CN118632867A - GLP-1/GIP receptor co-agonist prodrugs and uses thereof - Google Patents

GLP-1/GIP receptor co-agonist prodrugs and uses thereof Download PDF

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CN118632867A
CN118632867A CN202380017655.7A CN202380017655A CN118632867A CN 118632867 A CN118632867 A CN 118632867A CN 202380017655 A CN202380017655 A CN 202380017655A CN 118632867 A CN118632867 A CN 118632867A
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compound
formula
amide
pharmaceutically acceptable
glp
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P·J·克奈尔
B·P·芬南
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Novo Nordisk AS
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Novo Nordisk AS
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Abstract

Prodrug compounds of GLP-1/GIP receptor co-agonists are provided, wherein the GLP-1/GIP receptor co-agonist has been modified by dipeptide linking to the GLP-1/GIP receptor co-agonist via an amide bond. Prodrugs disclosed herein have an extended half-life and are converted to active GLP-1/GIP receptor co-agonists under physiological conditions by a non-enzymatic reaction driven by chemical instability.

Description

GLP-1/GIP receptor co-agonist prodrugs and uses thereof
Technical Field
The present invention relates to 2, 5-Diketopiperazine (DKP) based prodrugs of compounds that are co-agonists of the glucagon-like peptide 1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor, which have a prolonged spectrum of action, suitable for oral administration to humans, and their therapeutic uses.
Incorporation by reference of the sequence listing
The present application is presented with a sequence listing in electronic form. The entire contents of this sequence listing are incorporated herein by reference.
Background
Many therapeutically active agents have low bioavailability after oral administration due to malabsorption or susceptibility to first pass metabolism (e.g.) ,Salama N.N.,Fasano A.,Thakar M.,Eddington N.D.,The impact ofΔG on the oral bioavailability of low bioavailable therapeutic agents,J.Pharmacol.Exp.Ther.,2005,312,199-205).
Prodrugs are therapeutic agents that are themselves nearly inactive but predictably converted to active molecular entities (e.g., testa b., mayer J.M, hydrolysis in Drug and Prodrug Metabolism, wiley-VCH,2003, page 4). Prodrug chemistry provides the opportunity to precisely control the onset and duration of action of a drug after it has cleared from the site of administration and reached equilibrium in plasma at highly defined concentrations. Most prodrugs in clinical use today require enzymatic catalysis to be converted to the active drug. For drugs that need to be released into the bloodstream following gastrointestinal absorption, prodrug approaches catalyzed by enzymes are commonly employed. One common method of prodrugs is to use ester derivatives of the drug which are readily converted to the active drug by esterase-catalyzed hydrolysis (one disadvantage of enzymatic-based cleavage such as ,Yu L.X.,Straughn A.B.,Faustion P.J.,Yang Y.,Parekh A.,Ciavarella A.B.,Asafu-Adjaye E.,Mehta M.U.,Conner D.P.,Lesko L.J.,Hussain A.S.The effect of food on the relative bioavailability of rapidly dissolving immediate-release solid oral products containing highly solubledrugs.Mol.Pharm.2004,1,357-362). is inter-patient variability, there may be significant variability in enzyme levels between individuals, resulting in biological variability in activation of the prodrug by enzymatic cleavage, enzyme levels may also vary from site to site of administration for example, it is well known that in the case of subcutaneous injections, certain areas of the body produce more predictable therapeutic effects than other areas, non-enzymatic cleavage or intramolecular catalysis is of particular interest in order to reduce such unpredictable effects (e.g., tersta b., mayer J.M, hydrolysis in Drug and Prodrug Metabolism, wiley-VCH,2003, page 5).
DKP-based prodrug technology is based on chemical transformations, in which a partial cyclization consisting of two α -amino acids forms a six-membered ring, and simultaneously releases the active drug. DKP-based prodrug technology has been previously described. For example, WO2010/071807, WO2010080605, WO2011/163012 and WO 2011/162968 describe various peptide-based prodrugs linked by an amide bond to, for example, glucagon superfamily peptides or other known agents. WO2014152460 and WO2016049174 describe peptide-based prodrugs of glucagon superfamily peptides and insulin, which have an extended half-life.
Dual activation of GLP-1 and GIP receptors, for example by combining the actions of GLP-1 and GIP in one formulation, has been described to lead to a therapeutic principle that has significantly better blood glucose level reduction, insulin secretion increase and weight loss in mice with type 2 diabetes (T2D) and obesity (e.g., V AGault et al, clin Sci (Lond), 121,107-117,2011) than the commercially available GLP-1 agonist liraglutide. It has been demonstrated in humans that native GLP-1 and GIP interact in a cumulative manner after co-infusion, with significantly enhanced insulinotropic effects compared to GLP-1 alone (M A Nauck et al, J.Clin.Endocrinol. Metab.,76,912-917,1993).
GLP-1/GIP receptor co-agonists and their potential medical uses are described in several patent applications, such as WO 2010/011439、WO 2013/164483、WO 2014/192284、WO 2015/067715、WO 2015/022420、WO 2015/086728、WO 2015/086729、WO 2016/111971、WO 2020/023386、US 9745360、US2014/162945 and US2014/0357552. Patent applications disclosing oral delivery of GLP-1 derivatives are described, for example, in WO 2011/080103, WO 2012/080471, WO 2013/189988 and WO 2019/149880.
However, there remains a desire for compounds that have agonist activity at GIP and GLP-1 receptors and are suitable for oral administration. It would be desirable to find compounds that have an extended duration of action on both GIP and GLP-1 receptors, such that the frequency of administration of such compounds is low. Thus, there is a need for longer acting GLP-1/GIP receptor co-agonists to fully exploit their potential in the treatment of diseases such as T2D.
Disclosure of Invention
Peptide-based drugs are highly potent drugs, but have relatively short duration of action and variable therapeutic indices. Prodrug technology can be employed to optimize the properties of the drug in a manner that makes it suitable for a particular dosing regimen (e.g., once a week dosing). The prodrug is converted by enzymatic or non-enzymatic chemical processes resulting in the slow release of the biologically active drug molecule (referred to herein as the active drug) in the body.
The present disclosure relates to GLP-1/GIP receptor co-agonist prodrugs having desirable properties, for example, for once-weekly oral administration. The prodrugs described herein are designed to delay onset of action and extend the half-life of the active drug (i.e., GLP-1/GIP receptor co-agonist). Delaying the onset of action is advantageous because it allows systemic distribution of the prodrug prior to activation. Thus, administration of the prodrug may eliminate complications caused by peak activity at the time of administration and increase the therapeutic index of the active drug. The intact prodrug does not exert biological activity to a significant extent compared to the active drug. Once the active agent is released upon prodrug conversion, the biological activity is brought about by the active agent. The reduced biological activity of the prodrug compared to the released active drug is advantageous because it allows administration of relatively large amounts of the prodrug without concomitant side effects and risk of overdosing.
The present invention relates to GLP-1/GIP receptor co-agonist prodrugs. Additionally, or alternatively, the present invention relates to GLP-1/GIP receptor co-agonist prodrugs having an extended in vivo terminal half-life.
In a first aspect, the invention relates to compounds that are prodrugs of GLP-1/GIP receptor co-agonists. In some embodiments, the compound comprises formula I: B-Z, wherein Z is a GLP-1/GIP receptor co-agonist (active agent), B is a dipeptide ("dipeptide B"), and wherein the N-terminal amino group of the GLP-1/GIP receptor co-agonist is linked to B by a peptide bond. In some embodiments, the dipeptide B comprises a covalently linked moiety, e.g., a substituent that is capable of forming a non-covalent binding interaction with a mammalian plasma protein, such as mammalian serum albumin. In some embodiments, the dipeptide B is capable of cleaving from the active agent Z by an intramolecular reaction, thereby releasing the active agent (i.e., GLP-1/GIP receptor co-agonist) and forming 2, 5-Diketopiperazine (DKP) as a byproduct. In some embodiments, the intramolecular reaction occurs under physiological conditions. Additionally, or alternatively, in some embodiments, the intramolecular reaction occurs without enzymatic activity.
In a second aspect, the invention relates to a pharmaceutical composition comprising a compound as described herein.
In a third aspect, the present invention relates to a compound as described herein or a pharmaceutical composition comprising a compound as described herein for use as a medicament.
In one functional aspect, the invention provides prodrugs (e.g., compounds as described herein) having a conversion half-life suitable for once-weekly administration. Additionally, or alternatively, in another functional aspect, the present invention provides prodrugs having an observed terminal half-life that is suitable for once-weekly dosing. Additionally, or alternatively, in another functional aspect, the present invention provides prodrugs with surprisingly high oral bioavailability.
Another aspect of the invention relates to the medical use of the compounds described herein. Additionally, or alternatively, the present invention relates to the use of a compound as described herein for the prevention and/or treatment of type 2 diabetes. Additionally, or alternatively, the present invention relates to the use of the compounds described herein for the prevention and/or treatment of obesity. Additionally, or alternatively, the present invention relates to the use of a compound as described herein for the prevention and/or treatment of liver diseases.
Another aspect of the invention relates to methods of treating a disease by administering a compound described herein to a patient in need thereof. In some embodiments, the disease is type 2 diabetes. In some embodiments, the disease is overweight. In some embodiments, the disease is obesity.
The present invention may also address other issues as will be apparent from the disclosure of the exemplary embodiments.
Detailed Description
Hereinafter, greek letters may be represented by their symbols or corresponding written names, for example: α=alpha; beta = beta; epsilon = epsilon; gamma = gamma; omega = omega; etc. Furthermore, the greek letter μmay also be denoted by "u", e.g. μl=ul or μm=um. Symbols in the chemical diagramsRepresenting the connection point with the adjacent part. In the following, unless otherwise indicated in the specification, terms in the singular also include the plural, for example, when referring to "a compound" it is to be understood that this includes all individual variants falling within the broad definition of the compound. As used herein, a "means" one (species) or more (species) ". The present invention relates to compounds that are prodrugs of GLP-1/GIP receptor co-agonists, e.g., prodrugs of GLP-1/GIP receptor co-agonists that possess desirable properties, e.g., for once-a-week oral administration. These compounds are converted under physiological conditions in a controlled manner to active GLP-1/GIP receptor co-agonists (active drugs).
In a first aspect, the present invention relates to a compound that is a prodrug of a GLP-1/GIP receptor co-agonist, said compound comprising formula I: B-Z, wherein Z is a GLP-1/GIP receptor co-agonist (active agent) which is released from B upon prodrug conversion. In a second aspect, the invention relates to a pharmaceutical composition comprising a compound described herein. Another aspect of the invention relates to the medical use of the compounds described herein. Additionally, or alternatively, the present invention relates to the use of a compound as described herein for the prevention and/or treatment of type 2 diabetes. Additionally, or alternatively, the present invention relates to the use of the compounds described herein for the prevention and/or treatment of obesity. Additionally, or alternatively, the present invention relates to the use of a compound as described herein for the prevention and/or treatment of liver diseases.
General definition
The term "compound" relates to a prodrug of a GLP-1/GIP receptor co-agonist. The compounds of the present invention may be referred to as "compounds" and the term "compound" is also intended to encompass pharmaceutically relevant forms thereof, i.e. pharmaceutically acceptable salts, amides or esters thereof.
The term "polypeptide" or "polypeptide sequence" as used herein refers to a series of two or more amino acids that are linked to each other by an amide bond (e.g., a peptide bond). The term polypeptide may be used interchangeably with the term "peptide" and the term "protein".
The term "derivative" generally refers to a chemically modified polypeptide (e.g., a GLP-1/GIP receptor co-agonist) or dipeptide in which one or more substituents are covalently attached to the amino acid sequence of the polypeptide or dipeptide, e.g., through a bond to the epsilon amino group of Lys. In some embodiments, the compounds of the invention comprise derivatives (e.g., derivatives of GLP-1/GIP receptor co-agonists and/or derivatives of dipeptides) that have been modified such that one or more substituents having protracting properties are covalently linked to the amino acid sequence of the polypeptide or dipeptide.
The term "dipeptide derivative" means that the dipeptide has been chemically modified such that it bears at least one substituent (e.g., substituent b, as described herein). In some embodiments, two or more substituents may be present.
The term "GLP-1/GIP receptor co-agonist derivative" refers to a GLP-1/GIP receptor co-agonist that has been chemically modified to have a substituent. For example, such GLP-1/GIP receptor co-agonist derivatives may comprise one or more substituents covalently linked to the amino acid sequence of the polypeptide, e.g., through a linkage to the epsilon amino group of Lys.
The term "amino acid conjugated to a fatty acid" refers to any proteinogenic (proteinogenic) or nonproteinaceous (non-proteinogenic) amino acid having a functional group that has been chemically modified to be conjugated to the fatty acid by a covalent bond to the fatty acid or preferably by a linker. Examples of such functional groups include amino (e.g., lys), sulfhydryl (e.g., cys), and carboxyl (e.g., glu or Asp). In some embodiments, the conjugated amino acid is Lys. When the fatty acid is conjugated to the functional group-bearing proteinogenic or non-proteinogenic amino acid, a fatty acid precursor such as a dicarboxylic acid (e.g. CO 2H-(CH2)-CO2 H, where n=10-22) may be used.
The term "fatty acid" refers to an optionally substituted carboxylic acid having an aliphatic or cyclic hydrocarbon chain, wherein the aliphatic chain is saturated or unsaturated. In some embodiments, the fatty acid is a C 12-C24 saturated carboxylic acid, such as a C 16-C22 saturated carboxylic acid. In some embodiments, the fatty acid comprises additional functional groups.
The term "lipophilic moiety" as used herein refers to a moiety comprising aliphatic and/or cyclic hydrocarbon moieties, which together have more than 6 and less than 30 carbon atoms, preferably more than 8 and less than 20 carbon atoms. In some embodiments, the lipophilic moiety comprises a carbon chain containing at least 8 consecutive-CH 2 -groups. In some embodiments, the lipophilic moiety comprises at least 10 consecutive-CH 2 -groups, such as at least 12 consecutive-CH 2 -groups, at least 14 consecutive-CH 2 -groups, at least 16 consecutive-CH 2 -groups, or at least 18 consecutive-CH 2 -groups. In some embodiments, the lipophilic moiety may comprise any number between 6 and 30 consecutive-CH 2 -groups (e.g., 6, 7, 8, 9, etc.).
The term "distal carboxylic acid" as used herein in the context of a lipophilic moiety refers to a carboxylic acid that is attached to the most distal (terminal) point of the lipophilic moiety relative to the point of attachment of the lipophilic moiety to an adjacent moiety, e.g., in the compounds described herein, the lipophilic moiety (e.g., chemical formula 1) having a distal carboxylic acid is an extension and the carboxylic acid is attached to the most distal (terminal) point of the lipophilic moiety relative to the point of attachment of the lipophilic moiety to an adjacent linker element (e.g., chemical formula 2, chemical formula 3, chemical formula 4, or chemical formula 5). A non-limiting example of a lipophilic moiety having a distal carboxylic acid is chemical formula 1.
The term "therapeutic index" describes the ratio between the blood concentration at which a drug becomes toxic and the blood concentration at which the drug is effective. The larger the Therapeutic Index (TI), the safer the drug. If TI is small (the difference between the two concentrations is very small), the drug must be carefully administered and the person receiving the drug should be closely monitored for any signs of drug toxicity.
Amino acids
The term "amino acid" as used herein refers to any amino acid, i.e., both proteinogenic and non-proteinogenic amino acids. The term "proteinogenic amino acids" as used herein refers to the 20 standard amino acids encoded by the human genetic code. The term "non-proteinogenic amino acid" as used herein refers to any amino acid that does not qualify as a proteinogenic amino acid. Non-protein amino acids are either not present in the protein or are not produced by standard cellular mechanisms (e.g., they may have undergone post-translational modification). Non-limiting examples of non-protein amino acids are Aib (alpha-amino isobutyric acid or 2-amino isobutyric acid), norleucine, norvaline, and D-isomers of protein amino acids.
Generally, amino acid residues as used herein (e.g., in the context of a polypeptide sequence) can be represented by their full name, their single letter code, and/or their three letter code. These three ways are fully equivalent and are used interchangeably. Hereinafter, each amino acid of the peptides described herein, for which optical isomers are not specified, should be understood to mean the L-isomer (unless otherwise indicated). Examples of non-proteinogenic amino acids that may be incorporated into the compounds of the invention are listed in table 1.
Table 1. Non-limiting examples of non-protein amino acids that may be incorporated into the compounds of the present invention.
Prodrugs
The term "prodrug" as used herein refers to a compound that undergoes chemical conversion in vivo by enzymatic or non-enzymatic chemical processes resulting in the release of an active drug. The term "active agent" as used herein refers to a pharmacologically active compound that is released from a prodrug upon conversion of the prodrug. Non-limiting examples of active agents are parent compounds 1-5 as described herein. The term "conversion" as used herein in the context of a prodrug refers to a process in which the prodrug is enzymatically or non-enzymatically converted resulting in the release of the active drug. The rate at which transformation occurs can be quantified by the "transformation half-life". "conversion half-life" is the length of time required for the prodrug concentration to halve as a result of conversion. "conversion half-life" may also be referred to as "prodrug to drug conversion half-life" or "prodrug to active drug conversion half-life".
The prodrug does not exert the desired pharmacological activity to a significant extent, e.g., it does not exert the desired pharmacological activity to such an extent that it is incompatible with the desired therapeutic regimen. Once the active agent is released, pharmacological activity associated with the intended treatment of the prodrug is generated from the active agent. When the active agent is released from the prodrug, it is referred to as "free form". Prodrugs can achieve the desired conversion following intramolecular cyclization of a dipeptide-based terminal amide extension which is then cleaved from the active agent, resulting in release of the active agent in free form. Such intramolecular cyclization can occur under physiological conditions as an enzyme-independent process, for example, by the formation of 2, 5-Diketopiperazine (DKP). In prodrugs that are converted to active agents by intramolecular cyclization to form DKP, the moiety that releases the active agent upon conversion is referred to as the "DKP moiety". The "DKP moiety" comprises a dipeptide moiety (e.g., a dipeptide or dipeptide derivative). In some embodiments, the prodrug may have a temporary amide bond, such as a peptide bond, between the dipeptide portion of the DKP moiety and the aliphatic amine group of the active drug. In some embodiments, the DKP moiety is linked to the GLP-1/GIP receptor co-agonist through the α -amino group of the amino acid at position 1 of the GLP-1/GIP receptor co-agonist backbone, i.e., an amide bond formed between the carboxylic acid group of the DKP moiety and the α -amino group of Tyr at position 1 of the GLP-1/GIP receptor co-agonist backbone. In some embodiments, the DKP moiety is linked to the amino group of Tyr1 in the GLP-1/GIP receptor co-agonist backbone by acylation, i.e., by an amide bond formed between the carboxylic acid group of the DKP moiety and the α -amino group of Tyr in the GLP-1/GIP receptor co-agonist backbone.
The half-life of transformation may be affected by the structural nature of the DKP moiety. For example, a desirable half-life for transformation can be obtained by using dipeptide B as exemplified in the present application. The half-life of the transformation may be affected by the structural nature of the amino acid of the active drug to which the DKP moiety is attached. In some embodiments, a desirable half-life of transformation can be obtained by using the N-terminal amino acid residues of the active agents exemplified in the present application. In some embodiments, the DKP moiety is a dipeptide-based extension linked to the active agent. In some embodiments, the DKP moiety comprises additional structural elements, such as substituents covalently linked to the dipeptide (also referred to herein as "dipeptide derivatives"). DKP is formed as a by-product after prodrug conversion and active drug release. DKP may be inactive or may be associated with pharmacological activity. In some embodiments, the conversion of the prodrugs described herein occurs primarily in a non-enzymatic manner. In some embodiments, the conversion of the prodrugs described herein occurs only in a non-enzymatic manner.
In some embodiments, the compounds described herein are prodrugs or pharmaceutically acceptable salts, esters, or amides thereof. In some embodiments, the prodrug is a compound according to formula I, wherein B is a dipeptide optionally comprising a substituent B, wherein substituent B comprises or consists of an extension and an optional linker. In some embodiments, Z is a GLP-1/GIP receptor co-agonist bearing the substituent Z; and wherein the N-terminal amino group of the GLP-1/GIP receptor co-agonist is linked to B via a peptide bond. In some embodiments of the invention, B is a DKP moiety.
In some embodiments, the compounds described herein comprise a DKP moiety. In some embodiments, the compounds described herein comprise a prodrug comprising a DKP moiety and an active agent. In some embodiments, the active agent is a GLP-1/GIP receptor co-agonist (e.g., active agent Z). In some embodiments, the DKP moiety comprises a dipeptide (e.g., dipeptide B), optionally with one or more substituents.
Examples of nomenclature used for compounds described herein, also referred to herein as "prodrugs," comprising a DKP moiety and GLP-1/GIP receptor co-agonists as active agents are provided below: k [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH. In this compound, the DKP moiety comprises a Lys residue and a Sar residue linked to each other by an amide bond. The (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl moiety is covalently linked to the epsilon-nitrogen atom of the Lys residue of the dipeptide by an amide bond, while the 2- [2- [2- [ [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] amino ] ethoxy ] acetyl moiety is covalently linked to the epsilon-nitrogen atom of the Lys residue of the GLP-1/GIP receptor co-agonist by an amide bond. The carboxyl group of the Sar residue is covalently linked to the N-terminal amino group of the amino acid sequence of a GLP-1/GIP receptor co-agonist via an amide bond. The complete structure of this compound is shown below:
Substituent group
The term "substituent" as used herein refers to a moiety that is covalently linked to an amino acid of a dipeptide or polypeptide (e.g., a GLP-1/GIP receptor co-agonist). In some embodiments, substituent z is linked to a GLP-1/GIP receptor co-agonist by Lys. In some embodiments, substituent B is linked to an amino acid residue of a DKP moiety (e.g., a dipeptide moiety (e.g., dipeptide B) present in a compound of the invention) of a GLP-1/GIP receptor co-agonist, thereby forming part of the DKP moiety. A polypeptide or dipeptide is said to be "substituted" if the substituent is attached to the polypeptide or dipeptide. When a substituent is covalently attached to a polypeptide or amino acid residue, the polypeptide or amino acid is said to be "bearing" the substituent. Substituents may comprise a series of individually defined moieties; these moieties may be referred to as "substituent elements". Non-limiting examples of "substituent elements" are "extenders" and "linkers".
The substituents may be capable of forming a non-covalent binding interaction with albumin, thereby promoting circulation of the compound in the blood stream, thereby having the effect of extending the time of presence of the compound in the blood stream, as the aggregates of the compound bearing the substituents and albumin disintegrate only slowly to release the free form of the compound; thus, the substituent as a whole may also be referred to as an "albumin binding moiety" and the substituent may be referred to as having "protracting effect". The substituents may comprise moieties that are particularly associated with albumin binding and thus with action extension, which may be referred to as "extenders" or "extension moieties". The terms "extension" and "extension" are used interchangeably herein. The "extension" may be a lipophilic moiety (e.g., a fatty acid). The "extension" may be a fatty acid (e.g., a C 16-C22 carboxylic acid). A non-limiting example of an "extension" is shown in table 2. In the chemical formula 1, the chemical formula is shown in the drawing,Are used to describe the point of attachment to a linker or polypeptide by covalent bonds.
Table 2: non-limiting examples of "extenders".
The substituent may comprise a moiety located between the extension moiety and the point of attachment to an amino acid residue of the polypeptide, which moiety may be referred to as a "linker". The linker may comprise several "linker elements". The linker elements may be selected such that they improve the overall properties of the molecule, e.g., such that they improve oral bioavailability, conversion half-life, or prolongation, thereby improving the overall exposure profile of the compound following oral administration.
Non-limiting examples of connector elements are listed in table 3. In the chemical formula 2-5, a compound having a structure of,Are used to describe the point of attachment to an extender or polypeptide.
TABLE 3 non-limiting examples of connector elements
In some embodiments, the substituent is L-P (formula III), wherein P comprises or consists of a lipophilic moiety having a distal carboxylic acid, and P has protracting properties. In some embodiments, P is formula 1. In some embodiments, P is formula 1 and L is a linker comprising linker element a 1-A5:
L-P (formula III),
Wherein P is formula 1, and wherein L is formula IV:
A 1-A2-A3-A4-A5 (formula IV),
Wherein A 1 is covalently bound to an amino acid of a dipeptide or GLP-1/GIP receptor co-agonist and is selected from formula 2, formula 3, formula 4, and formula 5; wherein a 5 is covalently bound to P and is formula 2; wherein a 2、A3 and a 4 are each independently selected from formula 2, formula 3, formula 4, and formula 5, or are absent, provided that if a 2、A3、A4 and a 5 are absent, a 1 is also covalently bound to P.
Non-limiting examples of substituents comprising lipophilic moieties are listed in table 4. In some embodiments, the substituents are selected from table 4.
Table 4. Non-limiting examples of substituents.
If a substituent is attached to a dipeptide portion of a compound described herein (e.g., dipeptide B), the substituent is referred to herein as "substituent B". In some embodiments, substituent b comprises an extension according to formula 1, wherein n is 14, 16, or 18; and optionally a linker, wherein the linker comprises one or more γglu (formula 2) and/or one or more Ado (formula 3) and/or one or more Gly (formula 4) and/or one or more epsilon Lys (formula 5). In some embodiments, substituent b is selected from formula 16, formula 17, formula 18, formula 19, formula 20, formula 21, and formula 22.
If a substituent is attached to a GLP-1/GIP receptor co-agonist (e.g., active agent Z) of a compound described herein, the substituent is referred to herein as "substituent Z". In some embodiments, substituent z comprises or consists of an extension according to formula 1 (wherein n is 14, 16 or 18) and a linker, wherein the linker comprises or consists of one or more yglu (formula 2) and/or one or more Ado (formula 3) and/or one or more epsilon Lys (formula 5). In some embodiments, substituent z is selected from formula 7, formula 8, formula 10, and formula 11.
In some embodiments, the compounds of the present invention comprise substituent b and/or substituent z. GLP-1/GIP receptor co-agonists
As used herein, a "GLP-1/GIP receptor co-agonist" is a compound that is a GLP-1 receptor agonist and a GIP receptor agonist. GLP-1/GIP receptor co-agonists described herein comprise or consist of a polypeptide and optionally a defined substituent z. In some embodiments, the GLP-1/GIP receptor co-agonist exhibits an extended half-life, which is obtained by conjugation of the amino acid residue of the co-agonist with a C 16-C22 fatty acid (optionally via a linker), as described above.
In some embodiments, the carboxyl terminus of the peptide has a-CO 2 H group. In some embodiments, the compounds may optionally comprise an amide group (C (=o) -NH 2) at the C-terminus, which is a modification to replace-OH with-NH 2, as seen for example in parent compound No. 5.
In some embodiments, the GLP-1/GIP receptor co-agonist is
YX2EGTX6TSDYSX12X13LX15X16X17AX19X20X21FX23X24WLX27X28GX30X31X32X33X34X35X36X37X3 8X39(SEQ ID NO.:1)
Having an optional C-terminal amide modification, wherein
X 2 is Aib or A
X 6 is F or V
X 12 is I or Y
X 13 is Y, A, L, I or Aib
X 15 is D or E
X 16 is K or E
X 17 is Q or I
X 19 is A or Q
X 20 is Q, R, E, H or K
X 21 is A or E
X 23 is I or V
X 24 is E, Q or N
X 27 is L or I
X 28 is A or R
X 30 is G or is absent
X 31 is P or is absent
X 32 is E, S or is absent
X 33 is S, K or is absent
X 34 is G or is absent
X 35 is A or is absent
X 36 is P or is absent
X 37 is P or is absent
X 38 is P or is absent
X 39 is S or absent;
And optionally wherein the substituent z comprising a lipophilic moiety such as a fatty acid (e.g., C 16-C22 carboxylic acid) is attached to the GLP-1/GIP receptor co-agonist via lysine (K) at position 16, 20 or 33.
In some embodiments, substituent z comprises or consists of an extension according to formula 1, wherein n is 14, 16 or 18, and optionally a linker, wherein the linker comprises or consists of one or more yglu (formula 2) and/or one or more Ado (formula 3) and/or one or more Gly (formula 4) and/or one or more epsilon Lys (formula 5). In some embodiments, substituent z is selected from formula 7, formula 8, formula 10, and formula 11.
Dipeptide B
In some embodiments, dipeptide B is a DKP moiety. In some embodiments, dipeptide B may be referred to as X-Y (formula II), wherein X and Y are alpha-amino acids. In some embodiments, the conformation of the amide bond between X and Y is preferably cis to promote DKP formation by locating the α -amino group of X in suitable proximity to the α -carbonyl group of Y for nucleophilic attack. In some embodiments, dipeptide B carries substituent B. In some embodiments, Y is an N-alkylated α -amino acid. In some embodiments, Y is an N-alkylated α -amino acid linked to B through an amide bond formed between the α -carboxylic acid group of Y and the amine of active drug Z. An "N-alkylated α -amino acid" is any α -amino acid substituted at the α -amino group of the amino acid with an alkyl group, such as a C 1-C12 alkyl group or such as a C 1-C6 alkyl group, wherein the alkyl group may be linear or cyclic and may be unsubstituted or substituted with additional functional groups, such as amino groups (as in N- (2-aminoethyl) glycine). In some embodiments, the alkyl is selected from the group consisting of methyl, ethyl, 2-aminoethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, n-hexyl. In some embodiments, the alkyl group is selected from the group consisting of methyl, ethyl, 2-aminoethyl, n-propyl, sec-butyl, and n-hexyl. In some embodiments, the alkyl group is methyl. In some embodiments, Y is selected from Sar、N-secBu-Gly、Pro、Pro(4-OH)、N-Me-Glu、N-Me-NOr、N-Me-homoAla、N-Me-Ala、N-Me-Lys、Aeg、N-Hex-homoAla、N-Pr-Ala、homoPro、N-Et-Gly、N-Pr-Gly and N-Me-Phe. In some embodiments, Y is Aeg or Sar.
In some embodiments, X is any α -amino acid. In some embodiments, X is any α -amino acid linked to Y through an amide bond formed between the α -carboxylic acid group of X and the α -amino group of Y. In some embodiments, X is selected from Lys, phe (4-NH 2), D-Lys, ala, gly, pro, D-Val, homoPro, D-Pro, D-homoPro, D-Ala, and Aze. In some embodiments, X is selected from Gly, asp, leu, lys, D-Lys and Pro.
In some embodiments, Y is selected from Sar、N-sBu-Gly、Pro、Pro(4-OH)、N-Me-Glu、N-Me-NOr、N-Me-homoAla、N-Me-Ala、N-Me-Lys、N-Hex-homoAla、N-Pr-Ala、homoPro、N-Pr-Gly、N-Et-Gly and N-Me-Phe, and X is selected from Lys, phe (4-NH 2), and D-Lys. Additionally or alternatively, in some embodiments, Y is selected from Sar and Aeg, and X is selected from Ala, gly, D-Lys, pro, D-Val, homoPro, D-Pro, D-homoPro, D-Ala, and Aze.
In some embodiments, the dipeptide optionally comprising substituent b is selected from table 5a.
In some embodiments, the dipeptide derivative is selected from table 5b.
Non-limiting examples of dipeptide derivatives of dkp moieties.
Non-limiting examples of substituted dipeptides of dkp moieties are shown in table 5b.
Active drug Z
The active agent Z described herein is a GLP-1/GIP receptor co-agonist comprising a substituent Z as defined above, wherein the substituent Z is attached to the GLP-1/GIP receptor co-agonist by an amino acid residue.
Functional properties
The therapeutic use of pharmacologically active compounds may be hindered by unsuitable pharmacokinetic properties, for example, because the pharmacokinetic properties are unsuitable for achieving the desired exposure after administration of the compound. Prodrug technology can be used to improve pharmacokinetic properties, for example, to make it suitable for once-weekly oral administration. The level of exposure of the active agent following administration of the prodrug depends on the conversion half-life of the prodrug to the agent, so obtaining a suitable conversion half-life may adapt the compound to a particular dosing regimen (e.g., once a week dosing). The level of exposure of the active agent following administration of the prodrug depends on the observed terminal half-life of the active agent, so obtaining a suitable terminal half-life may adapt the compound to a particular dosing regimen (e.g., once a week dosing). The suitability of prodrugs for oral administration depends on their ability to reach the systemic circulation after absorption in the gastrointestinal tract, so obtaining suitable oral bioavailability may render the compounds suitable for oral administration (e.g., once a week oral administration).
According to the first functional aspect, the compounds as described herein do not exert to any significant extent the expected efficacy at human GLP-1 and/or GIP receptors compared to the active drug. Additionally, or alternatively, in a second functional aspect, a prodrug as described herein is converted to an active drug under physiological conditions. Additionally, or alternatively, in a third functional aspect, a prodrug as described herein has improved pharmacokinetic properties, such as an extended terminal half-life, following intravenous, subcutaneous, and/or oral administration.
Functional receptor activation activity
According to the first functional aspect, the prodrug of the invention does not activate the human GlP-1 receptor and/or the human GIP receptor to any significant extent compared to the active drug. Functional activity of a GLP-1/GIP receptor agonist as described herein can be tested in vitro as described herein as "general method for measuring functional efficacy in vitro".
The term half maximal effective concentration (EC 50) generally refers to the concentration that induces a response halfway between the baseline and maximum values, with reference to the dose response curve. EC 50 was used as a measure of the potency of the compound and represents the concentration at which 50% of its maximum effect was observed.
Thus, the in vitro potency of a compound can be determined as described herein, and the lower the EC 50.EC50 value, the better the potency.
In order to characterize such compounds, it may also be necessary to consider the in vitro potency of the natural hormone relative to each receptor.
For example, in vitro efficacy may be determined in media containing membranes expressing the appropriate GLP-1 and/or GIP receptor, and/or in assays using whole cells expressing the appropriate GLP-1 and/or GIP receptor.
For example, the functional response of human GLP-1 and/or GIP receptor can be measured in a reporter assay, e.g., in a stably transfected BHK cell line that expresses human GLP-1 and/or GIP receptor and contains CAMP Response Element (CRE) DNA coupled to a promoter and a firefly luciferase (CRE luciferase) gene. This in turn results in expression of luciferase when cAMP is produced due to activation of GLP-1 and/or GIP receptors. Luciferase may be determined by the addition of luciferin, which is converted by the enzyme to oxidized luciferin and produces bioluminescence, which is measured as a reporting indicator of in vitro efficacy. An example of such an assay is described in example 5 described herein. Since the compounds may contain one or more substituents intended to bind albumin, care must also be taken that receptor activity may be affected by the presence or absence of Human Serum Albumin (HSA) in the assay medium. An increase in EC 50 as compared to EC 50 in the absence of HSA indicates a decrease in potency of the compound in the presence of HSA, indicates interaction of the compound with HSA and predicts an increase in duration of action in vivo.
In one embodiment, the active agent has an effective in vitro effect of activating human GLP-1 and GIP receptors.
In one embodiment, the parent compound is capable of activating human GLP-1 and GIP receptors in vitro, and EC 50 is less than 20pM when performed in the absence of HSA in a CRE luciferase reporter assay as described in example 2 herein.
In one embodiment, the parent compound has in vitro potency against human GLP-1 and GIP receptors, as determined using the method of example 2, corresponding to EC 50 at or below 100pM, more preferably below 50pM, or most preferably below 20 pM.
In one embodiment, EC 50 in both human GLP-1 and GIP receptor assays is 1-25pM, such as 1-20pM, such as 1-15pM, or such as 1-10pM.
Half-life of transformation
According to a second functional aspect, the prodrugs of the invention have a surprisingly good half-life for conversion.
The rate at which conversion of the prodrug to the active drug occurs can be quantified by the conversion half-life. The term "conversion half-life" as used herein refers to the length of time required for the prodrug concentration to halve as a result of conversion.
For prodrugs intended for once-weekly oral administration in humans, the desirable conversion half-life may be 24-500 hours, such as 50-400 hours, such as 75-300 hours, or such as 100-200 hours, when measured at pH 7.4 and 37 ℃, as described in the general methods for measuring conversion half-life herein.
Prodrugs can achieve the desired transformation following intramolecular cyclization of the DKP moiety, which in turn is cleaved from the active agent, resulting in release of the active agent. Such intramolecular cyclization can occur under physiological conditions as an enzyme-independent process, for example, by the formation of 2, 5-Diketopiperazine (DKP). In prodrugs that can be converted to active agents by DKP formation, the moiety that releases the active agent upon conversion is referred to as the DKP moiety. The conversion half-life is dependent inter alia on the nature of the DKP moiety, and thus, for example, by molecular design of the DKP moiety, adapting the nature of the prodrug to a particular dosing regimen (e.g., once a week oral dosing) can improve the conversion half-life (e.g., adapt it to once a week oral dosing).
In some embodiments, the conversion half-life is suitable for once daily dosing. In some embodiments, the conversion half-life is suitable for once weekly dosing. In some embodiments, the conversion half-life is >24 hours. In some embodiments, the conversion half-life is >50 hours. In some embodiments, the conversion half-life is >75 hours. In some embodiments, the conversion half-life is >100 hours. In some embodiments, the transformation half-life is <500 hours. In some embodiments, the transformation half-life is <400 hours. In some embodiments, the transformation half-life is <300 hours. In some embodiments, the transformation half-life is <200 hours.
In some embodiments, the transformation half-life is 24-500 hours. In some embodiments, the transformation half-life is 50-400 hours. In some embodiments, the conversion half-life is 75-300 hours. In some embodiments, the conversion half-life is 100-200 hours.
Observed terminal half-life
Many drugs exhibit a dual-stage plasma profile that initially follows a steep slope followed by a gentle slope. The phase following the slow slope may be referred to as the "end phase". The term "terminal half-life" as used herein refers to the time required for the plasma concentration of a compound to halve in the terminal phase. The terminal half-life of a drug when administered in its free form differs from the terminal half-life of the drug when administered as a prodrug because when administered as a prodrug, sustained release of the drug in free form occurs upon in vivo conversion of the prodrug.
The plasma concentration of the active agent administered as a prodrug is especially a result of the elimination of the active agent from the blood stream and the gradual conversion of the prodrug to the active agent. The gradual conversion of the prodrug ensures a sustained supply of the active drug, thus reducing the number of administrations required to reach the desired level of exposure compared to when the drug is administered in free form. The sustained supply of active drug into the bloodstream is reflected in the observed terminal half-life (i.e., measurable terminal half-life) which is longer when administered as a prodrug than when administered in free form.
The pharmacokinetic properties of the prodrugs or active drugs of the prodrugs of the invention may be suitably determined by in vivo pharmacokinetic studies. Such studies were conducted to assess how drug compounds are absorbed, distributed and eliminated in the body over time, and how these processes affect the concentration of the compounds in the body. In the discovery and preclinical stages of drug development, such characterization can be performed using animal models such as mice, rats, monkeys, dogs, mini-pigs, or pigs. Any of these models can be used to test the pharmacokinetic properties of the prodrugs of the invention. In such studies, single doses of the drug are typically administered intravenously (i.v.), subcutaneously (s.c.), or orally (p.o.) to animals in the form of related formulations. Blood samples were withdrawn at predetermined time points after administration and analyzed for drug concentration by a related quantitative determination. Based on these measurements, plasma concentration curves of the compounds under study are plotted and so-called non-compartmental pharmacokinetic analysis of the data is performed. For most compounds, the final portion of the plasma concentration curve will be straight when plotted in a semilogarithmic graph, indicating that the drug is removed from the body at a constant fractional rate. The rate (lambda Z or lambda z) is equal to the negative of the slope of the final part of the plot. From this rate, the terminal half-life can also be calculated at t1/2 = ln (2)/lambda z (see, e.g., johan Gabrielsson and DANIEL WEINER: pharmacokinetics and Pharmacodynamic Data analysis. Protocols & Applications, 3 rd edition, swedish Pharmaceutical Press, stock holm (2000)). When studying active agents administered as prodrugs, the terminal half-life of the active agent is affected by the sustained supply of active agent caused by the gradual conversion of the prodrug, as the prodrug acts as a reservoir for the slow-release drug. Thus, analysis of the terminal half-life of an active drug administered as a prodrug is most conveniently referred to as the "observed terminal half-life" because it is different from when the active drug is administered in free form.
In some embodiments, the terminal half-life of a prodrug of the invention is determined as described herein in the general method of measuring terminal half-life in minipigs. The observed terminal half-life suitable for once weekly oral administration in humans may be >50 hours, or preferably >70 hours, or most preferably >90 hours, when measured in minipigs. 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 minipigs. The observed terminal half-life suitable for once weekly oral administration in humans may be in the range of 50-250 hours, or may preferably be in the range of 90-180 hours, when measured in minipigs.
Oral bioavailability
Oral treatment 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 fraction of the dose of the compound that reaches the systemic circulation after administration. It is desirable for a drug for oral administration to have high oral absorption (i.e., high absorption from the gastrointestinal tract after oral administration) because this can reduce the dosage required to reach the desired systemic concentration of the drug, thus, for example, reducing the tablet size and reducing manufacturing costs.
The term "oral bioavailability" as used herein refers to the ability of a compound to reach the systemic circulation after oral administration. Oral bioavailability reflects the extent to which a compound is absorbed in the gastrointestinal tract following 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 following oral administration. Oral bioavailability may be measured in beagle dogs as co-formulation with the absorption enhancer sodium N- (8- [ 2-hydroxybenzoyl ] amino) caprylate (SNAC), as described in WO 2019/149880.
Oral bioavailability may be measured as described herein in the general method of measuring oral bioavailability in beagle dogs. In some embodiments, the compounds as described herein have high oral bioavailability. In some embodiments, the compounds as described herein have similar oral bioavailability as the active drug. In some embodiments, the compounds as described herein have an oral bioavailability that is no inferior to that of the active drug. In some embodiments, the compounds as described herein have an oral bioavailability that is at least as high as the active drug. In some embodiments, a compound as described herein has an oral bioavailability suitable for once-a-week oral administration to a human. In some embodiments, a compound as described herein has an oral bioavailability as measured in beagle dogs as Cmax/dose [ kg/L ]. In some embodiments, a compound as described herein has an oral bioavailability measured as Cmax/dose [ kg/L ] in beagle; wherein Cmax/dose [ kg/L ] >0.10, preferably >0.15, most preferably >0.20. In some embodiments, a compound as described herein has an oral bioavailability as measured in beagle dogs as AUC/dose [ kg hr/L ]. In some embodiments, a compound as described herein has an oral bioavailability as measured in beagle dogs as AUC/dose [ kg hr/L ]; wherein AUC/dose [ kg hr/L ] >2.0, preferably >5.0, most preferably >10.0.
Pharmaceutical composition
Pharmaceutical compositions comprising a prodrug described herein, or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients, may be prepared as known in the art.
The term "adjuvant" broadly refers to any component other than the active therapeutic ingredient. The auxiliary materials can be inert substances, inactive substances and/or non-pharmaceutically active substances. Adjuvants may be used for a variety of purposes, for example as carriers, vehicles, fillers, binders, lubricants, glidants, disintegrants, flow control agents, crystallization inhibitors, solubilizers, stabilizers, colorants, flavorants, surfactants, emulsifiers or combinations thereof, and/or for improving dosing and/or improving absorption of the active substance. The amount of each adjuvant used may vary within the usual limits of the art. Techniques and adjuvants useful in formulating oral dosage forms are described in the following documents: handbook of Pharmaceutical Excipients (e.g., release 8, sheskey et al, publication 2017 of ,American Pharmaceuticals Association and Pharmaceutical Press,Royal Pharmaceutical Society of Great Britain, and any subsequent versions); and Remington, THE SCIENCE AND PRACTICE of Pharmacy (e.g., 22 nd edition, remington and Allen, eds., pharmaceutical Press (2013), and any subsequent versions).
Pharmaceutical compositions comprising the compounds described herein may be in several dosage forms, such as solutions, suspensions, tablets, and capsules. The pharmaceutical composition comprising the prodrug of the present invention may be administered to several sites, e.g., local sites, such as skin or mucosal sites, of a patient in need thereof; bypass the site of absorption, such as in an artery, vein or heart; and sites involved in absorption, such as in the skin, under the skin, in muscles, in the mouth or in the abdomen. The dosage administered may comprise from 0.1ug/kg to 100mg/kg of the compound of the invention.
In some embodiments, the pharmaceutical composition may be a solid formulation, e.g., a freeze-dried or spray-dried composition, which may be used as such, or to which a solvent and/or diluent is added by a physician or patient prior to use. In one embodiment, the pharmaceutical composition is in the form of a tablet. In a further embodiment, the pharmaceutical composition may be a solid formulation comprising or consisting of a prodrug of the invention, a salt of N- [8- (2-hydroxybenzoyl) amino ] caprylic acid, such as sodium N- [8- (2-hydroxybenzoyl) amino ] caprylate (SNAC), and one or more other excipients known in the art, for example using any one or more of the formulations described in WO 2012/080471, WO 2013/189988 or WO 2019/149880. In one embodiment, the pharmaceutical formulation is a tablet comprising a prodrug of the invention, SNAC and one or more additional excipients.
Or the pharmaceutical composition is a liquid formulation, such as an aqueous formulation. Liquid compositions suitable for injection may be prepared using conventional techniques in the pharmaceutical industry, including dissolving and mixing the ingredients as appropriate to obtain the desired end product. Thus, according to one procedure, the compounds according to the invention are dissolved in a suitable buffer of suitable pH. For example, the composition may be sterilized by sterile filtration.
Pharmaceutically acceptable salts
In some embodiments, the prodrug as described herein is in the form of a pharmaceutically acceptable salt. For example, salts are formed by chemical reactions between bases and acids, such as: 2NH 3+H2SO4→(NH4)2SO4. The salt may be a basic salt, an acid salt, or it may be neither (i.e., a neutral salt). In water, the basic salt produces hydroxide ions and the acid salt produces hydronium ions. Salts of prodrugs can be formed by adding cations or anions between the anions or cationic groups, respectively. These groups may be located in the peptide, and/or in substituents of the derivative.
Non-limiting examples of anionic groups include any free carboxylic acid groups in the substituents (if any) as well as in the peptide. The peptide may contain a C-terminal free carboxylic acid group (if present), as well as any free carboxylic acid groups of amino acid residues such as aspartic acid and glutamic acid.
Non-limiting examples of cationic groups include any free amino groups in the substituents (if any) as well as in the peptide. The peptide may comprise the free amino group at the N-terminus (if present), as well as any free imidazole, guanidine, or amino group of amino acid residues such as histidine, arginine, and lysine.
In certain embodiments, the prodrugs of the invention are in the form of pharmaceutically acceptable salts.
Pharmaceutical indications
Another aspect of the invention relates to a compound as described herein for use as a medicament. As used herein, the term "treatment" refers to medical treatment of any human subject in need thereof. The treatment may be prophylactic, preventative, palliative, symptomatic and/or curative. The timing and purpose of the treatment may vary from individual to individual, depending on the health status of the subject.
In some embodiments, the compounds described herein are used in the following medical treatments:
(i) Preventing and/or treating all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes mellitus, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reducing HbA1C;
(ii) Delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of Impaired Glucose Tolerance (IGT) to type 2 diabetes requiring insulin, delaying or preventing insulin resistance, and/or delaying the progression of type 2 diabetes without insulin to type 2 diabetes requiring insulin;
(iii) For example, preventing and/or treating eating disorders such as overweight or obesity by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of antipsychotics or steroids; reducing gastric motility; delaying gastric emptying; increase physical activity; and/or preventing and/or treating co-morbidities of obesity, such as osteoarthritis and/or urinary incontinence;
(iv) Weight maintenance after successful weight loss (whether drug induced or diet and exercise induced) -i.e., preventing weight gain after successful weight loss.
(V) Preventing and/or treating liver disorders such as liver steatosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver inflammation or fatty liver.
In some embodiments, the compounds are used in methods of preventing and/or treating diabetes and/or obesity. In some embodiments, the compounds are used in methods of treating diabetes and/or obesity.
In some embodiments, the compounds are used in methods of treating or preventing type 2 diabetes. In some embodiments, the compounds are used in methods of treating type 2 diabetes. In some embodiments, the compounds are used in methods of treating or preventing obesity. In some embodiments, the compounds are used in methods of treating obesity. In some embodiments, the compounds are used in methods of weight management. In some embodiments, the compounds are used in methods of reducing appetite. In some embodiments, the compounds are used in methods of reducing food intake.
Production process
For example, prodrugs of the invention (or fragments thereof) may be produced by classical peptide synthesis, e.g., solid phase peptide synthesis using t-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 A"Organic Synthesis on Solid Phase", wiley-VCH VERLAG GmbH,2000; and "Fmoc Solid PHASE PEPTIDE SYNTHESIS", oxford University Press,2000, edited by W.C. Chan and P.D. white.
Specific examples of methods of preparing prodrugs are included in the experimental section.
In some embodiments, the methods of making the compounds described herein comprise a solid phase peptide synthesis step. The dipeptide moiety and/or substituent can be sequentially constructed as part of a solid phase peptide synthesis, or separately generated and attached via appropriate functional groups of the peptide after peptide synthesis.
In one embodiment, the compound is produced by a two-step process whereby two peptide fragments are linked after attachment of a substituent to one of the peptide fragments.
Description of the embodiments
The invention is further described by the following non-limiting examples:
1. A compound of formula I:
B-Z (formula I)
Or a pharmaceutically acceptable salt, ester or amide thereof,
Wherein B is a dipeptide, optionally comprising a substituent B;
Wherein Z is a GLP-1/GIP receptor co-agonist, said GLP-1/GIP receptor co-agonist optionally comprising the substituent Z; and
Wherein the N-terminal amino group of the GLP-1/GIP receptor co-agonist is linked to B via a peptide bond.
2. The compound of embodiment 1, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the GLP-1/GIP receptor co-agonist has an amino acid sequence of
YX2EGTX6TSDYSX12X13LX15X16X17AX19X20X21FX23X24WLX27X28GX30X31X32X33X34X35X36X37X3 8X39(SEQ ID NO.:1), Wherein the method comprises the steps of
X 2 is Aib or A
X 6 is F or V
X 12 is I or Y
X 13 is Y, A, L, I or Aib
X 15 is D or E
X 16 is K or E
X 17 is Q or I
X 19 is A or Q
X 20 is Q, R, E, H or K
X 21 is A or E
X 23 is I or V
X 24 is E, Q or N
X 27 is L or I
X 28 is A or R
X 30 is G or is absent
X 31 is P or is absent
X 32 is E, S or is absent
X 33 is S, K or is absent
X 34 is G or is absent
X 35 is A or is absent
X 36 is P or is absent
X 37 is P or is absent
X 38 is P or is absent
X 39 is S or absent.
3. The compound of embodiment 1, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the GLP-1/GIP receptor co-agonist has an amino acid sequence of
Y-Aib-EGTFTSDYSIX13LX15X16X17AX19X20X21FX23X24WLX27AGGP SX33GAPPPS(SEQ ID NO.:2), Wherein the method comprises the steps of
X 13 is L or Aib,
X 15 is D or E, and the total number of the components is D or E,
X 16 is K or E, and the total number of the components is,
X 17 is Q or I, and the total number of the components is,
X 19 is A or Q, and the total number of the components is H,
X 20 is R or K
X 21 is A or E
X 23 is I or V
X 24 is E or Q
X 27 is L or I;
x 33 is S or K.
4. The compound of embodiment 1, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the GLP-1/GIP receptor co-agonist has an amino acid sequence of
Y-Aib-EGTFTSDYSILLEX 16QAAREFIEWLLAGGPSX33 GAPPPS (SEQ ID NO: 3), wherein
X 16 is K or E, and the total number of the components is,
X 33 is S or K.
5. The compound according to any one of embodiments 2 to 4, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X 16 is E and X 33 is K.
6. The compound according to any one of embodiments 2 to 4, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X 16 is K and X 33 is S.
7. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the GLP-1/GIP receptor co-agonist has an amino acid sequence of Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS (SEQ ID No.: 4).
8. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is selected from Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS (SEQ ID No.: 4),
Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5) and Y-Aib-EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 6).
9. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the GLP-1/GIP receptor co-agonist has an amino acid sequence of Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID No.: 5) or Y-Aib-EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID No.: 6).
10. The compound of any one of embodiments 1 to 9, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the GLP-1/GIP receptor co-agonist comprises a substituent z, and wherein the substituent z is linked to the GLP-1/GIP receptor co-agonist through lysine (K).
11. The compound according to any one of embodiments 2 to 10, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X 16 and/or X 20 and/or X 33 are lysine.
12. The compound of any one of embodiments 2, 3, 11 or 6, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X 16 is lysine.
13. The compound of any one of embodiments 2, 11 or 7, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein X 20 is lysine.
14. The compound of any one of embodiments 2, 3, 11 or 5, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X 33 is lysine.
15. The compound of any one of embodiments 1 to 14, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the substituent z is linked to the GLP-1/GIP receptor co-agonist through lysine (K) at position 16, 20, or 33.
16. The compound of any one of embodiments 1 to 15, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the GLP-1/GIP receptor co-agonist has a C-terminal amide modification.
17. The compound of any one of embodiments 5, 8 or 9, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the lysine at position 33 is chemically modified by conjugation with the epsilon-amino group of the lysine side chain with chemical formula 8, chemical formula 7, or chemical formula 10.
18. The compound of any one of embodiments 6, 8 or 9, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the lysine at position 16 is chemically modified by conjugation with the epsilon-amino group of the lysine side chain with chemical formula 7.
19. The compound of embodiment 7 or embodiment 8, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the lysine at position 20 is chemically modified by conjugation with the epsilon-amino group of the lysine side chain using formula 11.
20. The compound according to any one of embodiments 1 to 19, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide B is of formula II:
X-Y (formula II),
Wherein X is any alpha-amino acid linked to Y through an amide bond formed between the alpha-carboxylic acid group of X and the alpha-amino group of Y,
Wherein Y is an N-alkylated alpha-amino acid linked to Z by a peptide bond formed between the alpha-carboxylic acid group of Y and the N-terminal amino group of the GLP-1/GIP receptor co-agonist.
21. The compound according to any one of embodiments 1 to 16, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide B is of formula II:
X-Y (formula II),
Wherein X is any alpha-amino acid linked to Y through an amide bond formed between the alpha-carboxylic acid group of X and the alpha-amino group of Y,
Wherein Y is an N-alkylated alpha-amino acid linked to Z by a peptide bond formed between the alpha-carboxylic acid group of Y and the N-terminal amino group of Z.
22. The compound of embodiment 20 or embodiment 21, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamic acid, N-methylnorleucine, N-methyl homoalanine, N-methylalanine, N-methyllysine, N- (2-aminoethyl) glycine, N-hexyl homoalanine, N-propyl alanine, homoproline, N-propyl glycine, N-ethyl glycine, and N-methyl phenylalanine.
23. The compound according to any one of embodiments 20 to 22, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from lysine, 4-aminophenylalanine, D-lysine, alanine, glycine, proline, D-valine, homoproline, D-proline, D-homoproline, D-alanine and azetidine-2-carboxylic acid.
24. The compound of any one of embodiments 20 to 23, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamic acid, N-methylnorleucine, N-methyl homoalanine, N-methylalanine, N-methyllysine, N-hexyl homoalanine, N-propylalanine, homoproline, N-propylglycine, N-ethylglycine, and N-methylphenylalanine.
25. A compound according to any one of embodiments 20 to 24, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is sarcosine or N- (2-aminoethyl) glycine.
26. The compound according to any one of embodiments 20 to 25, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from lysine, D-lysine, alanine, leucine, glycine, proline and aspartic acid.
27. A compound according to any one of embodiments 20 to 26, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from lysine, D-lysine and glycine.
28. The compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the dipeptide is capable of undergoing intramolecular cyclization to form 2, 5-Diketopiperazine (DKP) such that the amide bond between a and Z is broken.
29. The compound of any one of embodiments 1 to 27, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the dipeptide is capable of undergoing intramolecular cyclization to form 2, 5-Diketopiperazine (DKP) such that the peptide bond between a and Z is broken.
30. The compound of any one of embodiments 1 to 29, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the dipeptide comprises substituent b.
31. The compound of any one of embodiments 1 to 29, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the dipeptide carries substituent b.
32. The compound of any one of embodiments 1 to 29, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the dipeptide has substituent b.
33. The compound of any one of embodiments 21 to 30, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein substituent b is covalently attached to X, optionally through an amide bond.
34. The compound of any one of embodiments 21 to 30, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein substituent b is covalently attached to Y, optionally through an amide bond.
35. The compound of any one of embodiments 1 to 34, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the substituent b is an albumin binding moiety.
36. The compound of any one of embodiments 1 to 35, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the substituent b comprises or consists of an extender and an optional linker.
37. The compound of embodiment 36, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the extender is a fatty acid, such as a C 16-C22 carboxylic acid.
38. The compound of embodiment 36 or embodiment 37, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the extension is formula 1.
39. The compound of any one of embodiments 1 to 38, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the substituent comprises a linker, and optionally wherein the linker comprises or consists of a linker element.
40. The compound of any one of embodiments 38 to 39, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the linker is of formula IV
A 1-A2-A3-A4-A5 (formula IV),
Wherein a 1 is covalently bound to the amino acid of the dipeptide via an amide bond, and optionally also to the extension via an amide bond, and is selected from formula 2, formula 3, formula 4, and formula 5; wherein a 5 is covalently bound to formula 1 and is formula 2 or absent; wherein a 2、A3 and a 4 are each independently selected from formula 2, formula 3, formula 4, and formula 5, or are absent.
41. The compound of any one of embodiments 1 to 40, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the substituent b is selected from formula 16, formula 17, formula 18, formula 19, formula 20, formula 21, and formula 22.
42. The compound according to any one of embodiments 1 to 41, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound is selected from the group consisting of:
Compound No. 1
Compound No. 2
Compound No. 3
Compound No. 4
Compound No. 5
Compound No. 6
Compound No. 7
Compound No. 8
Compound No. 9
Compound No. 10
Compound 11
Compound No. 12
Compound No. 13
Compound No. 14
Compound No. 15
Compound No. 16
Compound No. 17
And
Compound No. 18
43. The compound according to any one of embodiments 1 to 42, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is selected from compounds No.1, 2, 3, 9 and 10.
44. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound is compound No. 1.
45. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound is compound No. 2.
46. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound is compound No. 3.
47. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound is compound No. 4.
48. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound is compound No. 5.
49. The compound according to any one of embodiments 1 to 48, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is a prodrug and does not exert any significant efficacy in vitro.
50. The compound of any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound is a prodrug and has a conversion half-life.
51. The compound of embodiment 50, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the conversion half-life is measured in vitro at pH 7.4 and 37 ℃.
52. The compound of embodiment 50 or 51, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the conversion half-life is measured as described herein as "general method of measuring conversion half-life".
53. The compound according to any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life is suitable for once daily administration.
54. The compound according to any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life is suitable for once weekly administration.
55. The compound of any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the conversion half-life measured in vitro is 90-4300 hours.
56. The compound of any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the conversion half-life measured in vitro is 90-4300 hours.
57. The compound of any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is 300-1100 hours.
58. The compound of any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is 450-650 hours.
59. The compound of any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the conversion half-life measured in vitro is at least 100 hours.
60. The compound of any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the conversion half-life measured in vitro is at least 200 hours.
61. The compound of any one of embodiments 50 to 52, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the conversion half-life measured in vitro is at least 300 hours.
62. The compound of any one of embodiments 1 to 61, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound has a terminal half-life.
63. The compound of any one of embodiments 1 to 61, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound has a terminal half-life and the terminal half-life is suitable for once daily administration.
64. The compound of any one of embodiments 1 to 61, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound has a terminal half-life and the terminal half-life is suitable for once weekly administration.
65. The compound according to any one of embodiments 62 to 64, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is determined in a minipig and is determined according to the general method for measuring terminal half-life in minipigs herein.
66. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is >90 hours when assayed in a minipig.
67. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is >110 hours when assayed in a minipig.
68. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is >250 hours when assayed in a minipig.
69. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is >180 hours when assayed in a minipig.
70. The compound of any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is 80-240 hours when assayed in a minipig.
71. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is 110-191 hours when assayed in a minipig.
72. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 71 and at least one pharmaceutically acceptable adjuvant.
73. The pharmaceutical composition of embodiment 72, wherein the pharmaceutical composition is a liquid formulation.
74. The pharmaceutical composition of embodiment 72, wherein the pharmaceutical composition is a solid formulation.
75. The pharmaceutical composition of embodiment 72, wherein the pharmaceutical composition is for oral administration.
76. The pharmaceutical composition according to any one of embodiments 74-76, wherein the composition is in the form of a tablet.
77. The pharmaceutical composition according to any one of embodiments 74-77, wherein at least one pharmaceutically acceptable excipient is a salt of N- (8- (2-hydroxybenzoyl) amino) caprylic acid, e.g., N- (8- (2-hydroxybenzoyl) amino) caprylic acid is sodium N- (8- (2-hydroxybenzoyl) amino) caprylate (SNAC).
78. The pharmaceutical composition according to any one of embodiments 74-78, further comprising a lubricant, such as magnesium stearate.
79. A tablet comprising a compound of any one of embodiments 1-71, a salt of N- (8- (2-hydroxybenzoyl) amino) octanoic acid, a lubricant, and optionally one or more pharmaceutically acceptable excipients.
80. The tablet of embodiment 79, wherein the salt of N- (8- (2-hydroxybenzoyl) amino) caprylic acid is sodium N- (8- (2-hydroxybenzoyl) amino) caprylate (SNAC).
81. The tablet of embodiment 79 or embodiment 80, wherein the lubricant is magnesium stearate.
82. A compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 for use as a medicament.
83. A compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 for use in the treatment of type 2 diabetes.
84. A compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 for use in the treatment of obesity.
85. The compound according to any one of embodiments 1 to 71 or the pharmaceutical composition according to any one of embodiments 72 to 78 or the tablet according to any one of embodiments 79 to 81 for use in the treatment of liver diseases such as liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation and/or fatty liver.
86. Use of a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 in the manufacture of a medicament for
A. Preventing and/or treating liver diseases such as liver steatosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver inflammation and/or fatty liver;
b. preventing and/or treating obesity; and/or
C. preventing and/or treating type 2 diabetes.
87. Use of a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 in the manufacture of a medicament for the treatment of type 2 diabetes.
88. Use of a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 in the manufacture of a medicament for the treatment of obesity.
89. A method of preventing and/or treating type 2 diabetes comprising administering to a subject in need thereof a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81.
90. A method of preventing and/or treating obesity comprising administering to a subject in need thereof a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81.
91. A method of preventing and/or treating liver diseases such as liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation and/or fatty liver comprising administering to a subject in need thereof a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81.
The invention is further described by the following further non-limiting embodiments:
1. A compound of formula I:
B-Z (formula I)
Or a pharmaceutically acceptable salt, ester or amide thereof,
Wherein Z is a GLP-1/GIP receptor co-agonist or derivative thereof;
Wherein B is a dipeptide of formula II:
X-Y (formula II),
Wherein X is any alpha-amino acid linked to Y through an amide bond formed between the alpha-carboxylic acid group of X and the alpha-amino group of Y,
Wherein Y is an N-alkylated alpha-amino acid linked to Z by an amide bond formed between the alpha-carboxylic acid group of Y and the amine of Z.
2. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamic acid, N-methylnorleucine, N-methyl homoalanine, N-methylalanine, N-methyllysine, N- (2-aminoethyl) glycine, N-hexyl homoalanine, N-propyl alanine, homoproline, N-propyl glycine, N-ethyl glycine, and N-methyl phenylalanine.
3. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from lysine, 4-aminophenylalanine, D-lysine, alanine, glycine, proline, D-valine, homoproline, D-proline, D-homoproline, D-alanine and azetidine-2-carboxylic acid.
4. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamic acid, N-methylnorleucine, N-methyl homoalanine, N-methylalanine, N-methyllysine, N-hexylhomoalanine, N-propylalanine, homoproline, N-propylglycine, N-ethylglycine, and N-methylphenylalanine.
5. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is sarcosine or N- (2-aminoethyl) glycine.
6. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from lysine, D-lysine, alanine, leucine, glycine, proline and aspartic acid.
7. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from lysine, D-lysine and glycine.
8. The compound of any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the dipeptide is capable of undergoing intramolecular cyclization to form 2, 5-Diketopiperazine (DKP) such that the amide bond between B and Z is broken.
9. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide comprises substituent b.
10. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide has substituent b.
11. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein substituent b is covalently linked to X, optionally through an amide bond.
12. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent b comprises or consists of an extender and an optional linker.
13. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the extension is formula 1.
14. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the substituent b is selected from formula 16, formula 17, formula 18, formula 19, formula 20, formula 21, and formula 22.
15. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the GLP-1/GIP receptor co-agonist has an amino acid sequence of YX2EGTX6TSDYSX12X13LX15X16X17AX19X2 0X21FX23X24WLX27X28GX30X31X32X33X34X35X36X37X38X39(SEQ ID NO.:1), wherein
X 2 is Aib or A
X 6 is F or V
X 12 is I or Y
X 13 is Y, A, L, I or Aib
X 15 is D or E
X 16 is K or E
X 17 is Q or I
X 19 is A or Q
X 20 is Q, R, E, H or K
X 21 is A or E
X 23 is I or V
X 24 is E, Q or N
X 27 is L or I
X 28 is A or R
X 30 is G or is absent
X 31 is P or is absent
X 32 is E, S or is absent
X 33 is S, K or is absent
X 34 is G or is absent
X 35 is A or is absent
X 36 is P or is absent
X 37 is P or is absent
X 38 is P or is absent
X 39 is S or absent.
16. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is
Y-Aib-EGTFTSDYSIX13LX15X16X17AX19X20X21FX23X24WLX27AGGP SX33GAPPPS(SEQ ID NO.:2), Wherein the method comprises the steps of
X 13 is L or Aib,
X 15 is D or E, and the total number of the components is D or E,
X 16 is K or E, and the total number of the components is,
X 17 is Q or I, and the total number of the components is,
X 19 is A or Q, and the total number of the components is H,
X 20 is R or K
X 21 is A or E
X 23 is I or V
X 24 is E or Q
X 27 is L or I;
x 33 is S or K.
17. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is
Y-Aib-EGTFTSDYSILLEX 16QAAREFIEWLLAGGPSX33 GAPPPS (SEQ ID NO: 3), wherein
X 16 is K or E, and the total number of the components is,
X 33 is S or K.
18. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X 16 is E and X 33 is K.
19. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X 16 is K and X 33 is S.
20. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is selected from Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS (SEQ ID No.: 4),
Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO: 5) and Y-Aib-EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO: 6).
21. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the GLP-1/GIP receptor co-agonist comprises a substituent z, and wherein the substituent z is linked to the GLP-1/GIP receptor co-agonist through lysine (K).
22. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent z is linked to the GLP-1/GIP receptor co-agonist through lysine (K) at position 16, 20 or 33.
23. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the compound is selected from compound No. 1, compound No. 2, compound No. 3, compound No. 4, compound No. 5, compound No. 6, compound No. 7, compound No. 8, compound No. 9, compound No. 10, compound No. 11, compound No. 12, compound No. 13, compound No. 14, compound No. 15, compound No. 16, compound No. 17, and compound No. 18.
24. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is selected from compounds No.1, 2, 3, 9 and 10.
25. A pharmaceutical composition comprising a compound according to any one of the preceding embodiments and at least one pharmaceutically acceptable excipient.
26. The pharmaceutical composition according to any of the preceding embodiments, wherein the pharmaceutical composition is a liquid formulation.
27. The pharmaceutical composition according to any of the preceding embodiments, wherein the pharmaceutical composition is a solid formulation.
28. The pharmaceutical composition according to any of the preceding embodiments, wherein the pharmaceutical composition is for oral administration.
29. The pharmaceutical composition according to any of the preceding embodiments, wherein the pharmaceutical composition is for parenteral administration.
30. The pharmaceutical composition according to any of the preceding embodiments, wherein the pharmaceutical composition is in the form of a tablet.
31. A compound according to any one of the preceding embodiments for use as a medicament.
32. A compound according to any one of the preceding embodiments for use in the prevention and/or treatment of type 2 diabetes.
33. A compound according to any one of the preceding embodiments for use in the prevention and/or treatment of obesity.
34. A compound according to any one of the preceding embodiments for use in the prevention and/or treatment of liver diseases, such as liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation and/or fatty liver.
Examples
The experimental section starts with a list of abbreviations followed by a section of the general method for compound preparation and a section of the measurement method for exposure profile related properties. Many specific examples are included in each section to illustrate the invention. All exemplified compounds were prepared according to the general methods described herein. Chemical names of substituents were generated using ACCELRYS DRAW version 4.1SP1 software and IUPAC nomenclature, as appropriate.
Abbreviations (abbreviations)
The following alphabetically listed abbreviations are used hereinafter:
ado: 8-amino-3, 6-dioxaoctanoic acid
Aeg: n- (2-aminoethyl) glycine
Aib: alpha-aminoisobutyric acid
Alloc: allyloxycarbonyl group
API: atmospheric pressure ionization
AUC: area under curve
BHK: baby hamster kidney
Boc: boc-group
Cl-HOBt: 6-chloro-1-hydroxybenzotriazole
DCM: dichloromethane (dichloromethane)
DIC: diisopropylcarbodiimide
DIPEA: n, N-diisopropylethylamine
DKP:2, 5-diketopiperazine
DMEM: dulbecco's modified Eagle's Medium
DPBS: dulbecco's phosphate buffered saline
EDTA: ethylenediamine tetraacetic acid
ELISA: enzyme-linked immunosorbent assay equiv: molar equivalent
FBS: fetal bovine serum
Fmoc: 9-fluorenylmethoxycarbonyl
GIP: glucose-dependent insulinotropic polypeptides
GIPR: glucose-dependent insulinotropic polypeptide receptor GLP-1: glucagon-like peptide 1GLP-1R: glucagon-like peptide 1 receptor h: hours of
HEPES:4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid
HFIP: HPLC of 1, 3-hexafluoro-2-propanol or hexafluoroisopropanol: high performance liquid chromatography
HSA: human serum albumin
I.v. intravenous
LCMS: liquid chromatography mass spectrometry
MeCN: acetonitrile
MeOH: methanol
MM: millimolar concentration of
Mmol: millimoles (milli)
Min: minute (min)
Mtt: 4-Methyltrityl radical
NMP: 1-methyl-pyrrolidin-2-one
OtBu: tert-butyl ester
OxymaCyano-hydroxy imino-acetic acid ethyl ester
Pbf:2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
PBS: phosphate buffered saline
PK: pharmacokinetics of
PM: picomolar concentration
P.o.: oral cavity
Rpm: revolutions per minute
Rt: retention time
Sar: sarcosine
S.c.: subcutaneous tissue
SNAC: n- [8- (2-hydroxybenzoyl) amino ] caprylic acid sodium salt
SPPS: solid phase peptide synthesis
TBu: tert-butyl group
T2D: type 2 diabetes mellitus
TFA: trifluoroacetic acid
TIS: triisopropylsilane
Trt: triphenylmethyl or trityl
UPLC: ultra-high performance liquid chromatography
General Process for the preparation of the Compounds of the invention
The solid phase peptide synthesis method (SPPS method, including amino acid deprotection method, method of cleaving peptide from resin, and purification method thereof), and method of detecting and characterizing the resulting peptide (LCMS method) are described below.
The resin used to prepare the C-terminal peptide amide is H-RINK AMIDE-ChemMatrix resin (e.g., loaded with 0.5 mmol/g). The resin used to prepare the C-terminal peptide acid is Wang-polystyrene resin preloaded with a suitably protected C-terminal amino acid derivative (e.g., loaded with 0.5 mmol/g). All the operations described below were carried out on a synthetic scale ranging from 0.1 to 1.0 mmol. Unless specifically stated otherwise, the Fmoc protected amino acid derivatives used are recommended standards: such as Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gly-OH、Fmoc-His(Trt)-OH、Fmoc-Ile-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Met-OH、Fmoc-Phe-OH、Fmoc-Pro-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Trp(Boc)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Val-OH、Fmoc-Lys(Mtt)-OH、Fmoc-Aib-OH provided by AAPPTEC, anaspec, bachem, chemImpex, iris Biotech, midwest Biotech, gyros Protein Technologies, or Novabiochem, among others. In the absence of any other description, the L-form amino acid of the protein type is used. For coupling the N-terminal amino acid of each compound, a reagent with Boc protection at the α -amino group was used.
In the case of dipeptide attachment using SPPS, the following appropriately protected building blocks are used, such as, but not limited to Alloc-Aeg(Fmoc)-OH、Boc-Ala-OH、Boc-Asp(OtBu)-OH、Boc-Gly-OH、Boc-Leu-OH、Boc-Lys(Fmoc)-OH、Boc-D-Lys(Fmoc)-OH、Boc-Pro-OH、Fmoc-Aeg(N3)-OH and Fmoc-Sar-OH. In the case of substituent attachment using SPPS, suitably protected building blocks such as, but not limited to, fmoc-8-amino-3, 6-dioxaoctanoic acid (Fmoc-Ado-OH), boc-Lys (Fmoc) -OH, fmoc-Glu-OtBu, fmoc-Gly-OH, mono-tert-butyl hexadecanedioate, mono-tert-butyl octadecanedioate or mono-tert-butyl eicosadioate are used.
1. Synthesis of protected peptide backbone bound to resin
Universal SPPS_A
SPPS was performed on Protein Technologies SymphonyX solid phase peptide synthesizer using Fmoc-based chemistry with minor modifications using the protocol provided by the manufacturer. The mixing was performed by bubbling nitrogen from time to time. The step assembly was performed using the following steps: 1) Preswelling the resin in DMF; 2) Fmoc-deprotection was performed by using 20% (v/v) piperidine in DMF with or without 1% (v/v) TFA for two treatments of 10min each; 3) Washing with DMF to remove piperidine; 4) By adding Fmoc-amino acids, oxyma, of each 3-12equivAnd DIC in DMF with or without 2,4, 6-trimethylpyridine, followed by mixing for at least 30min for Fmoc-amino acid coupling; 4) Washing with DMF to remove excess reagent; 5) The final wash was performed with DCM at the completion of the assembly. Some amino acids, such as, but not limited to, amino acids following a sterically hindered amino acid (e.g., aib), are coupled for an extended reaction time (e.g., 4 hours or overnight) to ensure reaction completion.
The method comprises the following steps: SPPS_B
SPPS was performed using Fmoc-based chemistry on Applied Biosystems A solid phase peptide synthesizer using the general Fmoc protocol provided by the manufacturer. Mixing was performed by vortexing and bubbling nitrogen from time to time. The stepwise assembly is completed using the following steps: 1) Fmoc-amino acids were activated by dissolving the solid Fmoc-acids of each 1O equiv in a 1M solution of Cl-HOBt in NMP, then adding a 1M solution of 10equiv of DIC in NMP, then mixing simultaneously with steps 2-3; 2) Fmoc deprotection was performed by treatment with 20% (v/v) piperidine in NMP for 3min once followed by 15min for the second treatment; 3) Washing with NMP to remove piperidine; 4) Adding the activated Fmoc-amino acid solution to the resin, and then mixing for at least 45min; 4) Washing with NMP to remove excess reagent; 5) The final wash was performed with DCM at the completion of the assembly. Some amino acids, such as, but not limited to, amino acids following a sterically hindered amino acid (e.g., aib), are coupled for an extended reaction time (e.g., 4 hours), and/or repeatedly treated with fresh coupling reagents to ensure reaction completion.
2. Attachment of dipeptides and substituents to protected peptide backbones bound to resins
The method comprises the following steps: DS_A
For compounds containing N-terminal Lys or D-Lys with substituents, SPPS was continued using the same protocol as in spps_a to attach the amino acid of dipeptide B and the element of substituent B.
The method comprises the following steps: DS_B
For compounds containing Aeg with substituents within dipeptide B, SPPS was continued using the same protocol as in spps_a to attach Fmoc-Aeg (N 3) -OH and nα -Boc protected N-terminal amino acids. By using 5-10equiv of tris (2-carboxyethyl) phosphine at 9: a solution in 1 DMF/water was treated with the resin-bound peptide for 2-3h to reduce the azido protecting group to an amine. The resin was drained and dried with 9:1 DMF/water and DMF washes followed by the elements of attached substituent b using the same scheme as in SPPS_A.
The method comprises the following steps: DS_C
For compounds containing Aeg with substituents within dipeptide B, as an alternative to ds_b, SPPS was continued using the same scheme as in spps_a to attach elements of Alloc-Aeg (Fmoc) -OH and substituent B. The Alloc protecting group was removed by treating the resin bound peptide with a 10equiv borane dimethylamine complex and 20equiv morpholine in DMF for 5min under an argon atmosphere, followed by the addition of 0.1equiv palladium-tetrakis (triphenylphosphine) in DMF for an additional 30 min. The resin was drained and washed with DCM, DMF, meOH, water and DMF. N α -Boc-protected N-terminal amino acids were then attached using the same protocol as in SPPS_A.
The method comprises the following steps: DS_D
For attachment of substituent z, the N epsilon-Mtt protection of the Lys with the substituent was removed by washing the resin with 30% hfip in DCM, twice for 45min each, or with 80% hfip in DCM for 5min, 10min, 15min, 20min and 30 min. The resin was drained and washed with DCM, DMF, 10% DIPEA/DCM, DCM and DMF. SPPS continues using the same scheme as in spps_a to attach the elements of substituent z.
3. Cleavage and purification of resin-bound peptide:
The method comprises the following steps: cp_a
After completion of side chain synthesis, the peptidyl resin was washed with DCM and dried, then with 95:2.5:2.5 (V/V) TFA/water/TIS or 92.5:5:2.5 (V/V/V) TFA/water/TIS treatment for 2-3h followed by precipitation with diethyl ether. The precipitate is separated (e.g., by filtration or centrifugation), washed with diethyl ether, dissolved in a suitable solvent (e.g., 2:1 water/MeCN), and allowed to stand until all of the labile adducts have been decomposed. In Phenomenex Luna C (2) column (10 μm particle size,Pore size, 250x 21.2mm size) or Phenomenex Gemini-NX C18 column (5 μm pore size,Pore size, 250x50mm size) was purified by reverse phase preparative HPLC. The separation of impurities and elution of the product was accomplished using a gradient of MeCN gradually increasing in water containing 0.1% tfa. The identity and purity of the relevant fractions were checked by analytical LCMS. Fractions containing the pure desired product were combined and lyophilized to give the peptide TFA salt as a white solid.
4. Salt exchange from TFA to sodium salt:
the method comprises the following steps: SX_A
The lyophilized peptide isolated from method cp_a is dissolved to 5-20mg/mL in a suitable aqueous buffer (e.g. 4:1 water/MeCN, 0.2M sodium acetate) and adjusted to pH 7-8 with 1M NaOH if necessary to achieve complete dissolution. Salt exchange of the peptide-containing buffer solution using a Sep-Pak C18 column (0.5-2 g): the column was first equilibrated with 4 column volumes of isopropanol, then with 4 column volumes of MeCN, then with 8 column volumes of water. The peptide solution was applied to the cartridge and the flow-through was reapplied to ensure complete peptide retention. The column is washed with 4 column volumes of water and then 10 column volumes of buffer solution (e.g., pH 7.5) containing, for example, but not limited to NaHCO 3, naOAc, or Na 2HPO4. The column was washed with 4 column volumes of water and the peptide eluted with 5-10 column volumes of 50-80% mecn in water. The peptide-containing eluate was freeze-dried to give the peptide sodium salt as a white solid, which was used as such.
Universal detection and characterization method
LCMS method:
the method comprises the following steps: lcms_a
By injecting an appropriate volume of sample into a Phenomenex Kinetex C column equilibrated at 37 c (2.6 μm particle size,Pore size, 4.6x75mm size), analysis was performed on an Agilent 1260Infinity series HPLC/MS system. Eluent a was 0.05% tfa in water; eluent B was 9:1 MeCN/0.05% TFA in water. Elution was achieved with a linear gradient of 20-100% eluent B over 10min at a flow rate of 1.0 mL/min. The UV detection was set at 214nm. MS ionization was run in API-ES mode and positive polarity with a scan mass range of 500-2000amu. The most abundant isotope per m/z is reported.
The method comprises the following steps: lcms_b
By injecting a suitable volume of sample into an ACQUITY UPLC BEH130 column equilibrated at 40 ℃ (1.7 μm particle size,Pore size, 2.1x150mm size), analysis was performed on Waters ACQUITY UPLC/MS system. Eluent a was 0.05% tfa in water; eluent B was 0.05% tfa in MeCN. Elution was achieved with the following gradient and flow rate: for ultraviolet detection, the linear gradient of eluent B is 5-95% within 16min, and the flow rate is 0.4mL/min; for MS detection, the linear gradient of eluent B was 5-60% over 4min, with a flow rate of 0.45mL/min. The UV detection was set at 214nm. MS ionization was run in API-ES mode and positive polarity with a scan mass range of 100-2000amu. The most abundant isotope per m/z is reported.
Example 1: synthesis of Compounds
Compounds are described below using single letter amino acid codes, except Aeg, aib, D-Lys and Sar. Each substituent is included in brackets after the residue to which it is attached.
Parent compound No. 1
Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [ [ (4S) _4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
SEQ ID NO. 5; substituents: chemical formula 8 attached to Lys33
The synthesis method comprises the following steps: SPPS_A; ds_d; cp_a
Calculated molecular weight (average): 4901.4Da
Lcms_a: rt=6.3 min; actual measurement [ M+3H ] 3+1634.6,[M+4H]4+ 1226.1
Parent compound No. 2
Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] hexanoyl ] -GAPPPS-OH
SEQ ID NO. 5; substituents: chemical formula 10 attached to Lys33
The synthesis method comprises the following steps: SPPS_A; ds_d; cp_a
Calculated molecular weight (average): 4867.5Da
Lcms_a: rt=6.1 min; actual measurement [ M+3H ] 3+1623.1,[M+4H]4+ 1217.6
Parent compound No. 3
Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -GAPPPS-OH
SEQ ID NO. 5; substituents: chemical formula 7 attached to Lys33
The synthesis method comprises the following steps: SPPS_A; ds_d; cp_a
Calculated molecular weight (average): 4895.5Da
Lcms_a: rt=6.3 min; actual measurement [ M+3H ] 3+1632.4,[M+4H]4+ 1224.6
Parent compound No. 4
Y-Aib-EGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAREFIEWLLAGGPSSGAPPPS-OH
SEQ ID NO. 6; substituents: chemical formula 7 attached to Lys16
The synthesis method comprises the following steps: SPPS_A; ds_d; cp_a
Calculated molecular weight (average): 4853.5Da
Lcms_a: rt=5.9 min; actual measurement of [ M+3H ] 3+1618.5,[M+4H]4+ 1214.2 th parent Compound
Y-Aib-EGTFTSDYSI-Aib-LDKIAQK [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynonacarbonamido) butanoyl ] amino ] ethoxy ] acetyl ] -AFVQWLIAGGPSSGAPPPS-NH 2
SEQ ID NO. 4 with C-terminal amide modification; substituents: chemical formula 11 attached to Lys20
The synthesis method comprises the following steps: SPPS_A; ds_d; cp_a
Calculated molecular weight (average): 4813.5DaLCMS_A: rt=6.2 min; actual measurement [ M+3H ] 3+1605.2,[M+4H]4+ 1204.3
Compound No. 1
(D-Lys) [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K- [2- [2- [2- [ [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x = D-Lys; y=sar; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 8 attached to Lys33 of Z.
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5498.2DaLCMS_A: rt=6.8 min; actual measurement [ M+3H ] 3+1833.4,[M+4H]4+ 1375.3
Compound No. 2
G-Aeg [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=gly; y= Aeg; substituent b: chemical formula 16 attached to Y; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_B; ds_d; cp_a
Calculated molecular weight (average): 5456.1Da
Lcms_a: rt=7.0 min; actual measurement of [ M+3H ] 3+1819.4,[M+4H]4+ 1364.8 Compound No. 3
K [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=lys; y=sar; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5498.2Da
Lcms_a: rt=6.9 min; actual measurement [ M+3H ] 3+1833.7,[M+4H]4+ 1375.6
Compound No. 4
A-aeg [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=ala; y= Aeg; substituent b: chemical formula 16 attached to Y; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_B; ds_d; cp_a
Calculated molecular weight (average): 5470.1Da
Lcms_a: rt=7.0 min; actual measurement [ M+3H ] 3+1823.8,[M+4H]4+ 1368.2
Compound No. 5
L-Aeg [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=leu; y= Aeg; substituent b: chemical formula 16 attached to Y; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_B; ds_d; cp_a
Calculated molecular weight (average): 5512.2Da
Lcms_a: rt=7.1 min; actual measurement [ M+3H ] 3+1838.1,[M+4H]4+ 1378.9
Compound No. 6
P-Aeg [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=pro; y= Aeg; substituent b: chemical formula 16 attached to Y; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_B; ds_d; cp_a
Calculated molecular weight (average): 5496.2DaLCMS_A: rt=7.0 min; actual measurement [ M+3H ] 3+1832.4,[M+4H]4+ 1374.6
Compound No. 7
D-Aeg [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=asp; y= Aeg; substituent b: chemical formula 16 attached to Y; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_A; DS_C; ds_d; cp_a
Calculated molecular weight (average): 5514.1Da
Lcms_b: rt=10.2 min; actual measurement [ M+3H ] 3+1838.8,[M+4H]4+ 1379.3.
Compound No. 8
K [ (4S) -4-carboxy-4- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] butanoyl ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=lys; y=sar; substituent b: chemical formula 18 attached to X; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5627.3Da
Lcms_a: rt=6.9 min; actual measurement of [ M+3H ] 3+1876.3,[M+4H]4+ 1407.6 Compound No. 9
K [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] acetyl ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=lys; y=sar; substituent b: chemical formula 19 attached to X; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5555.2DaLCMS_A: rt=6.9 min; actual measurement [ M+3H ] 3+1852.3,[M+4H]4+ 1389.7
Compound 10 (D-Lys) [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadec-arbon) acylamino) butanoyl ] amino ] ethoxy ] acetyl ] amino ] ethoxy ] acetyl-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x = D-Lys; y=sar; substituent b: chemical formula 21 attached to X; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5788.5DaLCMS_A: rt=6.9 min; actual measurement [ M+3H ] 3+1930.3,[M+4H]4+ 1447.8
Compound 11
(D-Lys) [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] hexanoyl
1-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x = D-Lys; y=sar; substituent b: chemical formula 20 attached to X; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5626.4Da
Lcms_a: rt=6.7 min; actual measurement [ M+3H ] 3+1876.2,[M+4H]4+ 1407.2
Compound No. 12
K [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl
The radical ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] hexanoyl ] -GAPPPS-OH
Z: parent compound No. 2; x=lys; y=sar; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 10 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5464.2Da
Lcms_a: rt=6.6 min; actual measurement [ M+3H ] 3+1822.2,[M+4H]4+ 1366.7
Compound No. 13
K [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] hexanoyl ] -GAPPPS-OH
Z: parent compound No. 2; x=lys; y=sar; substituent b: chemical formula 17 attached to X; substituent z: chemical formula 10 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_B; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5492.3Da
Lcms_a: rt=6.8 min; actual measurement [ M+3H ] 3+1831.4,[M+4H]4+ 1373.7
Compound No. 14
K [ (2S) -2, 6-bis [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] hexanoyl ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] hexanoyl ] -GAPPPS-OH
Z: parent compound No. 2; x=lys; y=sar; substituent b: chemical formula 22 attached to X; substituent z: chemical formula 10 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5989.9Da
Lcms_a: rt=7.0 min; actual measurement [ M+3H ] 3+1997.1,[M+4H]4+ 1498.0
Compound No. 15
K [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] -Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -GAPPPS-OH
Z: parent compound No. 3; x=lys; y=sar; substituent b: chemical formula 17 attached to X; substituent z: chemical formula 7 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5520.3Da
Lcms_a: rt=7.0 min; actual measurement [ M+3H ] 3+1840.9,[M+4H]4+ 1380.9
Compound No. 16
K [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Sar-Y-Aib-EGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAREFIEWLLAGGPSSGAPPPS-OH
Z: parent compound No. 4; x=lys; y=sar; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 7 attached to Lys16 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5450.2Da
Lcms_a: rt=6.3 min; actual measurement [ M+3H ] 3+1817.3,[M+4H]4+ 1363.1
Compound No. 17
G-Aeg [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Y-Aib-EGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecanoylamino) butanoyl ] amino ] hexanoyl ] -QAAREFIEWLLAGGPSSGAPPPS-OH
Z: parent compound No. 4; x=gly; y= Aeg; substituent b: chemical formula 16 attached to Y; substituent z: chemical formula 7 attached to Lys16 of Z
The synthesis method comprises the following steps: SPPS_A; DS_C; ds_d; cp_a
Calculated molecular weight (average): 5408.2Da
Lcms_b: rt=9.5 min; actual measurement of [ M+3H ] 3+1803.6,[M+4H]4+ 1353.0 Compound No. 18
K [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl
Phenyl ] -Sar-Y-Aib-EGTFTSDYSI-Aib-LDKIAQK [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynonacarbonamido) butanoyl ] amino ] ethoxy ] acetyl ] -AFVQWLIAGGPSSGAPPPS-NH 2
Z: parent compound No. 5; x=lys; y=sar; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 11 attached to Lys20 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5410.2Da
Lcms_a: rt=6.6 min; actual measurement [ M+3H ] 3+1804.0,[M+4H]4+ 1353.5
Non-transforming compound No. 1
K [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl
Phenyl ] -Val-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [2- [2- [2- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -GAPPPS-OH
Z: parent compound No. 1; x=lys; y=val; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 8 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5526.2Da
Lcms_a: rt=6.9 min; actual measurement [ M+3H ] 3+1842.8,[M+4H]4+ 1382.2
Non-transforming compound No. 2
K [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Val-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] amino ] hexanoyl ] -GAPPPS-OH
Z: parent compound No. 2; x=lys; y=val; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 10 attached to Lys33 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5492.3DaLCMS_A: rt=6.6 min; actual measurement [ M+3H ] 3+1831.4,[M+4H]4+ 1373.9
Non-transforming compound No. 3
K [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl
The radical ] -Val-Y-Aib-EGTFTSDYSILLE-K [ (2S) -2-amino-6- [ [ (4S) -4-carboxy-4- (17-carboxyheptadecylamino) butanoyl ] amino ] hexanoyl ] -QAAREFIEWLLAGGPSSGAPPPS-OH
Z: parent compound No. 4; x=lys; y=val; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 7 attached to Lys16 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5478.3DaLCMS_A: rt=6.1 min; actual measurement [ M+3H ] 3+1826.7,[M+4H]4+ 1370.3
Non-transforming compound No. 4
K [ (4S) -4-carboxy-4- (15-carboxypentadecanoylamino) butanoyl ] -Val-Y-Aib-EGTFTSDYSI-Aib-LDKIAQK [2- [2- [2- [ [ (4S) -4-carboxy-4- (19-carboxynonadecanoylamino) butanoyl ] amino ] ethoxy ] acetyl ] -AFVQWLIAGGPSSGAPPPS-NH 2
Z: parent compound No. 5; x=lys; y=val; substituent b: chemical formula 16 attached to X; substituent z: chemical formula 11 attached to Lys20 of Z
The synthesis method comprises the following steps: SPPS_A; DS_A; ds_d; cp_a
Calculated molecular weight (average): 5438.3Da
Lcms_a: rt=6.6 min; actual measurement [ M+3H ] 3+1813.4,[M+4H]4+ 1360.2
General method for measuring half-life of transformation
This assay was performed to investigate the conversion half-life of the prodrug of the invention to the drug. After incubation at 37 ℃, the transformation half-life was studied in vitro at pH 7.4.
Preparation and sampling of formulations
The test compound was dissolved in phosphate buffered saline (140mM NaCl,2.07mM KCl,8.05mM Na 2HPO4,1.96mM KH2PO4, pH 7.4) to a concentration of 100 μm. After dissolution the pH of the solution was measured and, if necessary, adjusted to pH 7.4 with aqueous NaOH. The solution was filtered through a 0.22 μm syringe filter and then incubated in a 37 ℃ water bath. Aliquots are removed at defined time points (e.g., every 24-72 hours) for LCMS analysis.
Analysis and calculation
LCMS analysis was performed using the procedure defined in lcms_a above.
Uv detection at 214nm was used to determine the AUC of the prodrug, active drug and DKP and calculate the ratio of prodrug AUC to the total AUC of prodrug plus active drug plus DKP. The negative natural logarithm of the ratio is plotted against time, and then the relationship is linearly regressed. The transformation half-life, i.e. the time at which AUC ratio = 0.5, was calculated from this linear regression.
Example 2
The conversion half-life of a prodrug of a compound of the invention to a drug is measured as described in the general method for measuring conversion half-life. The results are presented in table 6. The compounds of the invention have a surprisingly long conversion half-life, which can be modulated by minor modifications to the chemical structure of the DKP moiety.
Table 6: conversion half-life of prodrug in PBS buffer at pH 7.4 and 37℃
Numbering of compounds Conversion half-life [ h ]
1 244
2 363
3 111
4 97
5 74
6 440
7 38
8 125
9 132
10 195
11 182
12 129
13 137
14 166
15 127
16 117
18 101
General method for measuring terminal half-life in miniature pigs
The purpose of this method is to determine the in vivo half-life of the derivatives of the invention after intravenous administration to minipigs, i.e. the prolongation of their time in vivo, and hence of their duration of action. This was done in Pharmacokinetic (PK) studies to determine the terminal half-life of the derivatives in question. By terminal half-life is generally meant the length of time it takes to halve a certain plasma concentration, measured after an initial distribution phase.
Study of
Females used in the studyPig from ELLEGAARDMinipigs (Dalmose, denmark) obtained at about 7-14 months of age and weighing about 16-35kg. Mini-pigs were housed individually and were restricted to feeding SDS mini-pig diet once daily (SPECIAL DIETSSERVICES, essex, UK).
After 3 weeks of acclimation, two permanent central venous catheters were implanted into the vena cava caudalis of each animal. Animals were allowed to recover for 1 week after surgery and then were subjected to repeated pharmacokinetic studies with appropriate washout periods between successive derivative administrations.
Animals were fasted for approximately 18 hours prior to dosing and O to 4 hours post dosing, but were given ad libitum throughout the time period.
The sodium salt of the compound of example 1 was prepared using method sx_a in "general method for preparing compounds of the invention". The resulting sodium salt was dissolved in pH 7.4 buffer containing 0.007% polysorbate 20, 50mM sodium phosphate, 70mM sodium chloride to a concentration of 50-300nmol/mL. The compound is injected intravenously through one catheter (the volume corresponds to typically 1-20nmol/kg, e.g. 0.02-0.05 mL/kg) and blood samples are collected at predetermined time points for up to 21 days after administration (preferably through another catheter). Blood samples (e.g., 0.8 mL) were collected in 8mM EDTA buffer and then centrifuged at 1942g for 10 minutes at 4 ℃.
Sampling and analysis
Plasma was pipetted into a micro tube on dry ice and kept at-20 ℃ until the plasma concentration of the compounds was analyzed using ELISA or similar antibody-based assays or LCMS. Each plasma concentration-time curve was analyzed by a non-compartmental model in Phoenix WinNonLin ver.6.4 (Pharsight inc., mountain View, CA, USA) and the resulting terminal half-life (harmonic mean) was determined.
Example 3
The measured and/or observed terminal half-lives are shown in table 7 according to the general method for measuring terminal half-life in mini-pigs herein. The parent compounds No. 1, 2 and 4 administered in free form have surprisingly high observed terminal half-lives that can be further prolonged by the addition of non-transforming dipeptides and substituents (e.g., non-transforming compound No. 1). The use of a conversion prodrug (e.g., compounds No. 1,3, 9, and 10) results in a shorter half-life than the non-conversion counterpart (e.g., non-conversion compound No. 1) because conversion aids in the elimination of the conversion prodrug, but does not aid in the elimination of the non-conversion compound, and conversion is indistinguishable from other elimination mechanisms. This provides evidence that conversion of the prodrug to the parent compound is occurring in vivo. Due to the transformation's contribution to the terminal half-life of the prodrug, the terminal half-life of the prodrug may be faster or slower than the corresponding parent compound.
Table 7: terminal half-life measured after intravenous administration in minipigs
General method for measuring oral bioavailability in beagle dogs
The purpose of this method is to determine the in vivo terminal half-life and plasma exposure of the compounds of the invention after oral administration to beagle dogs, i.e. the terminal half-life and concentration of the test substance that reaches circulation over time. This was done in Pharmacokinetic (PK) studies to determine these parameters for the compounds in question. By terminal half-life is generally meant the length of time it takes to halve a certain plasma concentration, measured after an initial distribution phase.
Preparation of tablet compositions
Tablet compositions comprising the test compound obtained from example 1 and SNAC (sodium N- (8- (2-hydroxybenzoyl) amino) caprylate were prepared according to methods known to those skilled in the art by mixing the test substance with rolled SNAC and magnesium stearate as described, for example, in WO 2019/149880. Each tablet consisted of 7.7mg magnesium stearate, 2-4mg each test compound and 300mg SNAC.
Animals, administration and sampling
The study included male beagle dogs between 1-7 years of age and weighing 9-17kg during the study period. Dogs were dosed in a fasted state. Dogs were housed in pens in groups (12 hours light: 12 hours dark) and individually and restrictively fed Royal Canin Medium Adult dog foods (Royal Canin Products, china Branch, or Brogaarden A/S, denmark) once daily. These dogs were used for repeated PK studies with appropriate washout periods between consecutive administrations. Appropriate adaptation period was given prior to the initiation of the first PK study. All treatments, administrations and blood sampling of animals were performed by trained and skilled personnel. Dogs were fasted overnight prior to the study and 0 to 4 hours after dosing. Dogs were restricted to drinking water 1 hour prior to dosing up to 4 hours post dosing, but were given ad libitum throughout the rest of the period.
The compositions were administered by a single oral administration to a group of 6-8 dogs. The tablets were applied in the following manner: approximately 3nmol/kg of SEQ ID NO.7 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNT) may be subcutaneously administered to dogs 10 minutes prior to tablet administration, and then the tablets placed in the back of the dogs' mouth to prevent chewing. The mouth was then closed and 10mL of tap water was administered by syringe or gavage to facilitate swallowing of the tablet.
One blood sample is withdrawn prior to administration and additional samples are withdrawn at predetermined time points after administration, for example for up to 600 hours, to adequately cover the complete plasma concentration-time absorption profile of the test substance. For each blood sample time point, approximately 0.8mL of whole blood was collected in a 1.5mL EDTA-coated tube, which was gently turned to mix the sample with EDTA. Blood samples were collected in EDTA buffer (8 mM) and then centrifuged at 2000g for 10 min at 4 ℃. The plasma was pipetted into a micro tube on dry ice and stored at-20 ℃ or lower until analysis. Blood samples were taken as appropriate, for example venflon in the cephalic vein of the anterior leg for the first 2 hours, then from the jugular vein using a syringe for the remaining time points. The first few drops were drained from venflon to avoid heparin saline from venflon in the sample.
All blood samples were collected into tubes containing EDTA for stabilization and stored on ice until centrifugation. Plasma was separated from whole blood by centrifugation and stored at-20 ℃ or lower until analysis.
Analysis and calculation
The plasma is analyzed for test substances using LC-MS (liquid chromatography-mass spectrometry) as known to the person skilled in the art. The system consists of any one of the following: thermo Fisher QExactive mass spectrometer equipped with 10 valve interface module TurboFlow system, CTC HTS PAL autosampler, accela 1250 pump and Hot Pocket column incubator; or Thermo Fisher QExactive Plus mass spectrometer equipped with a valve interface module TurboFlow system, triPlus RSI autosampler, dionex UltiMate 3000 pump, and Hot Pocket column incubator. Use 1: a linear gradient of 1 acetonitrile/methanol in 1% aqueous formic acid solution achieved reverse phase HPLC separation, wherein either of the following conditions was used: phenomenex Onyx Monolithic C18 column (50 x 2.0 mm) and flow rate of 0.8mL/min, 30 ℃; or Agilent Poroshell SB-C18 column (50X2.1 mm,2.7 μm), a flow rate of 0.4mL/min, 60 ℃. The mass spectrometer operates in either positive ionization SIM mode or positive ionization PRM mode.
For each individual animal, the plasma concentration-time curve was analyzed by a non-compartmental model in PHARSIGHT PHOENIX winnonlink.6.4 software or other related software for PK analysis, and the resulting terminal half-life (t 1/2), maximum plasma concentration per dose (C max/D), maximum plasma concentration time (t max), and area under the curve (AUC/D) per dose to infinity were determined. Summarized statistics of pharmacokinetic results are presented as median (for t max), harmonic mean (t 1/2) or arithmetic mean (C max, AUC).
Example 4
The pharmacokinetic properties measured as described herein in the general method of measuring oral bioavailability in beagle dogs are shown in table 8. Since the concentration of the compound in plasma was detected after oral administration (C max/D >0 and AUC/D > 0), all tested compounds of the invention showed exit-oral bioavailability in this model. All compounds tested also exhibited a surprisingly high observed terminal half-life, as observed in example 3.
Table 8: pharmacokinetic parameters measured after oral administration in beagle dogs
* Average data of three experiments
General method for measuring in vitro functional efficacy
The purpose of this example is to test compounds for their functional activity or efficacy at human GLP-1 and GIP receptors in vitro. In vitro functional efficacy is a measure of target receptor activation in whole cell assays. The potency of the parent compounds 1-5 of example 1 was determined as follows. Human GLP-1 (7-37) (HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G; SEQ ID NO: 8) and human GIP (YAEGT FISDY SIAMD KIHQQ DFVNW LLAQK GKKND WKHNI TQ; SEQ ID NO: 9) were included as reference compounds in the appropriate assays for comparison.
Principle of
In vitro functional efficacy was determined by measuring the response of target receptors in a reporter gene assay in a separate cell line. The assay was performed in a stably transfected BHK cell line expressing one of the following G protein-coupled receptors: a human GLP-1 receptor or a human GIP receptor; and wherein each cell line contains CAMP Response Element (CRE) DNA coupled to a promoter and a firefly luciferase (CRE luciferase) gene. When the respective receptor is activated, it leads to the production of cAMP, which in turn leads to the expression of luciferase protein. When the assay incubation is complete, a luciferase substrate (luciferin) is added, resulting in enzymatic conversion of luciferin to oxidized luciferin and the generation of bioluminescence. The luminescence is measured as a readout of the assay.
Cell culture and preparation
The cell line used in these assays was BHK cells with BHKTS as the parent cell line. The cell line is derived from a clone containing the CRE luciferase element and is established by further transfection with the respective human receptor to obtain the relevant cell line: BHK CRE luc2PhGLP-1R or BHK CRE luc2P hGIPR. Cells were cultured in cell culture medium at 37 ℃ at 5% CO 2. Cells were aliquoted and stored in liquid nitrogen. Cells were maintained in continuous culture and inoculated one day prior to each assay.
Material
The following chemicals were used in this assay: pluronic F-68 10% (Gibco 2404), human serum albumin (HSA; sigma A9511), 10% fetal bovine serum (FBS; invitrogen 16140-071), egg white ovalbumin (Sigma A5503), phenol red free DMEM(Gibco21063-029)、DMEM(Gibco 12430-054)、1M Hepes(Gibco 15630)、Glutamax 100x(Gibco 35050)、G418(Invitrogen 10131-027)、 hygromycin (Invitrogen 10687-010) and STEADYLITE PLUS (Perkinelmer 6016757).
Buffer solution
GLP-1R cell culture medium consisted of DMEM medium containing 10% FBS, 500 μg/mL G418 and 300 μg/mL hygromycin. GIPR cell culture medium consisted of DMEM medium containing 10% FBS, 400. Mu.g/mL G418 and 300. Mu.g/mL hygromycin. The assay buffer consisted of phenol red free DMEM, 10mM Hepes, 1 XGlutamax, 1% ovalbumin and 0.1% Pluronic F-68, with twice the final assay concentration of HSA added. The assay buffer is mixed with an equal volume of test compound 1:1 in the assay buffer to give the final assay concentration of HSA.
Program
1) Cells were plated at 5000 cells/well and incubated overnight in assay plates.
2) Cells were washed once in DPBS.
3) Stock solutions of test compound and reference compound at concentrations ranging from 100 to 300 μm were diluted 1:150 in assay buffer. The compounds were then diluted 1:10 in column 1 of a 96 deep well dilution plate, and then starting from this row, a 3.5-fold, 12-point dilution curve was generated.
4) To each well of the assay plate was added assay buffer (50 μl aliquots) with or without HSA.
5) Aliquots of 50 μl of compound or blank were transferred from the dilution plate to assay plates containing assay buffer with or without HSA.
6) The assay plates were incubated in a 5% CO 2 incubator at 37℃for 3h.
7) Cells were washed once with DPBS.
8) To each well of the assay plate, 100 μl aliquots of DPBS were added.
9) A100. Mu.l aliquot of STEADYLITE PLUS reagent (photosensitive) was added to each well of the assay plate.
10 Each assay plate was covered with aluminum foil to protect from light and shaken at 250gm for 30min at room temperature.
11 Reading each assay plate in a microtiter plate reader.
Calculation and results
Data from the microtiter plate reader were first regressed in Excel to calculate x-axis, logarithmic scale concentrations from the stock concentrations of the individual test compounds and the dilutions of the assay. This data is then transferred to GRAPHPAD PRISM software for mapping and statistical analysis. The software performs nonlinear regression (log (agonist) versus response). EC 50 values calculated with this software and reported in pM are shown in table 9 below. A minimum of two replicates were measured for each sample. The reported values are the average of the repeated measurements. The compounds of the invention exhibit potent functional activation of human GLP-1R and human GIPR receptors under given conditions.
Example 5
The measured GLP-1 and GIP receptor functional potency as described herein as "general method of measuring in vitro functional potency" is shown in table 9. The parent compounds 1-5 administered in free form exhibited potent functional activation of the human GLP-1 receptor and the human GIP receptor under the given conditions.
Table 9: functional efficacy on human GLP-1R and GIPR in the presence of 0% and 1% HSA.
Nd=not determined.
While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true scope of the invention.

Claims (15)

1. A compound of formula I:
B-Z (formula I)
Or a pharmaceutically acceptable salt, ester or amide thereof,
Wherein B is a dipeptide or derivative thereof;
wherein Z is a GLP-1/GIP receptor co-agonist or derivative thereof; wherein the method comprises the steps of
Wherein the GLP-1/GIP receptor co-agonist has an amino acid sequence of YX2EGTX6TSDYSX12X13LX15X16X17AX19X2 0X21FX23X24WLX27X28GX30X31X32X33X34X35X36X37X38X39(SEQ ID NO.:1), wherein
X 2 is Aib or A,
X 6 is F or V, and the total number of the components is H,
X 12 is I or Y, and the total number of the components is,
X 13 is Y, A, L, I or Aib,
X 15 is D or E, and the total number of the components is D or E,
X 16 is K or E, and the total number of the components is,
X 17 is Q or I, and the total number of the components is,
X 19 is A or Q, and the total number of the components is H,
X 20 is Q, R, E, H or K,
X 21 is A or E, and the total number of the components is,
X 23 is I or V, and the total number of the components is H,
X 24 is E, Q or N,
X 27 is L or I, and the total number of the components is L or I,
X 28 is A or R,
X 30 is G or is absent,
X 31 is P or is not present,
X 32 is E, S or is absent,
X 33 is S, K or is absent,
X 34 is G or is absent,
X 35 is A or is absent,
X 36 is P or is not present,
X 37 is P or is not present,
X 38 is P or is not present,
X 39 is S or absent; and
Wherein the N-terminal amino group of the GLP-1/GIP receptor co-agonist is linked to B via a peptide bond.
2. The compound of claim 1, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the GLP-1/GIP receptor co-agonist has an amino acid sequence of Y-Aib-EGTFTSDYSILLEX 16QAAREFIEWLLAGGPSX33 GAPPPS (SEQ ID No.: 3), wherein
X 16 is K or E, and the total number of the components is,
X 33 is S or K.
3. The compound of claim 1 or claim 2, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein X 16 is E and X 33 is K.
4. The compound of claim 1 or claim 2, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein X 16 is K and X 33 is S.
5. The compound of any one of claims 1-4, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the GLP-1/GIP receptor co-agonist comprises a substituent z, and wherein the substituent z is linked to the GLP-1/GIP receptor co-agonist through lysine (K) at position 16 or 33.
6. The compound of claim 5, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the substituent z is selected from formula 7, formula 8, formula 10, or formula 11.
7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the dipeptide B is of formula II:
X-Y (formula II),
Wherein X is any alpha-amino acid linked to Y through an amide bond formed between the alpha-carboxylic acid group of X and the alpha-amino group of Y,
Wherein Y is an N-alkylated alpha-amino acid linked to Z by a peptide bond formed between the alpha-carboxylic acid group of Y and the N-terminal amino group of the GLP-1/GIP receptor co-agonist.
8. The compound of claim 7, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamic acid, N-methylnorleucine, N-methyl homoalanine, N-methylalanine, N-methyllysine, N- (2-aminoethyl) glycine, N-hexyl homoalanine, N-propyl alanine, homoproline, N-propyl glycine, N-ethyl glycine, and N-methyl phenylalanine.
9. The compound of claim 7 or claim 8, wherein X is selected from lysine, 4-aminophenylalanine, D-lysine, alanine, glycine, proline, D-valine, homoproline, D-proline, D-homoproline, D-alanine, and azetidine-2-carboxylic acid.
10. The compound of any one of claims 7-9, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein Y is sarcosine or N- (2-aminoethyl) glycine.
11. The compound according to any one of claims 7-10, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from lysine, D-lysine and glycine.
12. The compound of any one of claims 1-11, or a pharmaceutically acceptable salt, ester, or amide thereof, wherein the dipeptide carries a substituent b, and optionally wherein the substituent b is selected from formula 16, formula 17, formula 18, formula 19, formula 20, formula 21, and formula 22.
13. The compound of any one of claims 1-12, wherein the compound is selected from the group consisting of:
Compound No. 1
Compound No. 2
Compound No. 3
Compound No. 4
Compound No. 5
Compound No. 6
Compound No. 7
Compound No. 8
Compound No. 9
Compound No. 10
Compound 11
Compound No. 12
Compound No. 13
Compound No. 14
Compound No. 15
Compound No. 16
Compound No. 17
And compound No. 18
14. A compound according to any one of claims 1-13 for use as a medicament.
15. A compound according to any one of claims 1-13 for use in the prevention and/or treatment of type 2 diabetes.
CN202380017655.7A 2022-01-20 2023-01-20 GLP-1/GIP receptor co-agonist prodrugs and uses thereof Pending CN118632867A (en)

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