Prodrugs of GLP-1 polypeptides and uses thereof

Abstract

The present invention relates to DKP-based prodrugs that release therefrom a parent drug, such as semeglutide, after transformation under in vivo conditions. The invention also relates to the use of a DKP-based prodrug.

Description

Technical Field

[0001] The present invention relates to DKP-based prodrugs and their therapeutic uses.

[0002] Sequence Listing

[0003] This application is submitted with a sequence listing in electronic form. The entire contents of the sequence listing are incorporated herein by reference. Background Art

[0004] Prodrug technology can be used to generate compounds with properties suitable for specific dosing frequencies. Prodrugs based on diketopiperazine (DKP) have been previously described (e.g., Arnab De, Richard D. DiMarchi, Investigation of the Feasibility of an Amide-based Prodrug Under Physiological Conditions, International Journal of Peptide Research and Therapeutics, 2008, Vol 14, 3, pp255–262). The technology is based on chemical transformation, in which the part consisting of two amino acids is cyclized to form a six-membered ring, followed by release of active drugs.

[0005] WO 2010 / 071807 is said to disclose prodrug formulations of glucagon superfamily peptides, wherein the peptide has been modified by attachment to a dipeptide via an amide bond.

[0006] WO 2010 / 080605 is said to disclose non-enzymatic self-cleavable dipeptide elements linked to known pharmaceutical agents via amide bonds.

[0007] WO 2011 / 089216 is said to disclose dipeptide-based prodrugs for aliphatic amine-containing drugs.

[0008] WO 2011 / 163012 is said to disclose prodrug formulations of glucagon superfamily peptides, wherein the peptide has been modified by attachment to a dipeptide via an amide bond.

[0009] WO2013 / 127779 is said to disclose ester prodrugs of insulinotropic peptides.

[0010] WO 2014 / 152460 is said to disclose peptide-based prodrugs with significantly extended half-lives.

[0011] WO2016 / 049174 purportedly discloses prodrug formulations of insulin and insulin analogs, wherein the insulin peptide has been modified by amide bond attachment of a dipeptide prodrug element. Summary of the invention

[0012] GLP-1 receptor agonists are widely used to treat chronic diseases. Currently available oral GLP-1 receptor agonist drugs must be administered once a day. Treatment regimens with a dosing frequency lower than once a day may lead to improved patient convenience and improved patient compliance, so the development of oral GLP-1 receptor agonists suitable for administration at a frequency lower than once a day will be a significant improvement in available treatment options. Prodrug technology can be used to optimize the properties of the drug in a manner that makes it suitable for a specific dosing regimen (e.g., once a week dosing). The present invention relates to prodrugs having desired properties, such as for once-weekly oral administration.

[0013] In a first aspect, the present invention relates to a prodrug of formula I: XYZ, wherein Z is a parent drug and wherein XY is a DKP forming portion, which undergoes a chemical transformation under in vivo conditions, resulting in the release of the parent drug from the DKP forming portion. In a second aspect, the present invention relates to a prodrug for use as a drug. In one functional aspect, the present invention provides a prodrug having a conversion half-life suitable for once-weekly administration. Additionally or alternatively, in another functional aspect, the present invention provides a prodrug having an observed terminal half-life suitable for once-weekly administration. Additionally or alternatively, in another functional aspect, the present invention provides a prodrug having a surprisingly high oral bioavailability. The present invention can also solve other problems that will be apparent from the disclosure of the exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Dose-corrected plasma concentration (vs. time) curves of the compounds of the present invention after oral administration in beagle dogs. DETAILED DESCRIPTION

[0015] In the following, Greek letters may be represented by their symbols or corresponding written names, for example: α=alpha; β=beta; γ=gamma; δ=delta; ε=epsilon; ω=omega; and so on. In addition, the Greek letter μ can also be represented by "u", for example in μl=ul or in μM=uM. The symbol * in a chemical formula or chemical diagram indicates the point of connection with an adjacent part. In the following, unless otherwise specified in the specification, terms presented in the singular also include plural cases, for example, when referring to a "compound", it should be understood that this includes all individual variations falling within the broad definition of the compound.

[0016] The present invention relates to a prodrug having desirable properties, such as for once-weekly oral administration. In a first aspect, the present invention relates to a prodrug comprising formula I: XYZ, wherein X is an amino acid, wherein Y is selected from Thz and D-Thz, and wherein Z comprises a GLP-1 polypeptide; or a pharmaceutically acceptable salt, ester or amide of said prodrug. In a second aspect, the present invention relates to a prodrug of the present invention for use as a medicament.

[0017] General Definition

[0018] The term "compound" as used herein refers to a molecular entity and therefore, in addition to having the minimum elements defined for each compound or group of compounds, may also have different structural elements. The term compound can be used interchangeably with the term "construct". The term "compound" may be used to describe the prodrugs of the present invention. The compounds of the present invention may be referred to as "compounds", while the term "compound" is also intended to cover its pharmaceutically relevant forms, i.e., the present invention relates to compounds as defined herein or pharmaceutically acceptable salts, amides or esters thereof.

[0019] As used herein, the term "polypeptide" or "polypeptide sequence" refers to a compound comprising a series of two or more amino acids interconnected by amide (or peptide) bonds. The term polypeptide can be used interchangeably with the term "peptide" and the term "protein".

[0020] As used herein, the term "analog" generally refers to a polypeptide whose sequence has one or more amino acid changes compared to a reference amino acid sequence. The amino acid changes may include amino acid additions, amino acid deletions and / or amino acid substitutions. Amino acid substitutions, deletions and / or additions may also be referred to as "mutations". In specific embodiments, an analog "comprises" a specified change. In other specific embodiments, an analog "consists of" or "has" a specified change. When the terms "comprising" or "containing" are used in connection with an amino acid change in an analog, it should be understood that the analog may have further amino acid changes compared to its reference sequence. When the terms "consisting of" or "having" are used in connection with an amino acid change in an analog, it should be understood that the specified amino acid mutation is the only amino acid change in the analog compared to the reference sequence.

[0021] The term "derivative" generally refers to a chemically modified polypeptide in which one or more substituents are covalently linked to the amino acid sequence of the polypeptide, for example, via a bond to the ε-amino group of Lys. In one embodiment, the compounds of the invention include derivatives that have been modified so that one or more substituents having an extended nature are covalently linked to the amino acid sequence of the polypeptide.

[0022] As used herein, the term "sequence identity" refers to the degree to which two amino acid sequences (e.g., polypeptides) have the same residue at the same position in the comparison. This may also be referred to as "identity". Sequence identity is conveniently expressed as a percentage, i.e., if 85 amino acids in 100 alignment positions are identical between two sequences, the degree of identity is 85%. For the present invention, the sequence identity between two amino acid sequences is determined by using simple handwriting and visual, and / or standard protein or peptide alignment programs, such as "align" based on the Needleman-Wunsch algorithm. The algorithm is described in Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48:443-453, and the align program is described by Myers and W.Miller in "Optimal Alignments in Linear Space" CABIOS (computer applications in the biosciences) (1988) 4:11-17. For the alignment, the default scoring matrix BLOSUM62 and the default identity matrix can be used, and the penalty for the first residue in a gap can be set to -12, or preferably -10, and the penalty for additional residues in a gap can be set to -2, or preferably -0.

[0023] Amino Acids

[0024] As used herein, the term "amino acid" refers to any amino acid, i.e., both proteinogenic and non-proteinogenic amino acids. As used herein, the term "proteinogenic amino acid" refers to the 20 standard amino acids encoded by the human genetic code. As used herein, the term "non-proteinogenic amino acid" refers to any amino acid that does not qualify as a proteinogenic amino acid. Generally, as used herein, an amino acid residue (e.g., in the context of a polypeptide sequence) can be represented by its full name, its single-letter code, and / or its three-letter code. These three methods are completely equivalent and can be used interchangeably. In the following, each amino acid of the peptide of the present invention that does not specify its optical isomer should be understood to mean the L-isomer (unless otherwise specified). Examples of non-proteinogenic amino acids incorporated into the compounds of the present invention are listed in Table 1. It should be understood that when a moiety is referred to as being attached to the "side chain amino group" of an amino acid, it is attached to the amino group located on the side chain of that amino acid, e.g., if a moiety is attached to the side chain amino group of Lys or D-Lys, it is attached to the ε-amino group, if a moiety is attached to the side chain amino group of Orn, it is attached to the δ-amino group, and if a moiety is attached to the side chain amino group of Dab, it is attached to the γ-amino group.

[0025] Table 1: Non-limiting examples of non-proteinogenic amino acids incorporated into the compounds of the invention

[0026]

[0027] GLP-1 peptide

[0028] The term "GLP-1 polypeptide" as used herein refers to a polypeptide that is capable of binding to a GLP-1 receptor and / or activating a GLP-1 receptor. In other words, a GLP-1 polypeptide is a polypeptide that is referred to as having "GLP-1 activity". A GLP-1 polypeptide may bind to and / or activate other types of receptors, i.e., as long as the polypeptide binds to and / or activates a GLP-1 receptor, it qualifies as a GLP-1 polypeptide, regardless of any other receptor interactions that may be involved therewith. In addition to the amino acid residues responsible for the GLP-1 receptor interaction, the GLP-1 polypeptide may also contain other amino acid residues that are not involved in the GLP-1 receptor interaction.

[0029] As used herein, the term "GLP-1 receptor agonist" refers to a compound that is capable of binding to a GLP-1 receptor and / or activating a GLP-1 receptor. In other words, a GLP-1 receptor agonist is said to have "GLP-1 activity". A GLP-1 receptor agonist may be based on any type of molecular scaffold, such as a small molecule, a polypeptide, and an antibody, or any combination thereof. A GLP-1 receptor agonist may comprise one or more moieties that are capable of activating a GLP-1 receptor.

[0030] As used herein, the term "GLP-1 analogue" refers to an analogue (or variant) of human glucagon-like peptide-1 (GLP-1 (7-37)). The amino acid sequence of human GLP-1 (7-37) is included in the sequence list as SEQ ID NO: 1. Compared with GLP-1 (7-37), the amino acid sequence of the GLP-1 analogue has one or more amino acid changes. The amino acid changes may include amino acid additions, amino acid deletions and / or amino acid substitutions. The amino acid sequence of semaglutide is a non-limiting example of a GLP-1 analogue.

[0031] As used herein, the term "GLP-1 derivative" refers to a chemically modified GLP-1 polypeptide in which one or more substituents have been covalently attached to the GLP-1 polypeptide. For example, a GLP-1 derivative is a GLP-1 analog to which one or more substituents have been covalently attached. A non-limiting example of a GLP-1 derivative is semaglutide.

[0032] In one embodiment, the compound of the present invention comprises a GLP-1 polypeptide. In one embodiment, the GLP-1 polypeptide is the amino acid sequence of semaglutide. In one embodiment, the compound of the present invention comprises a GLP-1 polypeptide, wherein the GLP-1 polypeptide is a GLP-1 analog; and wherein the GLP-1 analog has a maximum of 3 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1). In one embodiment, the compound of the present invention comprises a GLP-1 polypeptide, wherein the GLP-1 polypeptide is a GLP-1 analog, and wherein the GLP-1 analog has a maximum of 2 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1). In one embodiment, the compound of the present invention comprises a GLP-1 derivative, and in a preferred embodiment, the GLP-1 derivative is semaglutide.

[0033] Substituents

[0034] As used herein, the term "substituent" refers to a moiety covalently linked to a polypeptide, such as a moiety that is linked to a GLP-1 polypeptide or to a dipeptide extension of a GLP-1 polypeptide, such as a dipeptide extension present in a compound of the invention, thereby constituting part of a DKP-forming moiety. If a substituent is linked to a polypeptide or dipeptide, the polypeptide or dipeptide is referred to as "substituted". When a substituent is covalently linked to a polypeptide or amino acid residue, the polypeptide or amino acid is referred to as "carrying" a substituent. A substituent may contain a series of separately defined moieties; these moieties may be referred to as "substituent elements".

[0035] The substituent may be capable of forming a non-covalent bond with albumin, thereby promoting the circulation of the compound in the bloodstream, thereby having the effect of prolonging the presence of the compound in the bloodstream, since the aggregate of the fused compound and albumin only slowly disintegrates to release the free form of the compound; therefore, the substituent as a whole may also be referred to as an "albumin binding moiety", and the substituent may be referred to as having a "prolonging effect". The substituent may include a portion that is particularly associated with albumin binding, and thus with prolonging the effect, which portion may be referred to as a "prolonging body" or "prolonging moiety". The substituent may be a lipophilic moiety with a distal carboxylic acid.

[0036] Substituents may include a portion located between the prolonging portion and the point of attachment to the polypeptide, which portion may be referred to as a "linker". A linker may include several "linker elements". Linker elements may be selected such that they improve the overall properties of the molecule, for example, such that they improve oral bioavailability, conversion half-life, or prolongation of action, thereby improving the overall exposure profile of the compound after oral administration.

[0037] The nomenclature used to describe the extension, linker and other structural elements is as commonly used in the art, for example, *-CO-* refers to a carbonyl group, -CH2- refers to a methylene group, -COOH refers to a carboxylic acid, and "-" refers to a covalent bond. Non-limiting examples of substituent elements are listed in Table 2.

[0038] Table 2: Non-limiting examples of substituent elements

[0039]

[0040]

[0041] As used herein, the term "lipophilic moiety" refers to a moiety comprising an aliphatic and / or cyclic hydrocarbon moiety having 6-30 carbon atoms, preferably more than 6 and less than 20 carbon atoms. As used herein in the context of a lipophilic moiety, the term "distal carboxylic acid" refers to a carboxylic acid that is connected to the farthest (terminal) point of the lipophilic moiety relative to the point of connection of the lipophilic moiety with an adjacent moiety, for example, in the compounds of the present invention, a lipophilic moiety with a distal carboxylic acid (e.g., Formula 1 and Formula 2) is an extended moiety, and relative to the point of connection of the lipophilic moiety with an adjacent linker element (e.g., Formula 3 and Formula 4), the carboxylic acid is connected to the farthest (terminal) point of the lipophilic moiety. Non-limiting examples of lipophilic moieties with distal carboxylic acids are Formula 1 and Formula 2.

[0042] In one embodiment, the prodrug of the present invention comprises a substituent connected to the dipeptide prodrug portion. In one embodiment of the present invention, the substituent has a prolonging effect. In one embodiment of the present invention, the substituent comprises a lipophilic portion having a distal carboxylic acid. In one embodiment of the present invention, the lipophilic portion having a distal carboxylic acid is selected from Chemical Formula 1 and Chemical Formula 2. In one embodiment, n of Chemical Formula 1 is 12, 14, 16 or 18. In one embodiment, n of Chemical Formula 1 is 14 or 16. In one embodiment of the present invention, the substituent comprises a portion selected from Chemical Formula 3 and Chemical Formula 4. In one embodiment of the present invention, the substituent comprises a portion of Formula II: A5-A4-A3-A2-A1-* (Formula II). In one embodiment of the present invention, * represents a point of connection with X. In one embodiment of the present invention, A1 is selected from Chemical Formula 3, Chemical Formula 4, Chemical Formula 5, Chemical Formula 6 and Chemical Formula 7, or is absent. In one embodiment of the present invention, A2 and A3 are each individually selected from Chemical Formula 3, Chemical Formula 4 and Chemical Formula 5, or are absent. In one embodiment of the present invention, A4 is Chemical Formula 3 or Chemical Formula 4. In one embodiment of the present invention, A5 is selected from Chemical Formula 1 and Chemical Formula 2. In one embodiment of the present invention, the residues A5, A4, A3, A2, A1 are linked to each other via an amide bond.

[0043] Prodrug

[0044] The term "prodrug" as used herein refers to a compound that undergoes chemical conversion in vivo by an enzymatic or non-enzymatic chemical process, thereby causing the release of the parent drug. The term "parent drug" as used herein refers to a pharmacologically active compound released from a prodrug when the prodrug is converted. The term "conversion" as used herein in the context of a prodrug refers to a process in which a prodrug is converted enzymatically or non-enzymatically, thereby causing the release of the parent drug. The rate at which the conversion occurs can be quantified by a "conversion half-life." The "conversion half-life" is the length of time required for the prodrug concentration to be halved due to the conversion. The "conversion half-life" may also be referred to as a "prodrug to drug conversion half-life" or a "prodrug to parent drug conversion half-life."

[0045] The complete prodrug will not exert the expected pharmacological activity to a significant extent, for example, it will not exert the expected pharmacological activity to an extent that makes it incompatible with the expected treatment regimen. Once the parent drug is released, the pharmacological activity associated with the expected treatment of the prodrug is obtained from the parent drug. When the parent drug is released from the prodrug, it is referred to as the "free form". The prodrug can achieve the desired conversion after the intramolecular cyclization of the amide extension based on the terminal dipeptide, and the extension is then cut off from the parent drug, resulting in the release of the parent drug in free form. Such intramolecular cyclization can occur as an enzyme-independent process under physiological conditions, such as through the formation of diketopiperazine (DKP). In the prodrug converted by DKP formation, the part that releases the parent drug during conversion is called the "DKP forming part". The prodrug of the present invention can have a temporary amide bond between the dipeptide part of the DKP forming part and the aliphatic amine group of the parent drug. The conversion half-life may be affected by the structural properties of the DKP forming part. For example, the desired conversion half-life can be obtained by using the dipeptide of the DKP forming part illustrated in the present application. The conversion half-life may be affected by the structural properties of the aliphatic amino acids of the parent drug to which the DKP forming part is connected. For example, the desired conversion half-life can be obtained by using the N-terminal amino acid residue of the parent drug illustrated in the present application. The DKP forming part can be a dipeptide-based extension connected to the parent drug. The DKP forming part can also contain other structural elements in addition to the dipeptide, such as substituents covalently linked to the dipeptide. The DKP forming part can be inactive or can be associated with pharmacological activity. The conversion of the prodrug of the present invention occurs mainly in a non-enzymatic manner. In one aspect of the present invention, the prodrug of the present invention comprises a DKP forming part.

[0046] An example of nomenclature used for a compound of the present invention comprising a DKP forming moiety and semaglutide as the parent drug is provided below: N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(3-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide. In this compound, the DKP forming moiety comprises a Lys residue and a Thz residue. The moiety '[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-[10-(3-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]' is attached to the ε-amino group of the Lys residue forming the DKP moiety. The parent drug is an analog of GLP-1-(7-37) substituted with Aib at position 8, with Arg at position 34, and with the moiety '[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]' attached to the ε-amino group of the Lys residue at position 26. The complete structure of the compound is shown below:

[0047]

[0048] In one embodiment, the compound of the present invention is a prodrug or a pharmaceutically acceptable salt, ester or amide thereof. In one embodiment, the compound of the present invention comprises Formula I: XYZ (Formula I). ​​In one embodiment of the present invention, Z is a parent drug. In one embodiment of the present invention, XY is a DKP forming part. In one embodiment of the present invention, X is an amino acid. In one embodiment of the present invention, X is selected from Ala, Arg, Asn, Asp, His, Leu, Lys, D-Lys, Phe, Ser, Orn and Dab. In one embodiment of the present invention, X is an amino acid. In one embodiment of the present invention, X is selected from Lys, D-Lys, Orn and Dab. In one embodiment of the present invention, X is an amino acid. In one embodiment of the present invention, X is selected from Asp, Lys and D-Lys. In one embodiment of the present invention, Y is selected from Thz and D-Thz. In one embodiment of the present invention, Z comprises a GLP-1 polypeptide. In one embodiment of the present invention, the N-terminal amino group of the GLP-1 polypeptide is connected to Y via an amide bond. In one embodiment of the present invention, the N-terminal residue of the GLP-1 polypeptide is His. In one embodiment of the present invention, the GLP-1 polypeptide is a GLP-1 analog. In one embodiment of the present invention, the GLP-1 analog has a maximum of 3 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1). In one embodiment of the present invention, the GLP-1 analog has a maximum of 2 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1). In one embodiment of the present invention, Z is a GLP-1 derivative. In one embodiment of the present invention, Z is semaglutide. In one embodiment, X optionally carries a substituent, provided that if X carries a substituent, X is selected from Lys, D-Lys, Dab and Orn. ​​In one embodiment, X is selected from Lys, D-Lys, Dab and Orn, and X carries a substituent.

[0049] In one embodiment, the compound of the present invention is selected from Formula 8, Formula 9, Formula 10, Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, Formula 16, Formula 17, Formula 18, Formula 19, Formula 20, Formula 21, Formula 22, Formula 23, Formula 24, Formula 25, Formula 26, Formula 27, Formula 28, Formula 29, Formula 30, Formula 31, Formula 32, Formula 33, Formula 34 and Formula 35; or a pharmaceutically acceptable salt, ester or amide thereof. In one embodiment, the compound of the present invention is selected from Formula 8, Formula 9, Formula 10, Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, Formula 16, Formula 17, Formula 18, Formula 19, Formula 20, Formula 21, Formula 22 and Formula 23; or a pharmaceutically acceptable salt, ester or amide thereof. In one embodiment, the compound of the present invention is Formula 8.

[0050] In one embodiment, the compound of the present invention is in Formula 9. In one embodiment, the compound of the present invention is in Formula 10. In one embodiment, the compound of the present invention is in Formula 11. In one embodiment, the compound of the present invention is in Formula 12. In one embodiment, the compound of the present invention is in Formula 13. In one embodiment, the compound of the present invention is in Formula 14. In one embodiment, the compound of the present invention is in Formula 15. In one embodiment, the compound of the present invention is in Formula 16. In one embodiment, the compound of the present invention is in Formula 17. In one embodiment, the compound of the present invention is in Formula 18. In one embodiment, the compound of the present invention is in Formula 19. In one embodiment, the compound of the present invention is in Formula 20. In one embodiment, the compound of the present invention is in Formula 21. In one embodiment, the compound of the present invention is in Formula 22. In one embodiment, the compound of the present invention is in Formula 23. In one embodiment, the compound of the present invention is in Formula 24. In one embodiment, the compound of the present invention is in Formula 25. In one embodiment, the compound of the present invention is in Formula 26. In one embodiment, the compound of the present invention is in Formula 27. In one embodiment, the compound of the present invention is in Formula 28. In one embodiment, the compound of the present invention is in Formula 29. In one embodiment, the compound of the present invention is in Formula 30. In one embodiment, the compound of the present invention is of Formula 31. In one embodiment, the compound of the present invention is of Formula 32. In one embodiment, the compound of the present invention is of Formula 33. In one embodiment, the compound of the present invention is of Formula 34. In one embodiment, the compound of the present invention is of Formula 35.

[0051] Semaglutide

[0052] Semaglutide is a GLP-1 derivative. Compared to human GLP-1 (7-37), semaglutide has Aib at position 8, Arg at position 34, and a substituent covalently linked to the side chain of Lys at position 26. The amino acid sequence of semaglutide is included in the sequence listing and may be described herein as "[Aib8, Arg34]-GLP-1-(7-37)-peptide". The amino acid sequence of semaglutide is a GLP-1 polypeptide. The amino acid sequence of semaglutide is a GLP-1 receptor agonist. The amino acid sequence of semaglutide is a GLP-1 analog with two amino acid changes compared to human GLP-1 (7-37). The amino acid sequence of semaglutide is included in the sequence listing as SEQ ID NO: 2.

[0053] The chemical name of semaglutide is N-ε 26 -[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37).

[0054] Semaglutide has the following structure:

[0055]

[0056] Lau et al.: "Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide", Journal of Medicinal Chemistry, vol. 58, no. 18 (2015), p. 7370-7380 describes the development of semaglutide. and and for the treatment of chronic weight management Semaglutide can be prepared using methods known to those skilled in the art, such as those described in WO2006 / 097537.

[0057] The terminal half-life of semaglutide in humans is approximately one week. of active drugs, It is an injectable prescription medicine for adults with type 2 diabetes that, along with diet and exercise, can improve blood sugar levels. The dosing frequency is once a week. of active drugs, It is an oral prescription medicine for adults with type 2 diabetes that, along with diet and exercise, can improve blood sugar levels. Administered as a once-daily oral tablet. A treatment regimen that adopts once-weekly oral administration rather than once-daily oral administration can result in improved patient convenience and patient compliance. The properties of semaglutide are not optimal for once-weekly oral administration. If semaglutide is administered as a suitable prodrug that is converted to semaglutide at a suitable rate once absorbed in the body, then semaglutide may be compatible with once-weekly oral administration. Designing such a semaglutide prodrug would be a significant improvement in available treatment options. In one embodiment, the parent drug of the prodrug of the present invention is semaglutide.

[0058] Functional properties

[0059] The therapeutic use of pharmacologically active compounds may be hindered by inappropriate pharmacokinetic properties, for example, because pharmacokinetic properties are not suitable for reaching the required exposure after the compound is administered. Prodrug technology can be used to improve pharmacokinetic properties, for example, making it suitable for oral administration once a week. The exposure level of the parent drug after the administration of the prodrug depends on the conversion half-life of the prodrug to the drug, so obtaining a suitable conversion half-life can make the compound suitable for a specific dosing regimen (such as once a week administration). The exposure level of the parent drug after the administration of the prodrug depends on the observed terminal half-life of the parent drug, so obtaining a suitable terminal half-life can make the compound suitable for a specific dosing regimen (such as once a week administration). The suitability of the prodrug for oral administration depends on their ability to reach the systemic circulation after absorption in the gastrointestinal tract, so obtaining a suitable oral bioavailability can make the compound suitable for oral administration (such as once a week oral administration).

[0060] According to the first functional aspect, the compounds of the present invention have a desired conversion half-life, such as suitable for once-weekly dosing in humans. According to the second functional aspect, the compounds of the present invention are related to the observed terminal half-life desired for the parent drug, such as suitable for once-weekly dosing in humans. According to the third functional aspect, the compounds of the present invention have a desired oral bioavailability, such as suitable for oral dosing in humans.

[0061] Transformation half-life

[0062] The rate at which a prodrug is converted to a drug can be quantified by a conversion half-life. As used herein, the term "conversion half-life" refers to the length of time required for the prodrug concentration to be halved due to conversion. When measured in vitro at pH 7.4 and 37° C., a suitable conversion half-life of a semaglutide prodrug intended for oral administration once weekly to humans is 3.0-21 days. When measured in vitro at pH 7.4 and 37° C., a preferred conversion half-life of a semaglutide prodrug intended for oral administration once weekly to humans is 3.0-14 days.

[0063] Prodrugs can achieve the desired conversion after intramolecular cyclization of the terminal dipeptide-based amide extension, which is then cut off from the parent drug, resulting in the release of the parent drug in free form. Such intramolecular cyclization can occur as an enzyme-independent process under physiological conditions, such as through the formation of diketopiperazine (DKP). In the prodrug converted by DKP formation, the part that releases the parent drug during conversion is called the DKP forming part. The conversion half-life is particularly dependent on the properties of the DKP forming part, so, for example, by molecular design of the DKP forming part, the properties of the prodrug are suitable for a specific dosing regimen (e.g., oral administration once a week), and the conversion half-life can be improved (e.g., making it suitable for oral administration once a week).

[0064] The conversion half-life can be measured in vitro, for example, at pH 7.4 and 37°C. The conversion half-life of a prodrug to a drug can be measured as described in the general method for measuring the conversion half-life. In one embodiment, the compound of the present invention is a prodrug. In one embodiment of the present invention, when measured in vitro at pH 7.4 and 37°C, the conversion half-life of the prodrug to the parent drug of the prodrug is at least 3.0 days. In one embodiment of the present invention, when measured in vitro at pH 7.4 and 37°C, the conversion half-life of the prodrug to the parent drug of the prodrug is 3.0-21 days, preferably 3.0-14 days.

[0065] Observed terminal half-life

[0066] Many drugs show a biphasic plasma distribution curve, which initially follows a steep slope and then follows a slow slope. The stage following a slow slope can be referred to as a "terminal stage". As used herein, the term "terminal half-life" refers to the time required for the plasma concentration of a compound to be halved in the terminal stage. The terminal half-life of a drug when administered in its free form is different from the terminal half-life of the drug when administered as a prodrug, because when administered as a prodrug, the sustained release of the drug in free form occurs when the prodrug is converted in vivo. Therefore, the prodrug acts as a depot that slowly releases the drug. When administered as a prodrug, the terminal half-life of the parent drug can also be referred to as the "observed terminal half-life". It should be understood that if the term "observed terminal half-life" is used in the context of a prodrug, it refers to the observed terminal half-life of the parent drug released when the prodrug is converted.

[0067] When measured in miniature pigs, the observed terminal half-life suitable for once weekly oral administration in humans may be >80 hours, or preferably >90 hours, or most preferably >100 hours. When measured in miniature pigs, the observed terminal half-life suitable for once weekly oral administration in humans may be <250 hours, or may preferably be <180 hours. When measured in miniature pigs, the observed terminal half-life suitable for once weekly oral administration in humans may be in the range of 90-250 hours, or may preferably be in the range of 100-180 hours.

[0068] The observed terminal half-life can be determined in miniature pigs. The observed terminal half-life can be measured as described in the general method for measuring the terminal half-life. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is >80 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is >90 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is >100 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is >110 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is >120 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is <200 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is <190 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is <180 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is <170 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is <160 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is 80-200 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is 90-180 hours when measured in miniature pigs. In one embodiment of the invention, the observed terminal half-life of the prodrug of the present invention is 120-160 hours when measured in miniature pigs.

[0069] Oral bioavailability

[0070] Oral therapy with pharmacologically active compounds may be hampered by poor bioavailability. The term "bioavailability" refers to the ability of a compound to reach the systemic circulation after administration, and it can be quantified as the fractional extent to which a dose of the compound reaches the systemic circulation after administration. It is desirable for drugs intended for oral administration to have high oral absorption (i.e., high absorption from the gastrointestinal tract after oral administration) because this can reduce the dose required to achieve the desired systemic concentration of the drug, thereby, for example, reducing tablet size and reducing manufacturing costs.

[0071] The term "oral bioavailability" as used herein refers to the ability of a compound to reach systemic circulation after oral administration. Oral bioavailability reflects the degree of absorption of a compound in the gastrointestinal tract after oral administration. In other words, high oral bioavailability is associated with high oral absorption. The high oral bioavailability of a drug is associated with high drug exposure after oral administration. The high oral bioavailability of a prodrug is associated with the high absorption of the prodrug, resulting in high exposure of the parent drug after the prodrug is converted into the parent drug in vivo. For example, as described in WO2019 / 149880, oral bioavailability can be measured in beagle dogs in the form of a co-formulation with an absorption enhancer, N-(8-[2-hydroxybenzoyl]amino) sodium caprylate (SNAC).

[0072] Oral bioavailability can be measured as described in the general method for measuring oral bioavailability. In one embodiment, the compounds of the present invention have high oral bioavailability. In one embodiment, the compounds of the present invention have oral bioavailability similar to that of semaglutide. In one embodiment, the compounds of the present invention have oral bioavailability that is not inferior to that of semaglutide. In one embodiment, the compounds of the present invention have oral bioavailability that is at least as high as that of semaglutide. In one embodiment, the compounds of the present invention have oral bioavailability suitable for oral administration once a week in humans. In one embodiment, the compounds of the present invention have oral bioavailability determined in beagle dogs and measured as Cmax / dose [kg / L]. In one embodiment, the compounds of the present invention have oral bioavailability measured in beagle dogs as Cmax / dose [kg / L]; wherein Cmax / dose [kg / L]>0.10, preferably>0.15, preferably>0.20, preferably>0.25, most preferably>0.30. In one embodiment, the compounds of the present invention have oral bioavailability determined in beagle dogs and measured as AUC / dose [kg*hr / L]. In one embodiment, the compounds of the invention have an oral bioavailability determined in beagle dogs and measured as AUC / dose [kg*hr / L]; wherein AUC / dose [kg*hr / L]>2.0, preferably>5.0, preferably>10.0, preferably>15.0, most preferably>20.0.

[0073] GLP-1 activity

[0074] As used herein, the term "GLP-1 activity" refers to the ability of a compound to activate the GLP-1 receptor. Thus, GLP-1 activity may also be referred to as "GLP-1 potency". GLP-1 activity may be measured as in vitro potency, i.e., performance in a functional GLP-1 receptor assay, more particularly the ability to stimulate cAMP formation in a cell line expressing a cloned human GLP-1 receptor. GLP-1 activity may be expressed as EC50 The ability of a compound to bind to the GLP-1 receptor can also be used as a measure of GLP-1 activity. In this case, the GLP-1 activity can be referred to as "GLP-1 receptor affinity", and the activity can be expressed as IC 50 Methods for studying GLP-1 activity are well known in the art and are described, for example, in WO2011 / 073328, WO2011 / 080102 and WO2012 / 062803.

[0075] Drug Indications / Medical Uses

[0076] The present invention also relates to compounds of the present invention for use as medicaments. As used herein, the term "treatment" refers to medical treatment of any human subject in need. The treatment may be preventive, prophylactic, palliative, symptomatic and / or curative. The timing and purpose of the treatment may vary from individual to individual, depending on the subject's health status.

[0077] In one embodiment, the compounds of the invention are useful for treating and / or preventing (i) all forms of diabetes, (ii) obesity, (iii) non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), (iv) cardiovascular disease, (v) neurodegenerative disorders, (vi) chronic kidney disease (CKD), (vii) diabetic nephropathy (DKD), (viii) peripheral arterial disease (PAD), and / or (ix) heart failure (HF).

[0078] In one embodiment, the invention relates to a method of treating one or more of (i), (ii), (iii), (iv), (v), (vi), (vii), (viii) and (ix) comprising administering to a patient in need thereof an effective amount of a compound of the invention, optionally in combination with one or more additional therapeutically active compounds.

[0079] In one embodiment, the compounds of the invention are useful for the treatment and / or prevention of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes, MODY (maturity-onset diabetes of the young) and gestational diabetes, or for diseases where the reduction of HbA1C is a therapeutic goal. In one embodiment, the compounds are useful for the treatment of cardiovascular diseases, such as syndrome X, atherosclerosis, myocardial infarction, coronary heart disease, reperfusion injury, stroke, cerebral ischemia, early heart disease or early cardiovascular disease, left ventricular hypertrophy, coronary artery disease, hypertension, essential hypertension, acute hypertensive emergency, cardiomyopathy, cardiac insufficiency, exercise intolerance, acute and / or chronic heart failure, arrhythmias, arrhythmias, syncope, angina pectoris, reocclusion of heart bypass and / or stent, intermittent claudication (occlusive arteriosclerosis), diastolic dysfunction and / or systolic dysfunction; and / or blood pressure reduction, such as systolic blood pressure reduction. In one embodiment, the compound is used to treat dyslipidemia and / or diseases with one or more of the following clinical outcomes as treatment targets: lowering serum total lipids; increasing HDL; lowering small dense LDL; lowering VLDL; lowering triglycerides; lowering cholesterol; lowering plasma levels of lipoprotein a (Lp(a)) in humans; inhibiting the production of apolipoprotein A (apo(A)). In one embodiment, the compound is useful for treating non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In one embodiment, the compounds of the present invention are used to treat and / or prevent all forms of HF, such as heart failure with reduced ejection fraction (HFrEF), heart failure with mid-range ejection fraction (HFmrEF), and / or heart failure with preserved ejection fraction (HFpEF).

[0080] In one embodiment, the compounds of the invention are used to treat obesity and / or eating disorders with one or more of the following clinical outcomes as treatment goals: reducing food intake, increasing energy expenditure, reducing body weight, suppressing appetite, inducing satiety. In one embodiment, the compounds are used to treat neurodegenerative disorders.

[0081] Treatment with the compounds of the invention may also be combined with one or more additional pharmacologically active substances, for example, the latter are selected from cardiovascular agents, antidiabetic agents and / or anti-obesity agents. Examples of these pharmacologically active substances are: cardiotonic agents, beta-adrenergic receptor blockers, HMG-CoA reductase inhibitors, angiotensin II receptor antagonists, angiotensin converting enzyme inhibitors, calcium channel blockers, endothelin antagonists, renin inhibitors, diuretics, aldosterone receptor blockers, endothelin receptor blockers, aldosterone synthase inhibitors, CETP inhibitors, relaxin, PCSK9 inhibitors, BNP and NEP inhibitors, GLP-1 analogs, insulin, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV inhibitors, SGLT2 inhibitors. Treatment with the compounds of the invention may also be combined with cardiac surgery.

[0082] Pharmaceutical composition

[0083] The present invention also relates to pharmaceutical compositions (also referred to as pharmaceutical preparations) comprising the compounds of the present invention. In one embodiment, the pharmaceutical composition comprising the compound comprises at least one pharmaceutically acceptable excipient.

[0084] Pharmaceutical compositions comprising a compound of the present invention or a pharmaceutically acceptable salt, amide or ester thereof and a pharmaceutically acceptable excipient can be prepared as known in the art.

[0085] The term "adjuvant" refers broadly to any component other than the active therapeutic ingredient. An adjuvant can be an inert substance, an inactive substance and / or a non-pharmaceutically active substance. An adjuvant can be used for various purposes, for example as a carrier, a vehicle, a diluent, a tablet adjuvant, and / or to improve the absorption of administration and / or active substances. The formulation of pharmaceutical active ingredients and various adjuvants is known in the art, referring to, for example, Remington: The Science and Practice of Pharmacy (e.g., the 19th edition (1995) and any subsequent editions). Other optional ingredients of pharmaceutical compositions include, for example, wetting agents, emulsifiers, antioxidants, fillers, metal ions, oily vehicles, proteins. Non-limiting examples of adjuvants are: solvents, diluents, buffers, preservatives, tonicity regulators, chelating agents, surfactants and stabilizers.

[0086] The pharmaceutical composition comprising the compounds of the present invention can be several dosage forms, such as solutions, suspensions, tablets and capsules. Preferably, the pharmaceutical composition comprising the compounds of the present invention is suitable for oral administration, for example, in a preferred embodiment, the pharmaceutical preparation comprising the compounds of the present invention is prepared in the form of tablets, wherein the compound is co-formulated with an absorption enhancer N-(8-[2-hydroxybenzoyl]amino) sodium caprylate (SNAC), for example, as described in WO2019 / 149880 or WO2019 / 215063.

[0087] In a preferred embodiment, the pharmaceutical composition comprising the compound of the invention is used for the same pharmaceutical indication for which the compound is indicated.

[0088] Preparation process

[0089] The compounds of the invention (or fragments thereof) can be prepared by classical peptide synthesis, such as solid phase peptide synthesis using t-Boc or Fmoc chemistry or other established techniques, see, for example, Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza et al. "Organic Synthesis on solid Phase", Wiley-VCH Verlag GmbH, 2000, and "Fmoc Solid Phase Peptide Synthesis", edited by WC Chan and PD White, Oxford University Press, 2000. In addition or alternatively, the compound (or its fragment) can be produced in whole or in part by recombinant methods, i.e., by culturing a host cell containing a DNA sequence encoding the analog and capable of expressing the peptide in a suitable nutrient medium under conditions that allow the expression of the peptide. Non-limiting examples of host cells suitable for expressing these peptides are: Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines. For example, those derivatives of the present invention containing non-encoded amino acids can be produced as described in the experimental section. Or see, for example, Hodgson et al.: "The synthesis of peptides and proteins containing non-natural amino acids", Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430.

[0090] Specific examples of methods for preparing derivatives of the invention are included in the experimental section.

[0091] List of implementation plans

[0092] 1. A compound comprising formula I:

[0093] XYZ (Formula I)

[0094] Wherein X is an amino acid;

[0095] wherein Y is selected from Thz and D-Thz;

[0096] wherein Z comprises a GLP-1 polypeptide;

[0097] or a pharmaceutically acceptable salt, ester or amide thereof.

[0098] 2. A compound according to any preceding embodiment, wherein X is selected from Ala, Arg, Asn, Asp, His, Leu, Lys, D-Lys, Phe, Ser, Orn and Dab.

[0099] 3. The compound according to any of the preceding embodiments, wherein X is selected from Lys, D-Lys, Orn and Dab.

[0100] 4. The compound according to any of the preceding embodiments, wherein X is selected from Lys, Orn and Dab.

[0101] 5. A compound according to any preceding embodiment, wherein the N-terminal amino group of the GLP-1 polypeptide is linked to Y via an amide bond.

[0102] 6. The compound according to any preceding embodiment, wherein the N-terminal residue of the GLP-1 polypeptide is His.

[0103] 7. The compound according to any preceding embodiment, wherein the GLP-1 polypeptide is a GLP-1 analog.

[0104] 8. A compound according to any preceding embodiment, wherein the GLP-1 polypeptide is a GLP-1 analog; and wherein the GLP-1 analog has a maximum of 3 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1).

[0105] 9. A compound according to any preceding embodiment, wherein the GLP-1 polypeptide is a GLP-1 analog; and wherein the GLP-1 analog has a maximum of 2 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1).

[0106] 10. The compound according to any preceding embodiment, wherein Z is a GLP-1 derivative.

[0107] 11. The compound according to any preceding embodiment, wherein Z is semaglutide.

[0108] 12. The compound according to any preceding embodiment, wherein X is linked to Y via an amide bond.

[0109] 13. A compound according to any preceding embodiment, wherein X is optionally substituted, provided that if X is substituted, X is selected from Lys, D-Lys, Dab and Orn.

[0110] 14. A compound according to any preceding embodiment, wherein X bears a substituent, provided that if X bears a substituent, X is selected from Lys, D-Lys, Dab and Orn.

[0111] 15. The compound according to any of the preceding embodiments, wherein X is selected from Lys, D-Lys, Dab and Orn, and wherein X bears a substituent.

[0112] 16. A compound according to any preceding embodiment, wherein the substituent is linked to Lys via the ε-amino group, to D-Lys via the ε-amino group, to Dab via the γ-amino group, or to Orn via the δ-amino group.

[0113] 17. A compound according to any preceding embodiment, wherein the substituent has a prolonging effect.

[0114] 18. A compound according to any preceding embodiment, wherein the substituent comprises a prolonging moiety.

[0115] 19. A compound according to any preceding embodiment, wherein the substituent is linked to the side chain amino group of X via an amide bond.

[0116] 20. A compound according to any preceding embodiment, wherein the substituent comprises a lipophilic moiety having a distal carboxylic acid.

[0117] 21. The compound according to any preceding embodiment, wherein the substituent comprises a moiety selected from Formula 1 and Formula 2.

[0118] 22. The compound according to any of the preceding embodiments, wherein n of Chemical Formula 1 is 12, 14, 16 or 18.

[0119] 23. The compound according to any of the preceding embodiments, wherein n of Chemical Formula 1 is 14 or 16.

[0120] 24. The compound according to any preceding embodiment, wherein the substituent comprises a moiety selected from Formula 3 and Formula 4.

[0121] 25. A compound according to any of the preceding embodiments, wherein the substituent is of formula II:

[0122] A5-A4-A3-A2-A1-*(Formula II)

[0123] Where * indicates the connection point with X;

[0124] wherein A1 is selected from the group consisting of Chemical Formula 3, Chemical Formula 4, Chemical Formula 5, Chemical Formula 6 and Chemical Formula 7, or does not exist;

[0125] wherein A2 and A3 are each individually selected from Formula 3, Formula 4 and Formula 5, or are absent;

[0126] wherein A4 is chemical formula 3 or chemical formula 4;

[0127] wherein A5 is selected from Chemical Formula 1 and Chemical Formula 2.

[0128] 26. A compound according to any preceding embodiment, wherein the residues A5, A4, A3, A2, A1 are interconnected via an amide bond.

[0129] 27. The compound according to any of the preceding embodiments, wherein the compound is selected from Formula 8, Formula 9, Formula 10, Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, Formula 16, Formula 17, Formula 18, Formula 19, Formula 20, Formula 21, Formula 22, Formula 23, Formula 24, Formula 25, Formula 26, Formula 27, Formula 28, Formula 29, Formula 30, Formula 31, Formula 32, Formula 33, Formula 34, and Formula 35; or a pharmaceutically acceptable salt, ester, or amide thereof.

[0130] 28. The compound according to any of the preceding embodiments, wherein the compound is selected from Formula 8, Formula 9, Formula 10, Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, Formula 16, Formula 17, Formula 18, Formula 19, Formula 20, Formula 21, Formula 22, Formula 23; or a pharmaceutically acceptable salt, ester or amide thereof.

[0131] 29. A compound according to any preceding embodiment, wherein the compound is a prodrug and Z is the parent drug.

[0132] 30. A compound according to any preceding embodiment, wherein the compound is a prodrug and XY is a DKP forming moiety.

[0133] 31. A compound according to any preceding embodiment, wherein the compound is a prodrug, Z is the parent drug, and XY is a DKP forming moiety.

[0134] 32. A compound according to any preceding embodiment, wherein the compound has a transformation half-life.

[0135] 33. A compound according to any preceding embodiment, wherein the compound has a conversion half-life suitable for once-weekly dosing.

[0136] 34. A compound according to any preceding embodiment, wherein the compound has a long transformation half-life.

[0137] 35. The compound according to any preceding embodiment, wherein the conversion half-life is measured in vitro at 37°C and pH 7.4.

[0138] 36. A compound according to any preceding embodiment, wherein the conversion half-life is measured as described in the general method for measuring conversion half-life.

[0139] 37. A compound according to any preceding embodiment, wherein the conversion half-life is at least 3.0 days.

[0140] 38. A compound according to any preceding embodiment, wherein the conversion half-life is at least 4.0 days.

[0141] 39. A compound according to any preceding embodiment, wherein the conversion half-life is at least 5.0 days.

[0142] 40. A compound according to any preceding embodiment, wherein the conversion half-life is at least 6.0 days.

[0143] 41. A compound according to any preceding embodiment, wherein the conversion half-life is 3.0-21 days.

[0144] 42. A compound according to any preceding embodiment, wherein the conversion half-life is 3.0-14 days.

[0145] 43. A compound according to any preceding embodiment, wherein the conversion half-life is measured at 37°C and pH 7.4, wherein the conversion half-life is 3.0-21 days.

[0146] 44. A compound according to any preceding embodiment, wherein the conversion half-life is measured at 37°C and pH 7.4, wherein the conversion half-life is 3.0-14 days.

[0147] 45. A compound according to any preceding embodiment, wherein the conversion half-life is the conversion half-life of a prodrug to a drug.

[0148] 46. ​​A compound according to any preceding embodiment, wherein the parent drug has an observed terminal half-life after administration of the prodrug.

[0149] 47. A compound according to any preceding embodiment, wherein the parent drug has an observed terminal half-life after administration of the prodrug that is suitable for once-weekly dosing.

[0150] 48. A compound according to any preceding embodiment, wherein the parent drug has an observed long terminal half-life after administration of the prodrug.

[0151] 49. A compound according to any preceding embodiment, wherein the terminal half-life observed after administration of the prodrug is suitable for once weekly oral dosing in humans.

[0152] 50. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is >80 hours.

[0153] 51. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is >90 hours.

[0154] 52. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is > 100 hours.

[0155] 53. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is > 110 hours.

[0156] 54. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is > 120 hours.

[0157] 55. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is <200 hours.

[0158] 56. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is <190 hours.

[0159] 57. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is <180 hours.

[0160] 58. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is <170 hours.

[0161] 59. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is <160 hours.

[0162] 60. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is 80-200 hours.

[0163] 61. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is 90-180 hours.

[0164] 62. A compound according to any preceding embodiment, wherein the observed terminal half-life of the parent drug measured after administration of the prodrug in minipigs is 120-160 hours.

[0165] 63. A compound according to any preceding embodiment, wherein the compound is orally bioavailable.

[0166] 64. A compound according to any preceding embodiment, wherein the compound has high oral bioavailability.

[0167] 65. The compound according to any preceding embodiment, wherein the compound has an oral bioavailability similar to that of semaglutide.

[0168] 66. The compound according to any preceding embodiment, wherein the compound has an oral bioavailability that is not inferior to semaglutide.

[0169] 67. The compound according to any preceding embodiment, wherein the compound has an oral bioavailability at least as high as semaglutide.

[0170] 68. A compound according to any preceding embodiment, wherein the oral bioavailability is suitable for once-weekly oral administration in humans.

[0171] 69. A compound according to any preceding embodiment, wherein the oral bioavailability is determined in beagle dogs.

[0172] 70. A compound according to any preceding embodiment, wherein the oral bioavailability is determined in beagle dogs after administration of a tablet containing 3 mg of the compound, 300 mg of sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) and 7.7 mg of magnesium stearate.

[0173] 71. The compound according to any preceding embodiment, wherein the oral bioavailability is measured as Cmax / dose [kg / L].

[0174] 72. The compound according to any preceding embodiment, wherein the oral bioavailability is measured as AUC / dose [kg*hr / L].

[0175] 73. A compound according to any preceding embodiment, wherein the oral bioavailability is determined as described in the General Methods for Measuring Oral Bioavailability.

[0176] 74. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as Cmax / dose [kg / L] in beagle dogs; and wherein the Cmax / dose [kg / L]>0.10.

[0177] 75. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as Cmax / dose [kg / L] in beagle dogs; and wherein the Cmax / dose [kg / L]>0.15.

[0178] 76. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as Cmax / dose [kg / L] in beagle dogs; and wherein the Cmax / dose [kg / L]>0.20.

[0179] 77. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as Cmax / dose [kg / L] in beagle dogs; and wherein the Cmax / dose [kg / L]>0.25.

[0180] 78. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as Cmax / dose [kg / L] in beagle dogs; and wherein the Cmax / dose [kg / L]>0.30.

[0181] 79. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as AUC / dose [kg*hr / L] in beagle dogs; and wherein the AUC / dose [kg*hr / L]>2.0.

[0182] 80. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as AUC / dose [kg*hr / L] in beagle dogs; and wherein the AUC / dose [kg*hr / L]>5.0.

[0183] 81. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as AUC / dose [kg*hr / L] in beagle dogs; and wherein the AUC / dose [kg*hr / L]>10.0.

[0184] 82. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as AUC / dose [kg*hr / L] in beagle dogs; and wherein the AUC / dose [kg*hr / L]>15.0.

[0185] 83. A compound according to any preceding embodiment, wherein the oral bioavailability is measured as AUC / dose [kg*hr / L] in beagle dogs; and wherein the AUC / dose [kg*hr / L]>20.0.

[0186] 84. A compound according to any of the preceding embodiments, wherein semaglutide is N-ε 26 -[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)

[0187]

[0188] 85. A pharmaceutical composition comprising a compound according to any preceding embodiment and at least one pharmaceutically acceptable excipient.

[0189] 86. A pharmaceutical composition comprising a compound selected from Formula 8, Formula 9, Formula 10, Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, Formula 16, Formula 17, Formula 18, Formula 19, Formula 20, Formula 21, Formula 22, Formula 23, Formula 24, Formula 25, Formula 26, Formula 27, Formula 28, Formula 29, Formula 30, Formula 31, Formula 32, Formula 33, Formula 34 and Formula 35, or a pharmaceutically acceptable salt, ester or amide thereof; and at least one pharmaceutically acceptable excipient.

[0190] 87. A pharmaceutical composition comprising a compound selected from Formula 8, Formula 9, Formula 10, Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, Formula 16, Formula 17, Formula 18, Formula 19, Formula 20, Formula 21, Formula 22 and Formula 23; or a pharmaceutically acceptable salt, ester or amide thereof; and at least one pharmaceutically acceptable excipient.

[0191] 88. A compound according to any preceding embodiment for use as a medicament.

[0192] 89. A compound according to any preceding embodiment for use in treating (i) diabetes, (ii) obesity, (iii) non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), (iv) cardiovascular disease, (v) neurodegenerative disorders, (vi) chronic kidney disease (CKD), (vii) diabetic nephropathy (DKD), (viii) peripheral arterial disease (PAD), and / or (ix) heart failure (HF).

[0193] 90. A pharmaceutical composition according to any preceding embodiment for use as a medicament.

[0194] 91. A pharmaceutical composition according to any preceding embodiment, for use in treating (i) diabetes, (ii) obesity, (iii) non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), (iv) cardiovascular disease, (v) neurodegenerative disorders, (vi) chronic kidney disease (CKD), (vii) diabetic nephropathy (DKD), (viii) peripheral arterial disease (PAD), and / or (ix) heart failure (HF).

[0195] 92. A method of treating (i) diabetes, (ii) obesity, (iii) non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), (iv) cardiovascular disease, (v) neurodegenerative disorders, (vi) chronic kidney disease (CKD), (vii) diabetic nephropathy (DKD), (viii) peripheral arterial disease (PAD) and / or (ix) heart failure (HF) by administering to a subject in need thereof a pharmaceutically relevant amount of a compound according to any of the preceding embodiments.

[0196] 93. A pharmaceutical composition comprising a compound according to any preceding embodiment and at least one pharmaceutically acceptable excipient.

[0197] 94. A pharmaceutical composition comprising a compound according to any preceding embodiment and at least one pharmaceutically acceptable excipient for use in treating a disease selected from (i) diabetes, (ii) obesity, (iii) non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), (iv) cardiovascular disease, (v) neurodegenerative disorders, (vi) chronic kidney disease (CKD), (vii) diabetic nephropathy (DKD), (viii) peripheral arterial disease (PAD) and / or (ix) heart failure (HF).

[0198] 95. Use of a compound according to any preceding embodiment in the preparation of a medicament for treating a disease selected from (i) diabetes, (ii) obesity, (iii) non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), (iv) cardiovascular disease, (v) neurodegenerative disorders, (vi) chronic kidney disease (CKD), (vii) diabetic nephropathy (DKD), (viii) peripheral arterial disease (PAD) and / or (ix) heart failure (HF).

[0199] Example

[0200] This experimental section begins with a list of abbreviations, followed by a section on general methods for compound preparation and a section on methods for measuring properties related to exposure profiles. In each section, a number of specific examples are included to illustrate the invention. All example compounds were prepared according to the general methods described herein.

[0201] Abbreviations

[0202] Aib: alpha-aminoisobutyric acid

[0203] Boc: tert-butyloxycarbonyl

[0204] CAD: Charged Aerosol Detector

[0205] Dab: 2,4-diaminobutyric acid

[0206] DIPEA: Diisopropylethylamine

[0207] DCM: Dichloromethane

[0208] DIC: Diisopropylcarbodiimide

[0209] DKP: diketopiperazine

[0210] DMF: Dimethylformamide

[0211] D-PBS: Dulbecco's phosphate buffered saline

[0212] DTT: dithiothreitol

[0213] EDTA: Ethylenediaminetetraacetic acid

[0214] Fmoc: 9-fluorenylmethoxycarbonyl

[0215] Ado: 8-amino-3,6-dioxaoctanoic acid

[0216] HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol

[0217] HPLC: High Performance Liquid Chromatography

[0218] LC: Liquid chromatography

[0219] LCMS: Liquid chromatography mass spectrometry

[0220] MeCN: Acetonitrile

[0221] MQ: Milli-Q

[0222] MS: mass spectrometry

[0223] Mtt: 4-methyltrityl

[0224] Orn: Ornithine

[0225] OtBu: tert-butoxy

[0226] Oxyma Ethyl cyano-hydroxyiminoacetate

[0227] Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

[0228] PBS Phosphate Buffered Saline

[0229] RP: Reverse

[0230] RP-HPLC: Reversed-Phase High-Performance Liquid Chromatography

[0231] RT: Room temperature

[0232] Sar: creatine

[0233] SEC: Size Exclusion Chromatography

[0234] SNAC: Sodium N-(8-(2-hydroxybenzoyl)amino)caprylate

[0235] SPPS: Solid Phase Peptide Synthesis

[0236] tBu: tert-butyl

[0237] TCEP: Tris(2-carboxyethyl)phosphine

[0238] TFA: trifluoroacetic acid

[0239] Thz: Thiazolidine-4-carboxylic acid

[0240] TIPS: Triisopropylsilane

[0241] Trt: triphenylmethyl (trityl)

[0242] UPLC: Ultra-Performance Liquid Chromatography

[0243] UV: Ultraviolet

[0244] General Methods for Preparing Compounds of the Invention

[0245] In one aspect, the derivatives of the invention can be prepared as described in the examples herein. In one aspect, the derivatives of the invention can be prepared as known in the art, i.e., can be prepared by classical peptide synthesis, e.g., solid phase peptide synthesis using Boc or Fmoc chemistry or other established techniques, see, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza “Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH, 2000, and “Fmoc Solid Phase Peptide Synthesis” edited by WC Chan and PD White, Oxford University Press, 2000.

[0246] Fatty acids and special amino acid building blocks

[0247] The synthesis of octadecane dioic acid mono-tert-butyl ester was carried out as described in WO2010102886 (pages 27-28). The corresponding mono-tert-butyl esters of C14, C16 and C20 diacids were prepared accordingly. The synthesis of 10-(3-tert-butoxycarbonylphenoxy)decanoic acid was carried out as described in WO2011080103 (page 131) for 9-(4-tert-butoxycarbonylphenoxy)undecanoic acid.

[0248] Fmoc-Aib-OH, Boc-Dab(Fmoc)-OH, Fmoc-Glu(OH)-OtBu, Boc-Lys(Fmoc)-OH, Boc-Orn(Fmoc)-OH, Fmoc-Thz-OH, and Fmoc-D-Thz-OH can be obtained from Iris Biotech or Sigma-Aldrich.

[0249] Peptide synthesis

[0250] Peptides were prepared by SPPS using Fmoc-based chemistry on a Prelude or Symphony X solid phase peptide synthesizer from Protein Technologies. The Fmoc-protected amino acids used in the method are the recommended standards: for example, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied by Anaspec, Bachem, Iris Biotech or NovabioChem.

[0251] For the final backbone amino acids, alfa-Boc protected amino acids were used: for example Boc-Arg(Pbf)-OH, Boc-Asn(Trt)-OH, Boc-Asp(OtBu)-OH, Boc-His(Trt)-OH, Boc-Leu-OH, Boc-Lys(Ac)-OH, Boc-Lys(Boc)-OH, Boc-D-Lys(Boc)-OH, Boc-Phe-OH, Boc-Ser(tBu)-OH supplied by Bachem, Novabiochem, Iris Biotech or Sigma-Aldrich.

[0252] Wang resin preloaded with Fmoc-protected glycine (Fmoc-Gly-Wang) was used. Subsequent amino acids were introduced in a stepwise procedure using a Prelude or Symphony X peptide synthesizer according to the SPPS principle.

[0253] Fmoc deprotection was achieved with 20% piperidine in DMF over 2x 10 min. Introduction of substituents at the α-position of the N-terminal amino acid was accomplished using standard Fmoc-protected amino acids. Peptide coupling was performed with DIC / Oxyma Pure. First, the amino acid / Oxyma Pure solution (0.3M / 0.3M in DMF, 3-4 times molar excess) was added to the resin. Then, the same molar equivalent of DIC (0.6M in DMF) was added. The coupling time was 1.5 hours. In some cases, the coupling time was increased or the coupling steps were repeated to achieve a satisfactory coupling level. Subsequent end-capping steps were performed using 1M acetic anhydride in DMF and DIPEA.

[0254] Boc-Lys(Fmoc)-OH was used to introduce a substituent on the ε-nitrogen of lysine at the N-terminus of the sequence. Boc-Orn(Fmoc)-OH was used to introduce a substituent on the δ-nitrogen of ornithine at the N-terminus of the sequence. Boc-Dab(Fmoc)-OH was used to introduce a substituent on the γ-nitrogen of 2,4-diaminobutyric acid at the N-terminus of the sequence.

[0255] Fmoc-Lys(Mtt)-OH was used to introduce a substituent on the epsilon-nitrogen of lysine at position 26. The Mtt group was removed by treatment with HFIP / DCM / TIPS (75:22.5:2.5) (2 x 20 min) followed by washing with DCM and DMF before introducing a substituent on the epsilon-nitrogen of Lys.

[0256] General cutting method

[0257] The peptide was cleaved with TFA / TIPS / H2O / DTT (95:2:2:1) for 2 hours, after which the solution was drained into cold ether and centrifuged. The ether was decanted and the peptide was washed twice with ether.

[0258] General methods for purification and quantification of derivatives

[0259] The crude peptide was dissolved in MQ water containing 50% acetic acid and purified by reverse phase preparative HPLC (Waters Delta Prep 4000) on a column containing C18-silica gel. The gradient gradually increased with MeCN in MQ water containing 0.1% TFA was eluted. The relevant fractions were analyzed by UPLC. The fractions containing the pure target peptide were merged. The resulting solution was analyzed (UPLC, LCMS), and the peptide derivative was quantitatively analyzed using a CAD specific HPLC detector (Vanquish Thermo-Fischer HPLC-CAD). The product was dispensed into glass vials. The vials were capped with Millipore glass fiber prefilters. Freeze drying obtained the trifluoroacetate of the derivative as a white solid.

[0260] General LCMS Methods

[0261]

[0262]

[0263] Example 1

[0264] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(3-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0265]

[0266] Chemical formula 8; SEQ ID NO: 3

[0267] LCMS

[0268] Calculated mass: M / 3 = 1688.85; M / 4 = 1266.89; M / 5 = 1013.71

[0269] Measured mass: M / 3 = 1688.86; M / 4 = 1266.90; M / 5 = 1013.72

[0270] Example 2

[0271] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{γ}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butyryl]

[0272] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0273]

[0274] Chemical formula 9; SEQ ID NO: 4

[0275] LCMS

[0276] Calculated mass: M / 2 = 2362.72; M / 3 = 1575.48; M / 4 = 1181.86

[0277] Measured mass: M / 2 = 2362.71; M / 3 = 1575.48; M / 4 = 1181.87

[0278] Example 3

[0279] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{δ}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butyryl]Orn-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0280]

[0281] Chemical formula 10; SEQ ID NO: 5

[0282] LCMS

[0283] Calculated mass: M / 2 = 2369.73; M / 3 = 1580.15; M / 4 = 1185.36

[0284] Measured mass: M / 2 = 2369.70; M / 3 = 1580.15; M / 4 = 1185.37

[0285] Example 4

[0286] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butyryl

[0287] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0288]

[0289] Chemical formula 11; SEQ ID NO: 6

[0290] LCMS

[0291] Calculated mass: M / 3 = 1584.82; M / 4 = 1188.87; M / 5 = 951.29

[0292] Measured mass: M / 3 = 1584.82; M / 4 = 1188.87; M / 5 = 951.29

[0293] Example 5

[0294] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0295]

[0296] Chemical formula 12; SEQ ID NO: 7

[0297] LCMS

[0298] Calculated mass: M / 3 = 1681.54; M / 4 = 1261.40; M / 5 = 1009.32

[0299] Measured mass: M / 3 = 1681.62; M / 4 = 1261.46; M / 5 = 1009.55

[0300] Example 6

[0301] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butyryl]amino]butyryl]amino]butyryl

[0302] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0303]

[0304] Chemical formula 13; SEQ ID NO: 8

[0305] LCMS

[0306] Calculated mass: M / 3 = 1670.85; M / 4 = 1253.39; M / 5 = 1002.91

[0307] Measured mass: M / 3 = 1670.85; M / 4 = 1253.40; M / 5 = 1002.92

[0308] Example 7

[0309] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butyryl

[0310] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0311]

[0312] Chemical formula 14; SEQ ID NO: 9

[0313] LCMS

[0314] Calculated mass: M / 3 = 1584.82; M / 4 = 1188.87; M / 5 = 951.29

[0315] Measured mass: M / 3 = 1584.82; M / 4 = 1188.87; M / 5 = 951.30

[0316] Example 8

[0317] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butyryl]amino]butyryl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0318] Chemical formula 15; SEQ ID NO: 10

[0319] LCMS

[0320] Calculated mass: M / 3 = 1627.84; M / 4 = 1221.13; M / 5 = 977.10

[0321] Measured mass: M / 3 = 1628.09; M / 4 = 1221.07; M / 5 = 977.28

[0322] Example 9

[0323] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]

[0324] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0325]

[0326] Chemical formula 16; SEQ ID NO: 11

[0327] LCMS

[0328] Calculated mass: M / 3 = 1594.17; M / 4 = 1195.87; M / 5 = 956.90

[0329] Measured mass: M / 3 = 1594.17; M / 4 = 1195.88; M / 5 = 956.91

[0330] Example 10

[0331] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0332]

[0333] Chemical formula 17; SEQ ID NO: 12

[0334] LCMS

[0335] Calculated mass: M / 3 = 1690.88; M / 4 = 1268.41; M / 5 = 1014.93

[0336] Measured mass: M / 3 = 1690.92; M / 4 = 1268.43; M / 5 = 1014.94

[0337] Embodiment 11

[0338] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[(2S)-2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]-3-hydroxypropanoyl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0339] Chemical formula 18; SEQ ID NO: 13

[0340] LCMS

[0341] Calculated mass: M / 3 = 1623.18; M / 4 = 1217.63; M / 5 = 974.31

[0342] Measured mass: M / 3 = 1623.18; M / 4 = 1217.63; M / 5 = 974.32

[0343] Example 12

[0344] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0345] Chemical formula 19; SEQ ID NO: 14

[0346] LCMS

[0347] Calculated mass: M / 3 = 1637.18; M / 4 = 1228.13; M / 5 = 982.71

[0348] Measured mass: M / 3 = 1637.19; M / 4 = 1228.14; M / 5 = 982.72

[0349] Example 13

[0350] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]acetyl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0351]

[0352] Chemical formula 20; SEQ ID NO: 15

[0353] LCMS

[0354] Calculated mass: M / 3 = 1613.17; M / 4 = 1210.13; M / 5 = 968.30

[0355] Measured mass: M / 3 = 1613.18; M / 4 = 1210.13; M / 5 = 968.31

[0356] Embodiment 14

[0357] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[2-[[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]acetyl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0358]

[0359] Chemical formula 21; SEQ ID NO: 16

[0360] LCMS

[0361] Calculated mass: M / 3 = 1709.89; M / 4 = 1282.67; M / 5 = 1026.33

[0362] Measured mass: M / 3 = 1709.89; M / 4 = 1282.66; M / 5 = 1026.34

[0363] Embodiment 15

[0364] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-[(4S)-4-carboxy-4-(19-carboxynonadecaylamino)butyryl

[0365] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0366]

[0367] Chemical formula 22; SEQ ID NO: 17

[0368] LCMS

[0369] Calculated mass: M / 3 = 1603.51; M / 4 = 1202.88; M / 5 = 962.51

[0370] Measured mass: M / 3 = 1603.43; M / 4 = 1202.84; M / 5 = 962.48

[0371] Example 16

[0372] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-([(2S)-4-carboxy-2-(13-carboxytridecanoylamino)butanoyl]Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0373]

[0374] Chemical formula 23; SEQ ID NO: 18

[0375] LCMS

[0376] Calculated mass: M / 3 = 1575.48; M / 4 = 1181.86; M / 5 = 945.69

[0377] Measured mass: M / 3 = 1575.40; M / 4 = 1181.82; M / 5 = 945.67

[0378] Embodiment 17

[0379] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0380] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0381]

[0382] Chemical formula 24; SEQ ID NO: 19

[0383] LCMS

[0384] Calculated mass: M / 2 = 2171.58; M / 3 = 1448.05; M / 4 = 1086.29

[0385] Measured mass: M / 2 = 2171.56; M / 3 = 1448.06; M / 4 = 1086.30

[0386] Embodiment 18

[0387] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0388] [Aib8, Arg34]-Lys-Thz-[Aib8, Arg34]-GLP-1-(7-37)-peptide

[0389]

[0390] Chemical formula 25; SEQ ID NO: 20

[0391] LCMS

[0392] Calculated mass: M / 3 = 1452.41; M / 4 = 1089.55; M / 5 = 871.84

[0393] Measured mass: M / 3 = 1452.34; M / 4 = 1089.53; M / 5 = 871.83

[0394] Embodiment 19

[0395] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0396] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0397]

[0398] Chemical formula 26; SEQ ID NO: 21

[0399] LCMS

[0400] Calculated mass: M / 3 = 1452.41; M / 4 = 1089.55; M / 5 = 871.84

[0401] Measured mass: M / 3 = 1452.34; M / 4 = 1089.53; M / 5 = 871.84

[0402] Embodiment 20

[0403] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0404] [Aib8, Arg34]-D-Lys-Thz-[Aib8, Arg34]-GLP-1-(7-37)-peptide

[0405]

[0406] Chemical formula 27; SEQ ID NO: 22

[0407] LCMS

[0408] Calculated mass: M / 3 = 1452.41; M / 4 = 1089.55; M / 5 = 871.84

[0409] Measured mass: M / 3 = 1452.34; M / 4 = 1089.53; M / 5 = 871.83

[0410] Embodiment 21

[0411] N{ε26}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-(acetyl)Lys-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0412]

[0413] Chemical formula 28; SEQ ID NO: 23

[0414] LCMS

[0415] Calculated mass: M / 3 = 1466.41; M / 4 = 1100.06; M / 5 = 880.25

[0416] Measured mass: M / 3 = 1466.34; M / 4 = 1100.02; M / 5 = 880.24

[0417] Embodiment 22

[0418] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0419] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0420]

[0421] Chemical formula 29; SEQ ID NO: 24

[0422] LCMS

[0423] Calculated mass: M / 3 = 1433.39; M / 4 = 1075.29; M / 5 = 860.43

[0424] Measured mass: M / 3 = 1433.33; M / 4 = 1075.27; M / 5 = 860.42

[0425] Embodiment 23

[0426] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0427] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0428]

[0429] Chemical formula 30; SEQ ID NO: 25

[0430] LCMS

[0431] Calculated mass: M / 3 = 1461.74; M / 4 = 1096.56; M / 5 = 877.45

[0432] Measured mass: M / 3 = 1461.67; M / 4 = 1096.52; M / 5 = 877.43

[0433] Embodiment 24

[0434] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0435] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0436]

[0437] Chemical formula 31; SEQ ID NO: 26

[0438] LCMS

[0439] Calculated mass: M / 3 = 1447.72; M / 4 = 1086.04; M / 5 = 869.03

[0440] Measured mass: M / 3 = 1447.67; M / 4 = 1086.02; M / 5 = 869.03

[0441] Embodiment 25

[0442] N{ε26}-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-His-Thz-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0443]

[0444] Chemical formula 32; SEQ ID NO: 27

[0445] LCMS

[0446] Calculated mass: M / 3 = 1455.39; M / 4 = 1091.80; M / 5 = 873.64

[0447] Measured mass: M / 3 = 1455.32; M / 4 = 1091.77; M / 5 = 873.63

[0448] Embodiment 26

[0449] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0450] [Aib8, Arg34]-GLP-1-(7-37)-peptide

[0451]

[0452] Chemical formula 33; SEQ ID NO: 28

[0453] LCMS

[0454] Calculated mass: M / 3 = 1447.40; M / 4 = 1085.80; M / 5 = 868.84

[0455] Measured mass: M / 3 = 1447.34; M / 4 = 1085.77; M / 5 = 868.83

[0456] Embodiment 27

[0457] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0458] [Aib8, Arg34]-Phe-Thz-[Aib8, Arg34]-GLP-1-(7-37)-peptide

[0459]

[0460] Chemical formula 34; SEQ ID NO: 29

[0461] LCMS

[0462] Calculated mass: M / 3 = 1458.73; M / 4 = 1094.30; M / 5 = 875.64

[0463] Measured mass: M / 3 = 1458.68; M / 4 = 1094.27; M / 5 = 875.63

[0464] Embodiment 28

[0465] N{ε26}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl

[0466] [Aib8, Arg34]-Ser-Thz-[Aib8, Arg34]-GLP-1-(7-37)-peptide

[0467]

[0468] Chemical formula 35; SEQ ID NO: 30

[0469] LCMS

[0470] Calculated mass: M / 3 = 1438.72; M / 4 = 1079.29; M / 5 = 863.63

[0471] Measured mass: M / 3 = 1438.66; M / 4 = 1079.27; M / 5 = 863.62

[0472] Example 29 - Reference Compounds

[0473] N{ε 26}-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-N{ε}-(octadecanoyl)D-Lys-Sar-[Aib8,Arg34]-GLP-1-(7-37)-peptide

[0474]

[0475] Chemical formula 36; SEQ ID NO: 31

[0476] LCMS

[0477] Calculated mass: M / 3 = 1526.50; M / 4 = 1145.13; M / 5 = 916.30

[0478] Measured mass: M / 3 = 1526.76; M / 4 = 1145.32; M / 5 = 916.46

[0479] General method for measuring transformation half-life

[0480] This assay was performed to study the prodrug to drug conversion half-life of the prodrugs of the invention. The conversion half-life was studied in vitro at pH 7.4 after incubation at 37°C.

[0481] Peptide stock solutions were prepared by dissolving the lyophilized powder in PBS buffer to a target of 200 μM. PBS buffer is Dulbecco's phosphate buffered saline without CaCl2 and MgCl2, gibco 14190-094, adjusted to pH = 7.4. The pH of the peptide stock solution was adjusted to 7.4 with 0.02M HCl or 0.02M NaOH. The samples were loaded into Agilent HPLC vials with fixed inserts. The vials were capped to prevent evaporation. The HPLC vials were incubated at 37°C and samples were removed at different time points within a 2-week period, flash frozen at -80°C, and stored at -20°C until analysis.

[0482] Sample analysis was performed using UPLC combined with UV detection at 215 nm and MS (UPLC-UV-MS). 1 μl of sample was injected onto a Waters Acquity UPLC with a flow-through needle injection system and a Waters Acquity CSH C18 column (1*150 mm) with a particle size of 1.7 μm and maintained at 55°C. A binary solvent management pump was used to deliver a flow rate of 100 μl / min, with 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B. Gradient elution was performed by using 15-32% B from 0 to 4 min, then 32-48% B from 4 to 54 min.

[0483] The identity of the prodrug was confirmed by MS and the peak purity - area % - from the UV signal at 215 nm was plotted as the natural logarithm versus time and the slope (k) was used to calculate the first order half-life (T1 / 2) = T1 / 2 = Ln(2) / k.

[0484] Embodiment 30

[0485] The half-life of the conversion of the prodrug of the compound of the present invention to the drug is as follows: General method for measuring transformation half-life The results are presented in Table 3. All compounds of the invention have a conversion half-life of 3.3 days or more. The compounds of the invention are associated with surprisingly high conversion half-lives.

[0486] Table 3: Transformation half-life

[0487]

[0488]

[0489] General Methods for Measuring Terminal Half-Life

[0490] This assay is performed to study the terminal half-life of a drug administered in free form or to study the observed terminal half-life of a drug administered as a prodrug (ie, the parent drug).The terminal half-life was studied in minipigs.

[0491] Three (3) heads Minipigs (about 25kg) are equipped with two central catheters. One catheter is used for intravenous administration, for example, 10nmol / kg (0.05ml / kg) of the test compound prepared in suitable formulations (pH 7.4) such as phosphate, propylene glycol and polysorbate, and rinsed with saline. After administration, blood samples (0.8ml) are collected via the second catheter at a predetermined time point (0-3 weeks). The sample is centrifuged, and 0.2ml of blood plasma is used for bioanalysis.

[0492] Bioanalysis is performed as follows: plasma samples are crushed by protein precipitation and analyzed by turboflowLCMS. Calibrators are prepared by spiking test compounds (usually in the range of 0.5 to 500nM) in blank plasma from relevant species. Calibrators, plasma blanks and research samples are precipitated with 3 volumes of ethanol and then centrifuged at 6300rpm for 30 minutes at 4°C. In the matrix where higher background interference is observed, acetonitrile is used for the second precipitation in a ratio of 1:1. The supernatant is diluted with water containing 1% formic acid in a ratio of 1:2 (or 1:1). Samples are analyzed by turboflow LCMS using Cycloneturboflow columns (TurboFlow Cyclone 0.5x 50mm, Thermo Fischer Scientific) at room temperature, and samples are analyzed using Aeris Peptide 3.6μm XB-C18 analytical columns (2.1x50mm, Phenomenex) at 60°C. Gradient elution was performed using mobile phase A (consisting of milli-Q water containing 1% formic acid and 5% methanol / acetonitrile (50 / 50)) and mobile phase B (consisting of methanol / acetonitrile (50 / 50) containing 1% formic acid and 5% milli-Q water). A QExactive Plus mass spectrometer was used as the detector in single ion monitoring mode. A linear calibration curve (weighted 1 / x2) was used to calculate the concentration in plasma samples.

[0493] The plasma concentration (vs. time) profiles of the test compounds were evaluated by noncompartmental analysis (NCA) and standard pharmacokinetic parameters were estimated using WinNonlin Phoenix 64 (version 8.10, CERTARA). 2The best-fit model for the estimated terminal half-life and / or observed terminal half-life was constructed using the NMLE add-on to Phoenix 64 (version 8.10, CERTARA) to fit the data.

[0494] Embodiment 31

[0495] The terminal half-life and / or observed terminal half-life are measured as described in the general method for measuring terminal half-life. The terminal half-life of semaglutide administered in free form in minipigs is 69 hours. The observed terminal half-life of two compounds of the invention and the reference compound are presented in Table 4. The observed terminal half-life (of released semaglutide) of the compounds of the invention is at least 100 hours. The compounds of the invention are associated with surprisingly high observed terminal half-lives, which constitute a proof of concept for the prodrug technology claimed herein.

[0496] Table 4: Terminal half-lives observed in minipigs

[0497] Compound Observed terminal half-life [hours] Test 1 154 Test 2 121 Chemical formula 36 (reference compound) 78

[0498] General Methods for Measuring Oral Bioavailability

[0499] This assay is performed to measure the oral bioavailability of compounds. This assay determines the exposure of test compounds following oral administration in beagle dogs as described by relevant pharmacokinetic parameters and plasma concentration profiles.

[0500] Preparation of tablets for oral administration: Tablets containing test compounds for the assays described herein are SNAC-based immediate release tablets. The test compounds were spray dried as neutral sodium salts (pH 7-8). Dry granulation was performed by roller compaction on a Gerteis MINI-PACTOR. Tablets containing 3 mg of test compound, 300 mg of sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) and 7.7 mg of magnesium stearate were produced using a 7.2x12 mm punch on a Kilian Style One.

[0501] Determination of absorption after oral administration: Eight (8) male beagle dogs, aged 1 to 5 years at the start of the study, weighing approximately 10-12 kg, were used. The dogs were housed in groups in pens (12 hours light: 12 hours dark) and fed Royal Canin Medium Adult dog food (Royal Canin Products, China Branch, or Brogaarden A / S, Denmark) once a day individually and restrictively. Exercise and group socialization were allowed every day whenever possible. Repeated pharmacokinetic studies were performed using these dogs with appropriate washout periods between consecutive doses. An appropriate adaptation period was given before the start of the first pharmacokinetic study. All handling, dosing, and blood sampling of the animals were performed by trained and skilled personnel. Prior to the study, the dogs were fasted overnight and fasted from 0 to 4 hours after dosing. In addition, water was restricted to the dogs from 1 hour before dosing until 4 hours after dosing, and water was available ad libitum for the rest of the period.

[0502] Tablets containing the test compound are administered in the following manner: 10 min before tablet administration, about 3 nmol / kg of SEQ ID NO:32 is administered subcutaneously to the dog. The tablet is placed in the back of the dog's oral cavity to prevent chewing. The mouth is then closed and 10 mL or 50 mL of tap water is administered by syringe to facilitate the swallowing of the tablet. Blood samples are collected at predetermined time points and continue until 336 hours after administration to fully cover the complete plasma concentration-time absorption curve of the prodrug. For each blood sampling time point, about 1.2 mL of whole blood is collected in a 1.5 mL EDTA-coated tube, and the tube is gently rotated to mix the sample with EDTA. Then, the blood sample is kept on ice until centrifugation (4 min, 4 ° C, 4000 rpm). The plasma is pipetted into a Micronic tube on dry ice and kept at -20 ° C until analysis. Blood samples were obtained as appropriate, for example from venflon in the cephalic vein of the foreleg for the first 2 hours and then from the jugular vein using a syringe for the remaining time points (the first few drops were allowed to drain from the venflon to avoid heparinized saline from the venflon in the sample).

[0503] Bioanalysis was performed as follows: plasma concentrations of test compounds were determined by plasma protein precipitation and analyzed by liquid chromatography-mass spectrometry (LC-MS). Calibrators were prepared by spiking blank canine plasma with analytes to a final concentration typically in the range of 2 to 200 nM. Calibrators, plasma blanks, or study samples were prepared for LC-MS by protein precipitation with the addition of 3 volumes of ethanol followed by centrifugation at 4000 rpm for 1 h at 4°C. The supernatant was diluted with 2 volumes of Milli-Q water containing 1% formic acid prior to injection into the LC-MS system. The system used was a Transcend II Interface Module SRD3200 system from Thermo Scientific (Waltham, MA, USA) coupled to an Orbitrap Exploris 240 mass spectrometer from Thermo Scientific. The LC was equipped with a Cyclone column (CH-953288, Thermo Scientific) as the first dimension trapping column and a Poroshell 120SB-C18 2.7 μm (2.1 x 50 mm, from Agilent, Santa Clara, CA, USA) as the analytical column. The mobile phase composition of the loading pump was as follows: mobile phase A consisted of 95% milli-Q water, 2.5% acetonitrile, 2.5% methanol, and 0.1% formic acid; mobile phase B consisted of 47.5% acetonitrile, 47.5% methanol, 5% milli-Q water, and 0.1% formic acid. The analytes of interest were loaded from the Turbo flow column to the second dimension analytical column at 30% B. Mobile phase A (95% milli-Q water, 2.5% acetonitrile, 2.5% methanol and 0.1% formic acid) and mobile phase B (47.5% acetonitrile, 47.5% methanol, 5% milli-Q water and 0.1% formic acid) were used for gradient elution with an elution pump with a gradually rising gradient from 0% mobile phase B to 70% mobile phase B in 0.25 minutes, from 70% mobile phase B to 80% mobile phase B in 1.17 minutes, and then from 80% mobile phase B to 95% mobile phase B in 1.17 minutes. The Orbitrap Exploris240 was operated in parallel reaction monitoring (PRM) scanning mode in positive ionization mode. Linear calibration curves (weighted 1 / x2) were used to calculate the concentration of the test compound in the plasma samples to determine the maximum plasma concentration (Cmax). Quality control samples for the analytes were included. The deviation between the nominal concentration and the calculated concentration in the calibrator and quality control samples was less than 15%, while the LLOQ sample was less than 20%. The plasma concentration (vs. time) profiles of the test compounds were evaluated by non-compartmental analysis (NCA) using WinNonlin Phoenix 64 (version 8.10, CERTARA) and standard pharmacokinetic parameters were estimated.Results are reported as dose-corrected plasma concentration (vs. time) curves as well as dose-corrected maximum plasma concentration (Cmax / dose) and dose-corrected area under the curve (AUC / dose).

[0504] Embodiment 32

[0505] Oral bioavailability was determined as described in the General Methods for Measuring Oral Bioavailability. The relevant pharmacokinetic parameters for the two compounds of the invention and the reference compound are presented in Table 5. The compounds of the invention are associated with surprisingly high Cmax / dose. The dose-corrected plasma concentration (vs. time) curves for the two compounds of the invention and the reference compound are shown in Table 5. Figure 1 The compounds of the invention are associated with surprisingly high exposure as determined by dose-normalized plasma concentration curves. The compounds of the invention are associated with surprisingly high oral bioavailability.

[0506] Table 5: Pharmacokinetic parameters after oral administration in beagle dogs

[0507] Compound Cmax / dose [kg / L] AUC / dose [kg*hr / L] Test 1 0.35 24.0 Test 2 0.32 24.2 Chemical formula 36 (reference compound) 0.10 1.6

Claims

1. A compound comprising formula I: XYZ (Formula I) Wherein X is an amino acid; wherein Y is selected from Thz and D-Thz; wherein Z comprises a GLP-1 polypeptide; or a pharmaceutically acceptable salt, ester or amide thereof.

2. The compound according to claim 1, wherein X is selected from Ala, Arg, Asn, Asp, His, Leu, Lys, D-Lys, Phe, Ser, Orn and Dab.

3. The compound according to claims 1-2, wherein the N-terminal amino group of the GLP-1 polypeptide is linked to Y via an amide bond.

4. The compound according to claims 1-3, wherein the N-terminal residue of the GLP-1 polypeptide is His.

5. The compound according to claims 1-4, wherein the GLP-1 polypeptide is a GLP-1 analogue having at most 2 amino acid changes compared to GLP-1 (7-37) (SEQ ID NO: 1).

6. The compound according to claims 1-5, wherein Z is semaglutide.

7. The compound according to claims 1-6, wherein X bears a substituent, provided that if X bears a substituent, X is selected from Lys, D-Lys, Dab and Orn.

8. The compound of claim 7, wherein the substituent comprises a lipophilic moiety having a distal carboxylic acid.

9. The compound according to claim 8, wherein the lipophilic moiety having a distal carboxylic acid is of Formula 1 or Formula 2: Chemical formula 1: Where n = 12-18, Chemical formula 2: Where m=9.

10. The compound according to claims 7-9, wherein the substituent comprises a moiety selected from Chemical Formula 3 and Chemical Formula 4: Chemical formula 3: Chemical formula 4:

11. The compound according to claim 7-10, wherein the substituent is of formula II: A5-A4-A3-A2-A1-*(Formula II) Where * indicates the connection point with X; Wherein A1 is selected from Chemical Formula 3, Chemical Formula 4, Chemical Formula 5, Chemical Formula 6 and Chemical Formula 7, or does not exist: Chemical formula 3: Chemical formula 4: Chemical formula 5: Chemical formula 6: Chemical formula 7: wherein A2 and A3 are each individually selected from Formula 3, Formula 4 and Formula 5, or are absent; wherein A4 is chemical formula 3 or chemical formula 4; Wherein A5 is selected from Chemical Formula 1 and Chemical Formula 2: Chemical formula 1: Where n = 12-18; Chemical formula 2: Where m=9.

12. The compound according to claim 11, wherein the residues A5, A4, A3, A2, A1 are linked to each other via an amide bond.

13. The compound according to claim 1, wherein the compound is selected from Formula 8, Formula 9, Formula 10, Formula 11, Formula 12, Formula 13, Formula 14, Formula 15, Formula 16, Formula 17, Formula 18, Formula 19, Formula 20, Formula 21, Formula 22, Formula 23, Formula 24, Formula 25, Formula 26, Formula 27, Formula 28, Formula 29, Formula 30, Formula 31, Formula 32, Formula 33, Formula 34 and Formula 35; or a pharmaceutically acceptable salt, ester or amide thereof.

14. A compound according to claims 1-13 for use as a medicament.

15. A compound according to claims 1-14 for use in the treatment of (i) diabetes, (ii) obesity, (iii) non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), (iv) cardiovascular disease, (v) neurodegenerative disorders, (vi) chronic kidney disease (CKD), (vii) diabetic nephropathy (DKD), (viii) peripheral arterial disease (PAD), and / or (ix) heart failure (HF).

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