-cutaneous-skeletal syndrome), digital abnormalities, brachydactyly, camptodactyly, polydactyly, syndactyly, segmental dysplasia, enchondrosis, fibrous dysplasia, hereditary multiple osteochondromas, hypophosphatemic rickets, Jaffe-Liechtenstein syndrome, Marfan syndrome, McCune-Albright syndrome, osteopetrosis, and bone poikilosis.

52. 51. The use according to claim 50, wherein the disease is an ophthalmic disease.

53. 52. The use according to claim 50 or 51, wherein the disease is achondroplasia.

54. Use of a CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 46, or a pharmaceutical composition according to claim 47 or 48, for the manufacture of a medicament for treating a disease that can be treated with CNP.

55. The diseases include achondroplasia, hypochondroplasia, short stature, dwarfism, chondro-osteodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, homozygous achondroplasia, kyphotic dysplasia, congenital lethal hypophosphatasia, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, proximal short-limbed chondrodysplasia punctata, Jansen metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia syndrome, osteogenesis imperfecta, diastrophic dysplasia, congenital femoral brachyosteosis, Langer metapodial dysplasia, Niebergelt metapodial dysplasia, Robinow syndrome, Reinhardt syndrome, acroostosis imperfecta, peripheral osteogenesis imperfecta, Kniest dysplasia, fibrochondromatosis, Roberts syndrome, distal metapodial dysplasia, brachymelia, Morquio syndrome, Kniest syndrome, metamorphic dysplasia, vertebral epiphyseal bone 55. The use of claim 54, wherein the condition is selected from the group consisting of metacarpophalangeal dysplasia, neurofibromatosis, Legius syndrome, Leopard syndrome, Noonan syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Legius syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, cleidocranial dysostosis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzon-cutaneous-skeletal syndrome), digital abnormalities, brachydactyly, camptodactyly, polydactyly, syndactyly, segmental dysplasia, enchondrosis, fibrous dysplasia, hereditary multiple osteochondromas, hypophosphatemic rickets, Jaffe-Liechtenstein syndrome, Marfan syndrome, McCune-Albright syndrome, osteopetrosis, and bone poikilosis.

56. 55. The use according to claim 54, wherein the disease is an ophthalmic disease.

57. 56. The use according to claim 54 or 55, wherein the disease is achondroplasia.

58. 49. A method of administering the CNP prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 46, or the pharmaceutical composition according to claim 47 or 48, comprising the step of administering the CNP prodrug or a pharmaceutically acceptable salt thereof by topical, enteral, or parenteral administration, or by topical, injection, or infusion methods including intra-articular, peri-articular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular, intraorbital, intravitreal, intratympanic, intravesical, intracardiac, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, intraventricular, or intrasternal injection and infusion, direct delivery to the brain by an implantable device enabling delivery of the invention to brain tissue or brain fluid, direct intraventricular injection or infusion, injection or infusion into the brain or brain-related regions, injection into the subchoroidal space, retro-orbital injection, or eye instillation.

59. 49. The CNP prodrug of any one of claims 1 to 46 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 47 or 48, for use in treating achondroplasia by subcutaneous injection.

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