METHOD OF TREATING SKELETAL MUSCLE STEM CELL PATHOLOGIC DYSFUNCTION

CROSS REFERENCE TO RELATED APPLICATIONS

This application is PCT application Serial No PCT/CA2023/0* filed on July 17, 2023 and published in English under PCT Article 21 (2), which itself claims benefit of U.S. provisional application Serial No. 63/368,535, filed on July 15, 2022. All documents above are incorporated herein in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N.A.

FIELD OF THE DISCLOSURE

The present disclosure relates to a method of treating a skeletal muscle stem cell (MuSC) pathologic dysfunction and impaired skeletal muscle regeneration. It is more particularly concerned with a method of treating pathologic MuSC dysfunctions and regenerative failure such as in muscular dystrophies.

REFERENCE TO SEQUENCE LISTING

Pursuant to 37 C.F.R. 1.821 (c), a sequence listing is submitted herewith as an ASCII compliant text file named G14692-00102.xml, that was created on July 17, 2023 and having a size of 195 kilobytes. The content of the aforementioned file named G14692-00102.xml is hereby incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

Muscular dystrophies (MDs) are a heterogeneous group of rare genetic diseases usually diagnosed in children and often characterized by skeletal muscle wasting and a progressive loss of ambulation. The heterogeneity of genetic defects causing MD represents a major challenge for the development and manufacturing of genome targeted therapeutics.

Present treatment options for MDs are limited and no effective curative therapies exist. Several non-genetic approaches for the treatment of MD have been discovered (Gawlik, 2018). Most common are glucocorticoids, which stabilize muscle strength and prolong ambulation in Duchenne MD patients by reducing inflammation and fibrosis (McDonald et al., 2018). In addition, drugs that modulate autophagy, reduce oxidative stress, and boost mitochondrial function have been shown to improve myofiber survival and thereby slow disease progression (Gawlik, 2018). In preclinical models of certain forms of congenital MD, anti-apoptotic agents have shown beneficial effects and underwent clinical trials (Girgenrath et al., 2009; Erb et al., 2009).

Many recent clinical trials for experimental treatments have not met all endpoints. Further, emerging treatments are applicable to small fractions of patients presenting specific mutations.

There is a need for less-personalized, more mutation-independent treatment methods targeting common pathological features of MDs. The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE DISCLOSURE

The present disclosure shows that defects in the endothelial cell (EC) compartment of the perivascular stem cell niche in three different types of MD are associated with inefficient mobilization of MuSCs following tissue damage. The 13 amino acid form of the peptidic hormone apelin (AP-13), ELABELA (Epiboly LAte Because of Endoderm Late), both ligands of the apelin receptor (APJ), and derivatives thereof were identified as being able to stimulate skeletal muscle ECs and thereby restore MuSC function and markedly slow disease progression. In all tested dystrophic mice models, administration of AP-13 generated a pro-myogenic EC-rich niche that supports MuSC function and markedly improves tissue regeneration, muscle strength, and physical performance. Moreover, EC specific knockout of the AP-13 receptor leads to regenerative defects that phenocopy major pathological features of MD.

The present disclosure provides in-vivo evidence of enhancing endogenous repair by targeting the perivascular niche through through apelinergic signaling for treating MD.

More specifically, in accordance with the present disclosure, there is provided a method of preventing or treating a muscle stem cell (MuSC) pathologic disfunction and/or impaired skeletal muscle regeneration in a subject in need thereof, comprising administering a therapeutically effective amount of a modulator of an apelin receptor. In a specific embodiment the MuSC pathologic disfunction is an MD. There is also provided a method of preventing or treating an MD with a direct stimulator or indirect stimulator (e.g., through ECs stimulation) of MuSC function.

In accordance with another aspect of the present disclosure, there are provided the following items:

Item 1 . A method of preventing or treating muscular dystrophy in a subject in need thereof comprising administering a therapeutically effective amount of a stimulator of muscle stem cell (MuSC) function to the subject.

Item 2. The method of item 1, wherein the stimulator of MuSC function is a stimulator of skeletal muscle endothelial cell function.

Item 3. The method of item 1 , wherein the stimulator is apelin-13, or a structural or functional analogue thereof.

Item 4. The method of item 3, wherein the stimulator is a compound of any one of formula (l)-(XV) and (Illa).

Item 5. The method of item 3, wherein the stimulator is a compound of any one of formula (I ll)-(XV) and (Illa) (e.g., any one of AM03-68, AM02-123, KT01-129 and KT03-69).

Item 6. The method of item 3, wherein the stimulator is a compound of any one of formula (I) and (lll)-(XI).

Item 7. A pharmaceutical composition comprising (a) a stimulator of muscle stem cell (MuSC) function and (b) (i) a pharmaceutically acceptable carrier; and/or (ii) another agent for preventing or treating muscular dystrophy.

Item 8. The composition of item 7, wherein the stimulator is as defined in any one of items 2 to 6.

Item 9. The composition of item 7 or 8, comprising another agent for preventing or treating muscular dystrophy. Item 10. A kit for preventing or treating a muscular dystrophy in a subject in need thereof comprising (A) a stimulator of muscle stem cell (MuSC) function; and (B) (i) another agent for preventing or treating the muscular dystrophy; (ii) instructions for using the kit in the prevention or treatment of muscular dystrophy in a subject in need thereof; or (ii) a combination of (i) and (ii).

Item 11 . The kit of item 10, wherein the stimulator is as defined in any one of items 2 to 6.

Item 12. The kit of item 10 or 11 , comprising another agent for preventing or treating muscular dystrophy.

There is also provided a method preventing or of treating impaired skeletal muscle regeneration in a subject in need thereof using a modulator of an apelin receptor, direct stimulator or indirect stimulator (e.g., through ECs stimulation) of MuSC function or any compound or composition of the present disclosure.

Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIGs. 1A-Q: MD affects the proliferative capacity of MuSCs (FIG. 1A) Scheme outlining the MD models used herein and graphical overview of the experimental timeline, d 16 mice are a model for ColVI-related myopathy, mdx mice for Duchenne MD, and dyW mice for LAMA2 MD. C57BL/6 mice were used as background matched wild-type (wt) controls. FIG. 1 B, Representative haematoxylin and eosin-stained cross sections of the tibialis anterior (TA) muscle from wt controls (ctrl), mdx, d16 and dyW mice under uninjured (uninj.) and at 5- and 10-days post cardiotoxin injury (dpi). FIGs. 1C-D, Frequency distribution of minimal fiber feret classes in TA muscles of ctrl, mdx, d16 and dyW muscles under uninjured conditions (FIG. 1C) and at 10 dpi (FIG. 1 D). FIG. 1 E, Representative embryonic myosin heavy chain positive fibers (eMHC) co-stained with fibronectin in TA muscle sections from wt ctrl, mdx, d 16 and dyW mice under uninjured (uninj.) and at 5 and 10 dpi FIGs. 1 F-H and FIGs. J-L, quantification of eMHC and fibronectin area in TA muscle sections of ctrl, mdx, d 16 and dyW mice under uninjured conditions (FIGs. 1 F and J, respectively), and at 5 (FIGs. 1G and K, respectively) and 10 (FIGs. 1 H and L, respectively) dpi. Kinetics of eMHC positive fibers (FIG. 11) and fibronectin expression (FIG. 1 M) over the regenerative time course FIGs. 1 N-P: Quantification of Pax7 positive cells in TA muscle sections in ctrl, mdx, d16 and dyW mice under uninjured conditions (FIG. 1 N), and at 5 (FIG. 10) and 10 dpi (FIG. 1 P). Kinetics of Pax7 positive cells over the regenerative time course (FIG. 1Q). Results are expressed as means + sem. n > 3 mice per condition. Scale bars = 50 pm (b) and 100 pm (e and n). P values were calculated using one and two-way ANOVA with Tukey’s post-hoc test. Statistical symbols: * means dyw group differs from the control group; # means mdx group differs from the control group; & means d 16 group differs from the control group. 1 symbol P<0.05, 2 symbols P<0.01 , 3 symbols P<0.001 , 4 symbols P<0.0001.

FIGs. 2A-L: MD affects microvascular remodeling. (FIGs. 2A-C) Immunostaining and quantification of Pdgfra positive cells in TA muscle sections in ctrl, mdx, d 16 and dyW mice under uninjured conditions (FIG. 2A), and at 5 (FIG. 2B) and 10 dpi (FIG. 2C). (FIG. 2D) Kinetics of Pdgfra positive cells over the regenerative time course. (FIGs. 2E-G) Immunostaining and quantification of F4/80 positive cells in TA muscle sections in Ctrl, mdx, d16 and dyW mice under uninjured conditions (FIG. 2E), and at 5 (FIG. 2F) and 10 dpi (FIG. 2G). (FIG. 2H) Kinetics of F4/80 positive cells over the regenerative time course. (FIGs. 2I-K) Immunostaining and quantification of CD31 positive cells in TA muscle sections in Ctrl, mdx, d16 and dyW mice under uninjured conditions (FIG. 2I), and at 5 (FIG. 2J) and 10 dpi (FIG. 2K). (FIG. 2L) Kinetics of CD31 positive cells over the regenerative time course. Results are expressed as means + sem. n > 3 mice per condition. Scale bars 100 pm. P values were calculated using one and two-way ANOVA with Tukey’s post-hoc test. Statistical symbols: * means dyw group differs from the control group; # means mdx group differs from the control group; & means d16 group differs from the control group. 1 symbol P<0.05, 2 symbols P<0.01 , 3 symbols P<0.001, 4 symbols P<0.0001.

FIGs. 3A-J: Heat map of the top 20 most expressed GPCRs in ECs under uninjured conditions (FIG. 3A) and at 5 dpi (FIG. 3B) and their cognate ligands based on single cell transcriptomics of TA muscles. UMI = Unique molecular identifiers. (FIGs. 3C-D) Immunostaining and quantification of APJ levels co-stained with CD31 , Pax7, F4/80, and PDGFRa in TA muscle cross sections of wt mice at 5 dpi. (FIGs. 3E-F) Chemical structure (FIG. 3E) and amino acid sequence (SEQ ID NO: 159) of the pyr-apelin-13 peptide (AP-13) (FIG. 3F). (FIGs. 3G-I) Proliferation of ECs (FIG. 3G) and MuSC derived myoblasts (FIG. 3H) in response to increasing concentrations of AP-13. (FIG. 3I) Proliferation of ECs (Human Umbilical Vein Endothelial Cells (HUVEC)) in response to AM02-123 and KT03-69. (FIG. 3J) Total number of human skeletal muscle derived cells in response to increasing doses of AP-13, Elabela, AM03-68 and KT01-129. Results are expressed as means + sem. n > 3 mice per condition. Scale bars = 100 pm. P values were calculated using one way ANOVA with Tukey’s post-hoc test (FIGs. 3G-H). Two-way ANOVA (FIG. 3J) or ANOVA with Bonferroni correction (FIG. 3I). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

FIG. 4: Synthesis of AP-13. Experimental scheme depicting the chemical synthesis of AP-13. rt = room temperature, Fmoc = 9-Fluorenylmethoxycarbonyl, DIPEA = A/./V-diisopropylethylamine, HATU = O-(7-Azabenzotriazol-1-yl)- A/,A/,A/',A/'-tetramethyluronium hexafluorophosphate, TFA = Trifluoroacetic acid, TIPS = triisopropylsilane, EDT = ethanedithiol;

FIGs. 5A-R: AP-13 stimulates the perivascular MuSC niche and improves endogenous repair in MD. (FIG. 5A) Scheme outlining the AP-13 treatment strategy of MD mice models. (FIG. 5B) Quantification of CD31 positive cells in TA muscle cross sections of dyW mice treated with vehicle (veh) or AP-13. (FIG. 5C) Quantification of Pdgfra positive cells in TA muscle cross sections of dyW mice treated with veh or AP-13. (FIG. 5D) Quantification of F4/80 positive cells in TA muscle cross sections of dyW mice treated with veh or AP-13. (FIG. 5E) Quantification of IgG positive fibers in TA muscle cross sections of dyW mice treated with veh or AP-13. (FIG. 5F) Quantification of the fibronectin positive area in TA muscle cross sections of dyW mice treated with veh or AP-13. (FIG. 5G) Quantification of Pax7 positive cells in TA muscle cross sections of dyW mice treated with veh or AP-13. (FIG. 5H) Quantification of Myogenin (MyoG) positive cells in TA muscle cross sections of dyW mice treated with veh or AP-13. (FIG. 5I) Quantification of eMHC positive fibers in TA muscle cross sections of dyW mice treated with veh or AP-13. (FIG. 5J) Western blot quantification of activated cleaved Casp-3 in TA muscle lysates of dyW mice treated with veh or AP-13. (FIGs. 5K-L) Quantification of CD31 positive cells in TA muscle cross sections of d16 mice under uninjured conditions (FIG. 5K), and at 5 dpi (FIG. 5L) treated with vehicle (veh) or AP-13. (FIGs. 5M-N) Quantification of CD31 positive cells in TA muscle cross sections of mdx mice under uninjured conditions (FIG. 5M), and at 5 dpi (FIG. 5N) treated with vehicle (veh) or AP-13. (FIGs. 5O-P) Quantification of Pax7 positive cells in TA muscle cross sections of d16 mice under uninjured conditions (FIG. 50), and at 5 dpi (FIG. 5P) treated with vehicle (veh) or AP-13. (FIGs. 5Q- R) Quantification of Pax7 positive cells in TA muscle cross sections of mdx mice under uninjured conditions (FIG. 5Q), and at 5 dpi (FIG. 5R) treated with vehicle (veh) or AP-13. Results are expressed as means + sem. n 2 3 mice per condition. Scale bars = 100 pm. P values were calculated using students t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

FIGs. 6A-K: Systemic AP-13 treatment slows disease progression in MD. (FIG. 6A) Body weight evolution of dyW mice over the veh or AP-13 treatment time-course. (FIG. 6B) Cumulative body weight gain of veh and AP-13 treated dyW mice. (FIG. 6C) Survival curve of veh or AP-13 treated dyW mice over the treatment time-course. (FIGs. 6D-E) Scheme outlining the treatment strategy of dyW mice with veh or AP-13 (FIG. 6D) and the set-up for ex-vivo and in situ muscle force measurements (FIG. 6E). (FIG. 6F) Quantification of normalized ex-vivo force of extensor digitorum longus muscles of dyW mice treated with veh or AP-13. Black lines designate littermates. (FIG. 6G) Quantification of normalized in situ isometric torque force of posterior muscles of the lower leg of dyW mice treated with veh or AP-13. Black lines designate littermates. (FIG. 6H) Scheme depicting the rotarod, single beam suspension, and grid suspension fitness tests. (FIGs. 6I-K) Quantification of the normalized mean impulse (time to task failure x body mass) of dyW mice treated with veh or AP-13 in the rotarod assay (FIG. 6I), single beam challenge (FIG. 6J) and horizontal grid test (FIG. 6K). Results are expressed as means + sem. n>8 (FIGs. 6A-C), n>3 (FIGs. 6F-G), and n>5 (FIGs. 6J-K) mice per condition. P values were calculated using students t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

FIGs. 7A-D: AP-13 does not cause adverse effects on heart function. (FIG. 7A) Scheme outlining the treatment strategy of dyW mice with veh or AP-13. (FIG. 7B) Quantification of heart weight normalized to body weight of C57BL/6J wild-type (ctrl), and veh and AP-13 treated dyW mice. (FIG. 7C) Quantification of the cardiac index (cardiac output / body mass) of ctrl, and veh and AP-13 treated dyW mice. (FIG. 7D) Quantification of the heart fractional shortening of ctrl, and veh and AP-13 treated dyW mice. Results are expressed as means ± sem. n>3 per condition. P values were calculated using one-way ANOVA with Tukey post-hoc test. *P<0.05, **P<0.01, ***P<0.001.

FIG. 8: APJ expression in dyW skeletal muscle tissue. Quantification of APJ expression in TA muscle sections in 6- week-old ctrl and dyW mice under uninjured conditions. Results are expressed as means + sem. n>3 mice per condition. Scale bar = 100 pm. P values were calculated using student t-test. *P<0.05.

FIGs. 9A-O: EC specific knockout of APJ phenocopies MD features. (FIGs. 9A-B) Scheme outlining the breeding strategy to generate APJECKO mice and the muscle injury protocol. (FIGs. 9C-D) Quantification of CD31 positive cells in TA muscle cross sections of wt control (ctrl) and APJECKO mice at 5 (FIG. 9C) and 10 dpi (FIG. 9D). (FIG. 9E) Representative haematoxylin and eosin-stained cross sections of the TA muscle of Ctrl and APJECKO mice at 5 and 10 dpi. (FIGs. 9F-G) Frequency distribution of minimal fiber feret classes in TA muscles of Ctrl and APJECKO muscles 5 (FIG. 9F) and 10 dpi (FIG. 9G). (FIGs. 9H-I) Quantification of eMHC positive fibers in TA muscle sections in Ctrl and APJECKO mice at 5 (FIG. 9H) and 10 dpi (FIG. 9I). (FIGs. 9J-K) Quantification of the fibronectin positive area in TA muscle sections in Ctrl and APJECKO mice at 5 (FIG. 9J) and 10 dpi (FIG. 9K). (FIGs. 9L-M) Quantification of Pax7 positive cells in TA muscle sections in Ctrl and APJECKO mice at 5 (FIG. 9L) and 10 dpi (FIG. 9M). (FIGs. 9N-O) Quantification of MyoG positive cells in TA muscle sections in Ctrl and APJECKO mice at 5 (FIG. 9N) and 10 dpi (FIG. 90). Results are expressed as means + sem. n>3 per condition. Scale bars = 100pim. P values were calculated using student t-test (FIGs. 9C-D, H-O) and two-way ANOVA with Tukey’s post-hoc test (FIG. 9F-G). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 .

FIG. 10: Synthesis scheme for illustrative compounds of formula III, Illa, IV, V, VI, VII and VIII (e.g., compound 88, KT01-129 and 158, KT03-69 of Table I).

FIGs. 11A-B: Synthesis scheme for illustrative compounds of formula X-XI (e.g., compound 145, AM03-68 of Table I).

FIG. 12: Synthesis scheme for illustrative compounds of formula XII-XIII (e.g., compounds 146-155 of Table I).

FIGs. 13A-B: Synthesis scheme for illustrative compounds of formula XIV-XV (e.g., compounds 156-157 of Table I).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

DEFINITIONS

General definitions

Terms and symbols of genetics, molecular biology, biochemistry and nucleic acid used herein follow those of standard treatises and texts in the field, e.g. Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like. All terms are to be understood with their typical meanings established in the relevant art.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.

No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

Herein, the term "about" has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. of the disclosure

As used herein the term “stimulator of MuSC function”, “modulators of the apelin receptor’ or “compound of the present disclosure’ includes AP-13, AP-13 structural analogs, agonists of AP-13 function, agents that increase AP-13 endogenous levels, and AP-13 functional analogues (e.g., other modulators of the apelin receptor (apelin or APJ receptor)). In more specific embodiments, it refers to any such compound having a binding K to the Apelin receptor of 100 nM. In other embodiments, a binding Ki to the Apelin receptor of 90nM or less, 85nM or less, 80nM or less, 75nM or less, 70nM or less, 65nM or less, 60nM or less, 55nM or less, 50nM or less, 45nM or less, 40nM or less, 35nM or less, 30nM or less, 25nM or less, 20nM or less, 15nM or less, 10nM or less or 5nM or less.

As used herein the term “MuSC function” refers to skeletal muscle stem cell expansion, and skeletal muscle stem cell differentiation.

As used herein the term “AP-13 function” includes AP-13’s capacity to stimulate ECs (e.g., increase EC proliferation, improve skeletal muscle microvasculature remodeling, stimulate angiogenesis, increase capillary density in skeletal muscle), expand MuSC, promote endogenous repair, improve cardiac function (e.g., by stimulating stem cell function and by generating a pro-myogenic cellular niche), and improve force generation.

Apelin-13 (or AP-13) refers to the active form of Apelin containing 13 amino acids (Pyr-apelin-13) (see FIGs. 3E-F). Apelin is naturally produced as a 77-amino-acid precursor that is processed into active 36, 17, and 13 amino acid fragments (Marsault et al., 2019). Apelin 13 (Pyr-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe (SEQ ID NO: 159)), the smallest active form of apelin, has a molecular weight of 1.5 kDa and is naturally pyroglutamylated at its N- terminus (FIGs. 3E-F).

Without being so limited, structural analogues of AP-13 include AP-13 linear and cyclic (e.g., macrocyclic) analogues.

Without being so limited, AP-13 linear analogues of the present disclosure include those identified in Murza et al. 2015, Tran et al. 2018 and Tran et al. 2021 b. Without being so limited it includes C-terminal Phe (13) substituted AP-13 (substituted with e.g., a synthetic amino acid such as Tyr(Obn), 1 -naphtylalanine, 2-naphtylalanine, 4- benzoylphenylalanine as described in Murza et al. 2015 or conformationally restricted derivatives such as those described in Tran et al. 2021 b) and in formula (I) below. Examples of such AP-13 linear analogues are listed in Table I below. In a more specific embodiment, it includes linear analogues as described below such as AM02-123 (SEQ ID NO: 48) (FIG. 3I).

Without being so limited AP-13 cyclic structural analogues of the present disclosure include those identified in PCT publication no. WO2023108291 , Tran et al. 2022, Tran et al. 2018, Murza et al. 2017, US20130196899A1 , Hamada et al. 2008, Macaluso et al., 2011 , Macaluso et al., 2010, and Brame et al., 2015; and/or as described in any one of formula (III), (Illa), (IV), (V), (VI), (VII), (VIII) and (IX) described herein. Specific examples of such AP-13 macrocyclic compounds are listed in Table I below. In a more specific embodiment, it includes cyclic analogues as described below such as KT01-129 (Pyr-R-P-R-L-S-H-K-c[Allyl(o-nosylDab)-P-Nle-P-Allyl(A)] (SEQ ID NO:88)) (FIG. 3J) and KT03-69 (SEQ ID NO: 158) (FIG. 3I). In case of discrepancies herein between the name (list of residues) and structure (formula) mentioned herein for compounds of the disclosure or parts thereof, the structure (formula) shall prevail. In case of discrepancies herein between the compounds as described in the sequence listing and the name (list of residues) and/or structure (formula) mentioned herein for compounds of the disclosure or parts thereof, the name (list of residues) and/or structure (formula) shall prevail.

Agonists of AP-13 function of the present disclosure include CMF019 and other known in the art.

Agents that increase AP-13 endogenous levels of the present disclosure include neprilysin inhibitors such as sacubitril (Novartis Entresto).

AP-13 functional analogues include Elabela (i.e., a 54 amino acid peptide in human), Elabela precursors see e.g., accession number AHW47894 (MRFQQFLFAFFIFIMSLLLISGQRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 89)), Elabela fragments (e.g., such as natural fragment QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 161) used in Example 4 (FIG. 3J) herein, ELA23-32) and Elabela structural analogs.

Elabela structural analogues include linear analogues. Without being so limited, Elabela linear analogues of the present disclosure include those identified in Murza et al. 2016, and Tran et al., 2021 a and in formula (II) described herein. Examples of such Elabela linear compounds are listed in Table I below. Elabela structural analogues also include cyclic analogues such as those described in any one of formula (X) to (XV) described herein, in PCT publication no. WO2023108291 , Murza et al., 2016 and those more specifically described herein. Examples of such Elabela cyclic analogues are listed in Table I below. In a more specific embodiment, it refers to AM03-68 ([K-R-R-E]-Nle-C-L-H-C-Orn-V-P-F-P (SEQ ID NO: 145)) (FIG. 3J).

In specific embodiments, macrocyclic compounds of the present disclosure are developed from the cyclization of a synthetic peptide (generally made from natural and/or non-natural amino acids) derived from the C-terminal fragment of the AP-13 peptide (FIGs. 3E-F) or the Elabela peptide QRPVNLTMRRKLRKHNCLQRRCMPLHSRVPFP (SEQ ID NO: 161).

In specific embodiments, the cyclisation of the peptide is a side chain to side chain cyclisation (e.g., Table I compounds 63-88 and 144-158). In specific embodiments, the cyclisation of the synthetic peptide is achieved through a reaction of ring-closing metathesis of alkene (or alkyne) groups at the end of each of the side chains of the N- and of a central amino acid (or acid) moieties, the cyclisation resulting in a single carbon-carbon double bond (or single carbon-carbon triple bond if alkyne groups are used) (e.g., Table I compounds 63-88 and 158 such as KT01- 129 and KT03-69). The macrocycle may then further be modified to replace the double bond by a single bond through palladium-catalyzed hydrogenation.

In other specific embodiments, the cyclisation of the peptide is achieved through a macrolactamisation reaction between an amine at the end of the side chain of one of the N-terminal amino acids and a carboxylic acid at the end of the side chain of the amino acid residue used to close the cycle or the reverse. (Elabela macrocyclic analogues (i.e., AP-13 functional analogs) of Table I such as AM03-68).

In a specific embodiment, compounds of the present disclosure are of formula disclosed herein, or are stereoisomers or a mixture thereof, or pharmaceutically acceptable salts, esters or solvates thereof. In case of discrepancies herein between the name and structure presented of compounds or parts thereof, the structure shall prevail when both structures and names are shown.

References herein to amino acids or acids that are part of molecules of the present disclosure should be understood to designate amino acid or acid residues. At least one of their ends is linked to another amino acid or acid to form e.g., a peptide bond thereby losing a hydroxy group and/or one hydrogen of an amine group. Hence, for example, an amino acid or acid listed in any one of the definitions of X1, X2, X3, X4, X5 and X6, etc. should be understood to be the corresponding amino acid or acid residue.

AP-13 linear analogues

In another specific embodiment, the AP-13 linear analogue comprises or consists in the following formula (I):

X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, wherein:

X1 is Pyr or allylglycine

X2 is Arg or allylarginine (e.g., A/a-allyl-Arg); X3 is Pro or allylglycine;

X4 is Arg or allylarginine (e.g., A/a-allyl-Arg);

X5 is Leu or allylglycine;

X6 is Ser, allylglycine, allylserine (e.g., A/a-Allyl-Ser);

X7 is His or allylglycine;

X8 is Lys or allylglycine;

X9 is Gly or allylglycine;

X10 is Pro or allylglycine;

X11 is Met, Nle or allylglycine;

X12 is any natural amino acid, or is any synthetic amino acid, the side chain of which is H, a - (CH2)p-(C1 -C8)alkyl, - (CH2)p-(C1-C8)heteroalkyl, a — (CH2)p-(C3-C8)aryl, -(CH2)p-(C3-C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a - (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In another specific embodiment, it is Pro, Pro analogue, allylglycine, Hyp(4-OBn), Pc3Phe Tic, 1 Nal, 2Nal, Trp, or Aia.

X13 is any natural amino acid, or is any synthetic amino acid, the side chain of which is H, a - (CH2)p-(C2-C8)alkyl, - (CH2)p-(C1-C8)heteroalkyl, a — (CH2)p-(C3-C8)aryl, -(CH2)p-(C3-C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a - (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In another specific embodiment, it is a Phe, Leu, Vai, Tyr(Ome), Phe(4-me), (4-pyridyl)Ala, (3-pyridyl)Ala, Phg, hPhe, Tyr(OBn), Bpa, StyrylAla, dihydroAnthranylAla, (3-benzothienyl)Ala, (L-alpha-Me)Phe, (D-alpha-Me)Phe, aminoindane, AllylGly, (beta-MePhe)-OH, (beta-diMePhe)-OH, Tic-OH, (D-Tic)-OH, Db2g-OH, (4-Bip)-OH, (3-Bip)-OH, (2-Bip)-OH, (1-Ana- Gly)-OH, Aia-Gly-OH Tyr(OBn)-OH, Tic(7-OBn)-OH, cypTyr (OBn)-OH, decypTyr (OBn)-OH, dcypTyr(OH)-OH, m- Tyr(OBn)-OH, Hyp(4-OBn)-OH, -(L-alpha-MeTyr(OBn))-OH, -(D-alpha-MeTyr(OBn))-OH, (beta-MeTic)-OH, (beta- diMeTic)-OH, Tcc-OH, DB2g-OH, or dcypTyr(OH)-OH.

In specific embodiments, the compound of formula (I) is compound AM02-123 of Table I.

Elabela linear analogues

In another specific embodiment, the Elabela linear analogue comprises or consists in the following formula (II):

X1 -X2-X3-X4-X5-X6-X7-X8-X9-R-X10-X11 -X12-X13, wherein:

X1 is Pyr, Ala or absent; X2 is Arg, Ala or absent;

X3 is Arg, Ala or absent;

X4 is Cys, Ala, Ser or absent.

X5 is Met, Ala, Nle or DesaminoNle;

X6 is Pro or Ala;

X7 is any natural amino acid; or is any synthetic amino acid, the side chain of which is H, a - (CH ₂ )p-(C1 -C8)alkyl, - (CH2)p-(C1-C8)heteroalkyl, a — (CH2)p-(C3-C8)aryl, -(CH2)p-(C3-C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a - (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In a specific embodiment, it is a natural amino acid with a hydrophobic side chain, or any synthetic amino acid with a hydrophobic side chain. In another specific embodiment, it is Leu, Ala, a-Me-Leu, Vai, Tie, I, Cha, a-Me-Phe, or CycloLeu;

X8 is His, Ala, (Thiazol-5-yl)Ala, or W;

X9 is Ser or Ala;

X10 is Vai or Nva;

X11 is Pro or Ala;

X12 is any natural amino acid, or is any synthetic amino acid, the side chain of which is H or a — (CH2)p-(C1-C8)alkyl, — (CH2)p-(C1-C8)heteroalkyl, a — (CH2)p-(C3-C8)aryl, -(CH2)p-(C3-C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a - (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In another specific embodiment, it is Phe, Ala, Tyr(Obn), Bpa, 1 Nal, 2Nal, hPhe such as -HomoPhe or y-hPhe, or Cha;

X13 is absent or is any natural amino acid, or any synthetic amino acid, the side chain of which is H or a -(CH ₂ )p- (C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, a -(CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3-C8)heteroaryl, a -(CH ₂ )p-(C3- C8)cycloalkyl, or a - (CH ₂ )p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3- C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1- C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In another specific embodiment, it is Pro, D-Pro, Ala, Pro-NH ₂ , Hyp, Hyp(OBn), Tyr(OBn), Phe, Tic, 1 Nal, 2Nal, Leu, or Oic.

AP-13 cyclic analogues The present disclosure encompasses apelin 13 cyclic analogues such as those described in formula (lll)-(VIII) and (Illa).

In another specific embodiment, the macrocyclic compound comprises or consists in the following formula (III):

Pyr-R-X1 -R-X2-X3-X4-X5-c[X6-P-X7-P-X8]c, wherein:

X1 is Pro, Phe, Lys, Leu, or Ser;

X2 is Leu, Phe, or Thr;

X3 is Ser, Phe, Leu, or Glu;

X4 is His, Lys, Leu, Glu, or Ser;

X5 is Lys, Phe, Leu, or Ser;

X6 and X8 close the ring and are identical or different and are aliphatic residues, alkenyl residues, acid residues or a natural or non-natural amino acid, a peptoid, or a derivative thereof, these moieties are optionally substituted.

In specific embodiments, X6 is a N-allylated analogue of 4-(benzenesulfonamido)-2-aminobutyric acid; 4-(o-nosyl)- 2,4-diaminobutyric acid (Dab); 3-(o-nosyl)-2,3-diaminopropionic acid (Dap); N6-(o-nosyl)-ornithine; Nco-(o-nosyl)- lysine; 4-tosylamido-2-aminobutyric acid; 3-tosylamido-2-aminopropanoic acid (Dap); 4-benzamido-2-aminobutyric acid; 4-(2’-methylbenzamido)-2-aminobutyric; 4-(3’-methylbenzamido)-2-aminobutyric; 4-(4’-methylbenzamido)-2- aminobutyric; 4-(2’-bromobenzamido)-2-aminobutyric; 4-(3’-bromobenzamido)-2-aminobutyric; 4-(4’- bromobenzamido)-2-aminobutyric; 4-(phenylacetamido)-2-aminobutyric acid; O-allylserine, allylglycine, pentenylglycine, Nim-allylhistidine, 3-(N1 -allyl-tetrazol-5-yl)-2-aminopropanoic acid or their derivatives. In specific embodiments, X8 is an allylalanine, allylglycine, butenylglycine, butenylalanin, pentenylglycine, pentenylalanine, or a derivative thereof, this natural or non-natural amino acid or derivative thereof being optionally substituted.

In a specific embodiment, one of the end terminal non-natural amino acid residue used for closing the cycle (e.g., X6 - corresponding to position 3 in AP-13) is, before ring-closure, an alkenyl-Dab (e.g., X6 is an o-nosyl-allylDab) and the other end (e.g., X8) is, before ring-closure, an alkenyl residue or an acid (e.g., X8 is an allylglycine), an acid residue or a non-natural amino acid residue, this alkenyl, acid or amino acid residue comprising an alkene moiety at its end (end of its lateral chain in the case of an amino acid residue). After closure, the double bonds (alkenes) of each moiety have merged into a single carbon-carbon double bond using, for example, a ring-closing metathesis reaction. In specific embodiments, the pair X6-X8 can alternatively comprise one or two peptoids of any of the foregoing natural or non-natural amino acids, or derivative thereof. These natural or non-natural amino acids, peptoids or derivative thereof being optionally substituted. In all the foregoing combinations of two residues, they may be in the L, L; L-D; D, L; or D; D configurations. In specific embodiments, X6 and X8 are linked through their lateral chains. In specific embodiments, when X8 is an amino acid, its C-terminal is linked to one or more additional amino acids that do not form part of the ring.

X7 is any natural amino acid; or is any synthetic amino acid, the side chain of which is H, a - (CH2)p-(C1 -C8)alkyl, - (CH2)p-(C1-C8)heteroalkyl, a — (CH2)p-(C3-C8)aryl, — (CH2)p-(C3-C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a - (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In a specific embodiment, it is a norleucine (Nle), leucine, valine, isoleucine, D-1 Nal, D-2Nal, cyclohexylalanine (Cha), or a derivative thereof, this natural or non-natural amino acid or derivative thereof being optionally substituted.

In specific embodiments, the compound of formula (III) is compound KT01-129 of Table I.

In another embodiment, the macrocyclic compound comprises or consists in the following formula (Illa):

(X1 )r-c[X6-P-X7-P-X8]-(X2)p, wherein p is 0, 1, 2 or 3 and r is 5, 6, 7 or 8 (e.g., up to 10), wherein X6, X7 and X8 are as described in formula III; and

X1 and X2 are each independently any natural amino acid; or any synthetic amino acid, the side chain of which is H, a -(CH ₂ )p-(C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, a -(CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3-C8)heteroaryl, a -(CH ₂ )p- (C3-C8)cycloalkyl, or a — (CH ₂ )p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, O or 8.

In another specific embodiment, the macrocyclic compound comprises or consists in the following formula (IV): wherein X1, X2, X3, X4, X5 and X7are as defined in formula III or Illa; benzensulfonamido of formula ; or a benzamido of formula , wherein R is 0, P, m-alkyl, halogen or nitro; or an heterocycle of formula wherein X is CH or N; and

B is -(CH2)x, wherein x is 3, 4 or 5; or -(CH2)y-CH=CH-(CH2)y-, wherein y is 0, 1, 2 ou 3., and z is 1 , 2 or 3.

In specific embodiments, the compound of formula (IV) is compound 70, 71, 72 or 88 (KT01-129) of Table I below.

In another specific embodiment, the macrocyclic compound comprises or consists in the following formula (V): Pyr-R-P-R-L-S-H-K [X6-P-X7-P-X8], wherein X6, X7 and X8 are as described in any one of formula III, Illa and IV.

In specific embodiments, the compound of formula (V) is compound 70, 71 , 72 or 88 (KT01-129) of Table I below.

In another specific embodiment, the apelin 13 cyclic analogue comprises or consists in the following formula (VI):

X1 -X2-X3-X4-X5-X6-X7-X8-X9-X10-[X11 -X12-X13-X14-X15], wherein X1 is -(CH2)q-CH3 or -(CF2)q-CF3 wherein q is 0 to 11 , a natural amino acid, a synthetic amino acid, the side chain of which is H, a -(C1-C12)alkyl, -(CF2)q-CF3 wherein q is 0 to 11, -(C1-C8)heteroalkyl, a -(CH2)p-(C3- C8)aryl, — (CH2)p-(C3-C8)heteroaryl,— (CH2)p-(C3-C8)cycloalkyl, or — (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3- C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S, -(C1-C6)alkyl, -O-(C1-C6)alkyl, -(CH2)p-(C3-C8)aryl, -O-(CH2)p-(C3-C8)aryl, -(C3- C8)cycloalkyl, or -O-(C3-C8)cycloalkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In a specific embodiment, X1 is Pyr, -(CH2)q-CH3 or -(CF2)q-CF3 wherein q is 0 to 11. In a more specific embodiment, it is Pyr;

X2, X4, X7 and X8 are each independently a natural or synthetic amino acid, the side chain of which is -CH2- (CH2)p-NH2, -CH2-(CH2)p-guanidine, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3- C8)aryl, or — (CH2)p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, O or S, preferably N. In a specific embodiment, X2, X4, X7 and X8 are each independently an amino acid, the side chain of which is -CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or — (CH2)p- imidazole wherein p is 0 to 4. In a specific embodiment, X2, X4, X7 and X8 are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, hArg, or His. In a more specific embodiment, X2, X4, X7 and X8 are each independently Arg, His or Lys. In a more specific embodiment, X2, X4, and X8 are each independently an amino acid, the side chain of which is — CH2-(CH2)p-guanidine, or -CH2-(CH2)p-NH2, wherein p is 0 to 4; or are each independently Arg or Lys; and/or X7 is an amino acid, the side chain of which is— (CH2)p- imidazole wherein p is 0 to 4; or is His;

X3 is Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, Ala, Hyp or Hyp(OBn). In a specific embodiment, X3 is Pro; X5 and X6 are each independently any natural amino acid, or a synthetic amino acid, the side chain of which is H, - (CH ₂ )p-(C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, -(CH ₂ )p-(C3-C8)cycloalkyl, -(CH ₂ )p-(C3-C8)heterocycloalkyl, - (CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3-C8)heteroaryl, -CH ₂ -(CH ₂ )p-NH ₂ , -CH ₂ -(CH2)p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S or a (C1-C6)alkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)ary I , (C3-C8)heteroaryl, (03- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a specific embodiment, X5 and X6 are each independently a -(CH2)p-(C1- C8)alkyl, wherein p is 0 to 5, wherein the alkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl; or is independently serine, threonine, alanine, valine, isoleucine, leucine, alpha-methylleucine, norleucine, cycloleucine or tert-leucine. In a specific embodiment X5 is Leu; and/or X6 is Ser;

X9 is absent, or is a natural or synthetic amino acid, the side chain of which is -(CH2)p-C(O)OH, wherein p is 0 to 5. In a specific embodiment X9 is Glu or Asp. In a more specific embodiment, X9 is absent. In another more specific embodiment, X9 is Glu;

X10 and X12 are each independently absent or Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a more specific embodiment, X10 is not absent. In a specific embodiment, X10 and X12 are each independently absent or Pro. In a specific embodiment, X10 is absent and/or X12 is Pro;

X11 and X15 close the ring and are identical or different and are aliphatic residues, alkenyl residues, acid residues or a natural or non-natural amino acid, or a derivative thereof, these moieties being optionally substituted. In specific embodiments, they are residues bearing a terminal alkene or free carboxylic or amine function. In specific embodiments, they are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Asp, Glu, AllylGly, Na-allyl-arginine, Nrr-allyl-histidine, Ny-nosyl-Ny-allyl-a-amino-butanoic acid, Ny-allyl-Ny-nosyl- a.y-diamino-butanoic acid, Ny-allyl-a.y-diamino-butanoic acid, or Ny-allyl-Ny-methyl-a.Y-diamino-butanoic acid whereby the cycle is closed by an amide bridge or an alkene. In specific embodiments, at least one of X12 and X15 is independently allylglycine or D-allylglycine and the other is Ny-nosyl-Ny-allyl-a-amino-butanoic acid;

X13 is absent or is any natural amino acid, or a synthetic amino acid, the side chain of which is H, — (CH2)p-(C1- C8)alkyl, -(CH2)p-(C1-C8)heteroalkyl, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3- C8)aryl, — (CH2)p-(C3-C8)heteroaryl, -CH2-(CH2)p-NH2, -CH2-(CH2)p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S or a (C1-C6)alkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In specific embodiments, X13 is a — (CH2)p-(C1-C8)alkyl, wherein p is 0 to 5, wherein the alkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl; or is independently serine, threonine, alanine, valine, isoleucine, leucine, alpha-methylleucine, norleucine, cycloleucine or tert-leucine. In specific embodiments, X13 is Nle, alphamethylleucine, cycloleucine, tert-leucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine), alphamethylphenylalanine, preferably norleucine (Nle).

X14 is Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, X14 is Pro.

In a specific embodiment of compounds of formula VI, X9 and X10 are not absent. In a specific embodiment, if X11 is allylglycine, X15 is not allylglycine.

In specific embodiments, compounds of formula (VI) are any one of compounds 70-72 and 88 of Table I. In other specific embodiments, the compound of formula (VI) is compound 88 of Table I.

The present disclosure comprises compounds of Formula (VI), wherein each of X1 to X15 are independently defined using any of the more general or more specific definitions provided above for these residues in formula (VI).

In specific embodiments, the compound of formula (VI) is compound 70, 71, 72 or 88 (KT01-129) of Table I below.

In a specific embodiment, the apelin 13 cyclic analogue comprises or consists in the following formula (VII):

X1 -X2-Y-[X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12]-X13-X14-X15-X16-X17-X18, wherein

X1 is absent, -(CH2)q-CH3 or — (CF2)q-CF3, wherein q is 0 to 11 , or is any natural amino acid; or any synthetic amino acid, the side chain of which is H, -(C1-C 12) alkyl, -(CF2)q-CF3 wherein q is 0 to 11 , -(C1-C8)heteroalkyl, a - (CH2)p-(C3-C8)aryl, -(CH2)p-(C3-C8)heteroaryl, a -(CH2)p-(C3-C8)cycloalkyl, or a -(CH2)p-(C3- C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)ary I , (C3-C8) heteroaryl , (C3-C8)cycloal ky I or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S, -(C1-C6)alkyl, -O-(C1-C6)alkyl, — (CH2)p-(C3-C8)aryl, - 0— (CH2)p-(C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3-C8)cycloalkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In a specific embodiment, it is Pyr, or absent. In a specific embodiment, it is absent;

X2 and X7 are each independently absent, or a natural or synthetic amino acid, the side chain of which is -CH2- (CH2)p-NH2, — CH2-(CH2)p-guanidine, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p- (C3-C8)aryl, or — (CH2)p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S, preferably N. In a specific embodiment, X2 and X7 are each independently absent, or an amino acid, the side chain of which is -CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or -(CH2)p- imidazole wherein p is 0 to 4. In a specific embodiment, X2 and X7 are each independently Lys, Orn, Dab (2,4- diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, hArg, His or absent. In a more specific embodiment, X2 and X7 are each independently absent, -CH2-(CH2)p-guanidine, or -CH2-(CH2)p-NH2, wherein p is 0 to 4; or are each independently Arg or Lys. In a more specific embodiment, X2 and/or X7 are absent;

Y is H, Ac, Ac-NH, -NH2, guanidine or absent. In a more specific embodiment, Y is -NH2,

X3 and X12 close the ring and are identical or different and are aliphatic residues, alkenyl residues, acid residues or a natural or non-natural amino acid, or a derivative thereof, these moieties being optionally substituted. In specific embodiments, they are residues bearing a terminal alkene or free carboxylic or amine function. In specific embodiments, they are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Asp, Glu, AllylGly, Na-allyl-arginine, Nrr-allyl-histidine, Ny-allyl-Ny-nosyl-a.Y-diamino-butanoic acid, Ny-allyl-a.y- diamino-butanoic acid, or Ny-allyl-Ny-methyl-a.Y-diamino-butanoic acid whereby the cycle is closed by an amide bridge or an alkene. In specific embodiments, at least one or both of X3 and X12 are allylglycine. In specific embodiments, X3 and X12 are each allylglycine. In specific embodiments, if X3 is allylglycine, X12 is not allylglycine;

X4, X5 and X6 are each independently Ser, Thr, Asn, Gin, Asn-(8-aminooctanoic), Trp-(8-aminooctanoic) or absent. In a specific embodiment, X4, X5 and X6 are each independently Thr, Asn, Asn-(8-aminooctanoic), Trp-(8- aminooctanoic) or absent. In another specific embodiment, they are all absent;

X8 is absent or is Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, it is absent or Pro. In another specific embodiment, X8 is absent.

X9 is any natural amino acid; or a synthetic amino acid, the side chain of which is H, — (CH2)p-(C 1 -C8)alkyl, — (CH2)p- (C1-C8)heteroalkyl, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3-C8)aryl, -(CH2)p- (C3-C8)heteroaryl, -CH2-(CH2)p-NH2, -CH2-(CH2)p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S, -(C1-C6)alkyl, -O-(C1-C6)alkyl, -(CH2)p-(C3- C8)aryl, -0— (CH2)p-(C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3-C8)cycloalkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a more specific embodiment, X9 is absent, or any natural or synthetic amino acid, the side chain of which is -CH2-(CH2)p-NH2, -CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, -(CH2)p-(C3- C8)cycloalkyl, — (CH2)p-(C3-C8)heterocycloalkyl, — (CH2)p-(C3-C8)aryl, or — (CH2)p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (03- C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S, preferably N. In a more specific embodiment, X9 is an amino acid, the side chain of which is -CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or -(CH2)p-imidazole, preferably -CH2-(CH2)p-guanidine, or -CH2-(CH2)p-NH2, wherein p is 0 to 4. In a more specific embodiment, X9 is Nle, Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, or hArg. In a more specific embodiment, X9 is Arg;

X10 is any natural amino acid, or a synthetic amino acid, the side chain of which is H, — (CH2)p-(C1-C8)alkyl, - (CH2)p-(C1-C8)heteroalkyl, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3-C8)aryl, - (CH2)p-(C3-C8)heteroaryl, -CH2-(CH2)p-NH2, -CH2-(CH2)p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S or a (C1-C6)alkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In specific embodiments, X10 is a — (CH2)p-(C1-C8)alkyl, wherein p is 0 to 5, wherein the alkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl; or is independently serine, threonine, alanine, valine, isoleucine, leucine, alpha-methylleucine, norleucine, cycloleucine or tert-leucine. In a more specific embodiment, X10 is Leu, norleucine, alpha-methylleucine, cycloleucine, tert-leucine, cyclohexylalanine, alpha-methylphenylalanine, Ala, Vai, lie. In a more specific embodiment, it is Leu;

X11 is absent, or is any natural amino acid; or any synthetic amino acid, the side chain of which is H, — (CH2)p-(C1 - C8)alkyl, -(CH2)p-(C1-C8)heteroalkyl, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3- C8)aryl, — (CH2)p-(C3-C8)heteroaryl, -CH2-(CH2)p-NH2, -CH2-(CH2)p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S, -(C1-C6)alkyl, -O-(C1-C6)alkyl, — (CH2)p- (C3-C8)aryl, -0— (CH2)p-(C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3-C8)cycloalkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In specific embodiments, X11 is a — (CH2)p-(C1-C8)alkyl, wherein p is 0 to 5, wherein the alkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl; or is independently serine, threonine, or absent. In a more specific embodiment, X11 is Ser or absent, preferably Ser;

X13 is a natural or synthetic amino acid, the side chain of which is -CH2-(CH2)p-NH2, — CH2-(CH2)p-guanidine, - (CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3-C8)aryl, or -(CH2)p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a specific embodiment X13 is -CH2-(CH2)p- NH2, or -CH2-(CH2)p-guanidine, wherein p is 0 to 4. In a specific embodiment X13 is Lys, Orn, Dab, Dap, Arg, , or His. In a specific embodiment X13 is Lys;

X14 is Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, it is Gly;

X15 and X17 are each independently Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, X15 and X17 are each independently absent or Pro. In a specific embodiment, X15 is Pro and/or X17 is absent;

X16 is any natural amino acid; or a synthetic amino acid, the side chain of which is H, - (CH2)p-(C1-C8)alkyl, - (CH ₂ )p-(C1-C8)heteroalkyl, -(CH ₂ )p-(C3-C8)cycloalkyl, -(CH ₂ )p-(C3-C8)heterocycloalkyl, -(CH ₂ )p-(C3-C8)aryl, - (CH ₂ )p-(C3-C8)heteroaryl, -CH ₂ -(CH ₂ )p-NH ₂ , -CH ₂ -(CH ₂ )p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S, -(01 -06) alkyl, -O-(C1-C6)alkyl, -(CH ₂ )p-(C3- C8)aryl, -0— (CH ₂ )p-(C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3-C8)cycloalkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (03- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1, 2 or 3 N, 0 or S. In a specific embodiment, it is an amino acid, the side chain of which is - (CH ₂ )p-(C1-C8)alkyl, or — (CH ₂ )p-(C3-C8)aryl, wherein the aryl is optionally fused with one or two (C3-C8)aryl, and wherein the aryl is optionally substituted with one or more substituents, wherein each substituent is independently 0- (C1-C6)alkyl, -(CH ₂ )p-(C3-C8)aryl, -O-(CH ₂ )p-(C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3-C8)cycloalkyl.. In a more specific embodiment, X16 is Nle, alpha-methylleucine, cycloleucine, tert-leucine, cyclohexylalanine, alphamethylphenylalanine, Phe, Tic ((S)-N-Fmoc-tetrahydroisoquinoline-3-carboxylic acid), Tyr, 1 Nal, 2Nal, TyrOBn, cypTyr(OBn), dcypTyr(OBn), cypTyr(OCyp), cypTyr(OPr), D-1 Nal, D-2Nal, D-TyrOBn, or D-Tyr. In a specific embodiment X16 is cypTyr(OCyp);

X18 is absent; is any natural amino acid; or a synthetic amino acid, the side chain of which is H, — (CH2)p-(C1- C8)alkyl, -(CH2)p-(C1-C8)heteroalkyl, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3- C8)aryl, — (CH2)p-(C3-C8)heteroaryl, -CH2-(CH2)p-NH2, -CH2-(CH2)p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S, -(C1-C6)alkyl, -0-(C1-C6)alkyl, — (CH2)p- (C3-C8)aryl, -0— (CH2)p-(C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3-C8)cycloalkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a specific embodiment, it is absent or is an amino acid, the side chain of which is a — (CH2)p-(C3-C8)aryl, wherein the aryl is optionally substituted with one or more substituents, wherein each substituent is independently an halogen, amine, -OH, S or a -(C1-C6)alkyl. In another specific embodiment, it is Phe or an halogen substituted Phe. In another specific embodiment, X18 is absent. In another embodiment, it is Phe. In a specific embodiment of compounds of Formula (VII), when X3 is allylglycine, X12 is not allylglycine. In a specific embodiment of compounds of Formula (VII), when X17 and X18 are absent, X16 is not Ala. In specific embodiments, compounds of formula (VII) are any one of compounds 65 and 158 (KT03-69) of Table I. In other specific embodiments, compounds of formula (VII) are any one of compounds 13-29, and 35-46 of Table I of WO2023108291. In other specific embodiments, compounds of formula (VII) are any one of compounds 13, 15-16, 18-20, 28, and 42-44 of Table I of WO2023108291.

The present disclosure comprises compounds of Formula (VII), wherein each of X1 to X18 are independently defined using any of the more general or more specific definitions provided above for these residues in Formula (VII).

In another specific embodiment, the apelin 13 cyclic analogue comprises or consists in the following formula (VIII): wherein X1 is absent, or is X7-X8, wherein X7 is -(CH2)q-CH3 or -(CF2)q-CF3 wherein q is 0 to 11 , a natural amino acid, a synthetic amino acid, the side chain of which is H, a -(C1-C12)alkyl, -(CF2)q-CF3 wherein q is 0 to 11, -(C1-C8)heteroalkyl, a -(CH2)p-(C3- C8)aryl, — (CH2)p-(C3-C8)heteroaryl,— (CH2)p-(C3-C8)cycloalkyl, or — (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3- C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S, -(C1-C6)alkyl, -O-(C1-C6)alkyl, -(CH2)p-(C3-C8)aryl, -O-(CH2)p-(C3-C8)aryl, -(C3- C8)cycloalkyl, or -O-(C3-C8)cycloalkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, O or S. In a specific embodiment, X7 is Pyr; and

X8 is absent, or is a natural or synthetic amino acid, the side chain of which is -CH2-(CH2)p-NH2, -CH2-(CH2)p- guanidine, — (CH2)p-(C3-C8)cycloalkyl, — (CH2)p-(C3-C8)heterocycloalkyl, — (CH2)p-(C3-C8)aryl, or -(CH2)p-(C3- C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, O or S, preferably N. In a specific embodiment, X8 is an amino acid, the side chain of which is - C H2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or — (CH2)p- imidazole, preferably -CH2-(CH2)p-guanidine, or -CH2-(CH2)p-NH2, wherein p is 0 to 4. In a specific embodiment, X8 is Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, hArg, His or absent, preferably Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, or hArg. In a more specific embodiment, X8 is Arg. Y is absent, -NH2-, Ac-NH-, guanidine, or H;

A is -(CH2)n-, or -(CH2)nNH=C(NH2)N-CH2-CH=CH- (preferably Na-allyl-arginine), wherein n is 2, 3 or 4; or -CH=CH- (CH2)m, wherein m is 0, 1 or 2 (preferably allyl-glycine);

B is absent or wherein R is 0, P, m-alkyl, halogen or nitro and n is 1, 2, or 3; wherein R is H,

C3-C7 alkyl, benzyl or arylalkyl and n is 1, 2 or 3; wherein n is 1 , 2, 3 or 4 and m is 0 or 1 ; or wherein X9 is CH or N.

X2 and X3 are each independently absent, or a natural or synthetic amino acid, the side chain of which is -CH2- (CH2)p-NH2, — CH2-(CH2)p-guanidine, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p- (C3-C8)aryl, or -(CH2)p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1, 2 or 3 N, 0 or S, preferably N. In a specific embodiment, X2 and X3 are each independently an amino acid, the side chain of which is -CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or -(CH2)p-imidazole, preferably -CH2-(CH2)p-guanidine, or -CH2-(CH2)p-NH2, wherein p is 0 to 4. In a specific embodiment, X2 and X3 are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, hArg, His, Nle, alphamethylleucine, cycloleucine, tert-leucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine) or alphamethylphenylalanine. In specific embodiments, X2 and X3 are each independently Lys, Arg, hArg, Nle, Leu, Phe, or Cha. In a more specific embodiment, X2 and X3 are each independently Arg or Lys. In another more specific embodiment, X2 is Arg and/or X3 is Lys;

X4 is a natural or non-natural amino acid having a positively charged or uncharged sidechain. In specific embodiments, X4 is Gly, Phe, Leu, lie, Ser, Aib, Pro, Sar, Oic, Ala, Hyp or Hyp(OBn). In a specific embodiment, it is Gly; X5 is Gly, Phe, Leu, lie, Ser, Aib, Pro, Sar, Oic, pAla, Hyp or Hyp(OBn). In a specific embodiment, it is Pro;

X6 is X10-X11-X12, wherein

X10 is any natural amino acid; or a synthetic amino acid, the side chain of which is H, - (CH2)p-(C1-C8)alkyl, - (CH ₂ )p-(C1-C8)heteroalkyl, -(CH ₂ )p-(C3-C8)cycloalkyl, -(CH ₂ )p-(C3-C8)heterocycloalkyl, -(CH ₂ )p-(C3-C8)aryl, - (CH ₂ )p-(C3-C8)heteroaryl, -CH ₂ -(CH ₂ )p-NH ₂ , -CH ₂ -(CH ₂ )p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S, -(C1-C6)alkyl, -O-(C1-C6)alkyl, -(CH ₂ )p’-(C3- C8)aryl, -O-(CH ₂ )p’-(C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3-C8)cycloalkyl, wherein p’ is 0 to 5; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)ary I , (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a specific embodiment, X10 is an amino acid, the side chain of which is - (CH ₂ )p-(C1-C8)alkyl, or — (CH ₂ )p-(C3-C8)aryl, wherein p is 0 to 5, wherein the aryl is optionally fused with one or two (C3-C8)aryl, and wherein the aryl is optionally substituted with one or more substituents, wherein each substituent is independently, -OH, -O-(C1-C6)alkyl, -(CH ₂ )p-(C3-C8)aryl, -O-(CH ₂ )p-(C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3- C8)cycloalkyl, wherein p is 0 to 5. In a specific embodiment, it is not Ala. In a more specific embodiment, Xw is Nle, alpha-methylleucine, cycloleucine, tert-leucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine), alphamethylphenylalanine, Phe, Tic ((S)-N-Fmoc-tetrahydroisoquinoline-3-carboxylic acid), Tyr, 1 Nal, 2Nal, TyrOBn, cypTyr(OBn), dcypTyr(OBn), cypTyr(OCyp), cypTyr(OPr), D-1 Nal, D-2Nal, D-TyrOBn, or D-Tyr;

Xu is absent or Gly, Phe, Leu, lie, Ser, Aib, Pro, Sar, Oic, Ala, Hyp or Hyp(OBn). In a specific embodiment, it is absent or Pro. In a specific embodiment, it is absent; and

X12 is absent or Phe. In a specific embodiment, it is absent.

In specific embodiments, compounds of formula (VIII) is compound 65 or 158 (KT03-69) of Table I. In specific embodiments, compounds of formula (VIII) are any one of compounds 13-25, 27-29, 35, 36-37 and 42-45 of Table I in WO2023108291. In other specific embodiments, compounds of formula (VIII) are any one of compounds 13, 15- 16, 18-20, 28, and 42-44 of Table I of WO2023108291.

In a specific embodiment, X1 is Pyr-Arg, Y is -NH-, A is-CH2-CH2-, B is absent, X2 is Arg, X3 Lys, X4 is Gly, X5 is Pro, and X6 is Nle-Pro-Phe. , , , , , ,

In another specific embodiment, X1 is H, Y is -NH-, A is -CH=CH-, B is absent, X2 is Arg, X3 is Lys, X4 is Gly, X5 is Pro, X6 is Nle-Pro-Phe.

In another specific embodiment, X1 is absent, Y is -H, A is -CH=CH-, B is absent, X2 is Arg, X3 is Lys, X4 is Gly, X5 is Pro, X6 is Nle.

In another specific embodiment, X1 is H, Y is -NH-, A is -CH=CH-, B is absent, X2 is Nle, X3 is Lys, X4 is Gly, X5 is Pro, X6 is Nle.

In another specific embodiment, X1 is H, Y is -NH-, A is -CH=CH-, B is absent, X2 is Arg, X3 is Lys, X4 is Gly, X5 is Pro, X6 is Nle.

In another specific embodiment, X1 is H, Y is -NH-, A is -CH=CH-, B is absent, X2 is Arg, X3 is Lys, X4 is Gly, X5 is Pro, X6 is D-1 Nal.

In another specific embodiment, X1 is H, Y is -NH-, A is -CH=CH-, B is absent, X2 is Arg, X3 is Lys, X4 is Gly, X5 is Pro, X6 is D-2Nal.

The present disclosure comprises compounds of Formula (VIII), wherein each of the variables X1, X2, X3, X4, X5, X6, Y, A and B are independently defined using any of the more general or more specific definitions provided above for these residues in Formula (VIII).

In another specific embodiment, the apelin 13 cyclic analogue comprises or consists in the following formula (IX):

X1 -[X2-X3-X4-X5-X6-X7]-X8-X9-X10-X11 -X12-X13-X14-X15, wherein X1 is absent, -(CH2)q-CH3 or -(CF2)q-CF3 wherein q is 0 to 11 , a natural amino acid, a synthetic amino acid, the side chain of which is H, a -(C1-C12)alkyl, -(CF2)q-CF3 wherein q is 0 to 11, -(C1-C8)heteroalkyl, a - (CH2)p-(C3-C8)aryl, -(CH2)p-(C3-C8)heteroaryl,-(CH2)p-(C3-C8)cycloalkyl, or -(CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3- C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S, -(C1-C6)alkyl, -O-(C1-C6)alkyl, -(CH2)p-(C3-C8)aryl, -O-(CH2)p- (C3-C8)aryl, -(C3-C8)cycloalkyl, or -O-(C3-C8)cycloalkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In a specific embodiment, X1 is -NH2, Pyr, -(CH2)q-CH3 or -(CF2)q-CF3 wherein q is 0 to 11 . In a specific embodiment, X1 is -NH2;

X2 and X7 close the ring and are identical or different and are aliphatic residues, alkenyl residues, acid residues or a natural or non-natural amino acid, or a derivative thereof, these moieties being optionally substituted. In specific embodiments, they are residues bearing a terminal alkene or free carboxylic or amine function. In specific embodiments, they are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Asp, Glu, AllylGly, Na-allyl-arginine, Nrr-allyl-histidine, Ny-allyl-Ny-nosyl-a.Y-diamino-butanoic acid, Ny-allyl-a.y- diamino-butanoic acid, or Ny-allyl-Ny-methyl-a.Y-diamino-butanoic acid whereby the cycle is closed by an amide bridge or an alkene. In specific embodiments, at least one or both of X2 and X7 are allylglycine;

X3 is absent, Gly, Phe, Leu, lie, Ser, Aib, Pro, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, it is absent or Pro. In a specific embodiment, it is absent;

X4 is a natural or synthetic amino acid, the side chain of which is -CH2-(CH2)p-NH2, — CH2-(CH2)p-guanidine, - (CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3-C8)aryl, or -(CH2)p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S, preferably N. In a specific embodiment, X4 is an amino acid, the side chain of which is — CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or -(CH2)p-imidazole, preferably -CH2-(CH2)p-guanidine or -CH2-(CH2)p-NH2, wherein p is 0 to 4. In a specific embodiment, X4 is Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, or His. In a more specific embodiment, X4 is Arg or Lys. In a more specific embodiment, X4 is Arg.

X5 and X6 are each independently any natural amino acid, or a synthetic amino acid, the side chain of which is H, - (CH2)p-(C1-C8)alkyl, -(CH2)p-(C1-C8)heteroalkyl, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, - (CH2)p-(C3-C8)aryl, -(CH2)p-(C3-C8)heteroaryl, -CH2-(CH2)p-NH2, -CH2-(CH2)p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S or a (C1-C6)alkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)ary I , (C3-C8)heteroary I , (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a specific embodiment, X5 and X6 are each independently a -(CH2)p-(C1- C8)alkyl, wherein p is 0 to 5, wherein the alkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl; or is independently serine, threonine, alanine, valine, isoleucine, leucine, alpha-methylleucine, norleucine, cycloleucine or tert-leucine. In a specific embodiment, X5 is Leu and/or X6 is Ser;

X8 is absent or is any natural amino acid, or a synthetic amino acid, the side chain of which is H, -(CH2)p-(C1- C8)alkyl, -(CH2)p-(C1-C8)heteroalkyl, -(CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3- C8)aryl, -(CH2)p-(C3-C8)heteroaryl, -CH2-(CH2)p-NH2, -CH2-(CH2)p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S or a (C1-C6)alkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a specific embodiment, X8 is absent or Ser;

X9 is absent or is a natural or synthetic amino acid, the side chain of which is -CH2-(CH2)p-NH2, — CH2-(CH2)p- guanidine, — (CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3-C8)aryl, or -(CH2)p-(C3- C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S, preferably. In a specific embodiment, X9 is absent, or an amino acid, the side chain of which is -CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or -(CH2)p-imidazole, preferably -(CH2)p-imidazole, wherein p is 0 to 4. In a specific embodiment, X9 is absent, Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, or His. In a more specific embodiment, X9 is absent or His;

X10 is a natural or synthetic amino acid, the side chain of which is -CH2-(CH2)p-NH2, — CH2-(CH2)p-guanidine, - (CH2)p-(C3-C8)cycloalkyl, -(CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3-C8)aryl, or -(CH2)p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a more specific embodiment, X10 is an amino acid, the side chain of which is — CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or -(CH2)p-imidazole, preferably -CH2- (CH2)p-guanidine, or -CH2-(CH2)p-NH2, wherein p is 0 to 4. In a specific embodiment, X10 is Lys, Orn, Dab (2,4- diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, hArg, or His. In a more specific embodiment, X10 is Lys;

X11 , X12 and X14 are each independently Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, X11 is Gly. In a specific embodiment, X12 and/or X14 are Pro. In a specific embodiment, X12 is Pro and/or X14 is absent;

X13 is absent, Nle, alpha-methylleucine, cycloleucine, tert-leucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine), alpha-methylphenylalanine, preferably absent or Nle;

X15 is an amino acid, the side chain of which is -(CH2)p-(C3-C8)aryl, wherein the aryl is optionally substituted with one or more substituents, wherein each substituent is independently an halogen, amine, -OH, S, (C3-C6)cycloalkyl or a (C1-C6)alkyl. In specific embodiments, X15 is cypTyr(OCyp), Phe, or halogen substituted Phe. In specific embodiments, X15 is cypTyr(OCyp) or Phe.

The present disclosure comprises compounds of Formula (IX), wherein each of X1 to X15 are independently defined using any of the more general or more specific definitions provided above for these residues in Formula (IX). In specific embodiments, compounds of Formula (IX) are any one of compounds 3 and 4 of Table I of WO2023108291. In specific embodiments, compounds of formula (IX) are any one of compounds 65 and 158 (KT03-69) of Table I.

The present disclosure comprises compounds of Formula (IX), wherein each of X1 to X15 are independently defined using any of the more general or more specific definitions provided above for these residues in Formula (IX).

Elabela

In another specific embodiment, the macrocyclic compound comprises or consists in the following formula (X): c[X1 ’-R-R-X2’]c-X3’-X4’-c[C-X5’-X6’-X7’-C]c-X8’- X9’-X10’-X11 ’-X12’, wherein:

X1 ’ and X2’ are each independently Lys, Orn, Dab, Dap, Asp, Glu, or AllylGly where the cycle is closed by an amide bridge or an alkene;

X3’, X5’ and X9’ are each independently a - (CH2)p-(C1 -C8)alkyl, wherein p is 0 to 5, wherein the alkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl; or is independently alpha-methylleucine, valine, norleucine, cycloleucine, tert-leucine, cyclohexylalanine, or alpha-methylphenylalanine; or preferably X3’ is norleucine and/or X5’ is leucine and/or X9’ is valine;

X4’ and X6’ a are each absent or are each independently any natural amino acid; or any synthetic amino acid, the side chain of which is H, a - (CH2)p-(C1-C8)alkyl, - (CH2)p-(C1 -C8)heteroalkyl, a - (CH2)p-(C3-C8)aryl, — (CH2)p-(C3- C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a - (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)ary I , (C3-C8)heteroary I , (03- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. Preferably X4’ is absent and/or X6’ is absent;

X7’ and X8’ are each independently -CH2-(CH2)p-NH2, — CH2-(CH2)p-guanidine, -(CH2)p-(C3-C8)cycloalkyl, - (CH2)p-(C3-C8)heterocycloalkyl, -(CH2)p-(C3-C8)aryl, or -(CH2)p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)ary I , (C3- C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S; In a specific embodiment, each of X7’ and X8’ is independently an amino acid, the side chain of which is - CH2-(CH2)p-guanidine, -CH2-(CH2)p-NH2, or -(CH2)p-imidazole, preferably — CH2-(CH2)p-guanidine, or -CH2-(CH2)p-NH2, wherein p is 0 to 4; in a specific embodiment, each of X7’ and X8’ is independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, or His. In a specific embodiment, X7’ is -(CH2)p-imidazole, wherein p is 0 to 4 or His; and/or X8’ is — (CH2)p-(C3-C8)aryl . -CH2-(CH2)p- guanidine, or -CH2-(CH2)p-NH2, wherein p is 0 to 4 or is Arg, Orn, Lys, or 4-aminomethyl-phenylalanine, preferably Orn;

X10’ and X12’ are each independently Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, X12’ and/or X14’ is/are Pro; and

X11’ is an amino acid, the side chain of which is — (CH2)p-(C3-C8)aryl, wherein the aryl is optionally substituted with one or more substituents, wherein each substituent is independently an halogen, amine, -OH, S or a (01 -C6)alkyl. In specific embodiments, X14’ is a Phe or an halogen substituted Phe such as a bromophenyl.

In other specific embodiments of Formula (X), X3’, X5’, X7’, X’8, X9’, Xaa10’, X11’ and X12’ are each independently either as defined above or are any natural amino acid; or any synthetic amino acid, the side chain of which is H, a — (CH ₂ )p-(C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, a -(CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3-C8)heteroaryl, a -(CH ₂ )p- (C3-C8)cycloalkyl, or a — (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S.

In specific embodiments, the compound of Formula (X) is compound AM03-68.

In a specific embodiment, the elabela cyclic analogue comprises or consists in the following formula (XI): c[X1 ^, -X2 ^, -X3 ^, -X4 ^, ]c-X5 ^, -X6 ^, -c[C-X7 ^, -X8 ^, -X9’-C]c-X1 O’-X11 ’-X12’-X13’-X14’, wherein:

X1 ’ and X4’ close the ring and are identical or different and are aliphatic residues, alkenyl residues, acid residues or a natural or non-natural amino acid, or a derivative thereof, these moieties being optionally substituted. In specific embodiments, they are residues bearing a terminal alkene or free carboxylic or amine function. In specific embodiments, they are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Asp, Glu, AllylGly, Na-allyl-arginine, Nrr-allyl-histidine, Ny-allyl-Ny-nosyl-a.Y-diamino-butanoic acid, Ny-allyl-a.y- diamino-butanoic acid, or Ny-allyl-Ny-methyl-a.Y-diamino-butanoic acid whereby the cycle is closed by an amide bridge or an alkene. In specific embodiments, one of X1’ and X4’ is Glu and the other is Lys. In a specific embodiment, they are Lys and Glu or Glu and Lys;

X2’, X3’ and X9’ and X1 O’ are each independently a natural or synthetic amino acid, the side chain of which is -CH ₂ - (CH ₂ )p-NH2, — CH ₂ -(CH ₂ )p-guanidine, -(CH ₂ )p-(C3-C8)cycloalkyl, -(CH ₂ )p-(C3-C8)heterocycloalkyl, -(CH ₂ )p-(C3- C8)aryl, or — (CH ₂ )p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group;_wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(03- C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1, 2 or 3 N, 0 or S. In a specific embodiment, X2’, X3’, X9’ and X1 O’ are each independently absent or an amino acid, the side chain of which is -CH ₂ -(CH ₂ )p-guanidine, -CH ₂ -(CH ₂ )p-NH ₂ , or -(CH ₂ )p-imidazole, preferably -CH ₂ -(CH ₂ )p-guanidine, or - CH ₂ -(CH ₂ )p-NH ₂ , wherein p is 0 to 4. In a specific embodiment, X2’, X3’, X9’ and X1 O’ are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, His or absent. In a more specific embodiment, X2’ and X3’ are each independently -CH ₂ -(CH ₂ )p-guanidine, wherein p is 0 to 4; or Arg; and/or X9’ is - (CH ₂ )p-imidazole, wherein p is 0 to 4; and/or X10’ - (CH ₂ )p-(C3-C8)aryl. -CH ₂ -(CH ₂ )p-guanidine, or -CH ₂ -(CH ₂ )p- NH ₂ , wherein p is 0 to 4 or is Arg, Orn, Lys, or 4-aminomethyl-phenylalanine;

X12’ and X14’ are each independently Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, X12’ and/or X14’ is/are Pro;

X5’, X7’, and X11’, are each independently any natural amino acid; or any synthetic amino acid, the side chain of which is H, a -(CH ₂ )p-(C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, a -(CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3-C8)heteroaryl, a — (CH ₂ )p-(C3-C8)cycloalkyl, or a — (CH ₂ )p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5,_wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (03- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In other specific embodiments X5’, X7’, and X11’ are alpha-methylleucine, valine, norleucine, cycloleucine, tert-leucine, cyclohexylalanine, or alpha-methylphenylalanine; Trp, thiazol-5-yl-alanine, 3-(2-pyridyl)-alanine, 3-(3- pyridyl)-alanine, or 3-(4-pyridyl)-alanine. In specific embodiments, X5’, X7’, and X11’ are each independently - (CH ₂ )p-(C1-C8)alkyl or — (CH ₂ )p-(C3-C8)hydroxyalkyl wherein p is 0 to 5. In another specific embodiment, X5’, X7’, and X11’ are each independently Nle, Leu, Ala, Vai, alpha-methylleucine, cycloleucine, tert-leucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine) or alpha-methylphenylalanine, In a more specific embodiment, X5’ is Nle; and/or X7’ is Leu; and X11 ’ is Vai; and

X6’ and X8’ are each absent or are each independently any natural amino acid; or any synthetic amino acid, the side chain of which is H, a -(CH ₂ )p-(C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, a -(CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3- C8)heteroaryl, a — (CH ₂ )p-(C3-C8)cycloalkyl, or a -(CH ₂ )p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5,_wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)ary I , (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S.

In specific embodiments, the compound of Formula (XI) is compound AM03-68.

The present disclosure comprises compounds of Formula (XI), wherein each of XT to X14’ are independently defined using any of the more general or more specific definitions provided above for these residues Formula (XI).

In another specific embodiment, the compound comprises or consists in the following formula (XII):

X1 ’-X2’-X3’-X4’-c[X5’-X6’-X7’-X8’-X9’-X 1 O’]c-X11 ’-X12’-X13’-X14’-X15’-X16’, wherein:

XT is absent, -(CH ₂ )q-CH3 or -(CF ₂ )q-CF3 wherein q is 0 to 11 , a natural amino acid, a synthetic amino acid, the side chain of which is H, a -(C1-C 12) alkyl, -(CF ₂ )q-CF3 wherein q is 0 to 11, -(C1-C8)heteroalkyl, a -(CH ₂ )p-(C3- C8)aryl, — (CH ₂ )p-(C3-C8)heteroaryl,— (CH ₂ )p-(C3-C8)cycloalkyl, or — (CH ₂ )p-(C3-C8)heterocycloalkyl, wherein p is O to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3- C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S, -(C1-C6)alkyl, -O-(C1-C6)alkyl, -(CH ₂ )p-(C3-C8)aryl, -O-(CH ₂ )p-(C3-C8)aryl, -(C3- C8)cycloalkyl, or -O-(C3-C8)cycloalkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In a specific embodiment, X1 is Pyr, -(CH ₂ )q-CH3 or -(CF ₂ )q-CF3 wherein q is 0 to 11. In a more specific embodiment, it is Pyr.

X2’, X3’ and X13’ are each independently a natural or synthetic amino acid, the side chain of which is -CH ₂ -(CH ₂ )p- NH ₂ , — CH ₂ -(CH ₂ )p-guanidine, — (CH ₂ )p-(C3-C8)cycloalkyl, — (CH ₂ )p-(C3-C8)heterocycloalkyl, — (CH ₂ )p-(C3-C8)aryl, or — (CH ₂ )p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3- C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1, 2 or 3 N, 0 or S, preferably N. In a specific embodiment, X2’, X3’ and X13’ are each independently an amino acid, the side chain of which is -CH ₂ -(CH ₂ )p-guanidine, -CH ₂ -(CH ₂ )p-NH ₂ , or — (CH ₂ )p-imidazole, preferably -CH ₂ -(CH ₂ )p-guanidine, wherein p is 0 to 4, optionally substituted with e.g., an aryl. In a specific embodiment, X2’, X3’ and X13’ are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, hArg, or His. In a more specific embodiment, X2’, X3’ and X13’ are each independently -CH ₂ -(CH ₂ )p-guanidine, wherein p is 0 to 4, optionally substituted with e.g., an aryl. In a more specific embodiment, X2’ and X3’ are each independently Arg, aryl- substituted Arg (e.g., 4bromobenzoyl), hArg, Nle, Leu, Ala, Vai, alpha-methylleucine, cycloleucine, tert-leucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine) or alpha-methylphenylalanine. In a more specific embodiment, X2’, and/or X3’ are each independently Arg or hArg; and X13’ is Arg.

X4’, X6’, X8’ and X12’ are each independently absent or is any natural amino acid, or a synthetic amino acid, the side chain of which is H, -(CH ₂ )p-(C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, -(CH ₂ )p-(C3-C8)cycloalkyl, -(CH ₂ )p-(C3- C8)heterocycloalkyl, -(CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3-C8)heteroaryl, -CH ₂ -(CH ₂ )p-NH ₂ , -CH ₂ -(CH ₂ )p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S or a (C1-C6)alkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3- C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In a more specific embodiment, X4’, X6’, X8’ and X12’ are each independently a — (CH ₂ )p-(C1 -C8)alkyl or -(CH ₂ )p-(C3-C8)hydroxyalkyl wherein p is 0 to 5. In a more specific embodiment, X4’, X6’, X8’ and X12’ are each independently Leu, Nle, alpha-methylleucine, cycloleucine, tertleucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine), alpha-methylphenylalanine, Ala, Vai, lie, Ser or Thr. In a more specific embodiment, X4’, X6’, X8’ and X12’ are each independently Ser, Nle or Leu. In a more specific embodiment, X4’ and/or X12’ are Ser; and/or X6’ is Nle; and/or X8’ is Leu.

X5’ and X1 O’ close the ring and are identical or different and are aliphatic residues, alkenyl residues, acid residues or a natural or non-natural amino acid, or a derivative thereof, these moieties being optionally substituted. In specific embodiments, they are residues bearing a terminal alkene or free carboxylic or amine function. In specific embodiments, they are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Asp, Glu, AllylGly, Na-allyl-arginine, Nrr-allyl-histidine, Ny-allyl-Ny-nosyl-a.Y-diamino-butanoic acid, Ny-allyl-a.y- diamino-butanoic acid, or Ny-allyl-Ny-methyl-a.Y-diamino-butanoic acid whereby the cycle is closed by an amide bridge or an alkene. In a specific embodiment, there are each independently Lys, Orn, Dab, Dap, Asp, Glu, or AllylGly, wherein the cycle is closed by an amide bridge or an alkene. In specific embodiments, one of X1 ’ and X4’ is Glu and the other is Lys. In a specific embodiment, they are Lys and Glu or Glu and Lys;

X7’, X15’ and X17’ are each independently Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn). In a specific embodiment, X7’, X15’ and X17’ are each Pro.

X9’ and X11’ are each independently absent or a natural or synthetic amino acid, the side chain of which is -CH ₂ - (CH ₂ )p-NH ₂ . — CH ₂ -(CH ₂ )p-guanidine, -(CH ₂ )p-(C3-C8)cycloalkyl, -(CH ₂ )p-(C3-C8)heterocycloalkyl, -(CH ₂ )p-(C3- C8)aryl, or — (CH ₂ )p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3- C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1, 2 or 3 N, 0 or S, preferably N. In a specific embodiment, X9’ and X11’ are each independently absent or is an amino acid, the side chain of which is -CH ₂ -(CH ₂ )p-guanidine, -CH ₂ -(CH ₂ )p-NH ₂ , or -(CH ₂ )p-imidazole wherein p is 0 to 4. In a specific embodiment, X9’ is Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Arg, hArg, His or absent. In a more specific embodiment, X9’ and X11’ are each independently absent are -(CH2)p-imidazole wherein p is 0 to 4 or are His.

X14’ is any natural amino acid; or any synthetic amino acid, the side chain of which is H, a - (CH2)p-(C1-C8)alkyl, - (CH2)p-(C1-C8)heteroalkyl, a — (CH2)p-(C3-C8)aryl, -(CH2)p-(C3-C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a - (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In another specific embodiment, X14’ is Nle, Leu, Ala, lie, Vai, alpha-methylleucine, cycloleucine, tert-leucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine) or alpha-methylphenylalanine. In specific embodiments, X14’ is a - (CH2)p-(C1-C8)alkyl wherein p is 0 to 5, or is Ala, Vai, lie, Nle, or Leu; and

X16’ is an amino acid, the side chain of which is - (CH2)p-(C3-C8)aryl, wherein the aryl is optionally substituted with one or more substituents, wherein each substituent is independently an halogen, amine, -OH, S or a (C1-C6)alkyl. In specific embodiments, X14’ is a Phe or a halogen substituted Phe such as a bromophenyl.

In specific embodiments, the compound of formula (XII) is any one of compounds 146-155 in Table I below.

The present disclosure comprises compounds of Formula (XII), wherein each of XT to X16’ are independently defined using any of the more general or more specific definitions provided above for these residues in Formula (XII).

In another specific embodiment, the macrocyclic compound comprises or consists in the following formula (XIII):

X1 ’-X2’-X3’-X4’-c[X5’-X6’-X7’-X8’-X9’]c-X1 O’-X11 ’-X 12’-X13’-X14’-X15’-X16’, wherein:

XT is Pyr or 4BrBz;

X2’ and X3’ are each independently R or hR;

X5’ and X9’ are each independently Lys, Orn, Dab, Dap, Asp, Glu, or AllylGly . where the cycle is closed by an amide bridge or an alkene;

X6’, X7’, X8’, X10’, X11’, X12’, X13’, and X14’, are each independently any natural amino acid; or any synthetic amino acid, the side chain of which is H, a — (CH2)p-(C1-C8)alkyl, - (CH2)p-(C1-C8)heteroalkyl, a -(CH2)p-(C3- C8)aryl, — (CH2)p-(C3-C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a — (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (03- C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S. In specific embodiments, X6’ is Pro or Nle; and/or X7’ is Leu or Pro; and/or X10’ is Arg or His; and/or X1 T is Vai or Ser; and/or X12’ is Pro, Arg or Oic; and/or X13’ is Phe, 4BrF, Vai, and/or X14’ is Pro or Hyp(OBn)-OH; and

X4’, X8’, X15’ et X16’ are each absent or are each independently any natural amino acid; or any synthetic amino acid, the side chain of which is H, a - (CH2)p-(C1-C8)alkyl, - (CH2)p-(C1-C8)heteroalkyl, a - (CH2)p-(C3-C8)aryl, - (CH2)p-(C3-C8)heteroaryl, a — (CH2)p-(C3-C8)cycloalkyl, or a — (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3- C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S.

In specific embodiments, the compound of formula (XIII) is any one of compounds 146-155 in Table I below.

In another specific embodiment, the compound comprises or consists in the following formula (XIV): c[XT-X2’-X3’-X4’]c-X5’-X6’-X7’-X8’-X9’-X10� �-X1 T-X12’-X13’-X14’ wherein:

XTand X4’ close the ring and are identical or different and are aliphatic residues, alkenyl residues, acid residues or a natural or non-natural amino acid, or a derivative thereof, these moieties being optionally substituted. In specific embodiments, they are residues bearing a terminal alkene or free carboxylic or amine function. In specific embodiments, they are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3-diaminopropionic acid), Asp, Glu, AllylGly, Na-allyl-arginine, Nrr-allyl-histidine, Ny-allyl-Ny-nosyl-a.Y-diamino-butanoic acid, Ny-allyl-a.y- diamino-butanoic acid, or Ny-allyl-Ny-methyl-a.Y-diamino-butanoic acid whereby the cycle is closed by an amide bridge or an alkene. In a specific embodiment, there are each independently Lys, Orn, Dab, Dap, Asp, Glu, or AllylGly. where the cycle is closed by an amide bridge or an alkene In specific embodiments, one of X1 ’ and X4’ is Glu and the other is Lys. In a specific embodiment, they are Lys and Glu or Glu and Lys;

X2’, X3’, X8’ and X10’ are each independently a natural or synthetic amino acid, the side chain of which is -CH ₂ - (CH ₂ )P-NH ₂ , -CH ₂ -(CH ₂ )p-guanidine, -(CH ₂ )p-(C3-C8)cycloalkyl, -(CH ₂ )p-(C3-C8)heterocycloalkyl, -(CH ₂ )p-(C3- C8)aryl, or — (CH ₂ )p-(C3-C8)heteroaryl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with at least one amino or guanidino group; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(03- C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1, 2 or 3 N, 0 or S, preferably N. In a specific embodiment, X2’, X3’, X8’ and X10’ are each independently an amino acid, the side chain of which is -CH ₂ -(CH ₂ )p-guanidine, -CH ₂ -(CH ₂ )p-NH ₂ , or — (CH ₂ )p-imidazole, wherein p is 0 to 4. In a specific embodiment, X2’, X3’, X8’ and X10’ are each independently Lys, Orn, Dab (2,4-diaminobutyric acid), Dap (2,3- diaminopropionic acid), Arg, hArg, or His. In a more specific embodiment, X2’, X3’, X8’ and X10’ are each independently -CH ₂ -(CH ₂ )p-guanidine, or — (CH ₂ )p-imidazole, wherein p is 0 to 4. In another specific embodiment, X2’, X3’ and X10’ are each independently -CH ₂ -(CH ₂ )p-guanidine, wherein p is 0 to 4. In another specific embodiment, X2’, X3’ and X1 O’ are each independently Arg, or hArg. In another specific embodiment, X8’ is His. X5’, X7’, X9’, and X11’ are each independently a natural amino acid; or a synthetic amino acid, the side chain of which is H, -(CH ₂ )p-(C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, -(CH ₂ )p-(C3-C8)cycloalkyl, -(CH ₂ )p-(C3- C8)heterocycloalkyl, -(CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3-C8)heteroaryl, -CH ₂ -(CH ₂ )p-NH ₂ , -CH ₂ -(CH ₂ )p-guanidine, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amino group, guanidino group, -OH, S or a (C1-C6)alkyl; wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3- C8)aryl, (C3-C8)heteroaryl, (C3-C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is 1 , 2 or 3 N, 0 or S. In another specific embodiment, X5’, X7’, X9’, and X11’ are each independently a — (CH ₂ )p-(C1-C8)alkyl wherein p is 0 to 5. In another specific embodiment, X5’, X7’, X9’, and X11’ are each independently Leu, Nle, alpha-methylleucine, cycloleucine, tert-leucine, cyclohexylalanine (e.g., (3-cyclohexyl-L-alanine), alpha-methylphenylalanine, Ala, Vai, lie, Ser or Thr. In a more specific embodiment, X5’, X7’, X9’, and X11’ are each independently Ser, Nle, Leu or Vai. In a more specific embodiment, X5’ is Nle; and/or X7’ is Leu; and/or X9’ is Ser; and/or X11’ is Vai.

X6’, X12’ and X14’ are each independently Gly, Phe, Leu, lie, Ser, Pro, Aib, Sar, Oic, |3Ala, Hyp or Hyp(OBn), preferably Pro or Oic. In a specific embodiment, X6’, X12’ and X14’ are each Pro.

X13’ is an amino acid, the side chain of which is a -(CH ₂ )p-(C3-C8)aryl, wherein the aryl is optionally substituted with one or more substituents, wherein each substituent is independently an halogen, amine, -OH, S or a -(C1-C6)alkyl. In another specific embodiment, it is Phe or an halogen substituted Phe. In another embodiment, it is Phe.

In specific embodiments, the compound of formula (XIV) is any one of compounds 156-157 in Table I below.

In another specific embodiment, the macrocyclic compound comprises or consists in the following formula (XV): c[X1 ’-R-R-X2’]c-X3’-X4’-X5’-X6’-X7’-X8’-X9’-X1 O’-X11 ’-X12’ wherein:

XTand X2’ are each independently Lys, Orn, Dab, Dap, Asp, Glu, or AllylGly. where the cycle is closed by an amide bridge or an alkene; and

X3’ to X1 O’ are each independently any natural amino acid; or any synthetic amino acid, the side chain of which is H, a -(CH ₂ )p-(C1-C8)alkyl, -(CH ₂ )p-(C1-C8)heteroalkyl, a -(CH ₂ )p-(C3-C8)aryl, -(CH ₂ )p-(C3-C8)heteroaryl, a -(CH ₂ )p- (C3-C8)cycloalkyl, or a — (CH ₂ )p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)aryl, (C3-C8)heteroaryl, (03- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S.

X11’ and X12’ are each absent or are each independently any natural amino acid; or any synthetic amino acid, the side chain of which is H, a — (CH2)p-(C1-C8)alkyl, — (CH2)p-(C1 -C8)heteroalkyl, a — (CH2)p-(C3-C8)aryl, -(CH2)p-(C3- C8)heteroaryl, a - (CH2)p-(C3-C8)cycloalkyl, or a - (CH2)p-(C3-C8)heterocycloalkyl, wherein p is 0 to 5, wherein the cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally fused with one or two (C3-C8)ary I , (C3-C8)heteroaryl, (C3- C8)cycloalkyl or -(C3-C8)heterocycloalkyl; and wherein the alkyl, heteroaryl, aryl, cycloalkyl and heterocycloalkyl is optionally substituted with one or more substituents, wherein each substituent is independently e.g., an halogen, amine, -OH, S or a (C1-C6)alkyl, and wherein the heteroatom in the heteroalkyl, heteroaryl or heterocycloalkyl is a N, 0 or S.

In specific embodiments, the compound of formula (XV) is any one of compounds 156-157 in Table I below.

In another specific embodiment of formula (I) to (XV), one of the ring closing residues’ is Lys, Dap, Dab, Orn, and the other is Glu or Asp.

In specific embodiments, the compound is any one of compounds in Table I below.

In an embodiment, one of the end terminal (natural or non-natural) amino acid residue used for closing the cycle (e.g., XT or X5’ corresponding to position 19 and 23, respectively, in Elabela; or X2 or X9’ corresponding to position 22 and 27, respectively, in Elabela) is, before ring-closure, an (natural or non-natural) amino acid having an amine on its lateral chain, and the other end terminal is, before ring-closure, an (natural or non-natural) amino acid having a carboxylic acid on its lateral chain, so that the amine and the carboxylic acid react to form an amide through a macrolactamisation. More specifically, one of the end terminals (natural or non-natural) amino acid residue can be substituted before ring-closure. After closure, the lateral chain carboxylic acid, activated by a coupling agent, has reacted with the amino group of the lateral chain of the other residue to form a peptide bond using, for example, a macrolactamisation reaction.

In another specific embodiment of any one of formula X, XIII and XV, X5’ is an alpha-methylleucine, valine, norleucine, cycloleucine, tert-leucine, cyclohexylalanine, or alpha-methylphenylalanine.

In another specific embodiment of any one of X, XIII and XV, X6’ is a tryptophane, thiazol-5-yl-alanine, 3-(2-pyridyl)- alanine, 3-(3-pyridyl)-alanine, or 3-(4-pyridyl)-alanine.

In specific embodiments of compounds of formula (I) to (XV), at least 4 (or at least 5, 6, 7, 8, 9, or 10, or more.) of the residues (e.g., residues a positions Xn defined above), other than residues closing the cycle which differ from the corresponding residues in AP-13 or Ela, correspond to those in AP-13 or Ela, respectively. For example, compound KT01-129 has at least 10 residues corresponding to those in AP-13.

In other specific embodiments, compounds of the present disclosure correspond to macrocyclic analogs of Ap13 or Ela, wherein the compounds vary from Ap13 by at least two substitutions at the positions closing the cycle, and by at least one (or 2, 3, 4, 5, 6, 7 or 8) further substitution(s), deletion(s) and/or insertion(s). These substitutions, deletions and/or insertions are defined in the various Xn of formula (I) to (XV) above. The correspondence between these Xn and Ap13 or Ela is shown in Tables A and B below, wherein the “[“ and “]” symbols are used to denote the positions of the ring closure residues in formula (I) to (XV) and compounds of the disclosure satisfying these formula. In formula (I) to (XV) unless specific otherwise, the link between two amino acid residues (natural or non-natural) are peptide bonds.

In another specific embodiment of formula (I) to (XV) one of the ring closing residues’ is Lys, Dap, Dab, Orn, and the other is Glu or Asp. In an embodiment, one of the end terminal (natural or non-natural) amino acid residue used for closing the is, before ring-closure, an (natural or non-natural) amino acid having an amine on its lateral chain, and the other end terminal is, before ring-closure, an (natural or non-natural) amino acid having a carboxylic acid on its lateral chain, so that the amine and the carboxylic acid react to form an amide through a macrolactamisation. More specifically, one of the end terminals (natural or non-natural) amino acid residue can be substituted before ring-closure. After closure, the lateral chain carboxylic acid, activated by a coupling agent, has reacted with the amino group of the lateral chain of the other residue to form a peptide bond using, for example, a macrolactamisation reaction.

In specific embodiments, the compound is any one of compounds in Table I below.

Table A

Table B

As used herein, the term “substituted” in reference to above listed natural or unnatural amino acid or acid residues in the structures refers to a substitution by an halogen (e.g., Cl, F, Br, I), -OH, hydroxy(C1-C6)alkyl, (C1-C6)alkyl, (C3- C6)aryl, (C3-C6)aryl(C1-C6)alkyl, (C3-C6)cycloalkyl, hetero(C3-C6)aryl, hetero(C3-C6)aryl(C1-C6)alkyl, hetero(C3- C6)cyclo(C1-C6)alkyl, amino(C1-C6)alkyl, amino(C3-C6)aryl, amino(C3-C6)aryl(C1-C6)alkyl, amino(C3- C6)cycloalkyl, aminohetero(C3-C6)aryl, aminohetero(C3-C6)aryl(C1-C6)alkyl, or amino hetero(C3-C6)cyclo(C1- C6)alkyl.

In specific embodiments, the size of the macrocycle can be of 15 to 24-ring atoms (or 15 to 23, 16 to 22, 17-20). In specific embodiments, the size of the macrocycle can be of 17- to 20-ring atoms.

In all the foregoing compounds, the residues (e.g., X1 to Xn) may be in L or D configurations. In all the foregoing combinations of two residues, they may be in the L, L; L-D; D, L; or D; D configurations.

Without being so limited, specific compounds of the present disclosure encompass those satisfying formula (I) to (XV). In a more specific embodiment, compounds of the present disclosure encompass compounds listed in Table I.

Nle: Norleucine

C: disulfur bond.

Unatural amino acids in compounds of Table I.

The present disclosure encompasses compounds of the present invention satisfying any one of formula (I), (II), (III), (Illa), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV) and (XV). In another specific embodiment, it encompasses a compound of formula (I), wherein X12 is not Pro or AllylGly. In another specific embodiment, it encompasses a compound of formula (III), wherein XT to X4’ are absent. In another specific embodiment, it encompasses a compound of any one of formula (X) to (XV). In another specific embodiment, it encompasses any one of compounds 1-158 of Table I. In another specific embodiment, it encompasses any one of compounds 30-62, and 110-158. In another specific embodiment, it encompasses any one of compounds 144-157.

Chemical groups

As used herein, the term “alkyl” refers to a monovalent straight or branched chain, saturated or unsaturated aliphatic hydrocarbon radical having a number of carbon atoms in the specified range. Thus, for example, “(C1-C8)alkyl” (or “(C1 -C8)alkyl”) refers to any alkyl of up to 8 carbon atom, including of the hexyl alkyl and pentyl alkyl isomers as well as n-, iso-, sec- and t-butyl, n- and iso- propyl, ethyl, and methyl. As another example, “(C1 -4)alkyl” refers to n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl, and methyl. As another example, “C1-3 alkyl” refers to n-propyl, isopropyl, ethyl, and methyl. Alkyl includes unsaturated aliphatic hydrocarbon including alkyne (R-C=C-R); and/or alkene (R- C=C-R). In preferred embodiments, the (C 1 -C8)alkyls of the present disclosure are (C1-C7)alkyls, (C1-C6)alkyls or (C1-C5)alkyls.

The term "halogen" (or “halo”) refers to fluorine, chlorine, bromine and iodine (alternatively referred to as fluoro, chloro, bromo, and iodo). The term "haloalkyl" refers to an alkyl group as defined above in which one or more of the hydrogen atoms have been replaced with a halogen (i.e., F, Cl, Br and/or I). Thus, for example, “C1-10 haloalkyl” (or “C1-C6 haloalkyl”) refers to a C1 to C10 linear or branched alkyl group as defined above with one or more halogen substituents. The term “fluoroalkyl” has an analogous meaning except that the halogen substituents are restricted to fluoro. Suitable fluoroalkyls include the series (CH2)0-4CF3 (i.e., trifluoromethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoro-n- propyl, etc.).

The term "heteroalkyl" is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms is replaced with a heteroatom (e.g., oxygen, nitrogen, sulfur, or derivatives thereof, and the like). Examples of heteroalkyl groups include, but are not limited to, alkoxy, alkyl-substituted amino, thiol such as methionine side group. Up to two heteroatoms may be consecutive. When a prefix such as C1-8 is used to refer to a heteroalkyl group, the number of carbons (1-8, in this example) is meant to include the heteroatoms as well.

The term "aminoalkyl" refers to an alkyl group as defined above in which one or more of the hydrogen or carbon atoms has been replaced with a nitrogen or an amino derivative such as but not limited to guanidine. Thus, for example, “C1-6 aminoalkyl” (or “C1-C6 aminoalkyl”) refers to a C1 to C6 linear or branched alkyl group as defined above with one or more amino derivatives (e.g., NH, amide, diazirin, azide, etc.).

The term "thioalkyl" refers to an alkyl group as defined above in which one or more of the hydrogen or carbon atoms has been replaced with a sulfur atom or thiol derivative. Thus, for example, “C1-6 thioalkyl” (or “C1-C6 thioalkyl”) refers to a C1 to C6 linear or branched alkyl group as defined above with one or more sulfur atoms or thiol derivatives (e.g., S, SH, etc.).

Aminoalkyl and thioalkyls are specific embodiments of and encompassed by the term “heteroalkyl” or substituted alkyl depending on the heteroatom replaces a carbon atom or an hydrogen atom.

The term "cycloalkyl" refers to saturated alicyclic hydrocarbon consisting of saturated 3-8 membered rings optionally fused with additional (1-3) aliphatic (cycloalkyl) or aromatic ring systems, each additional ring consisting of a 3-8 membered ring. It includes without being so limited cyclopropyl, cyclobutyl, cyclopentyl (cyp) (e.g., compounds 38-41 and 63-65), cyclohexyl and cycloheptane.

The term "heterocyclyl" refers to (i) a 4- to 7-membered saturated heterocyclic ring containing from 1 to 3 heteroatoms independently selected from N, 0 and S, or (ii) is a heterobicyclic ring (e.g., benzocyclopentyl, octahydroindol (e.g., compound 166)). Examples of 4- to 7-membered, saturated heterocyclic rings within the scope of this disclosure include, for example, azetidinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, oxazolidinyl, isoxazolidinyl, pyrrolidinyl, pyridine, imidazolidinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrazolidinyl, hexahydropyrimidinyl, thiazinanyl, thiazepanyl, azepanyl, diazepanyl, tetrahydropyranyl, tetrahydrothiopyranyl, and dioxanyl. Examples of 4- to 7-membered, unsaturated heterocyclic rings within the scope of this disclosure include mono-unsaturated heterocyclic rings corresponding to the saturated heterocyclic rings listed in the preceding sentence in which a single bond is replaced with a double bond (e.g., a carbon-carbon single bond is replaced with a carbon-carbon double bond).

The term "C(O)" refers to carbonyl. The terms "S(O)2" and "SO2" each refer to sulfonyl. The term "S(O)" refers to sulfinyl.

The term "aryl" refers to aromatic (unsaturated) compounds consisting of 3-8 membered rings, optionally fused with additional (1-3) aliphatic (cycloalkyl) or aromatic ring systems, each additional ring consisting of 3-8 membered ring (such as anthracene, indane, Tic, 3-benzothienylalanine, or dihydroindol. In a specific embodiment, it refers to phenyl, benzocyclopentyl, or naphthyl.

The term "heteroaryl" refers to (i) a 3-, 4-, 5- , 6-, 7- or 8-membered heteroaromatic ring (more specifically 3-7 or 3-6 membered ring) containing from 1 to 4 heteroatoms independently selected from N, 0 and S, such as thiophenyl, thienyl, pyridine, or (ii) is a heterobicyclic ring selected from indolyl, quinolinyl, isoquinolinyl, Tic, dihydroindolylglycine and quinoxalinyl. Suitable 3-, 4-, 5- and 6-membered heteroaromatic rings include, for example, diazirin, pyridyl (also referred to as pyridinyl), pyrrolyl, diazine (e.g., pyrazinyl, pyrimidinyl, pyridazinyl), triazinyl, thienyl, furanyl, imidazolyl, pyrazolyl, triazolyl (e.g., 1 , 2, 3 triazolyl), tetrazolyl (e.g., 1 , 2, 3, 4 tetrazolyl), oxazolyl, iso- oxazolyl, oxadiazolyl, oxatriazolyl, thiazolyl, isothiazolyl, and thiadiazolyl. Heteroaryls of particular interest are pyrrolyl, imidazolyl, pyridyl, pyrazinyl, quinolinyl (or quinolyl), isoquinolinyl (or isoquinolyl), and quinoxalinyl. Suitable heterobicyclic rings include indolyl.

The term “aralkyl” and more specifically “(C4-C 14)aralkyl” or “C4-14 aralkyl” refers herein to compounds comprising a 3-7 ring-member aryl substituted by a 1 to 7 alkyl. In specific embodiments, it refers to a benzyl or a phenetyl. As used herein, and unless otherwise specified, the terms “alkyl”, "haloalkyl", "aminoalkyl", "cycloalkyl", "heterocyclyl", “aryl”, “heteroalkyl” and “heteroaryl” and the terms designating their specific embodiments (e.g., butyl, fluoropropyl, aminobutyl, cyclopropane, morpholine, phenyl, pyrazole, etc.) encompass the substituted (i.e., in the case of haloalkyl and aminoalkyl, in addition to their halogen and nitrogen substituents, respectively) and unsubstituted embodiments of these groups. Hence for example, the term “phenyl” encompasses unsubstituted phenyl as well as fluorophenyl, hydroxyphenyl, methylsulfonyl phenyl (or biphenyl), diphenyl, trifluoromethyl-diazirin- phenyl, isopropyl-phenyl, trifluorohydroxy-phenyl. Similarly, the term pyrazole, encompass unsubstituted pyrazole as well as methylpyrazole. The one or more substituents may be an amine, halogen, hydroxyl, C1-6 aminoalkyl, C1-6 heteroalkyl, C1-6 alkyl, C3-8 cycloalkyl, C1-6 haloalkyl, aryl, heteroaryl and heterocyclyl groups (etc.).

It is understood that the specific rings listed above are not a limitation on the rings which can be used in the present disclosure. These rings are merely representative.

Unless expressly stated to the contrary in a particular context, any of the various cyclic rings and ring systems described herein may be attached to the rest of the compound at any ring atom (i.e., any carbon atom or any heteroatom) provided that a stable compound results therefrom.

Isomers, tautomers and polymorphs

As used herein, the term “isomers” refers to stereoisomers including optical isomers (enantiomers), diastereoisomers as well as the other known types of isomers.

The compounds of the disclosure have at least 5 asymmetric carbon atoms and can therefore exist in the form of optically pure enantiomers (optical isomers), and as mixtures thereof (racemates). It is to be understood, that, unless otherwise specified, the present disclosure embraces the racemates, the enantiomers and/or the diastereoisomers of the compounds of the disclosure as well as mixtures thereof. Furthermore, certain macrocyclic compounds of the present invention comprise an alkene closing the cycle. Such compounds have Z and E isomers.

For further clarity, (S)-H or (S)-CH3 indicates that the stereogenic center bearing the H or CH3 substituent is of (S) stereochemistry.

In addition, the present disclosure embraces all geometric isomers. For example, when a compound of the disclosure incorporates a double bond or a fused ring, both the cis- and trans-forms, as well as mixtures, are embraced within the scope of the disclosure.

Within the present disclosure, it is to be understood that a compound of the disclosure may exhibit the phenomenon of tautomerism and that the formula drawings within this specification can represent only one of the possible tautomeric forms. It is to be understood that the disclosure encompasses any tautomeric form and is not to be limited merely to any one tautomeric form utilized within the formula drawings.

It is also to be understood that certain compounds of the disclosure may exhibit polymorphism, and that the present disclosure encompasses all such forms.

Salts The present disclosure relates to the compounds of the disclosure as hereinbefore defined as well as to salts thereof. The term “salt(s)”, as employed herein, denotes basic salts formed with inorganic and/or organic bases. Salts for use in pharmaceutical compositions will be pharmaceutically acceptable salts, but other salts may be useful in the production of the compounds of the disclosure. The term "pharmaceutically acceptable salts" refers to salts of compounds of the present disclosure that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these salts retain the biological effectiveness and properties of the anti-atherosclerosis compounds of the disclosure and are formed from suitable non-toxic organic or inorganic acids or bases.

For example, where the compounds of the disclosure are sufficiently acidic, the salts of the disclosure include base salts formed with an inorganic or organic base. Such salts include alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts and a cobalt salts; inorganic amine salts such as ammonium or substituted ammonium salts, such as e.g., trimethylammonium salts; and salts with organic bases (for example, organic amines) such as chloroprocaine salts, dibenzylamine salts, dicyclohexylamine salts, dicyclohexylamines, diethanolamine salts, ethylamine salts (including diethylamine salts and triethylamine salts), ethylenediamine salts, glucosamine salts, guanidine salts, methylamine salts (including dimethylamine salts and tri methyl amine salts), morpholine salts, morpholine salts, N,N'-dibenzylethylenediamine salts, N-benzyl-phenethylamine salts, N- methylglucamine salts, phenylglycine alkyl ester salts, piperazine salts, piperidine salts, procaine salts, t-butyl amines salts, tetramethylammonium salts, t-octylamine salts, tris-(2-hydroxyethyl)amine salts, and tris(hydroxymethyl)aminomethane salts. Preferred salts include those formed with sodium, lithium, potassium, calcium and magnesium.

Such salts can be formed routinely by those skilled in the art using standard techniques. Indeed, the chemical modification of a pharmaceutical compound (i.e., drug) into a salt is a technique well known to pharmaceutical chemists, (See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457, incorporated herein by reference). Salts of the compounds of the disclosure may be formed, for example, by reacting a compound of the disclosure with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

Esters

The present disclosure relates to the compounds of the disclosure as hereinbefore defined as well as to the esters thereof. The term “ester(s)”, as employed herein, refers to compounds of the disclosure or salts thereof in which a carboxylic acid has been hydroxy groups have been converted to the corresponding esters using an alcohol and a coupling reagent. Esters for use in pharmaceutical compositions will be pharmaceutically acceptable esters, but other esters may be useful in the production of the compounds of the disclosure.

The term "pharmaceutically acceptable esters" refers to esters of the compounds of the present disclosure that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these esters retain the biological effectiveness and properties of the anti-atherosclerosis compounds of the disclosure and act as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, cleave in such a manner as to produce the parent alcohol compounds.

Esters of the compounds of the present disclosure include among others the following groups (1) carboxylic acid esters obtained by esterification, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, ethyl, n-propyl, t-butyl, n-butyl, methyl, propyl, isopropyl, butyl, isobutyl, or pentyl), n-hexyl, alkoxyalkyl (for example, methoxymethyl, acetoxymethyl, and 2,2- dimethylpropionyloxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, C1-4 alkyl, or C1-4 alkoxy, or amino).

Further information concerning examples of and the use of esters for the delivery of pharmaceutical compounds is available in Design of Prodrugs. Bundgaard H ed. (Elsevier, 1985) incorporated herein by reference. See also, H. Ansel et. al., 1995 at pp. 108-109; Krogsgaard-Larsen, 1996 at pp. 152-191 ; Jarkko Rautio, 2008; and Pen-Wei Hsieh, 2009, all incorporated herein by reference.

The compounds of this disclosure may be esterified by a variety of conventional procedures including the esters are formed from the acid of the molecule by reacting with a coupling agent such as DIC (diisopropyl carbodiimide) and a base, such as NN-dimethylaminopyridine (DMAP), and an alcohol, such as methanol (methyl ester), ethanol, longer chain alcohols or benzyl alcohol (benzyl ester). One skilled in the art would readily know how to successfully carry out these as well as other known methods of esterification of acid.

Esters of the compounds of the disclosure may form salts. Where this is the case, this is achieved by conventional techniques as described above.

Solvates

The compounds of the disclosure may exist in unsolvated as well as solvated forms with solvents such as water, ethanol, and the like, and it is intended that the disclosure embrace both solvated and unsolvated forms.

“Solvate” means a physical association of a compounds of this disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates. Solvates for use in pharmaceutical compositions will be pharmaceutically acceptable solvates, but other solvates may be useful in the production of the compounds of the disclosure.

As used herein, the term “pharmaceutically acceptable solvates” means solvates of compounds of the present disclosure that are pharmacologically acceptable and substantially non-toxic to the subject to which they are administered. More specifically, these solvates retain the biological effectiveness and properties of the antiatherosclerosis compounds of the disclosure and are formed from suitable non-toxic solvents.

Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like, as well as hydrates, which are solvates wherein the solvent molecules are H2O.

Preparation of solvates is generally known. Thus, for example, Caira, 2004, incorporated herein by reference, describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by van Tonder, 2004; Bingham, 2001, both incorporated herein by reference.

A typical, non-limiting, process for preparing a solvate involves dissolving the inventive compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example IR spectroscopy, can be used to show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

Compositions, Combination and kits

Compositions

The present disclosure also relates to pharmaceutical compositions comprising the above-mentioned compounds of the disclosure or their pharmaceutically acceptable salts, esters and solvates thereof and optionally a pharmaceutically acceptable carrier.

As used herein, the terms “pharmaceutically acceptable” refer to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to subjects (e.g., humans). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compounds of the present disclosure may be administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin. The pharmaceutical compositions of the present disclosure may also contain excipients/carriers such as preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants.

Diseases

As used herein the term “muscular dystrophy” or “MD” refers to any muscular dystrophy including but not limited to laminin alpha-2 deficient muscular dystrophy (also called merosin-deficient congenital muscular dystrophy (MDCIA)), congenital muscular dystrophy, ColVI-related myopathy, Duchenne muscular dystrophy, Ullrich congenital muscular dystrophy (UCMD) and Becker muscular dystrophy (BMD).

As used herein “symptoms of muscular dystrophy” includes any one of frequent falls; difficulty rising from a lying or sitting position; trouble running and jumping; waddling gait; walking on the toes; large calf muscles; muscle pain and stiffness; learning disabilities; delayed growth and reduced survival. It further includes a reduced body weight, heart fibrosis; defective heart function; asynchronous, multi-focal skeletal muscle fiber degeneration; skeletal muscle inflammation and fibrosis; increased level of regeneration marker eMHC (embryonic or developmental myosin heavy chain); decrease in number and function of MuSCs in skeletal muscle tissue; lower skeletal muscle fiber numbers, delayed skeletal muscle fiber maturation, and decreased muscle strength.

Treatment and prevention

The terms “treat/treating/treatment” and “prevent/preventing/prevention” as used herein, refers to eliciting the desired biological response, i.e., a therapeutic and prophylactic effect, respectively. In accordance with the disclosure herein, the therapeutic effect comprises one or more of a decrease/reduction in the severity of MD, a decrease/reduction in the frequency, duration and/or severity of at least one symptom thereof, or an increase in frequency and/or duration of disease symptom-free periods following administration of the at least one compound of the present disclosure (e.g., AP-13, structural or functional analogues thereof), or of a composition comprising the compound of the present disclosure, alone or in combination with another agent for the prevention or treatment of a MD or of at least one symptom thereof. In accordance with the disclosure provided herein, in some embodiments, a prophylactic effect comprises a complete or partial avoidance/inhibition of MD or of at least one a symptom thereof following administration of the at least one compound of the present disclosure, or of a composition comprising the compound of the present disclosure, alone or in combination with another agent for the prevention or treatment of a MD or of at least one a symptom thereof.

In some embodiments, "therapeutically effective amount" or “effective amount” or "therapeutically effective dosage" of a compound of the present disclosure provided herein results in a treatment or prevention of MD or of at least one a symptom thereof in a subject.

As used herein the term “subject” is meant to refer to any animal, such as a mammal including human, mice, rat, dog, cat, pig, cow, monkey, horse, etc. In a particular embodiment, it refers to a human. “Subject in need thereof” is a subject that has an MuSC pathology such as a MD or is a likely candidate for an MuSC pathology such as a MD.

As used herein the term “likely candidate for muscular dystrophy muscular dystrophy” is meant to refer to subjects with genetic predisposition for MD and subjects with a family history comprising MD.

In some embodiments, compositions provided herein are administered by one or more routes of administration using one or more of a variety of suitable methods. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for compounds of the present disclosure (e.g., AP-13, agonist of AP-13 function or expression, etc.) for uses and methods herein include, but are not limited to, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. Alternatively, compounds of the present disclosure provided herein are administered by a non-parenteral route, such as oral (see e.g., US 7,875,648 B2 to Meier), a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds, in some embodiments, are prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers used in some embodiments, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. In some embodiments, therapeutic compositions are administered with medical devices known in the art. For example, in one embodiment, therapeutic compositions provided herein are administered with a needleless hypodermic injection device.

In certain embodiments, the compounds of the present disclosure (e.g., AP-13, structural or functional AP-13 analogues thereof) provided herein are formulated to ensure proper distribution in vivo.

Combination

Compounds of the present disclosure can be administered together with another therapy for the treatment or prevention of MD or at least one symptom thereof (e.g., therapeutic agent or non-pharmacological therapies such as orthopedic interventions). In some embodiments, the two are alternatively administered sequentially in either order; or administered simultaneously (in the same composition or in different compositions). In some embodiments, a compound of the present disclosure is administered to a subject who is also receiving therapy with another agent for preventing or treating MD or at least a symptom thereof. Other agents potentially useful for preventing or treating MD, or at least a symptom thereof include Ataluren, Drisapersen, Omigapil, glucocorticoids, Losartan, antioxidants, coenzyme Q10 derivative (e.g., Idebenone de Santera).

In some embodiments, the combination therapy regimen is additive. In some embodiments, the combination therapy regimen produces synergistic results (e.g., increase in muscular strength greater than expected for the combined use of the two agents). In some subjects, this allows reduction in the dosage of the other agent for preventing or treating MD. Compounds of the present disclosure, in some embodiments, are useful for subjects who are intolerant to therapy with the other active agent, or for whom therapy with the other active agent has produced inadequate results.

Similarly, as used herein, the term “increase” or “increasing” (e.g., MuSC function, or AP-13 activity) of at least 10% as compared to a control, in an embodiment of at least 20% higher, in a further embodiment of at least 30% higher, in a further embodiment of at least 40% higher, in a further embodiment of at least 50% higher, in a further embodiment of at least 60% higher, in a further embodiment of at least 70% higher, in a further embodiment of at least 80% higher, in a further embodiment of at least 90% higher, in a further embodiment of 100% higher, in a further embodiment of 200% higher, etc.

As used herein, the term “decrease” or “reduction” (e.g., MuSC function or a MD symptom) refers to a reduction of at least 10% as compared to a control, in an embodiment of at least 20% lower, in a further embodiment of at least 30% lower, in a further embodiment of at least 40% lower, in a further embodiment of at least 50% lower, in a further embodiment of at least 60% lower, in a further embodiment of at least 70% lower, in a further embodiment of at least 80% lower, in a further embodiment of at least 90% lower, in a further embodiment of 100% (complete inhibition).

The “control” for use as reference in the method disclosed herein is a cell (in the context of e.g., method of testing stimulators of MuSC function in vitro or in cellulo) or subject (human or model animal) not treated with a compound of the present disclosure). In the context of a method of preventing or treating laminin alpha2 deficient MD or of a symptom thereof may be e.g., a control subject (or model animal) that has MD, and that is not treated with a compound of the present disclosure.

Methods/assays to determine AP-13 activity are further described below.

Kits

Also provided is a kit for preventing or treating MD, comprising (a) the compound (e.g., of any one of the formula described herein), stereoisomer, mixture, pharmaceutically acceptable salt, ester or solvate thereof defined herein; and (b) (i) at least another compound (e.g., of any one of the formula described herein), stereoisomer, mixture, pharmaceutically acceptable salt, ester or solvate thereof defined herein; (ii) another agent for preventing or treating MD or a symptom thereof; (iii) a pharmaceutically acceptable carrier; (iv) instructions to use the kit in the prevention or treatment of MD or a symptom thereof; or (v) a combination of at least two of (i) to (iv).

In some embodiments, candidate compounds are tested for the ability to increase MuSC function (or AP-13 activity or expression). Each “dose-responses” experiment is done in triplicate for 4 to 6 different dosages.

Method of screening

The activity of identified compounds may be tested with methods known in the art such as that described in example 4.

The present disclosure is illustrated in further details by the following non-limiting examples. EXAMPLE 1 : Material and Methods

Mice and animal care

Mouse models of Duchenne MD (mdx), laminin alpha-2 deficient (LAMA2) MD (dyW) and Collagen VI (ColVI)- related myopathy (d16) were used in the present disclosure. DyW mice on C57BL/6N background die prematurely and display a global muscle wasting phenotype, including severe kyphosis and reduced body weight. Mdx and d16 mice are comparably healthy and skeletal muscle phenotypes only manifest strongly after experimental muscle injury (e.g., cardiotoxin). Through them, impaired expansion capacity of the stem cell pool and aberrant changes in the endothelial cell (EC) compartment of the niche are a common denominator of all three types of MD.

Husbandry and all experimental protocols using mice were performed in accordance with the guidelines established by the animal committee of the Universite de Sherbrooke and the Centre Hospitalier Universitaire Sainte-Justine of the Universite de Montreal, which are based on the guidelines of the Canadian Council on Animal Care, mdx (Jackson Laboratory, Stock No: 013141), d16 (Jackson Laboratory, Stock No: 024972), and dyW mice (Jackson Laboratory, Stock No: 013786) were generally analyzed at 6-8 weeks of age. Age matched C57BL/6 mice (Charles River, strain 027) were used as wt Ctrl animals. Physiological and functional testing of veh or AP-13 treated dyW mice was performed at 5 weeks of age. Female and male mice were included at equal proportions. To ensure optimal access to water and food, cages containing dyW mice were supplied with long-necked water bottles and wet food. For AP-13 or veh treatment, mice were implanted with equilibrated osmotic minipumps (Alzet, model 1004) at the age of 14 days (dyW mice) or 42 days (mdx and d16 mice) through a small incision at the level of the scapula. Surgery was performed under isoflurane anesthesia. Osmotic minipumps were loaded either with veh (100 pil of 0.9% NaCI) or AP-13 (1 mg/kg/day). APJECKO mice were generated by crossing Cdh5(PAC)CreERT2 (Taconic) and APJ flox mice (Charo 2009). To trigger genetic recombination, mice were injected with tamoxifen (Toronto Research Chemicals Inc, T006000-25) dissolved in corn oil (i.p., 2mg) during five consecutive days.

Muscle regeneration and histology

Muscle injury was induced by injection of 50 pl of 10 piM cardiotoxin (CTX, Latoxan, L8102) from Naja Mossambica, a snake venom that induces muonecrosis, into the tibialis anterior (TA) muscle of isoflurane-anesthetized animals that were treated with a single dose of buprenorphine for pain management. Following euthanasia by CO2, TA muscles were harvested and embedded in gum tragacanth (Sigma, G1128), snap-frozen in liquid nitrogen chilled isopentan, and stored at -80 °C. 10 pm thick muscle cryosections were stained using hematoxylin and eosin (Sigma, MHS16). Images have been acquired using a Nanozoomer™ scanner (Hamamatsu, C10730-12). For immunostaining, muscle cryosections were fixed with 4% paraformaldehyde (TCI America, P0018) for 10 min and then permeabilized with 0.5% Triton-X (Sigma, T8787) for 10 min. Sections were blocked in 5% bovine serum albumin (Thermo Fisher Scientific, BP9703100) for at least 1 h at room temperature. Primary antibodies (eMHC, DSHB, F1.652; laminin, Sigma-Aldrich, L9393; dystrophin, DSHB, MANDRA1-7A10; Pax7, DSHB; myogenin, Abeam, ab124800; CD31 , Thermo Fisher Scientific, 14-0311-082; fibronectin, Sigma-Aldrich F3648; PDGFRa, R&D systems, AF1062; F4/80, Biorad, MCA497RT; and APJ, ProteinTech 20341 -1-AP) were diluted in blocking solution and incubated overnight at 4 °C in a wet chamber. Appropriate secondary antibodies (Thermo Fisher Scientific) and Hoechst (Thermo Fisher Scientific, 62249) were applied for 2 h at room temperature, and mounted using Mowiol (Sigma, 81381) for image acquisition. For Pax7 staining, antigen retrieval using hot 10 mM sodium citrate buffer (Sigma, S4641) supplemented with 0.05% Tween 20 (Bio Basic Canada, TB0560) was performed for 20 min and Fab mouse antigen fragment (Jackson ImmunoResearch, 115-007-003) was added during the blocking step. For visualization, an lgG1 specific secondary antibody was used. For minimal fiber feret analysis, sections were stained with dystrophin and analyzed using the Open-CSAM Imaged macro (Desgeorgeset al., 2019).

Analysis of single-cell RNA-sequencinq data

A gene expression matrix of RNA-seq data in FPKM (GEO, GSE143437) from tissue resident endothelial cells was used to generate a gene list with the gene ontology tag GO: 0004930 “G protein-coupled receptor activity” using the Biomart™ mining tool of Ensembl. From the curated expression matrix containing only G protein-coupled receptor (GPCR) activity-related genes, Iog2 expression of the top expressed GPCRs with cognate ligands was extracted manually and mapped using GraphPad™ Prism. Colored Uniform Manifold and Projection (UMAP) plots were generated using Seurat version 4.04 based on single cell sequencing data of TA muscles at 5 dpi22,40,41 (GEO: GSE143437). Individual cells were colored based on their expression levels of genes APJ, Pax7 and CD31 .

Apelin-13 Synthesis

Pyr-apelin-13 (AP-13) was synthesized using Fmoc chemistry on solid support as previously described (Murza et al., 2015 and FIG. 4). Briefly, 2-chloro trityl chloride resin (2-CTC, Matrix Innovation, 2-401-1310) was mixed with a solution of amino acid (Fmoc-L-Phe-OH, 1.2 equiv, Chem-lmpex International, 02443), N, W-diisopropylethylamine (DIPEA, 2.5 equiv, Chem-lmpex International, 00141) in dichloromethane (DCM, Thermo Fisher Scientific, D37-20) overnight at room temperature. After removing excess reagents by filtration, the resin was washed consecutively with DCM, isopropanol (Thermo Fisher Scientific, A416-20), DCM, isopropanol, and DCM for 3 min for each solvent. This washing sequence was used to rinse the resin after every reaction (i.e., capping, deprotection or amino acid coupling). Unreacted groups were capped with a mixture of DCM, Methanol (Thermo Fisher Scientific, A412-20), and DIPEA (3.5:1 :0.5) for 1 h. The next amino acid was added to the peptide sequence in two steps: Fmoc deprotection and amino acid coupling. The Fmoc protecting group was removed by treating resin twice with 20% piperidine (Chem Impex International, 02351) in W,W-dimethylformamide (DMF, Thermo Fisher Scientific, D119-20) for 10 min. The coupling step was carried out using O-(7-Azabenzotriazol-1-yl)-A/,A/,A/',/\/-tetramethyluronium hexafluorophosphate (HATU, 5 equiv, Matrix Innovation, 1-063-0001), amino acid (5 equiv), and DIPEA (5 equiv) in DMF at room temperature for 30 min. These steps were repeated to build the full sequence of Pyr-apelin-13. Cleavage of the peptide from the resin and amino acid sidechain deprotection was carried out using a mixture of trifluoroacetic acid (TFA, Chem Impex International, 00289), triisopropylsilane (TIPS, Oakwook Chemical, S17975), ethanedithiol (EDT, Sigma, 8.00795), and water (92.5:2.5:2.5:2.5). The crude peptide was precipitated in tert-butyl methyl ether (TBME, ACROS Organics, AC378720025). After the supernatant was removed by centrifugation, the peptide was dissolved in 10% acetic acid and the aqueous layer was extracted, filtered, and purified by preparative HPLC (ACE5 C18 column 250 x 21.2 mm, 5 pm spherical particle size). The purity (> 95%) and authenticity of the peptide were confirmed by UPLC-MS Waters (Milford, USA, column Acquity UPLC CSH C18, 2.1 x 50 mm packed with 1.7 pirn particles) and high-resolution mass spectrometry (electrospray infusion on a maXis ESI-Q-Tof apparatus from Bruker, Billerica, USA).

General protocol for synthesis of macrocyclic compounds of Table I

Materials'. Fmoc-protected (L)-amino acids, 2-chlorotrityl chloride resin and [O-(7-azabenzotriazol-1-yl)-1 , 1 ,3,3- tetramethyluronium hexafluorophosphate] (HATU) were purchased from Matrix Innovation (Canada). N,N- diisopropylethylamine (DIPEA) and unnatural amino acids were purchased from Chem Impex (USA). Piperidine was purchased from ACP (Canada). All other solvents were purchased from Sigma-Aldrich (Canada) or Fisher Scientific (USA) and were of the highest commercially available purity. All reagents and starting materials were used as received. The peptide elongation was performed with a Symphony™ X peptide synthesizer from Gyros Protein Technology (USA).

Step 1: Loading of the 2-chlorotrityl chloride resin To load the first amino acid of the sequence, 2-chlorotrityl chloride resin (0.3 mmol/g, 300 mg) was treated with Fmoc-protected amino acid (1 equiv.), N, W-diisopropylethylamine (DIPEA, 2 equiv.), in dichloromethane (DCM, 4 mL). The mixture was shaken for 2 h on an orbital shaker at room temperature, then the resin was sequentially washed for 3-min periods with DCM (2 x 5 mL), 2-propanol (1 x 5 mL), DCM (1 x 5 mL), 2-propanol (1 x 5 mL), DCM (2 x 5 mL). A capping solution of DCM/MeOH/DIPEA (7/2/1 , 5 mL) was then added and the mixture shaken for 1 h at room temperature and washed with the above solvent sequence.

Step 2: Peptide elongation. The peptide synthesis was carried out with the typical Fmoc solid phase peptide synthesis (SPPS) procedure. 2-chlorotrityl chloride resin (0.3 mmol/g, 300 mg, loaded with the first amino acid of the sequence) was placed in a peptide synthesizer reactor and swell with A/,/V-dimethylformamide (DMF) (3 x 6 min, 4.5 mL). To be noted that the resin during coupling, deprotection and washing steps is mixed via N2 bubbling. The Fmoc group was then deprotected with 20% piperidine/DMF (2 x 5 min, 4.5 mL), then the subsequent Fmoc-protected amino acid (5 equiv.) was attached in the presence of HATU (5 equiv.), DIPEA (10 equiv.) in DMF/NMP (4.5 mL) and the reaction proceeded for 30 min. Then piperidine (20% in DMF) was used to deprotect the Fmoc group at every step. The resin was washed after each coupling and Fmoc deprotection step with DMF (4 x 1 min 30 s, 4.5 mL).

Step 3: Allyl / Aloe Deptrotection In a typical procedure, after coupling the last amino acid of the sequence, the Allyl /Aloe protecting groups were selectively deprotected with Tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) (0.2 equiv.) and Phenylsilane (PhSiHa) (20 equiv.) in Argon degassed DCM (5 mL) and the reaction proceeded during 30 min). The resin was then washed with DMF (3 x 1 min 30 s, 4.5 mL) and DCM (5 x 6 min, 4.5 mL).

Step 4: Macro-lactamization - Cleavage / deprotections Then, the macro-lactamization were carried out with 3- (diethoxyphosphoryloxy)-1 ,2,3-benzotriazin-4(3H)-one (DEPBT) (5 equiv.) and DIPEA (5 equiv.) in DMF (5 mL) during 16 h. After resin washings (DMF, 5 x 1 min 30 s, 4.5 mL), macrocycles were cleaved from the resin and the protecting groups were removed with a mixture of TFA (trifluoroacetic acid)/H2O/TIPS (triisopropylsilane) 95/2.5/2.5, v/v (2 mL / 0.2 g of resin) for 4 h at room temperature. The crude peptide was either precipitated in tert-butyl methyl ether (TBME) at 0°C, centrifuged and the supernatant removed, or the crude peptide was directly evaporated under vacuum. The crude peptide was then re-dissolved in 7:3 ^O/acetonitrile (ACN) and lyophilized before purification by reverse-phase HPLC.

Purification and characterization'. The crude peptide was re-suspended in 7:3 ^O/acetonitrile (ACN) and purified on a preparative HPLC-MS system from Waters (Milford, USA) (column XSELECT™ CSH™ Prep C18 (19 x 100 mm) packed with 5 pm particles, UV detector 2998, MS SQ Detector 2, Sample manager 2767 and a binary gradient module) using acetonitrile and water + 0.1 % formic acid as eluents. Pure fractions were lyophilized to give the final product as a white solid. For purity assessment, compounds were analyzed on an UPLC-MS system from Waters (Milford, USA) (column Acquity UPLC® CSH™ C18 (2.1 x 50 mm) packed with 1.7 pm particles) with the following gradient: acetonitrile and water with 0.1 % HCOOH (0— >0.2 min: 5% acetonitrile; 0.2— >1.5 min: 5%— >95%; 1.5— >1.8 min: 95%; 1.8^2.0 min: 95%^5%; 2.0^2.5 min: 5%).

Specific synthesis protocols for macrocyclic compounds of the present disclosure are shown or described in FIGs. 10, 11A-11 B, 12-13 and below.

Synthesis and characterization of C-terminal Apelin macrocyclic analogues

The synthesis of KT01-129 (Pyr-R-P-R-L-S-H-K-c[Allyl(o-nosylDab)-P-Nle-P-Allyl(G)] (SEQ ID NO: 88)) is presented in FIG. 10 and further described below.

Step 1 : Amino acid loading (0.1 mmol scale): in a 12 mL cartridge, Wang resin (loading max 0.85 mmol/g), Fmoc-L- allylglycine-OH (0.3 mmol) and triphenylphosphine (78 mg, 0.3 mmol) were suspended in 4 mL anhydrous tetrahydrofuran (THF) and mixed for 5 min. DIAD (60 pL, 0.3 mmol) was added dropwise (exothermic reaction) and this mixture was agitated overnight. The excess reagents were filtered and the resin was washed with DMF-DCM- iPrOH-DCM-iPrOH-DCM (5 mL solvent, 3 min cycles). This washing sequence was also used after every reaction (deprotection/coupling). Unreacted groups were capped using 5 mL of a solution of DCM-acetic anhydride-DIPEA (4:1 :0.2).

Step 2: Amino acid coupling: resin was treated with 20 % piperidine/DMF (5 mL, 2 x 10 min) to remove Fmoc group. The next amino acid was coupled using a mixture of Fmoc-protected amino acid (0.5 mmol), HATU (190 mg, 0.5 mmol) and DIPEA (87 piL, 0.5 mmol) in 5 mL DMF for 30 min. Deprotection and coupling steps were repeated until finishing the Fmoc-Dab(Alloc)-OH coupling.

Step 3:Alloc deprotection: dried resin (300 mg, 0.1 mmol peptide) was swelled with 3 mL DCM in a 10 mL microwave tube. This suspension was bubbled with Ar for 5 min. PheSiHa (0.3 mL, 2.5 mmol, 25 equiv.) was added and mixed for another 5 min. Pd(PPh3)4 (52 mg, 0.05 mmol, 0.5 equiv.) was added into the reaction mixture and stirred under Ar for 30 min at room temperature. Resin was filtered then washed to remove excess reagents.

Step 4: Nosylation - allylation. Nosylation, To the resin (0.1 mmol peptide), a solution of o-nosyl chloride (86 mg, 0.4 mmol; 4 equiv.) and sym-collidine (0.055 mL, 0.42 mmol, 4 equiv.) in 5 mL of NMP was added and agitated for 30 minutes at room temperature. Resin was filtered and washed with NMP. Those steps were repeated once. Upon completing the nosylation, the resin was washed with the washing sequence and dried in vacuo. Allylation. Well-dried resin was swelled and washed with 4 mL of anhydrous THF for 5 minutes. A mixture of allylic alcohol (0.068 mL, 1 mmol, 10 equiv.), PPha (106 mg, 0.4 mmol, 4 equiv.) in 3 mL of anhydrous THF was poured into the resin and mixed for 5 min. Finally, DIAD (0.079 mL, 0.4 mmol; 4 equiv.) was added dropwise and the mixture was agitated for 20 min at room temperature. Resin was filtered, washed with DCM. The reaction was monitored by UPLC and the allylation reaction was repeated until completed conversion (usually completed after 1 repeat reaction). The resin was washed with diethyl ether and dried in vacuo.

Step 5: Metathesis: dry resin (0.1 mmol peptide) was swelled with 3 mL 1 ,2-dichloroethane (DCE) in a 10 mL microwave tube equipped with a stirring bar. This suspension was bubbled with argon for 30 min. Subsequently, 15 mg (0.023 mmol, 0.23 equiv.) of Hoveyda-Grubbs II ^nd generation catalyst was added and the reaction mixture bubbled with argon for another 5 min. To initiate the reaction, the mixture was submitted to microwave irradiation in a Discover™SP microwave oven (CEM, Matthews, USA) with following parameters: reaction temperature 120 °C, time 10 min, maximum power 300 W. After the reaction, the resin was filtered and washed with the washing sequence.

Step 6: Peptide coupling: Similar to step 2. Repeated until completing the peptide sequence.

Step 7: Cleavage- purification: final cleavage from the resin and simultaneous sidechain deprotections were performed with 4 mL of a TFA-TIPS-H2O (95:2.5:2.5) mixture for 4 hours at room temperature. The cleavage solution was dropped slowly into 35 mL of TBME at 0°C. The mixture was centrifuged at 2000 rpm for 10 min at 4°C and crude peptide was isolated as a light brown solid after removal of the supernatant. Peptide purification. The crude product was re-suspended in acetic acid 10 %, the aqueous layer was separated and filtered. The peptide was purified on a preparative HPLC-MS system from Waters (Milford, USA) (column XSELECT™ CSH™ Prep C18 (19 x 100 mm) packed with 5 pm particles, UV detector 2998, MS SQ Detector 2, Sample manager 2767 and a binary gradient module) using acetonitrile and water + 0.1 % formic acid as eluents (gradient 13-28% acetonitrile in water for 15 min). Pure fractions were lyophilized to give the final product as a white solid powder.

Molecular weight: 1705.96 Da. Chemical formula: C75H116N24O20S. HRMS (ESI+): 853.4342 [M+2H] ² - (calc.: 853.4332). 0.1 mmol scale. Yield: 3.5 mg. Purity: 99%. 0.25 mmol scale. Yield: 52 mg. Purity: 96%.

Synthesis and characterization of apelin macrocyclic functional analogues

The synthesis of illustrative compound of formula X-XI, namely AM03-68 (c[K-R-R-E]-Nle-C-L-H-C-Orn-V-P-F-P (SEQ ID NO: 145))) is presented in FIGs. 11A-B and further described below.

Step 1 : (0.1 mmol scale), 2-chloro trityl chloride resin (2-CTC, 400 mg, 0.25 mmol/g loading) was shaken with a solution of amino acid (Fmoc-L-Pro-OH, 0.12 mmol, 1.2 equiv), diisopropylethylamine (DIPEA, 0.043 mL, 0.25 mmol, 2.5 equiv) in 4 mL dichloromethane (DCM) overnight at room temperature. After removing the excess reagents by filtration, the resin was washed consecutively with 5 mL DCM, 5 mL isopropanol, 5 mL DCM, 5 mL isopropanol, 5 mL DCM (3 min for each solvent). This washing sequence was used to clean the resin after every reaction (i.e. capping, deprotection or amino acid coupling). Non-reacted groups were capped with 5 mL solution DCM/MeOH/DIPEA (3.5: 1 :0.5) for 1 hour. The next amino acid was added to the peptide sequence by two steps: Fmoc deprotection (step 2) and amino acid coupling (Step 3).

Step 2: Fmoc protecting group was removed by treating resin with 5 mL solution of 20% piperidine in dimethylformamide (10 min x 2).

Step 3: The coupling step was carried out using [hexafluorophosphate of O-(7-azabenzotriazol-1-yl)-1 , 1,3,3- tetramethyluronium] (HATU, 190 mg, 0.5 mmol, 5 equiv), amino acid (0.5 mmol, 5 equiv), DIPEA (0.087 mL, 0.5 mmol, 5 equiv) in 5 mL dimethyl formamide (DMF) at rt for 30 min. Step 2 and 3 were repeated to build the full sequence of apelin analogue.

Step 4: Allyl/alloc deprotection, (similar to step 3 - section 1.2) dried resin (300 mg, 0.1 mmol peptide) was swelled with 6 mL DCM in a 35 mL microwave tube. This suspension was bubbled with Ar for 5 min. PheSiHa (0.19 mL, 1.5 mmol, 15 equiv.) was added and mixed for another 5 min. Pd(PPh3)4 (11 mg, 0.01 mmol, 0.1 equiv.) was added into the reaction mixture and stirred under Ar for 30 min at room temperature. Resin was filtered then washed to remove excess reagents.

Step 5: Lactamization. To the resin, a mixture of DEPBT (150 mg, 0.5 mmol, 5 quiv.), DIPEA (0.078 mL, 0.5 mmol, 5 equiv.) in 4 mL DMF was added. The resin was shaken over night at room temperature. The excess reagents were removed by filtration and resin was washed. The full conversion was confirmed by UPLC-MS.

Step 6: Cleavage: Fmoc protecting group was removed using piperidine 20%. Resin was washed with the washing sequence and dried. Peptide was cleaved from resin using 5 mL cocktail trifluoroacetic acid (TFA)Ztriisopropylsilane (TlPS)Zethanedithiol (EDT)Zwater (92.5:2.5:2.5:2.5) and precipitated in 30 mL t-butyl methyl ether (TBME). After the supernatant was removed by centrifugation, peptide was dissolved in 6 mL of acetic acid 10 % and the aqueous layer was extracted and lyophilized to remove residual EDT.

Step 7: Forming disulfide bridge: crude peptide was dissolved in AcOH 70% (2 mg/mL). A solution 10% l ₂ in MeOH was added dropwise until the appearance of yellow color persisted, indicating the complete conversion. The mixture was diluted with equal volume of water and lyophilized.

Purification: crude peptide was re-dissolved in AcOH 10%, filtered and purified on preparative HPLC-MS system from Waters (Milford, USA) (column XSELECT™ CSH™ Prep C18 (19 x 100 mm) packed with 5 pm particles, UV detector 2998, MS SQ Detector 2, Sample manager 2767 and a binary gradient module) using acetonitrile and water + 0.1 % formic acid as eluents (gradient 2-20% acetonitrile in water for 15 min).

Molecular weight: 1734.1250 Da. Chemical formula: C77H124N26O16S2. Scale 0.1 mmol - obtained 26.5 mg. Purity 99%.

Synthesis and characterization of compounds of Table I

Characterization of compounds 146-157

Apelin-13 proliferation assay

Cells were maintained in at 37°C in a 5% CO2 incubator. ECs (ATCC, CRL-1730) were cultured in endothelial cell growth medium MV2 (PromoCell, C-22022), MV2 supplement mix (PromoCell, C-39226), and 1 % Penicillin- Streptomycin solution (Wisent, 450-201-EL). MuSC-derived myoblast were maintained in growth media containing Ham’s F10 (Wisent, 318-050-CL), 20% FBS (Wisent, 2300040033), 1 % Penicillin-Streptomycin solution (Wisent, 450-201-EL), and 2,5 ng/mL bFGF (R&D systems, 3139-FB-025). To assess effects of AP-13 on proliferation, media were replaced by endothelial cell growth medium MV2 (PromoCell, C-22022) without supplement or myoblast growth medium without bFGF. Media were exchanged daily. After 3 days of treatment, cells were fixed with 4% paraformaldehyde (TCI America, P0018) for 10 min and then permeabilized with 0.5% Triton X-100 (Sigma, T8787) for 10 min and stained with Hoechst (Thermo Fisher Scientific, 62249). Images were acquired using a high- throughput Operetta microscope (Perkin Elmer). For each AP-13 concentration, 12 pictures of at least 3 biological replicates were quantified using the Harmony high-content imaging and analysis software (Perkin Elmer).

Elabela, AM03-68 and KT01-129 proliferation assay

Human skeletal muscle derived cells (Lonza) were plated in a 384-well plate with different doses of compounds. After 3 days of treatment, cells were fixed with 4% paraformaldehyde for 10’ minutes at room temperature, washed three times with PBS, permeabilized 5’ minutes with 0.5% Triton, washed three times with PBS, blocked 1 h with BSA 5% and then incubated with DAPI for 45’ minutes at 37 degrees. To determine cell numbers, the number of DAPI positive nuclei were quantified using an Operetta high content imager (PerkinElmer).

AM02-123 and KT03-69 proliferation assay

Pooled primary human umbilical vein endothelial cells (HuVECs; PCS-100-013, ATCC) were expanded and maintained in basal media (PCS-100-030, ATCC) supplemented with the Endothelial Cell Growth Kit-BBE (PCS-100- 040, ATCC). Mycoplasma testing was performed using a PCR kit (ABMG238, Sigma). Before exposure to peptidic compounds, the cells were plated into 96 well plates (353219, Falcon) at 2000 cells/per well and starved for 17-20hrs in RPM 1-1640 medium (350-000-CL, Wisent) containing 5% FBS (080450, Wisent). HuVECs were then switched to basal media (PCS-100-030, ATCC) plus 5% FBS (080450, Wisent), and the compounds that were solubilized in DMSO were added to the media at a final concentration of 1 pM. DMSO vehicle alone was used as negative control. Media was changed every 24hrs and the cells were cultured for 72hrs. Subsequently, the cells were then fixed with 4% ice cold PFA and stained with DAPI. Images were taken with a Perkin Elmer Operetta™ high throughput imager and quantification was performed with the Columbus software.

Skeletal muscle force measurements

Veh controls and AP-13 treated mice were anaesthetised with an intraperitoneal injection of pentobarbital (30 mg / kg). The in situ isometric torque tension was measured on the right hindlimb of anaesthetized mice placed on a 37°C preheated platform of the 1300A whole animal system (Aurora Scientific, Canada). The right hindlimb was first shaved, cleaned with 70% ethanol and fixed above the knee joint with a cone point set screw. Then, the foot was positioned at a 90° angle (neutral position) and stabilized with adhesive onto a footplate attached to a 300C-LR dualmode lever arm (Aurora Scientific, Canada), allowing the mice to push or pull under stimulation. Once the hindlimb was fixed, two sterile needle electrodes were subcutaneously inserted at either side of the tibial nerve to stimulate the posterior muscles of the lower leg, such as the gastrocnemius and the soleus, which are responsible of ankle plantar flexion. The stimulation current was tuned up to achieve a maximum twitch response, and then the leg was stimulated at different frequencies with a 2-min rest between each stimulation, until reaching the maximum torque tension (mN). For ex vivo measurements, EDL muscles were isolated by cutting the proximal and distal tendons, and were placed in an organ bath, maintained at 25°C, and filled with Krebs-Ringer's solution (137 mM NaCI, 5 mM KCI, 2 mM CaCfe, 24.7 mM NaHCOs, 2 mM MgSO4, 1.75 mM NabhPCU, and 2 g/l dextrose, pH 7.4) bubbled with carbogen (95% O2, 5% CO2). The proximal tendon was fixed in a stationary clamp with a 3-0 suture (Harvard Apparatus, St. Laurent, Canada), and the distal tendon was connected to a dual-mode level arm system 300C-LR (Aurora Scientific, Inc., Aurora, ON, Canada) that provided control of force and positioning of the motor arm. First, the muscle was initially set at a resting tension of 10 mN for 10 min. Then, the stimulation was delivered by a pair of platinum electrodes located on either side of the muscle using supramaximal 0.2 ms square wave pulses. Muscles were stimulated at different frequencies with 2 min rest between contraction to reach maximum isometric tetanic force (Po). The force generated by the muscle was measured and analyzed with a LabView-based DMC program (Dynamic Muscle Control and Data Acquisition; Aurora Scientific, Inc.). Optimal muscle length (Lo) was defined as the muscle length at which the maximal twitch force was elicited. The optimum fibre length (Lf) was determined by multiplying Lo by predetermined Lf/Lo ratios: 0.44 for EDL. The cross-sectional area (CSA) of muscle samples was then determined by dividing muscle mass (mg) by the product of Lf and 1 .06 mg / mm3, the density of mammalian muscle. Po values were normalized to the muscle cross-sectional area.

Fitness tests

Mice were tested using rotarod (Bioseb, LE8505) at a speed of 15 rounds per min. Grip tests were performed using a horizontal single beam and a grid engineered in-house. In order to calculate the normalized mean impulse, the time until task failure was measured and multiplied by the animals' body mass. For each test, three measurements have been recorded and the animals were rested for 5 min between each measurement.

Apoptosis assay

TA muscles were harvested and snap frozen in liquid nitrogen. Frozen samples were thawed on ice, weighted, and homogenized using a Potter-Elvehjem™ tissue grinder on ice in ice-cold incomplete radioimmunoprecipitation assay buffer (RIPA) containing 50 mM Tris-HCI pH 7.4 (Sigma-Aldrich, T1503) and 100 mM NaCI (Millipore, SX0420) supplemented with 1 mM 1,10-ortho-phenanthroline (Sigma, P9375), 10 piM 3,4-dicholoroisocoumarin (Sigma, D7910), 10 piM leupeptin (Sigma, L8511), and 10 piM E-64 (Sigma, E3132) for protease inhibition. 20 pil RIPA buffer was used per mg of tissue and the RIPA buffer was completed by addition of 0.1 % sodium dodecyl sulfate (Thermo Fisher Scientific, BP166), 0.5% sodium deoxycholate (Sigma, D6750), and 1 % Nonidet™ P-40 (Roche, 11754599001) to obtain a final volume of 30 pil. After lysis on ice for 1 h, cellular extracts were centrifuged at 18,000g for 15 min and the supernatants were recovered. 90 pig of protein was used for western blotting with cleaved caspase-3 (Cell Signaling Technologies, 9661), actin (Sigma, A3853), HRP-conjugated anti-mouse (Cell Signaling Technologies, 7076), and HRP-conjugated anti-rat (Cell Signaling Technologies, 7074) antibodies. Chemiluminescence was acquired with a VersaDoc™ 4000mp imaging system (BioRad) using the Immobilon Crescendo™ Western HRP substrate (Millipore, WBLUR0500) or Clarity Max™ Western ECL Substrate (BioRad, 1705062).

Echocardiography

Morphological and functional heart parameters have been assessed using a Vevo3100™ echocardiography system equipped with a MX400 ultrasound probe (FUJIFILM VisualSonics). Animals were anesthetized using isoflurane and parameters have been recorded at a heart rate near to 450 beats per minute. Cardiac function measurements were acquired in M-mode from a parasternal short-axis view of the left ventricle and analysed using Vevo LAB™ 3.1.1 (FUJIFILM VisualSonics).

Binding affinity assay

Binding experiments. Binding experiments were performed on isolated cell membranes of HEK293 stably expressing the YFP-tagged human apelin receptor (hAPJ). Cells were submitted to a freeze-thaw cycle and gently transferred to a falcon tube containing 4 mL EDTA solution (1 mM EDTA and 50 mM Tris-HCI, pH 7.4). To isolate cell membranes, this mixture was centrifuged at 3500 rpm/ 4°C for 15 minutes. The cell membranes were suspended in binding buffer (50 mM Tris-HCI, 0.2 % BSA, pH 7.4). Binding was carried out in 96-well plates: 15 pig of membrane proteins were incubated in the presence of 0.2 nM of radiolabeled [ ¹²⁵ l] [Nle ⁷⁵ , Tyr ⁷⁷ ]Pyr-apelin-13 (820 Ci/mmol) and tested ligands with a range of concentrations from 10 ⁵ to 10 ¹¹ M in a total volume of 200 piL for 1 h at room temperature. Unbound ligands were removed by filtration through a filter plate (pre-absorbed of PEI 0.5% for 2 h at 4°C), and the filtered membranes were washed three times with 170 piL binding buffer. The y emission was quantified using a y-counter 1470 Wizard from PerkinElmer (Waltham, USA) (80% efficiency). Non-specific binding, measured in the presence of 10 ⁵ M unlabeled Ape13, did not exceed 5% of total signal. Results were analyzed using GraphPad Prism 8 to determine the IC50 values, which represent the concentration of ligands displacing 50% of radiolabeled ligand from the APJ receptor. The Kd value of apelin determined by saturation binding assay is 1 .8 nM. Dissociation constant K, values were then calculated from the IC50 using the Cheng-Prusoff equation and results were displayed as mean ± SEM of two independent experiments each done in triplicates.

Quantification and statistical analysis

Except for animals that died a natural death during the course of the experiments, no mice were excluded from the study. Sample size determination was based on the expected effect size and variability that was previously observed for similar readouts in the investigators’ laboratories, in-vivo treatments were not blinded, but imaging readouts were analyzed in a blinded manner. Stained samples were analyzed using n>3 images per biological sample. Statistical analysis was performed using GraphPad™ Prism (GraphPad Software). Statistical significance for binary comparisons was assessed by a student’s t-test after verification that variances do not differ between groups or by a Welch correction when variance was observed. For comparison of more than two groups, one-way or two-way ANOVAs were used, according to the experimental design, and followed by Tukey or Dunnett’s post-hoc test. All data are expressed as means + sem.

EXAMPLE 2: Muscular dystrophy affects the proliferative capacity of MuSCs

To systematically assess stem cell function and the regenerative capacity of skeletal muscle across a spectrum of different types of MD, mouse models of CoM-related myopathy (d16), Duchenne MD (mdx), and LAMA2 MD (dyW), were compared to wild-type (wt) animals (FIG. 1A). In order to maximize tissue regeneration and MuSC activation, mice were injected with the snake venom cardiotoxin (CTX) and analyzed the tissue at 5 and 10 days post injury (dpi). Hematoxylin and eosin staining of muscle cross-sections revealed that at 5 and 10 dpi all three MD models displayed changes in tissue architecture including an increased interstitial volume and a higher abundance of mononuclear cells when compared to wt controls (FIG. 1 B). In the uninjured baseline, differences in fiber size were observed in all dystrophic models compared to control animals (FIG. 1C). mdx and d16 did not display differences in fiber size at 10 dpi (FIG. 1D). In contrast, the size distribution at 10 dpi was shifted significantly towards smaller fibers in dyW mice (FIG. 1D). Immunostaining for embryonic myosin heavy chain (eMHC), a marker of newly formed muscle fibers, showed that, following injury, tissue maturation in d16 and dyW mice was delayed compared to wt controls and remained increased with respect to the baseline in uninjured muscles (FIGs. 1 E-I). Moreover, d16 and dyW mice displayed an increased abundance of the fibrosis marker fibronectin at both 5 and 10 dpi (FIGs. 1 E and J- M). Staining for the MuSC marker Pax7 identified reduced stem cell numbers in all three MD models at 5 dpi, and in d16 and dyW mice at 10dpi (FIGs. 1 N-P). Importantly, relative to pre-injury levels, MuSC numbers in the dystrophic mouse models did not increase by the same magnitude as in wt mice (FIG. 1Q). The MuSC pool in dyW mice showed a particularly impaired expansion potential and did not change significantly compared to pre-injury levels. Thus, a reduced capacity for mobilization of the stem cell pool is a feature of multiple types of MD. Overall, the severity of the regenerative phenotype increases in from mdx to d16 mice and is most pronounced in the dyW model (Table below). All MD mice model display features of regenerative failure that are due to an impaired expansion of MuSCs following skeletal muscle fiber damage.

Table summarizing the severity of endogenous repair defects in dystrophic mouse models

- = decreased, - - strongly decreased, + = increased, + + strongly increased, = similar to Ctrl.

EXAMPLE 3: MD affects microvascular remodeling In order to study the cellular composition of the MuSC niche in the different types of MD, the number of fibro— adipogenic progenitors (FAPs), macrophages, and ECs were quantified under uninjured conditions, and at 5 and 10 dpi. Staining for Pdgrfa revealed increased FAP numbers at baseline in dyW mice (FIG. 2A). At 5 dpi, FAP numbers increased in mdx and dyW mice, while they decreased in d 16 muscles compared to wt controls (FIGs. 2B and D). In dyW mice, FAP numbers remained higher than in wt controls at 10 dpi (FIG. 2C). F4/80 positive macrophages showed an increased abundance at baseline and following injury in all MD models (FIGs. 2E-H). Compared to wt mice, mdx mice showed a particularly pronounced macrophage response at 5 dpi. Notably, respective to wt control muscles, the number of ECs was lower in mdx and dyW mice at baseline (FIG. 2I). Moreover, EC numbers were dramatically reduced at both 5 and 10 dpi in all three MD models (FIGs. 2J-L). These results support the notion that impaired microvascular remodeling is a pathologic hallmark of MD.

EXAMPLE 4: Identification of AP-13, and analogues thereof as skeletal muscle EC stimulatory molecules

The inventors sought to identify angiogenic factors with the potential to stimulate ECs. G protein-coupled receptors (GPCRs) represent the largest family of druggable targets in the human genome (De Micheli et al, 2020). The inventors compiled a list of GPCRs expressed by skeletal muscle ECs uninjured conditions and at 5 dpi (FIGs. 3A-B) was compiled based on the single cell atlas by De Micheli et al. The inventors observed that APJ, the receptor for the small peptidic hormone apelin, shows the second highest expression at 5 dpi. Mapping of APJ to the whole single cell transcriptome of skeletal muscle showed that its expression is highly specific to ECs and does not overlap with Pax7 positive MuSCs (Data not shown). APJ immunostaining of skeletal muscle sections of wt mice at 5 dpi confirmed a distinct colocalization with ECs, while MuSCs, macrophages, and FAPs did not express discernable levels of the receptor (FIGs. 3C-D).

The APJ ligand apelin is naturally produced as a 77-amino-acid precursor that is processed into active 36, 17, and 13 amino acid fragments found in the systemic circulation (Marsault et al., 2019). Apelin 13, the smallest active form of apelin, has a molecular weight of 1.5 kDa and is naturally pyroglutamylated at its N-terminus (FIGs. 3E-F). The pyroglutamylated apelin 13 (AP-13) fragment was produced using solid phase peptide synthesis to investigate potential stimulatory effects on ECs (Murza et al., 2015) (FIG. 4). Purity of AP-13 was confirmed to be >95% using analytical UPLC/MS (data not shown).

In vitro experiments revealed that AP-13 elicits a dose dependent proliferative response of ECs (FIG. 3G). Similarly, AP-13 derivatives AM02-123 and KT03-69 significantly increase the proliferation of endothelial cells in vitro (FIG. 3I). In contrast, MuSC derived primary myoblasts did not react with increased proliferation to AP-13 (FIG. 3H).

Further, AP-13, Elabela, and macrocyclic derivatives AM03-68 and KT01-129 increase the proliferation of human skeletal muscle derived cells (FIG. 3J).

EXAMPLE 5: AP-13 mediated stimulation of the perivascular niche improves endogenous repair

The ability of AP-13 to stimulate skeletal muscle ECs was assesses in MD mice models dyW, d16 and mdx. The dystrophic mice were implanted at the age of 14 days (dyW) or 42 days (d16 and mdx) days with osmotic pumps supplying AP-13 or PBS vehicle (veh) for four weeks (FIG. 5A). As predicted by in vitro results presented in Example 4, AP-13 treatment increased the number of Ecs in dyW muscles by 59% when compared to the veh condition (FIG. 5B). AP-13 also increased the number of ECs in d16 (FIGs. 5K-L) and mdx (FIGs. 5M-N) mice muscles at 5 dpi by 69.54% and 70.41 %, respectively, when compared to the veh condition (FIG. 5B) AP-13 caused a 14% reduction in the number of FAPs in dyW mice but did not affect macrophages (FIGs. 5C-D). No significant effect on fibrosis or fibers with membrane damage that stain for intracellular IgG were observed in dyW mice as a consequence of AP-13 treatment (FIGs. 5E-F). Moreover, western blot for cleaved caspase 3 revealed that apoptotic processes were not altered in the AP-13 group (FIG. 5J). Interestingly, AP-13 mediated stimulation of Ecs was accompanied by a 58% increase in Pax7 positive MuSCs and an 80% increase of differentiating myogenin (MyoG) positive cells (FIGs. 5G- H). AP-13 also increased the number of MuSCs in d16 (FIGs. 5O-P) and mdx (FIGs. 5Q-R) mice muscles at 5 dpi by 81.11 % and 58.22%, respectively, when compared to the veh condition. In agreement with increased MuSC proliferation and differentiation, AP-13 treatment led to a 72% increase in newly formed eMHC positive fibers (FIG. 5I).

Thus, AP-13 stimulation of skeletal muscle ECs promotes MuSC proliferation and function and endogenous repair in MD mice models but does not affect fibrosis and the survival or integrity of muscle fibers.

EXAMPLE 6: Systemic AP-13 treatment slows disease progression in MD

Cumulation over the four-week treatment course revealed that the AP-13 treated group of dyW mice displayed a 12% higher average body weight (FIGs. 6A-B). As opposed to veh treated dyW mice, not a single animal in the AP-13 group died before the study endpoint (FIG. 6C). To uncouple potential positive effects of AP-13 on skeletal muscle in dyW mice from effects on secondary tissues, ex-vivo and in situ muscle force measurements were performed (FIGs. 6D-E). This revealed that extensor digitorum longus (EDL) muscles isolated from dyW mice that were treated for 3 weeks with AP-13 were in average 109% stronger than muscles from the veh control group (FIG. 6F). Similarly, in situ stimulation of the posterior muscle compartment of the lower leg revealed a 69% increase in force production in the AP-13 group compared to the veh condition (FIG. 6G).

To determine whether AP-13 treated dyW mice show a global amelioration of disease progression compared to the veh group, dyW mice were challenged using a number of physical performance tests. This revealed a 180%, 118% and 166% increase in the mean impulse in AP-13 treated mice in the rotarod, single beam, and horizontal grid challenge respectively when compared to veh treated animals (FIGs. 6I-K). In summary, AP-13 mediated stimulation of endogenous repair in dyW muscles is accompanied by dramatic gains in muscle force and overall physical performance.

EXAMPLE 7: AP-13 treatment causes no adverse cardiovascular effects

MDs are frequently accompanied by cardiac dysfunction (Beynon et al., 2008). Normalized to their body weight, dyW mice in the veh group had 30% heavier hearts than C57BL/6N control (ctrl) mice (FIGs. 7A-B). This suggests the presence of hypertrophic compensatory growth or fibrosis. Compared to veh, AP-13 reversed this phenotype and the treated dyW mice had heart weights that were not different from the untreated Ctrl group. Moreover, echocardiography revealed that the cardiac index and fractional shortening of hearts in the AP-13 treated group were similar to untreated Ctrl hearts, while they were increased by 76% and 20% respectively in the veh group (FIGs. 7C- D). Therefore, it is concluded that AP-13 has no adverse effects on heart function. Moreover, the inventors observed no changes in histology in diverse organs of AP-13 treated mice (data not shown).

EXAMPLE 8: EC specific knockout of APJ phenocopies MD features

The foregoing shows that AP-13 not only holds therapeutic potential for MD but, given the high expression of APJ in skeletal muscle ECs, is also an important endogenous regulator of angiogenesis in this tissue. Indicative of an angiogenic response induced by the chronic de- and regenerative processes in dystrophic muscle, a 71% upregulation of APJ was observed in the microvasculature of uninjured dyW mice when compared to wt controls (FIG. 8). To address the role of endogenous APJ in ECs, mice carrying a CreERT2 cassette under the Cdh5 promoter with floxed alleles of APJ (APJECKO) were generated and, following tamoxifen mediated gene excision, analyzed them at 5 and 10 dpi (FIGs. 9A-B). Loss of APJ led to a 37% and 41 % reduction in CD31 positive cells at 5 and 10 dpi respectively when compared to the wt condition (FIGs. 9C-D). Interestingly, hematoxylin and eosin staining of skeletal muscle cross sections revealed that APJECKO mice displayed an increase in mononuclear cells and interstitial volume at both time-points after injury that resembles the regenerative phenotype observed in severe MD (FIG. 9E). Compared to wt controls, fiber size was significantly reduced in APJECKO mice at 10 dpi (FIGs. 9F- G). Indicating a delayed regenerative response in APJECKO mice, staining for eMHC showed a 6% decrease in eMHC positive fibers at 5 dpi and a 306% increase at 10 dpi (FIGs. 9H-I). Fibronectin staining also revealed an 43% increase of the fibrotic area 5 dpi and an increase of 53% at 10 dpi in APJECKO mice (FIGs. 9J-K). Supporting the notion that, similar to MD, microvascular defects in skeletal muscle affect the stem cell pool in APJECKO mice, the number of Pax7 positive MuSCs was reduced by 50% at 5 dpi compared to wt controls (FIGs. 9L-M). Moreover, correlating with delayed tissue regeneration, APJECKO mice showed a 30% reduction of differentiating MyoG positive MuSCs at 5 dpi and a 52% increase at 10 dpi (FIGs. 9N-O). The foregoing shows that EC specific loss of APJ leads to defective microvascular remodeling, an impaired expansion capacity of the MuSC pool, and a myopathic phenotype reminiscent of MD.

EXAMPLE 9: Binding affinity of Apelin-13, Elabela, and other compounds of the disclosure on APJ receptor

The dissociation constant, Ki, is reflective of the binding affinity of a ligand for its receptor and corresponds to the concentration of ligand that displaced 50 % of radiolabeled pyr-apelin-13 [Nle ⁷⁵ , Tyr ⁷⁷ ][ ¹²⁵ l]. T1/2 reflects the time needed to remove half of the molecule in a defined environment, here rat plasma in vitro. Data are expressed as mean ± SEM.

The scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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