Synthetic apelin mimetics for the treatment of heart failure

SYNTHETIC APELINE MIMETICS FOR TREATMENT

OF HEART INSUFFICIENCY

SEQUENCE LIST

This application contains a sequence listing, which has been submitted in ASCII format through the EFS-Web and is incorporated herein by reference in its entirety. This copy in ASCII, created on June 28, 2013, is called PAT054961-WO-PCT_SL.txt and is 85,581 bytes in size. FIELD OF THE INVENTION:

The invention relates to novel compositions comprising sequences of modified peptides and polypeptides designed to treat cardiovascular diseases in the subjects to whom they are administered, and which exhibit a greater resistance to degradation, and a bioactivity equivalent to or greater than their counterparts. wild type. The invention also relates to methods for the preparation of these compositions, and to the use of these compositions as pharmaceutically active agents for treating cardiovascular diseases.

BACKGROUND OF THE INVENTION:

The incidence of heart failure in the western world is approximately 1/100 adults after 65 years of age. The most common pathology is a chronic deficit in cardiac contractility and, therefore, in cardiac output, that is, the effective volume of blood expelled by any of the ventricles of the heart over time. Patients with chronic heart failure may have acute episodes of decompensation, that is, heart failure to maintain adequate blood circulation, where cardiac contractility declines further. There are approximately 500,000 hospitalizations per year for “acute decompensated heart failure” (ADHF) in the United States alone.

Current therapies for acute decompensated heart failure (ADHF) include diuretics, vasodilators, and inotropes, which directly increase cardiac contractility. Current intravenous inotropes (dobutamine, dopamine, milrinone, levosimendan) are used in the acute situation, despite their association with adverse events, such as arrhythmia and an increase in long-term mortality. These inconveniences have prevented its application in chronic heart failure. Digoxin is an oral inotrope, but is limited by a narrow therapeutic index, an increase in arrhythmogenic potential, and contraindication in renal failure.

There is an urgent need for a therapy for heart failure that increases cardiac contractility without arrhythmogenic or mortality problems for acute decompensated heart failure (ADHF), but which can also solve the enormous unmet medical need in chronic heart failure.

Apeline is the endogenous ligand for the previously orphaned G-protein coupled receptor (GPCR), APJ, also referred to as apelin receptor, 1 angiotensin type receptor, 1 angiotensin II type receptor, and the like. The apeline / APJ pathway is widely expressed in the cardiovascular system, and it has been shown that apeline has important beneficial cardiovascular effects in pre-clinical models. The administration of acute apeline in humans causes peripheral and coronary vasodilation, and increases cardiac output (Circulation. 2010; 121: 1818-1827). As a result, APJ agonism is emerging as an important therapeutic goal for patients with heart failure. It is thought that activation of the APJ apeline receptor increases cardiac contractility and provides cardioprotection, without the drawbacks of current therapies. However, native apelines exhibit a very short half-life and duration of action in vivo.

Therefore, it is desirable to identify peptides and polypeptides that mimic the function of apeline, but have an increase in its half-life and demonstrate an equivalent or greater bioactivity than naturally occurring apeline. Additionally, it is desirable to identify apeline analog peptides and polypeptides that exhibit an increase in conformational limitations, that is, the ability to achieve and maintain an active conformational state, such that the peptides and polypeptides can interact with their receptors and / or other path objectives, without the need for additional folding or repositioning. There is a need to use such analogs of peptides and polypeptides, of compositions comprising these analogs, and of methods for making and using these compositions as pharmaceutically active agents for treating diseases, such as cardiovascular diseases.

BRIEF DESCRIPTION OF THE INVENTION:

The objective of the present invention is to provide novel peptides and polypeptides that are useful as APJ agonists, and which also possess at least one of the following improvements over wild-type apeline and other known apeline analogs: increased half-life; greater immunity to degradation after administration and / or solubilization; and increased conformational limitations, all while exhibiting the same or greater biological activity than wild-type apeline. The peptides and polypeptides of this invention, therefore, are particularly useful for the treatment or prevention of cardiovascular diseases, such as heart failure, disorders and conditions associated with heart failure, and disorders and conditions that respond to the activation of the activity of the APJ receiver.

In one embodiment, the peptides and polypeptides of the invention are particularly useful for the treatment or prevention of a disorder or condition associated with heart failure, or of a disorder that responds to the activation (or agonism) of the receptor activity of APJ. In another embodiment, the peptides and polypeptides of the invention are useful in the treatment of acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic diseases cardiovascular, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral artery disease, strokes, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn), and preeclampsia.

The invention pertains to peptides and polypeptides, pharmaceutical compositions, and methods of making and using them, as described herein. Examples of the peptides and polypeptides of the invention include peptides and polypeptides according to any of formulas I to IX, or an amide, an ester or a salt thereof, as well as any peptides or polypeptides specifically listed herein. , including, but not limited to, experimental examples.

The invention, therefore, provides a peptide or a polypeptide of the formula (I '):

Χ1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X13 I-1 r where:

X1 is the N-terminus of the polypeptide and is absent or is selected from pE, R, Isn, Q, A, K, and 5-amino-valeric acid;

X2 is R, A, r, N-Me-R, K, H, hF, hK, F, E or Orn;

X3 is P, A, a, p, 4-PhP, K, D, pipecolic acid, or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X4 is R, A, r, N-Me-R, F, E or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X5 is L, Cha, A, DL, N-Me-L, K, D, 4-PhF or F;

X6 and X12 are independently a natural or unnatural amino acid selected from C, c, hC, D-hC, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked to each other by means of a covalent bond, forming any one of a monosulfide bond (-S-), a disulfide bond (-SS-), or an amide bond (-NHC (O) - or -C (O) -NH-), or a link of the formula -S-CH₂C (= Z) -CH₂-S-; where Z is O, NO-CH₂CABBAGE¹-R¹ or N-NHCH₂CABBAGE¹-R¹, where R¹ is a derivative of fatty acid, and L¹ it is a linker containing one or more amino acids and / or one or more units of -NH-alkylene of 2 to 6 carbon atoms-NH-;

or alternatively, X6 is K, X13 is absent, and X12 is F of, where the C term of X12 forms an amide bond with the amino side chain of X6;

X7 is H, h, A, N-Me-A, a, Aib, K, Nal, F, P, Dap, N, E or cysteine, where the cysteine ​​side chain forms a disulfide bond with the chain side of the cysteine ​​at position X3 or with the side chain of cysteine ​​at position X4;

X8 is K, k, F, f, A, hF, N-Me-R, E or 4-amino-lsn;

X9 is G, N-Me-G, A, D, L, R or Aib;

X10 is P, A, p, 4-PhP or pipecolic acid,

X11 is M, D-Nle, Nle, N-Me-Nle, M (O), A, F and, L, K, 3-PyA or Cha; and

X13 is the term C and is absent or selected from F, f, N-Me-F, Nal, D-Nal, 3-Br-F, (S) -B-3-F, I, A, a, K, Dap, H and E;

where:

Nle is L-norleucine;

D-hC is D-homocysteine ​​hC is L-homocysteine;

hF is L-homophenylalanine;

hK is L-lysine;

Nal is L-naphthylalanine;

Orn is ornithine;

Aib is a-amino-isobutyric acid;

Dab is (S) -diamino-butyric acid;

Dap is (S) -2,3-diamino-propionic acid;

M (O) is methionine sulfone;

Cha is (S) -B-cyclohexylalanine;

4-amino-lsn is 4-amino-piperidin-4-carboxylic acid;

Isn is isonipecotinoyl; pE is L-pyroglutamic acid;

3-PyA is 3- (3-pyridiI) -L-aanine;

4-PhF is 4-phenyl-L-phenylalanine;

wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine; and wherein the amino group in the side chain of K, Orn, Dab, Dap, hK or 4-amino-lsn is optionally linked to a lipophilic group by means of an amide bond;

or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto.

As explained further herein, the three letter or one letter abbreviations recognized in this field are used to represent the amino acid residues that constitute the peptides and polypeptides of the invention. Except when preceded with “D, the amino acid is an L-amino acid. When the abbreviation of a letter is an uppercase letter, it refers to the Lamino Acid. When the abbreviation of a letter is a lowercase letter, it refers to the D-amino acid.

Any of the amino acid residues listed above of formula I ', or their related formulas described herein, for example, formulas I, II to IX, may be substituted in a conservative manner, with the understanding that the peptide or Polypeptide of the invention still retain functional activity and structural properties (for example, extension of half-life, protection from degradation, conformational limitation). The principles and examples of permissible conservative amino acid substitutions are further explained herein.

The polypeptides of the invention, by activating the APJ receptor, have utility in the treatment of:

acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, cardiovascular ischemic diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including diabetes gestational), obesity, peripheral artery disease, strokes, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn), and preeclampsia.

In a preferred embodiment, the polypeptides of the invention are useful in the treatment of acute decompensated heart failure (ADHF).

In another embodiment, the invention pertains to a method for the treatment of a disorder or a disease that responds to the activation of the APJ receptor, in a subject in need of such treatment, which comprises: administering to the subject, an effective amount of a polypeptide according to any of the formulas

I to IX, or of an amide, an ester or a salt thereof, such that the disorder or disease that responds to the activation of the APJ receptor in the subject is treated.

In yet another embodiment, the invention pertains to pharmaceutical compositions comprising a polypeptide according to any of formulas I to IX, or an amide, an ester or salt thereof, and one or more pharmaceutically acceptable carriers.

In yet another embodiment, the invention pertains to combinations that include a polypeptide according to any of formulas I to IX, or an amide, an ester or a salt thereof, and the pharmaceutical combinations of one or more therapeutically active agents.

In another embodiment, the invention pertains to a method for activating the APJ receptor in a subject in need thereof, which comprises: administering to the subject, a therapeutically effective amount of a polypeptide according to any of the formulas

I to IX, or of an amide, an ester or a salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of interpreting this specification, the following definitions will apply unless otherwise specified, and whenever appropriate, the terms used in the singular will also include the plural and vice versa.

As used herein, "disorders or diseases that respond to APJ receptor modulation," disorders and conditions that respond to APJ modulation, "disorders and conditions that respond to APJ receptor activity modulation, "Disorders that respond to the activation (or agonism) of APJ receptor activity, and similar terms include:

acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, cardiovascular ischemic diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including diabetes gestational), obesity, peripheral artery disease, strokes, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn), and preeclampsia.

As used herein, "Activation of APJ receptor activity, or Activation of APJ receptor, refers to an increase in APJ receptor activity. Activation of the activity of the APJ receptor is also referred to as "agonism" of the APJ receptor, for example, by administration of the peptides and polypeptides of the invention.

As used herein, the terms "polypeptide and peptide" are used interchangeably to refer to two or more amino acids linked together. Except for the abbreviations for the non-common or unnatural amino acids stipulated in Table 1 below, the three-letter or one-letter abbreviations recognized in this field are used to represent amino acid residues that constitute the peptides and polypeptides of the invention. . Except when preceded with "D", the amino acid is an L-amino acid. When the abbreviation of a letter is an uppercase letter, it refers to the D-amino acid. When the abbreviation of a letter is a lowercase letter, it refers to the L-amino acid. The groups or cords or abbreviations of amino acids are used to represent peptides. Peptides are indicated with the term N on the left and the sequence is written from the term N to the term C.

The peptides of the invention contain unnatural amino acids (ie, compounds that do not occur in nature), and alternatively, other amino acid analogs can be employed as are known in the art.

Certain unnatural amino acids can be introduced by the technology described in Deiters et al., J Am Chem Soc 125: 1 1782-1 1783, 2003; Wang and Schultz, Science 301: 964-967, 2003; Wang et al., Science 292: 498-500, 2001; Zhang et al., Science 303: 371-373, 2004, or in United States Patent No. 7,083,970. Put briefly, some of these expression systems involve site-directed mutagenesis to introduce a nonsense codon, such as an amber TAG, into the open reading frame encoding a polypeptide of the invention. These expression vectors are then introduced into a host that can use a specific tRNA for the nonsense codon introduced, and loaded with the unnatural amino acid of choice. Particular non-natural amino acids that are beneficial for the purpose of conjugating the fractions with the polypeptides of the invention include those with acetylene and azido side chains.

One or more of the natural or unnatural amino acids in a peptide of the invention can be modified, for example, by the addition of a chemical entity, such as a carbohydrate group, a phosphate group, a farnesyl group, an isophanesyl group, a fatty acid group (C_thatH_{q + 1}CO)₂H where q is from 3 to 20), a linker for conjugation, functionalization, or other modification, etc. These modifications can be made in a site specific or non-site specific manner. In a preferred embodiment, peptide modifications lead to a more stable peptide (for example, one that exhibits a longer half-life in vivo). These modifications may include the incorporation of additional D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide, but these modifications may confer desirable properties, for example, a better biological activity, on the peptide.

These modifications improve the biological properties of the proteins of the invention in relation to wild-type proteins, as well as, in some cases, serve as binding points for, for example, brands and extension agents of the protein half-life. , and for the purposes of fixing these variants to the surface of a solid support.

In certain embodiments, these modifications, for example, site-specific modifications, are used to bind conjugates, for example, PEG groups to the polypeptides and / or peptides of the invention, for the purposes, for example, of extending the half-life. or otherwise improve the biological properties of said polypeptides and / or peptides. These techniques are further described herein.

In other embodiments, these modifications, for example, site-specific modifications, are used to bind other polymers and small molecules and recombinant protein sequences that extend the half-life of the polypeptide of the invention. One of these modalities includes the binding of fatty acids or albumin binding compounds specific to the polypeptides and / or peptides. In other embodiments, modifications are made to a particular type of amino acid, and can be linked at one or more sites on the polypeptides.

In other embodiments, these modifications, for example, site-specific modifications, are used as a binding means for the production of wild type multimers and / or variants, for example, dimers (homodimers or heterodimers) or trimers or tetramers. These multimeric protein molecules may additionally have groups, such as PEG, sugars, and / or PEG-cholesterol conjugates bound or to be fused either amino-terminally or carboxy-terminally with other proteins, such as Fe, human serum albumin ( HSA), etc.

In other embodiments, these site-specific modifications are used to produce proteins, polypeptides and / or peptides, wherein the position of the pyrrolysin or pyrrolysin analog specifically incorporated at the site, or naturally occurring amino acids (para- acetyl-Phe, para-azido-Phe), allows to have a controlled orientation and binding of these proteins, polypeptides and / or peptides on a surface of a solid support, or allows to have groups, such as PEG, sugars and / or conjugates of

PEG-cholesterol bound.

In other embodiments, these site-specific modifications are used to specifically cross-link the proteins, polypeptides and / or peptides at the site, thereby forming hetero-oligomers, including, but not limited to, heterodimers and heterotrimers. In other embodiments, these site-specific modifications are used to specifically cross-link proteins, polypeptides and / or peptides at the site, thereby forming protein-protein conjugates, protein polypeptide conjugates, protein-peptide conjugates, polypeptide conjugates. polypeptide, polypeptide-peptide conjugates or peptide-peptide conjugates. In other embodiments, a site-specific modification may include a branching point to allow more than one type of molecule to be attached at a single site of a protein, polypeptide or peptide.

In other embodiments, the modifications listed herein may be made in a non-site specific manner, and may result in protein-protein conjugates, protein-polypeptide conjugates, protein-peptide conjugates, polypeptide-polypeptide conjugates , polypeptide16 peptide conjugates or peptide-peptide conjugates of the invention.

In some embodiments, the present invention provides complexes comprising at least one peptide or polypeptide of any of formulas I to IX bound to an antibody, such as an antibody that specifically binds to a peptide or polypeptide as disclosed herein. I presented.

An ordinary expert in this field will appreciate that various amino acid substitutions can be made, for example, conservative amino acid substitutions, in the sequence of any of the polypeptides described herein, without necessarily decreasing their activity. As used herein, "amino acid commonly used as a substitute thereof" includes conservative substitutions (ie, amino acid substitutions of comparable chemical characteristics). For the purposes of conservative substitution, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan and methionine. Polar neutral (hydrophilic) amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, Usine and histidine. Negatively charged amino acids (acids) include aspartic acid and glutamic acid. Examples of amino acid substitutions include replacing an L-amino acid with its corresponding D-amino acid, replacing cysteine ​​with homocysteine ​​or other unnatural amino acids that have a thiol-containing side chain, replacing a lysine with homolysin, diamino-butyric acid, diamino-propionic acid, ornithine or other unnatural amino acids that have an amino-containing side chain, or the substitution of an alanine for norvaline, or similar.

The term "amino acid, as used herein, refers to naturally occurring amino acids, unnatural amino acids, amino acid analogs, and amino acid mimetics that function in a manner similar to the amino acids that occur. naturally, all in its stereoisomers D and L if its structure allows such stereoisomeric forms. Amino acids are referred to herein either by their name, their commonly known three-letter symbols, or by the one-letter symbols recommended by the lUPAC-lUB Biochemical Nomenclature Commission.

The term "naturally occurring" refers to materials that are found in nature and are not manipulated by man. In a similar way, "that does not occur naturally," unnatural, and the like, as used herein, refer to a material that is not found in nature or that has been structurally modified or synthesized by man. When used in relation to amino acids, the term "naturally occurring" refers to the conventional 20 amino acids (ie, alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or lie), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr)).

The terms "unnatural amino acid and unnatural amino acid, as used herein, are intended to represent in an interchangeable manner the amino acid structures that cannot be generated biosynthetically in any organism using unmodified or modified genes from any organism, whether Same or different. The terms refer to an amino acid residue that is not present in the naturally occurring apeline protein sequence (wild type), or in the sequences of the present invention. These include, but are not limited to, amino acids and / or modified amino acid analogs that are not one of the 20 naturally occurring amino acids, selenocysteine, pyrrolysine (PYL), or pyrroline-carboxylysine (Pcl, for example, as described in TCP Patent Publication Number WO201 0/48582). Such unnatural amino acid residues can be introduced by substituting naturally occurring amino acids, and / or by inserting unnatural amino acids into the naturally occurring apeline protein sequence (or wild-type) sequences of the invention The unnatural amino acid residue can also be incorporated in such a way that a desired functionality is imparted to the apeline molecule, for example, the ability to bind to a functional fraction (eg, PEG). When used in relation to amino acids, the symbol "U" shall mean "unnatural amino acid and unnatural amino acid, as used herein.

In addition, it is understood that these "unnatural amino acids" require a modified tRNA and a modified tRNA (RS) synthetase for incorporation into a protein. These "selected" orthogonal tRNA / RS pairs are generated by a selection process as developed by Schultz et al., Or by random or directed mutation. By way of example, pyrroline-carboxy-lysine is a "natural amino acid" because it is generated biosynthetically by genes transferred from an organism into host cells, and because it is incorporated into proteins using proteins. natural tRNA and tRNA synthetase genes, while pamino-phenylalanine (see, Generation of a bacterium with a 21 amino acid genetic code, Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG. J Am Chem Soc. 29January-2003; 1 25 (4): 935-9) is an "unnatural amino acid" because, although it is generated biosynthetically, it is incorporated into proteins by a pair of "selected" orthogonal tRNA / tRNA synthetase tRNA / tRNA.

Modified encoded amino acids include, but are not limited to, hydroxy-proline, γ-carboxy-glutamate, O-phosphoserine, azetidine carboxylic acid, 2-amino-adipic acid, 3-amino-adipic acid, beta-alanine, acid amino-propionic, 2-amino-butyric acid, 4-amino-butyric acid, 6-amino-caproic acid, 2-amino-heptanoic acid, 2-amino-isobutyric acid, 3-amino-isobutyric acid, 2-aminopimelic acid , terbutyl glycine, 2,4-diamino-isobutyric acid, desmosin, 2,2'-diamino-pimeric acid, 2,3-diamino-propionic acid, N-ethylglycine, N-methyl-glycine, N-ethyl-asparagine, homoproline, hydroxylysine, alo-hydroxylysine, 3-hydroxy-proline, 4-hydroxy-proline, isodesmosin, alo-isoleucine , N-methyl-alanine, N-methyl-glycine, N-methyl-isoleucine, Nm et i I - pe nti Ig I ic ina, N-methyl-valine, naphthylalanine, norvaline, norleucine, ornithine, pentyl-glycine, acid pipecolic and thioproline. The term "amino acid" also includes naturally occurring amino acids that are metabolites in certain organisms, but are not encoded by the genetic code for incorporation into proteins. These amino acids include, but are not limited to, ornithine, D-ornithine, and D-arginine.

The term "amino acid analogue, as used herein, refers to compounds that have the same basic chemical structure as an naturally occurring amino acid, by way of example only, an α-carbon that binds to a hydrogen , a carboxyl group, an amino group, and an R group. Amino acid analogs include natural and non-natural amino acids that are chemically blocked, in a reversible or irreversible manner, or that chemically modify their C-terminal carboxyl group, their N-terminal amino group, and / or their functional chain groups. side. These analogs include, but are not limited to, methionine sulfoxide, methionine sulfone, S- (carboxymethyl) -cysteine, S- (carboxymethyl) -cysteine ​​sulfoxide, S- (carboxymethyl) -cysteine-sulfone, (beta -methyl ester) of aspartic acid, N-ethylglycine, alanine-carboxamide, homoserine, norleucine, and methioninemethyl sulfonium.

Table 1

Unnatural or unnatural amino acids as described in the invention:

<td>Symbol</td><td>First name</td><td colspan="2">Structure</td>

<td>Aib</td><td>Α-amino acid isobutyric</td><td>&gt; H₂N</td><td>AND" 0</td>

<td>M (O)</td><td>Methionine Sulfone</td><td>^ORh₂n</td><td>V or</td>

<td></td><td></td><td></td><td></td>

<td>1 -Nal</td><td>1-Naphthylalanine</td><td>IL</td><td>ώ</td>

<td></td><td></td><td>h₂Item</td><td>.OH or</td>

<td>Symbol</td><td>First name</td><td>Structure</td>

<td>2-Nal</td><td>2-Naphthylalanine</td><td>JL OH H₂N or</td>

<td>Cha</td><td>β-cyclohexyl-alanine</td><td>JL oh^H2^N if or</td>

<td>Dab</td><td>Diamino Butyric Acid</td><td>Η₂Ν^/η X /^0Hh₂n γ 0</td>

<td>Dap</td><td>2,3-diamino acid propionic</td><td>H₂N And / °^HH₂rT γ 0</td>

<td>hC</td><td>Homocysteine</td><td>TO/^ohh₂n γ 0</td>

<td>Symbol</td><td>First name</td><td>Structure</td>

<td>hF</td><td>Homophenyl Alanine</td><td>Ph '^z'^Z^^Xi Λ /^ΟΗη₂γτ γ or</td>

<td>hK</td><td>Homolysin</td><td>h₂n K X /^0Hh₂n or</td>

<td>Nle</td><td>Norleucine</td><td>JL OH h₂n 0</td>

<td>Orn</td><td>Ornithine</td><td>JL OH h₂n yy 0</td>

<td>β-3-F</td><td>β-3-phenylalanine</td><td>V ....._ h₂n '^x^ oh</td>

<td>4-amino- Isn</td><td>4-amino-piperidine acid 4-carboxylic (the 4-amino group forms the peptide bond)</td><td>0 nh₂Ñ</td>

<td>Symbol</td><td>First name</td><td>Structure</td>

<td>Isn</td><td>Isonipecotinoic Acid</td><td> 0</td>

<td>PE</td><td>Pyroglutamic acid</td><td></td>

<td>4-PhP</td><td>4-phenyl-proline</td><td>XAoh Ph</td>

<td></td><td>Pipecolinic acid</td><td></td>

<td></td><td>5-amino-valeric acid</td><td>0 h₂n^{/ Z}^^^^^xoh</td>

<td>020c</td><td>8-amino-3,6- dioxa-octanoic</td><td>0 H / L · xx JL</td>

<td>Symbol</td><td>First name</td><td>Structure</td>

<td>3-PyA</td><td>3- (3-pyridyl) -alanine</td><td> 0</td>

<td>4-PhF</td><td>4-phenyl-phenylalanine</td><td> 0</td>

Nal refers to both 1-Naphthylalanine and 2-Naphthylalanine, preferably 2-naphthylalanine.

4-Phenylproline refers to both cis- and f / 'ans-4-phenyl-proline, preferably af /' ans-4-phenyl-proline.

As used herein, the term "amide" refers to an amide derivative of the carboxylic acid group in the term C (eg, -C (O) NH₂, -C (O) NH-alkyl of 1 to 6 carbon atoms, - C (O) NH-alkyl of 1 to 2 carbon atoms-phenyl, -C (O) NH-NHBn or -C (O) N (alkyl of 1 to 6 carbon atoms)₂).

The term "amide" also refers to a derivative of the amino group in the term N (for example, -NHC (O) -alkyl of 1 to 16 carbon atoms, -NHC (O) (CH₂)_nPh (n is an integer from 1 to 6),

-NHC (O) (CH₂)₂CO₂H, 4-CI-Ph- (CH₂)₃C (O) NH-, Ci 1 H₂₃C (O) NH- (CH₂)₂O- (CH₂)₂-O-CH₂-C (O) -NH-, Ci₃H₂₇C (O) NH- (CH₂)₂-OR- (CH₂)₂-O-CH₂C (O) -NH-; Ci₅H₂₇C (O) NH- (CH₂)₂-OR- (CH₂)₂-O-CH₂-C (O) NH-, Ph-CH₂

CH₂NHC (O) -NH- or CH₃(OCH₂CH₂)_mC (O) NH- (m is an integer from 1 to 12).

As used herein, the term "ester" refers to a form of an ester derivative of the carboxylic acid group in the term C (eg, -COOR), wherein R of the ester refers to the alkyl groups from 1 to 6 carbon atoms, such as methyl, ethyl, normal propyl, isopropyl, normal butyl, etc., cycloalkyl groups from 3 to 8 carbon atoms, such as cyclopentyl, cyclohexyl, etc., aryl groups of 6 at 10 carbon atoms, such as phenyl, α-naphthyl, etc., aryl groups of 6 to 10 carbon-alkyl atoms of 1 to 6 carbon atoms, for example, phenyl-alkyl groups of 1 to 2 carbon atoms, such as benzyl, phenethyl, benzhydryl, etc., and the groups α-naphthyl-alkyl of 1 to 2 carbon atoms, such as α-naphthyl methyl, and the like. Mention may also be made of pivaloyloxy methyl ester and the like, which are commonly used as esters for oral administration. When the polypeptides of the invention possess additional carboxy or carboxylate groups at different positions of the C term, those polypeptides, wherein these groups are amidated or esterified, also fall under the category of the polypeptide of the invention. In such cases, the esters, for example, may be the same classes of esters as the C27 terminal esters mentioned above.

The term "APJ" (also referred to as "apeline receptor," receptor 1 type angiotensin, "receptor 1 type angiotensin II, and the like), indicates a residue380, a transmembrane domain 7, a receptor coupled with Gi whose gene is located on the long arm of chromosome 11 in humans (NCBI reference sequence: NP 005152.1, and encoded by NCBI reference sequence: NM 005161). APJ was first cloned in 1993 from human genomic DNA using degenerate oligonucleotide primers (O'Dowd et al., Gene, 136: 355-60, 1993), and shares a significant homology with the angiotensin II type 1 receptor. Despite this homology, however, angiotensin II does not bind to APJ. Although he was an orphan for many years, the endogenous ligand was isolated and named as apeline (Tatemoto et al., Biochem Biophys Res Commun 251, 471-6 (1998)).

The term "apeline indicates a pre-protein of 77 residues (NCBI reference sequence: NP 0059109.3, and encoded by NCBI reference sequence: NM017413.3), which becomes processed in biologically active forms of apeline peptides, such as Apelina-36, Apelina-17, Apelina-16, Apelina-13, Apelina-12. The mature full-length peptide, referred to as "apeline-36, comprises 36 amino acids, but the most potent isoform is the pyroglutamate form of a 13mer of apeline (apeline-13), referred to as" Pyr-1-apeline-13 or Pyr¹-Apelina-13 ”. Different forms of apeline are described, for example, in United States Patent No. 6,492,324B1.

Polypeptides of the invention:

Different embodiments of the invention are described herein. It will be recognized that the characteristics specified in each modality can be combined with other characteristics specified to provide additional modalities.

In embodiment 1, the invention, therefore, provides a peptide or a polypeptide of the formula (I '):

Χ1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X13 I-1 I 'where:

X1 is the N-terminus of the polypeptide and is absent or is selected from pE, R, Isn, Q, A, K, and 5-amino-valeric acid;

X2 is R, A, r, N-Me-R, K, H, hF, hK, F, E or Orn;

X3 is P, A, a, p, 4-PhP, K, D, pipecolic acid, or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X4 is R, A, r, N-Me-R, F, E or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X5 is L, Cha, A, DL, N-Me-L, K, D, 4-PhF or F;

X6 and X12 are independently a natural or unnatural amino acid selected from C, c, hC, D-hC, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked to each other by means of a covalent bond, forming either a disulfide or amide bond;

or alternatively, X6 is K, X13 is absent, and X12 is F of, where the C term of X12 forms an amide bond with the amino side chain of X6;

X7 is H, h, A, N-Me-A, a, Aib, K, Nal, F, P, Dap, Ν, E or cysteine, where the cysteine ​​side chain forms a disulfide bond with the chain side of the cysteine ​​at position X3 or with the side chain of cysteine ​​at position X4;

X8 is K, k, F, f, A, hF, N-Me-R, E or 4-amino-lsn;

X9 is G, N-Me-G, A, D, L, R or Aib;

X10 is P, A, p, 4-PhP or pipecolic acid,

X11 is M, D-Nle, Nle, N-Me-Nle, M (O), A, F and, L, K, 3-PyA or Cha; and

X13 is the term C and is absent or selected from F, f, N-Me-F, Nal, D-Nal, 3-Br-F, (S) -B-3-F, I, A, a, K, Dap, H and E;

where:

Nle is L-norleucine;

D-hC is D-homocysteine ​​hC is L-homocysteine;

hF is L-homophenylalanine;

hK is L-lysine;

Nal is L-naphthylalanine;

Orn is ornithine;

Aib is a-amino-isobutyric acid;

Dab is (S) -diamino-butyric acid;

Dap is (S) -2,3-diamino-propionic acid;

M (O) is methionine sulfone;

Cha is (S) -B-cyclohexylalanine;

4-amino-lsn is 4-amino-piperidin-4-carboxylic acid;

Isn is isonipecotinoyl; pE is L-pyroglutamic acid;

3- PyA is 3- (3-pyridi I) -L-al an ina;

4- PhF is 4-phenyl-L-phenylalanine;

wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine; and wherein the amino group in the side chain of K, Orn, Dab, Dap, hK or 4-amino-lsn is optionally linked to a lipophilic group by means of an amide bond;

or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto.

In embodiment 2, the invention, therefore, provides a peptide or a polypeptide of the formula (I):

Χ1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X13 I-1 i where:

X1 is the N-terminus of the polypeptide and is absent or is selected from pE, R, Q, A, K, 5-amino-valeric acid and Isn;

X2 is R, A, r, N-Me-R, K, H, hF, hK or Orn;

X3 is P, A, a, p, 4-PhP, pipecolic acid, or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X4 is R, A, r, N-Me-R or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X5 is L, Cha, A, DL, N-Me-L or F;

X6 and X12 are independently a natural or unnatural amino acid selected from C, c, hC, D-hC, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked to each other by means of a covalent bond;

or alternatively, X6 is K, X13 is absent, and X12 is F of, where the C term of X12 forms an amide bond with the amino side chain of X6;

X7 is H, h, A, N-Me-A, a, Aib, K, Nal, F, P, Dap, N or cysteine, where the cysteine ​​side chain forms a disulfide bond with the side chain of cysteine ​​in position X3 or with the side chain of cysteine ​​in position X4;

X8 is K, k, F, f, A, hF, N-Me-R or 4-amino-lsn;

X9 is G, N-Me-G, A or Aib;

X10 is P, A, p, 4-PhP or pipecolic acid,

X11 is M, D-Nle, Nle, N-Me-Nle, M (O), A, F and, L, K or Cha; and

X13 is the term C and is absent or selected from F, f, N-Me-F, Nal, D-Nal, 3-Br-F, (S) -B-3-F, I, A, a, K, Dap where:

Nle is L-norleucine;

D-hC is D-homocysteine ​​hC is L-homocysteine; hF is L-homophenylalanine;

hK is L-lysine;

Nal is L-naphthylalanine;

Orn is ornithine;

Aib is a-amino-isobutyric acid;

Dab is (S) -diamino-butyric acid;

Dap is (S) -2,3-diamino-propionic acid;

M (O) is methionine sulfone;

Cha is (S) -B-cyclohexylalanine;

4-amino-lsn is 4-amino-piperidin-4-carboxylic acid;

Isn is isonipecotinoyl; pE is L-pyroglutamic acid;

wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine; and wherein the amino group in the side chain of K, Orn, Dab, Dap, hK or 4-amino-lsn is optionally linked to a lipophilic group by means of an amide bond;

or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto.

In one aspect of mode 1 or 2, the invention pertains to a peptide or polypeptide of the formula I or I ', wherein the amino group in the side chain of K, Orn, Dab, Dap, hK or 4-amino- lsn is optionally linked to a fatty acid via an amide bond.

In a further aspect of this embodiment, the fatty acid is selected from Lauroyl, Miristoyl or Palmitoyl, where Lauroyl is C uH₂₃C (O) -, Miristoílo is Οι₃Η₂₇Ο (Ο) - and Palmitoílo is Ci₅H₃₁CO)-. Alternatively, the CH₃ fatty acid terminal (such as Lauroyl, Miristoyl or Palmitoyl) can be oxidized to its corresponding carboxylic acid.

In one aspect of mode 1 or 2, the invention pertains to a peptide or polypeptide of the formula I or I ', wherein the amino group in the side chain of K, Orn, Dab, Dap, hK or 4-amino- lsn is optionally linked to a lipophilic group via an amide bond, wherein the lipophilic group is selected from a fatty acid as described above, and Lauroyl (O2Oc), Myristoyl (O2Oc), and Palmitoyl (O2Oc) , and where the Lauroyl (O2Oc) is CuH₂₃C (O) NH (CH2) 2-O- (CH₂) 2-O-CH₂CO)-; the Myristoyl (O2Oc) is Ci₃H₂₇C (O) NH (CH₂)₂-OR- (CH₂)₂-O-CH₂CO)-; the Palmitoílo (O2Oc) is Ci₅H₃₁C (O) NH (CH₂)₂-OR- (CH₂)₂-O-CH₂CO)-. Alternatively, the CH fraction₃ fatty acid terminal (such as Lauroyl, Miristoyl or Palmitoyl) can be oxidized to its corresponding carboxylic acid.

In embodiment 3, the invention pertains to a peptide or polypeptide of the formula I or I ':

Χ1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X13 I-1 i where:

X1 is the N-terminus of the polypeptide and is absent or is selected from pE, R, Q, A, K (Palmitoyl), K (Miristoyl), K (Lauroyl), 5-amino-valeric acid and Isn;

X2 is R, A, r, N-Me-R, K, H, hF, hK or Orn;

X3 is P, A, a, p, 4-PhP, pipecolic acid, or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X4 is R, A, r, N-Me-R or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X5 is L, Cha, A, DL, N-Me-L or F;

X6 and X12 are independently a natural or unnatural amino acid selected from C, c, hC, D-hC, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked to each other by means of a covalent bond;

or alternatively, X6 is K, X13 is absent, and X12 is F of, where the C term of X12 forms an amide bond with the amino side chain of X6;

X7 is H, h, A, N-Me-A, a, Aib, K (Lauroyl), K (Palmitoyl), Nal, F, P, Dap, N or cysteine, where the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain in the X3 position or with the cysteine ​​side chain in the position

X4;

X8 is K, k, F, f, A, hF, N-Me-R or 4-amino-lsn;

X9 is G, N-Me-G, A or Aib;

X10 is P, A, p, 4-PhP or pipecolic acid,

Χ11 is M, D-Nle, Nle, N-Me-Nle, M (O), A, F and, L, K (Palmitoyl) or Cha; and

X13 is the term C and is absent or selected from F, f, N-Me-F, Nal, D-Nal, 3-Br-F, (S) -B-3-F, I, A, a, K (Lauroílo), K (Miristoílo), K (Palmitoílo), K (O2Oc-Palmitoílo), Dap (Palmitoílo);

where:

K (Lauroyl) is Ν-ε-lauroyl-L-lysine;

K (Palmitoyl) is Ν-ε-palmitoyl-L-lysine;

K (Miristoyl) is Ν-ε-miristoiI-L-1isin;

K (O2Oc-Palmitoyl) is Ns- (O2Oc-Palmitoyl) -L-lysine; and Palmitoílo (020c) is Ci₅H3iC (O) NH- (CH2) 2-O- (CH2) 2-O-CH₂-CO)-;

Nle is L-norleucine;

D-hC is D-homocysteine ​​hC is L-homocysteine;

hF is L-homophenylalanine;

hK is L-lysine;

Nal is L-naphthylalanine;

Orn is ornithine;

Aib is a-amino-isobutyric acid;

Dab is (S) -diamino-butyric acid;

Dap is (S) -2,3-diamino-propionic acid;

M (O) is methionine sulfone;

Cha is (S) -Bc iclohexylalanine;

4-amino-lsn is 4-amino-piperidin-4-carboxylic acid;

Isn is isonipecotinoyl;

pE is L-pyroglutamic acid;

and wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine;

or an amide, an ester, or a salt of the polypeptide.

In modality 4, the invention pertains to a peptide or a polypeptide according to modality 1, 2 or 3, having the formula II (SEQ ID NO: 1):

X1-RPR-X5-X6-X7-X8-X9-P-X11 -X12-X13 I-1 II

X1 is absent, or is pE, R, Q or Isn;

X5 is L or Cha;

X7 is H, Aib, F, K (Lauroyl) or K (Palmitoyl);

X8 is K, F or 4-amino-lsn;

X9 is G or Aib;

X1 1 is Nle or Cha;

X13 is absent or is F, f, K (Lauroyl), K (Palmitoyl);

X6 and X12 are independently a natural or unnatural amino acid selected from C, c, hC, D-hC, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked to each other by means of a covalent bond; or alternatively, X6 is K,

X13 is absent, and X12 is F of, where the C term of X12 forms an amide bond with the amino side chain of X6; and wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine; or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto.

In modality 5, the invention pertains to a peptide or a polypeptide according to modality 4, wherein:

X1 is absent, or is pE, R, Q or Isn;

X5 is L or Cha;

X7 is H, Aib, F, K (Lauroyl) or K (Palmitoyl);

X8 is K, F or 4-amino-lsn;

X9 is G or Aib;

X1 1 is Nle or Cha;

X13 is absent or is F, f, K (Lauroyl), K (Palmitoyl);

X6 and X12 are independently a natural or unnatural amino acid selected from C, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked together by means of a covalent bond; or alternatively, X6 is K, X13 is absent, and X12 is F of, where the C term of X12 forms an amide bond with the amino side chain of X6; and wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine; or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto.

In modality 6, a further aspect of modality 4, the invention pertains to a peptide or polypeptide of formula II, wherein:

X1 is absent, or is pE, R, Q or Isn;

X5 is L or Cha;

Χ7 is Η, Aib, F, K (Lauroyl) or K (Palmitoyl);

X8 is K, F or 4-amino-lsn;

X9 is G or Aib;

X11 is Nle or Cha;

X13 is absent or is F, f, K (Lauroyl), K (Palmitoyl);

X6 and X12 are independently a natural or unnatural amino acid selected from C, c, hC, D-hC, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked to each other by means of a covalent bond; and wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine; or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto.

In still a further aspect of any of the above modalities, more specifically of any of modalities 1 to 6, the invention pertains to a peptide and polypeptide of the formula I, I 'or II, wherein X6 and X12 are independently an amino acid natural or unnatural selected from C, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked together by means of a covalent bond; or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto.

In modality 7, the invention pertains to a peptide or polypeptide of the formula I, I 'or II, according to any of the above modalities, more specifically of any of modalities 1 to 6, wherein X6 and X12 are selected independently from K, Orn, Dab, E and D, and where the side chains of X6 and X12 form an amide bond with each other; or an amide, an ester or a salt of the peptide or polypeptide. In a further aspect of this embodiment, X6 is K, Orn or Dab, and X12 is E or D, and the side chains of X6 and X12 form an amide bond. In yet another aspect of this mode, X6 is K, and X12 is E or D.

In modality 8, the invention pertains to a peptide or polypeptide of the formula I, I 'or II, according to any of the above modalities, more specifically of any of modalities 1 to 6, wherein X6 and X12 are independently C, c, D-hC or hC, wherein the side chains of X6 and X12 together form a disulfide bond; or an amide, an ester or a salt of the peptide or polypeptide. In a further aspect of this modality, X6 and X1 2 are C.

In modality 8A, the invention pertains to a peptide or polypeptide of the formula I, I 'or II, according to any of the above modalities, more specifically any of modalities 1 to 6, wherein X6 and X12 are independently C, c, D-hC or hC, where the side chains of X6 and X12 together form a monosulfide bond (-S-); or an amide, an ester or a salt of the peptide or polypeptide. In a further aspect of this modality, X6 and X12 are C.

In modality 8B, the invention pertains to a peptide or polypeptide of the formula I, I 'or II, according to any of the above modalities, more specifically any of modalities 1 to 6, wherein X6 and X12 are independently C, c, D-hC or hC, where the side chains of X6 and X12 together form a bond fraction of the formula -S-CH₂-C (= Z) -CH₂-S-; where = Z is = 0, = NO-CH₂CABBAGE¹-R¹ o = N-NH-CH₂CABBAGE¹-R¹, where R¹ is a derivative of fatty acid, and L¹ it is a linker containing one or more amino acids and / or one or more units of -NHalkylene of 2 to 6 carbon atoms-NH-; or an amide, an ester or a salt of the peptide or polypeptide. In a further aspect of this modality, X6 and X12 are C.

L1 is a suitable organic linker that connects a fatty acid derivative to the polypeptide by means of an ester or amide bond. Typically, the linker contains one or more amino acid fractions, such as, for example, a unit (O2Oc) or glutamic acid, or contains -NH-alkylene units of at 6 carbon atoms-NH-diamino, or a combination of the same. R¹ it is a property modifying group, such as a fatty acid derivative, which can be added to increase the half-life of the polypeptide. In one aspect of this embodiment, R1 is a fatty acid of the formula C_thatH_{q + 1}CO)₂H where q is 3 to 20. Optionally the CH unit₃ terminal can be oxidized to its corresponding carboxylic acid.

In a particular mode, -L¹-R¹ It is of the formula:

cooh o

OR

ΌΗ or

q is an integer from 3 to 20. Preferably, q is an integer from 12 to 18.

A peptide or polypeptide of the formula I, I 'or II, wherein X6 and X12 are independently C, c, D-hC or hC, wherein the side chains of X6 and X12 together form a binding fraction of the formula - S-CH₂-C (= Z) -CH₂-S-; where = Z is = O, = NO-CH₂CABBAGE¹-R¹ o = N-NH-CH₂CABBAGE¹-R¹, can be synthesized according to scheme 1:

<img file="CU20140097A7_D0001.tif" />

X13

Retiióiófi

TC £ P

O r Ί

LG 1G

<img file="CU20140097A7_D0002.tif" />

1C (Rt: adí fatty derived derivative) (snlaz.ssto 'Lía}

X1

<img file="CU20140097A7_D0003.tif" />

X '

XI3 or

L ·

Scheme 1

The reduction of the disulfide bond of the polypeptide in (1A) is followed by a nucleophilic reaction of the di-electrophilic compound (1B) forming the polypeptide (1C). The ketone functionality of the polypeptide (1C) was easily condensed with an oxiamine or hydrazine to generate the 1D polypeptide.

In mode 9, certain polypeptides of the invention include peptides or polypeptides according to any of modalities 1 to 6 and 8, which have the formula III (SEQ ID NO: 2):

X1-RPR-X5-C-X7-X8-X9-P-X11-C-X13

I-1 III;

or an amide, an ester, or a salt of the polypeptide. In the 9A mode, the invention pertains to the peptides and polypeptides of the formula III, wherein the 2 cysteines in positions 6 and 12 form a disulfide bond (-SS-), a monosulfide bond (-S-), or a link of the formula -S-CH₂-C (= Z) -CH₂-S-, where Z is as defined above. In a further aspect of mode 9 or 9A, the invention includes the peptides or polypeptides of formula III, wherein the 2 cysteines in positions 6 and 12 form a disulfide bond (-SS-).

In modality 10, certain peptides and polypeptides of the invention include peptides and polypeptides according to any of modalities 1 to 9, having the formula IV (SEQ ID NO: 3):

X1-RPR-X5-X6-X7-KGP-X11 -X12-X13

I-1 IV;

or an amide, an ester, or a salt of the polypeptide.

In modality 11, certain polypeptides of the invention include polypeptides according to any of modalities 1 to 6, and 8 to 10, having the formula V (SEQ ID NO: 4):

X1-RPR-X5-C-X7-KGP-X11-C-X13

I-1 V;

or an amide, an ester, or a salt of the polypeptide. In the 11A mode, the invention pertains to the peptides and polypeptides of the formula V, wherein the 2 cysteines in positions 6 and 12 form a disulfide bond (-SS-), a monosulfide bond (-S-) , or a link of the formula -S-CH₂-C (= Z) -CH₂-S-, where Z is as defined above. In a further aspect of mode 11 or 11 A, the invention includes peptides or polypeptides of formula V, wherein the 2 cysteines at positions 6 and 12 form a disulfide bond (-SS-).

In modality 12, the invention pertains to the bicyclic peptides or polypeptides of formulas I or I '(according to modality 1, 2 or 3), wherein X3 is cysteine, and where the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7. This modality is represented by the peptides and polypeptides of the formula VI (SEQ ID NO: 5):

X1-X2-C-X4-X5-CC-X8-X9-X10-X11-C-X13 I-1 VI or an amide, an ester, or a salt of the polypeptide.

In modality 13, the invention pertains to bicyclic peptides or polypeptides of the formula I or I '(according to modality 1, 2 or 3), wherein X4 is cysteine, and where the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7. This modality is represented by the peptides and polypeptides of the formula Vil (SEQ ID NO: 6):

X1-X2-X3-C-X5-CC-X8-X9-X10-Χ11-C-X13 I-1 Vil or an amide, an ester, or a salt of the polypeptide.

In embodiment 14, the invention pertains to a peptide or polypeptide of any of formulas I to V, according to any of the modalities 1 to 1 1; wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine; or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto. This modality is represented by the peptide or polypeptide having the formula VIII:

X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X 13

-ΓΠVIII where L is (G) r, G is glycine, and r is 1, 2, 3 or 4; or a salt of the polypeptide.

In mode 15, a further aspect of mode 14, the invention pertains to a peptide or polypeptide of formula VIII, wherein X1 is Q, X13 is F, and r is 2 or an ester, an amide or a salt thereof .

In mode 16, the invention pertains to a peptide or polypeptide according to formula I or I ', according to mode 1, 2 or 3, wherein X6 is K, X13 is absent, and X12 is F of, wherein the C term of X12 forms an amide bond with the amino side chain of X6. This modality is represented by a peptide or polypeptide of the formula IX:

Χ1-X2-X3-X4-X5-K-X7-X8-X9-X10-X11 -X12

I-1 IX, or an ester, an amide or a salt of the polypeptide.

Any of the amino acid residues listed above or below of formula I ', or their related formulas, and all the modalities described herein, for example, formulas I, II to IX, may be substituted in a conservative manner, with the understanding that the peptide or polypeptide of the invention still retains the properties of functional and structural activity (for example, the extension of the half-life,

<td>protection of</td><td>Degradation,</td><td>limitation</td><td colspan="2">conformational). The</td>

<td>principles and</td><td>examples of</td><td colspan="3">amino acid substitutions</td>

<td>conservative</td><td>permissible is</td><td>explain</td><td>Additionally</td><td>in the</td>

<td>I presented.</td><td></td><td></td><td></td><td></td>

<td colspan="2">The following modalities</td><td>can be</td><td>use one</td><td>way</td>

<td>Independent,</td><td>collective, or in</td><td>any</td><td>combination</td><td>or sub-</td>

combination:

In modality 17, the invention pertains to peptides and polypeptides according to any of formulas I ', I to Vil and IX, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 13 and 16) or an amide, an ester or a salt thereof, wherein X1 is pE.

In embodiment 18, the invention pertains to peptides and polypeptides according to any of formulas I to Vil and IX, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 13 and 16 ), or to an amide, an ester or a salt thereof, wherein X1 is absent; or to an amide, an ester, or a salt of the polypeptide. In one aspect of this embodiment, the N-terminus of the peptide is an amide. In modality 19, a further aspect of modality 18, the invention pertains to peptides and polypeptides according to any of formulas I to Vil and IX, or to any other classes and subclasses described above, or to an amide, an ester or a salt thereof, where X1 is absent, and the term N is an amide of the formula -NHR, and R is CH₃C (O) -, CH₃- (O-CH₂CH₂)_mC (O) -, Palmitoyl (O2Oc)_p, Myristoyl (O2Oc)_p, Lauroyl (O2Oc)_p or PhCH₂CH₂NHC (O) -, Acetyl, benzoyl, phenacyl, succinyl, octanoyl, 4-phenyl-butanoyl, 4-CI-Ph- (CH₂)₃C (O) -, or Ph-CH₂CH₂NHC (O) -; and where:

p is an integer from 1 to 4;

m is an integer from 1 to 12;

Lauroyl (O2Oc)_p is CnH₂₃C (O) [NH- (CH₂)₂-OR- (CH₂)₂-O-CH₂CO)]_P-;

Myristoyl (O2Oc)_p it's Ci₃H₂₇C (O) [NH- (CH₂)₂-OR- (CH₂)₂-O-CH₂CO)]_P-;

Palmitoyl (O2Oc)_p it's Ci₅H₃₁C (O) [NH- (CH₂)₂-OR- (CH₂)₂-O-CH₂CO)]_P-. In a particular aspect of this embodiment, R is acetyl, benzoyl, phenacyl, succinyl, octanoyl, 4-phenyl-butanoyl, 4-CI-Ph (CH₂)₃C (O) -, or Ph-CH₂CH₂NHC (O) -.

In embodiment 20, the invention pertains to a peptide or polypeptide according to any of formulas I to Vil and IX, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 13 and 16 ), or to an amide, an ester or a salt thereof, wherein the term N is an amide of the formula NHR1, wherein R1 is CH₃C (O) -, CH₃- (OCH₂CH₂)_m-C (O) -, Palmitoyl (O2Oc), Miristoyl (O2Oc), Lauroyl (O2Oc) or Ph-CH₂CH₂NHC (O) -; and where m, Lauroyl (O2Oc), Miristoyl (O2Oc), and Palmitoyl (020c) are defined above.

In embodiment 21, the invention pertains to peptides and polypeptides according to any of formulas I to Vil, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 13), or to a amide, an ester or a salt thereof, wherein X13 is F; or to an amide, an ester, or a salt of the polypeptide.

In embodiment 22, the invention pertains to peptides and polypeptides according to any of formulas I to Vil, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 13), or to a amide, an ester or a salt thereof, where X13 is absent; or to an amide, an ester, or a salt of the polypeptide. In modality 23, an aspect of modality 22, The term C is an amide. In mode 24, a further aspect of mode 23, the invention pertains to peptides and polypeptides according to any of formulas I to Vil, or to any other classes and subclasses described above, or to an amide, an ester or a salt thereof, wherein the term C is an amide of the formula -C (O) R2, and R2 is -NH₂, -NH-Me, -NH-NHBn, or -NH- (CH₂)₂-Ph. In a preferred aspect of embodiment 23, the invention pertains to peptides and polypeptides according to any of formulas I to Vil, or to any other classes and subclasses described above, or to an amide, an ester or a salt thereof, wherein the term C is an amide of the formula -C (O) R2, and R2 is -NH- (CH₂)₂-Ph.

In embodiment 25, the invention pertains to peptides and polypeptides according to any of formulas I to IX, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 25), or to a amide, an ester or a salt thereof, wherein X5 is L.

In modality 26, the invention pertains to peptides and polypeptides according to any of formulas I to V, VIII and IX, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 11 and 1425), or to an amide, an ester or a salt thereof, wherein X7 is H.

In modality 27, the invention pertains to peptides and polypeptides according to any of formulas I to III and VI to IX, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 9 and 12 to

26), or to an amide, an ester or a salt thereof, wherein X8 is K or F. In a further aspect of this embodiment, X8 is K.

In embodiment 28, the invention pertains to peptides and polypeptides according to any of formulas I to III and VI to IX, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 9 and 12 to

27), or to an amide, an ester or a salt thereof, wherein X9 is G.

In embodiment 29, the invention pertains to peptides and polypeptides according to any of formulas I to IX, or to any other classes and subclasses described above (ie, according to any of modalities 1 to 28), or to a amide, an ester or a salt thereof, wherein X11 is Nle.

In embodiment 30, the invention, therefore, provides a peptide or a polypeptide of the formula (I '):

Χ1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X13 I-1 r where:

X1 is the N-terminus of the polypeptide and is absent or is selected from pE, R, Isn and Q;

X2 is R, r, F or E;

X3 is P, p, K, D or 4-PhF; or cysteine, wherein the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

Χ4 is R, F, Ε or cysteine, where the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain at position X7;

X5 is L, K, D or 4-PhF;

X6 and X12 are independently a natural or unnatural amino acid selected from C, c, hC, D-hC, K, D, Orn, Dab or E, where the side chains of X6 and X12 are linked to each other by means of a covalent bond, forming either a disulfide or amide bond;

or alternatively, X6 is K, X13 is absent, and X12 is F of, where the C term of X12 forms an amide bond with the amino side chain of X6;

X7 is H, Aib, K, E, F or cysteine, where the cysteine ​​side chain forms a disulfide bond with the cysteine ​​side chain in the X3 position or with the cysteine ​​side chain in the X4 position ;

X8 is K, E, F or 4-amino-lsn;

X9 is G, D, L, R or Aib;

X10 is P or pipecolic acid,

X1 1 is Nle or 3-PyA; and

X13 is the term C and is absent or selected from F, f, K, H and E;

wherein the term N and the term C optionally form a ring together with 1, 2, 3 or 4 amino acids of glycine; and where the amino group in the side chain of K, Orn, Dab,

Dap, hK or 4-amino-lsn is optionally linked to a lipophilic group via an amide bond;

or an amide, an ester, or a salt of the polypeptide; or a polypeptide substantially equivalent thereto.

In modality 31, the invention pertains to a peptide or polypeptide of modality 1, 2 or 3, wherein three of amino acids X1 to X13 are different from the corresponding amino acids present in Pyr-1-Apeline-1 3. In the modality 32, the invention pertains to a peptide or polypeptide of mode 1, or 3, wherein four of amino acids X1 to X13 are different from the corresponding amino acids present in Pyr-1-apeline-1 3.

In another embodiment, amino acids X1, X2, X3, X4, X5, X6, X7,

X8 X9, X10, X11, X12 and X13 are those defined by amino acids X1, X2, X3, X4, X5, X6, X7, X8. X9, X10, X1 1, X12 and

X13 in the Examples section below.

In another embodiment, the individual polypeptides according to the invention are those listed in the Examples section below, or a pharmaceutically acceptable salt thereof.

Unless otherwise specified, the term "polypeptide of the present invention" refers to a polypeptide of the formula (I '), and of the sub-formulas thereof (formulas I, II to IX);

or to an amide, an ester or a salt thereof.

Unless otherwise specified, the terms "polypeptides of the present invention," peptides of the present invention, "apeline peptide agonists, and the like, refer to the peptides and polypeptides of the formula I 'and the sub-formulas thereof (formulas I, II, III, IV, V, VI, Vil, VIII or IX); or to an amide, an ester or a salt thereof. The peptides and polypeptides of the invention demonstrate a substantially equivalent or enhanced plasma activity and / or stability on the known apeline peptides and polypeptides described herein, including, but not limited to, wild-type apeline, apeline1, and pyr -1 -Apelina-1 3.

The peptides and polypeptides of the invention also encompass peptides and polypeptides that are at least about 95 percent identical to peptides and polypeptides according to any of formulas I ', I to IX, or to an amide, an ester or a salt thereof, as well as any peptides or polypeptides specifically listed herein, including, but not limited to, experimental examples.

As used herein, the phrase "homologous amino acid sequence, or variations thereof, refers to the sequences characterized by a homology, at the amino acid level, of at least a specified percentage, and is used in a interchangeable way with "sequence identity".

Homologous amino acid sequences include amino acid sequences that contain conservative amino acid substitutions and whose polypeptides have the same linkage and / or the same activity. In some embodiments, an amino acid sequence is homologous if it has at least 60 percent or more, and up to 99 percent identity with a comparison sequence. In some embodiments, an amino acid sequence is homologous if it shares one or more, and up to 60 amino acid substitutions, additions, or deletions with a comparison sequence. In some embodiments, homologous amino acid sequences have no more than 5 or no more than 3 conservative amino acid substitutions.

Homology can also be at the level of the polypeptide. The degree or percentage of identity of the peptides or polypeptides of the invention, or portions thereof, and different amino acid sequences, is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "sequence of the invention" or the "strange sequence", whichever is the shortest. The result is expressed as the percentage of identity.

A polypeptide comprising an amino acid sequence that has a homology of about 80 to 99.9 percent, preferably 90 to 99.9 percent with the amino acid sequence described in the specific examples, and that has a plasma stability greater than the Apelina-13 or pyr-1-Apelina13 falls under the category of the polypeptide of the invention. In one embodiment, the improvement in plasma stability is at least 2 times. In one embodiment, the polypeptide of the invention has a plasma stability of at least 30 minutes. In another embodiment, the polypeptide of the invention has a plasma stability of at least 60 minutes, preferably at least 100 minutes, and most preferably at least 1 50 minutes.

The term substantially equivalent means that the nature of the receptor binding activity, the signal transduction activity, and the like, is equivalent. Therefore, it is permissible for even differences between degrees, such as the strength of the receptor binding activity and the molecular weight of the polypeptide to be present.

A polypeptide as described herein, or a substantial equivalent thereof, by substitution, deletion, addition or insertion of one or more amino acids, can be mentioned as polypeptides containing a substantial equivalent of the amino acid sequence in the sense. previous. A polypeptide as described herein, or a substantial equivalent thereof, by substituting 1 to 5, preferably 1 to 3, and most preferably 1 or 2 amino acids, with natural or unnatural amino acids, It can be mentioned as polypeptides containing a substantial equivalent of the amino acid sequence in the above sense. Other modifications and alterations may include the replacement of an L-amino acid with a D-amino acid, or other variation, including, but not limited to, phosphorylation, carboxylation, alkylation, and the like, as long as the peptide's agonist agonist activity is maintained. or polypeptide of formulas I, II, III, IV, V, VI, Vil, VIII or IX, and that plasma stability on the pyroglutamate form of apeline-13 is improved. For example, D-amino acids are well tolerated with respect to the activity and stability of the polypeptide at position 2 (2 times), at position 3 (3 times), at positions 5, 6, 7 and 8 (X5, X6, X7 and X8), at position 10 (X10), and at position 13 (X1 3) of the cyclic peptides and polypeptides of formulas I, II, III, IV, V,

VI, Vil, VIII or IX.

As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the polypeptides of this invention, and which are typically not biologically or otherwise undesirable. In many cases, the polypeptides of the present invention are capable of forming acid and / or base salts by virtue of the presence of the amino and / or carboxyl groups, or of groups similar thereto.

Pharmaceutically acceptable acid addition salts may be formed with inorganic acids and with organic acids, for example, acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphor sulphonate, chloride salts. / hydrochloride, chlorteophyllonate, citrate, etandisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, iodide / iodide, isethionate, lactate, lactobionate, I au ri I - su I f ato, malate, maleate, malonate, Mandelate, mesylate, methyl sulfate, naphthoate, napsilate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / phosphate acid / diacid phosphate, polygalacturonate, propionate, stearate, succinate, sulphosalicylate, tartrate, tosrtluorolate and tartrate acetate.

Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid , mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the Periodic Table. In certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; Particularly suitable salts include the ammonium, potassium, sodium, calcium and magnesium salts.

The organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. . Certain organic amines include isopropyl amine, benzathine, colinate, diethanolamine, diethyl amine, lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound, a basic or acidic fraction, by conventional chemical methods. Generally speaking, these salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as hydroxide, carbonate, or bicarbonate of Na, Ca, Mg, or K, or the like. ), or by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid. These reactions are typically carried out in water or in an organic solvent, or in a mixture of both. In general terms, it is advisable to use non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, when practicable. Lists of additional suitable salts can be found, for example, in "Remington's Pharmaceutical Sciences," 20^to Edition, Mack Publishing Company, Easton, Pa., (1985); and in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial agents, anti-fungal agents), isotonic agents, absorption retarding agents , salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrating agents, lubricants, sweetening agents, flavoring agents, dyes, and the like, and combinations thereof, as would be known to those skilled in this field (see, for example, Remington's Pharmaceutical Sciences, 18^to Edition, Mack Printing Company, 1990, pages 1289-1329). Except as far as any conventional vehicle is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

The term a therapeutically effective amount of a polypeptide of the present invention refers to an amount of the polypeptide of the present invention that will cause the biological or medical response of a subject, for example, the mitigation of a symptom, the relief of a condition, slow down or slow the progress of the disease, or the prevention of a disease, etc.

In a non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the polypeptide of the present invention that, when administered to a subject, is effective for: (1) at least partially relieve, inhibit, prevent and / or mitigate a condition, disorder or disease or symptom thereof (i) that is mitigated by activation of the APJ receptor, or (ii) associated with APJ receptor activity, or (iii) characterized by an abnormal APJ receptor activity; or (2) activate the APJ receiver.

In another non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the polypeptide of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective for at least partially activating the APJ receiver. As will be appreciated by those of ordinary experience in this field, the absolute amount of a particular agent that is effective may vary depending on factors such as the desired biological endpoint, the agent to be supplied, the target tissue, etc. Those of ordinary experience in this field understand that "a therapeutically effective amount" can be administered in a single dose or can be achieved by administering multiple doses. For example, in the case of an agent for treating heart failure, an effective amount may be an amount sufficient to result in the patient's clinical improvement, for example, a greater tolerance / ability to exercise, an increase in blood pressure, a decrease in fluid retention, and / or better results in a quantitative test of cardiac functioning, for example, the ejection fraction, exercise capacity (time to exhaustion), etc.

As used herein, the term "subject" refers to an animal. Typically, the animal is a mammal. A subject also refers, for example, to primates (for example, to humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain modalities, the subject is a primate. In still other modalities, the subject is a human being.

As used herein, the term "inhibit", inhibition or inhibition "refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

As used herein, the term "treat", "treating or treating any disease or disorder, refers, in one embodiment, to mitigate the disease or disorder (ie, slow down or stop or reduce development of the disease or at least one of its clinical symptoms). In another embodiment, "treating", treating or treating refers to alleviating or mitigating at least one physical parameter, including those that cannot be discernible by the patient. In yet another embodiment, "treating", treating or treating refers to modulating the disease or disorder, whether physically (for example, the stabilization of a discernible symptom), physiologically (for example, the stabilization of a physical parameter), or both. In yet another embodiment, "treating", treating or treating refers to preventing or delaying the establishment or development or progress of the disease or disorder.

As used herein, the terms prevention, prevention and prevention refer to the prevention of recurrence, establishment, or development of one or more symptoms of a disorder in a subject, resulting from the administration of a therapy (by for example, of a therapeutic agent), or of the administration of a combination of therapies (for example, of a combination of therapeutic agents).

As used herein, a subject is "in need of" a treatment if this subject would benefit biologically, medically, or in their quality of life from such treatment.

As used herein, the term "one, one, the" and similar terms used in the context of the present invention (especially in the context of the claims), should be construed to cover both the singular and the plural, to unless otherwise indicated herein or clearly contradicted by the context.

All methods described herein can be carried out in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or of the example language (for example, such as ") provided herein, is intended merely to better illuminate the invention and does not present a limitation on the scope of the invention claimed otherwise.

The peptides and polypeptides of the present invention can be produced by the methods known per se for peptide synthesis. The methods for peptide synthesis can be any of a solid phase synthesis and a liquid phase synthesis. Accordingly, the peptide and polypeptide of interest can be produced by condensation of a partial peptide or amino acid capable of constituting the protein with the residual part thereof and, when the product has a protective group, this protective group is released, upon which, the desired peptide can be made. Known methods for condensation and deprotection include the procedures described in the following literature (1) to (5):

(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York, 1966, (2) Schroeder and Luebke, The Peptide, Academic Press,

New York, 1 965, (3) Nobuo Izumiya et al., Fundamentalis and Experiments in Peptide Synthesis, Maruzen, 1975, (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment Series 1, Protein Chemistry IV, 205, 1977, and (5 ) Haruaki Yajima (Editor), Development of DrugsContinued, 14, Peptide Synthesis, Hirokawa Shoten.

After the reaction, the peptide can be purified and isolated by a combination of conventional purification techniques, such as solvent extraction, column chromatography, liquid chromatography, and recrystallization. When the peptide isolated as above is a free compound, it can be converted to a suitable salt by the known method.

Conversely, when the isolated product is a salt, it can be converted to the free peptide by the known method.

The polypeptide amide can be obtained by using a resin for peptide synthesis, which is suitable for amidation. The resin includes chloro-methyl resin, hydroxy-methyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxy-benzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4- resin hydroxy-methyl-methylphenyl-acetamido-methyl, polyacrylamide resin, 4- (2 ', 4'-dimethoxy-phenyl-hydroxy-methyl) -phenoxy resin, 4- (2', 4'-dimethoxy-phenylFmoc- resin amino-methyl) -phenoxy, 2-chloro-trityl chloride resin, and so on. Using this resin, the amino acids whose amino groups and side chain functional groups have been adequately protected and condensed on the resin according to the sequence of the target peptide by various condensation techniques that are known per se. At the end of the series of reactions, the protected peptide or peptide is removed from the resin and the protecting groups are removed and, if necessary, disulfide bonds are formed to obtain the target polypeptide.

For the condensation of the protected amino acids mentioned above, a variety of activation reagents can be used for the synthesis of peptides, such as HATU, HCTU, or, for example, a carbodiimide. Carbodi-imide includes DCC, N, N'-di-isopropyl-carbodi-imide, and N-eti I-N '- (3-di methy I amino-propyl) -carbodi-imide. For activation with said reagent, a racemization inhibitor additive can be used, for example, HOBt or Pure Oxima. The protected amino acid can be added directly to the resin together with the activation reagents and the racemization inhibitor, or it can be previously activated as a symmetric acid anhydride, HOBt ester, or HOOBt ester, and then added to the resin . The solvent for the activation of protected amino acids or for condensation with the resin can be appropriately selected from among the solvents that are known to be useful for peptide condensation reactions. For example, N, N-dimethylformamide, N-methyl pyrrolidone, chloroform, trifluoroethanol, dimethyl sulfoxide (DMSO), Ν, Ν-dimethyl formamide (DMF), pyridine, dioxane, chloride methylene, tetrahydrofuran (THF), acetonitrile, ethyl acetate, or suitable mixtures thereof.

The reaction temperature can be selected from the range known so far as useful for the formation of the peptide bond, and is usually selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is generally used in a proportion of an excess of 1.5 to 4 times. If the condensation is found to be insufficient according to a test using the ninhydrin reaction, the condensation reaction can be repeated to achieve sufficient condensation without removing the protective group. If repeated condensation still fails to provide a sufficient degree of condensation, the unreacted group can be acetylated with acetic anhydride or with acetyl-imidazole.

The amino group protecting group for amino acid of the starting material includes Z, Boc, teramyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, 2-nit ro-f eni I - its I feni I o, diphenyl phosphino-thioyl, or Fmoc. The carboxyl protecting group that can be used includes, but is not limited to, alkyl groups of 1 to 6 carbon atoms, cycloalkyl of 3 to 8 carbon atoms and aryl of 6 to 10 carbon atoms-alkyl of 1 to 2 carbon atoms mentioned above, as well as 2-adamantyl, 4-nitro-benzyl,

4-methoxy-benzyl, 4-chloro-benzyl, phenacyl, benzyloxycarbonylhydrazide, terbutoxycarbonyl hydrazide, and trityl hydrazide.

The hydroxyl group of serine and threonine can be protected by esterification or etherification. The suitable group for said esterification includes carbon derived groups, such as lower alkanoyl groups, for example, acetyl etc., aroyl groups, for example, benzoyl etc., benzyloxycarbonyl, and ethoxycarbonyl. The group suitable for said etherification includes benzyl, tetrahydro-pyranyl, and terbutyl. The protecting group for the tyrosine phenolic hydroxyl group includes Bzl, Cl₂-Bzl, 2-nitro-benzyl, BrZ, and terbutyl.

The imidazole protecting group for histidine includes Tos, 4-methoxy-2,3,6-triethyl-benzenesulfonyl, DNP, benzyloxymethyl, Bum,

Boc, Trt, and Fmoc.

The activated carboxyl group of the starting amino acid includes the corresponding acid anhydride, azide and active esters, for example, esters with alcohols, such as pentachlorophenol, 2,4,5-trichloro-phenol, 2,4-dinitro -phenol, cyano-methyl alcohol, p-nitrophenol, HONB, N-hydroxy-succinimide, N-hydroxyphthalimide, HOBt, etc. The activated amino group of the starting amino acid includes the corresponding phosphoramide.

The method for removing protective groups includes catalytic reduction using hydrogen gas in the presence of a catalyst, such as palladium black or palladium on carbon, acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro-methane acid -sulfonic acid, trifluoroacetic acid, or a mixture of these acids, the basic treatment with di-isopropylethyl amine, triethyl amine, piperidine, piperazine, the reduction with sodium metal in liquid ammonia. The elimination reaction by the aforementioned acid treatment is generally carried out at a temperature of -20 ° C to 40 ° C, and can be conveniently conducted with the addition of a cation acceptor, such as anisole, phenol, thioanisole , m-cresol, p-cresol, dimethyl sulfide, 1,4-butandithiol, 1,2-ethanedithiol. The 2,4-dinitro-phenyl group used to protect the imidazole group from histidine can be removed by treatment with thiophenol, while the formyl group used to protect the indole group from tryptophan can be removed by alkaline treatment with a solution. diluted sodium hydroxide or with dilute aqueous ammonia, as well as by the aforementioned acid treatment, in the presence of 1,2-ethanedithiol or 1,4-butandithiol.

The method to protect the functional groups that should not take part in the reaction of the starting material, the protective groups that can be used, the method to remove the protective groups, and the method to activate the functional groups that will take part in the reaction, all can be selected with a good judgment from the known groups and methods.

Another method of obtaining the amide form of the polypeptide comprises first amidating the C-terminal amino acid-carboxy group, then extending the peptide chain to the N side to the desired chain length, and then selectively deprotecting the α-amino group. C-terminal peptide group and the α-carboxyl group of the amino acid or peptide that will form the rest of the target polypeptide, and condensing the two fragments whose α-amino group and side chain functional groups have been protected with the appropriate protecting groups mentioned above in a mixed solvent, such as that mentioned above herein. The parameters of this condensation reaction may be the same as described above herein. From the protected peptide obtained by condensation, all protecting groups are removed by the method described above, to thereby provide the desired crude peptide. This crude peptide can be purified by known purification procedures, and the main fraction can be lyophilized to provide the target amidated polypeptide. In order to obtain an ester of the polypeptide, the α-carboxyl group of the C-terminal amino acid is condensed with a desired alcohol to give an amino acid ester and then, the procedure described above for the production of the amide is followed.

The polypeptides of the present invention, or an amide, an ester or a salt thereof, can be administered in any of a variety of ways, including subcutaneously, intramuscularly, intravenously, intraperitoneally, by inhalation, etc. In particularly preferred embodiments of the invention, continuous intravenous administration of the polypeptides of the present invention, or of an amide, an ester, or a salt thereof, is employed. The polypeptides of the present invention can be administered as a bolus or as a continuous infusion for a period of time. An implantable pump can be used. In certain embodiments of the invention, intermittent or continuous administration of the polypeptides is continued for one to several days (for example, 2 to 3 or more days), or for longer periods of time, for example, weeks, months, or years. In some embodiments, intermittent or continuous administration of the polypeptide is provided for at least about 3 days. In other embodiments, intermittent or continuous administration of the polypeptides is provided for at least about a week.

In other embodiments, intermittent or continuous administration of the polypeptides is provided for at least about two weeks. It may be advisable to maintain an average plasma polypeptide concentration above a particular threshold value, either during administration or between administration of multiple doses. A recommended concentration can be determined, for example, based on the physiological condition of the subject, the severity of the disease, etc. These recommended values ​​can be identified by conducting conventional clinical studies.

In another aspect, the present invention provides a pharmaceutical composition, which comprises a polypeptide of the present invention or an amide, an ester or a salt thereof, and one or more pharmaceutically acceptable carriers. The pharmaceutical composition may be formulated for particular routes of administration, such as oral administration, parenteral administration, and rectal administration, etc. In addition, the pharmaceutical compositions of the present invention can be configured in a solid form (including, without limitation, capsules, tablets, pills, granules, lyophilisates, powders or suppositories), or in a liquid form (including, without limitation, solutions , suspensions or emulsions). The pharmaceutical compositions may be subjected to conventional pharmaceutical operations, such as aseptic processing, sterilization, and / or may contain conventional inert diluents, cake-forming agents, tonicity agents, lubricating agents, or regulating agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and pH regulators, etc.

Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (which are water soluble) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

For intravenous administration, suitable vehicles include physiological serum, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate regulated serum (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it can be easily passed through a syringe. Preferred pharmaceutical formulations are stable under processing and storage conditions, and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, the relevant vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propiIengIicoI, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, amino acids, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be caused by including in the composition an agent that retards absorption, for example, aluminum monostearate and gelatin.

Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are conveniently prepared from fatty emulsions or suspensions. These compositions may be sterilized and / or may contain adjuvants, such as preservatives, stabilizers, humectants or emulsifiers, solution promoters, salts for regulating osmotic pressure and / or pH regulators. In addition, they may also contain other therapeutically valuable substances. These compositions are prepared according to conventional mixing, granulation or coating methods, respectively, and contain from about 0.1 to 75 percent, or contain from about 1 to 50 percent, of the active ingredient.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with an ingredient or with a combination of ingredients listed above, as required, followed by sterilization by filtration. In general terms, the dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying, which provides a powder of the active ingredient plus any additional desired ingredient from a solution of the same previously filtered to sterilize.

Oral compositions in general terms include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients, and may be used in the form of tablets, troches, or capsules, for example, gelatin capsules. Oral compositions can also be prepared using a fluid vehicle to be used as a mouthwash. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient, such as starch or lactose, a disintegrating agent, such as alginic acid, Primogel, or corn starch; a lubricant, such as magnesium stearate or Sterotes; a skidder, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring. Formulations for oral delivery may conveniently incorporate agents to improve stability within the gastrointestinal tract and / or to improve absorption.

For administration by inhalation, the therapeutic agents of the invention are preferably supplied in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas, such as carbon dioxide, or a nebulizer. It is observed that the lungs provide a large surface area for the systemic supply of therapeutic agents.

The agents can be encapsulated, for example, in polymeric microparticles, such as those described in the

United States Patent Publication

Number 20040096403, or in association with any of a wide variety of other drug delivery vehicles that are known in the art. In other embodiments of the invention, the agents are supplied in association with a charged lipid as described, for example, in United States Patent Publication Number 20040062718. It is noted that the latter system has been used for the administration of a therapeutic polypeptide, insulin, demonstrating the usefulness of this system for the administration of peptide agents.

Systemic administration can also be by transmucosal or transdermal means.

Compositions suitable for transdermal application include an effective amount of a polypeptide of the invention with a suitable vehicle. Suitable vehicles for transdermal delivery include absorbable pharmacologically acceptable solvents to aid passage through the skin of the host. For example, the transdermal devices are in the form of a patch comprising a backing member, a reservoir containing the compound optionally with vehicles, optionally a speed control barrier to deliver the host skin compound at a controlled rate. and previously determined for a prolonged period of time, and elements to secure the device to the skin.

Compositions suitable for topical application, for example, to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, for example, for delivery by aerosol or the like. These topical delivery systems will be particularly suitable for dermal application. Accordingly, they are particularly suitable for use in topical formulations, including cosmetics, well known in this field. These may contain solubilizers, stabilizers, tonicity enhancing agents, regulators, and preservatives.

As used herein, a topical application may also belong to an inhalation or an intranasal application. They can be conveniently supplied in the form of a dry powder (either alone, as a mixture, for example, a dry mixture with lactose, or as a component particle mixed, for example, with phospholipids) from a Dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, sprinkler, atomizer or nebulizer, with or without the use of a suitable propellant.

The invention further provides pharmaceutical compositions and dosage forms comprising one or more agents that reduce the rate at which the compound of the present invention will decompose, as an active ingredient. These agents, which are referred to herein as stabilizers, include, but are not limited to, antioxidants, such as ascorbic acid, pH regulators, or salt regulators, etc.

Invention Method:

The apeline peptide family is the only family of natural ligands known for the G-protein coupled APJ receptor. The apeline gene encodes a 77 amino acid polypeptide, which becomes processed to biologically active forms of apeline peptides, such as apelina-36, apelina-17, apelina-16, apelina-13, apelina-12, and the form Apeline-13 modified by pyroglutamate (Pyr¹-Apelina-1 3). Any of these apeline peptides, after binding to the APJ receptor, transduces the signal by means of the Gi and Gq proteins. In cardiomyocytes, the coupling of Gi or Gq leads to changes in intracellular pH, in the activation of PLC, and in the production of IP3 that improves the calcium sensitivity of myofilaments, and finally results in an increase in the cardiac contractility Gi coupling inhibits activated Gs, adenylyl cyclase, and cAMP production, and increases pAkt levels, which leads to cardioprotection. In vascular endothelial cells, with the activation of APJ by means of Gi, pAKT leads to an increase in the production of nitric oxide (NO), which increases smooth muscle relaxation, which results in a global vasodilation.

Patients with chronic stable heart failure have occasional acute episodes of decompensation, where cardiac contractility declines further and symptoms worsen. These exacerbations are referred to as acute decompensated heart failure (ADHF). Current therapies for acute decompensated heart failure (ADHF) include diuretics, vasodilators, and inotropes, which directly increase cardiac contractility. Current intravenous inotropes (dobutamine, dopamine, milrinone, levosimendan) are well known for their adverse events, such as arrhythmia and an increase in long-term mortality. The synthetic apeline polypeptide analogs of the present invention provide a therapy for acute decompensated heart failure (ADHF) that increases cardiac contractility without arrhythmogenic or mortality problems, and that solves the enormous unmet medical need in chronic heart failure.

In fact, acute treatment with apeline (5 minutes) in humans results in coronary vasodilation and better cardiac output. However, native apelines exhibit a t¹/ 2 very short (seconds), and a very short duration of action (a few minutes) in vivo. The potent synthetic apeline peptide agonists of the present invention have longer half-lives compared to native apeline.

Activation of the APJ receptor in cardiomyocytes a) improves cardiac contractility through Gi / Gq, PLC and Ca2 +, and b) provides cardioprotection through Gi, activation of pAkt, but without increasing cAMP (as seen with other inotropes). In addition, APJ agonism in endothelial cells leads to arterial vasodilation, which further benefits heart failure by discharging labor from the left ventricle. Taken together, analogues of synthetic apeline polypeptides can improve overall cardiac function, reduce arrhythmogenesis, and provide the benefit of survival.

More recently, there have been a number of pre-clinical research publications that focus on the potential involvement of apeline in diabetes and insulin resistance. Apeline has been shown to: 1) lower blood glucose levels by improving glucose uptake in muscle, in adipose tissue, and in the heart, 2) protects pancreatic beta cells from ER stress and subsequent apoptosis,

3) decreases insulin secretion in beta cells, and

4) regulates catecholamine-induced lipolysis in adipose tissue. The activation of the pAKT path has been implicated in these processes.

Polypeptides according to any of formulas I to IX, or a pharmaceutically acceptable salt thereof, in free form or in a pharmaceutically acceptable salt form, exhibit valuable pharmacological properties, for example, agonism properties of the APJ receptor, for example, as indicated in the in vitro and in vivo tests provided in the following sections and, therefore, are indicated for therapy.

The polypeptides of the invention, or a pharmaceutically acceptable salt thereof, may be useful in the treatment of an indication selected from acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, cardiovascular ischemic diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral artery disease, strokes, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn), and preeclampsia.

Accordingly, as a further embodiment, the present invention provides the use of a polypeptide of any of formulas I to IX, or of an amide, an ester or a salt thereof, for the treatment of a disease that is associated with the APJ receptor activity. In an additional embodiment, the therapy is selected from a disease that responds to the agonism of the APJ receptor. In another embodiment, the disease is selected from the aforementioned list, in an appropriate manner acute decompensated heart failure. In yet another subset of this embodiment, the present invention provides the use of a polypeptide of any of formulas I to IX, or of an amide, ester or salt thereof, in the preparation of a medicament for the treatment of a disease. that is associated with the activity of the APJ receptor.

Accordingly, as a further embodiment, the present invention provides the use of a polypeptide of any of formulas I to IX, or of an amide, an ester or a salt thereof, in therapy. In a further embodiment, the therapy is selected from a disease that can be treated by activation (agonism) of the APJ receptor.

In another embodiment, the invention provides a method for the treatment of a disease that responds to APJ receptor agonism, which comprises the administration of a therapeutically acceptable amount of a polypeptide of any of formulas I to IX, or of an amide , an ester or a salt thereof. In a further embodiment, the disease is selected from the aforementioned list, in an appropriate manner acute decompensated heart failure.

In yet another subset of this embodiment, the invention provides a method for the treatment of a disease that is associated with the activity of the APJ receptor, which comprises the administration of a therapeutically acceptable amount of a polypeptide of any of formulas I to IX, or of an amide, an ester or a salt thereof.

The effective amount of a pharmaceutical composition or combination of the invention to be used therapeutically will depend, for example, on the therapeutic context and the objectives. One skilled in the art will appreciate that the dosage levels appropriate for the treatment will therefore vary depending, in part, on the molecule supplied, on the indication for which the fusion protein variant is being used, on the route of administration, and the size (body weight, body surface, or organ size) and condition (age and general health) of the patient. In accordance with the foregoing, the clinician can titrate and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage may be in the range of about 0.1 microgram / kilogram to about 100 milligrams / kilogram or more, depending on the factors mentioned above. In other embodiments, the dosage may be in the range of 0.1 microgram / kilogram to about 100 milligrams / kilogram; or from 1 microgram / kilogram to about 100 milligrams / kilogram.

The dosage frequency will depend on the pharmacokinetic parameters of the dual function protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition, therefore, can be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion by means of a device implantation or catheter. Further refinement of the appropriate dosage is routinely done by those of ordinary experience in this field, and is within the scope of the tasks that are routinely performed by them. Appropriate dosages can be asserted through the use of appropriate dose response data.

The activity of a polypeptide according to the present invention can be evaluated following the in vitro methods described below.

HAPJ calcium flow test:

The stable Chem-5 APJ cells (Millipore # HTS068C) were placed in a 384 well format with 10,000 cells / well in growth medium microliters, then grown for 24 hours in a tissue culture incubator at 37 ° C . One hour before the test, 25 microliters / Calcium 4 FLIPR dye well (Molecular Devices R8142) with 2.5 mM probenecid was added, and the cells were incubated for one hour in a 37 ° C tissue culture incubator. The peptides were solubilized in HBSS, HEPES, and 0.1% bovine serum albumin (BSA) regulators, and serially diluted 10 times, from 50 μΜ to 5 pM, in triplicate. FLIPR Tetra was used to add the peptide to the cells with the dye (1: 5, for concentrations of the final peptide in the range of 10 μΜ to 1 pM). The FLIPR dye inside the cells emitted fluorescence after calcium bonding, while fluorescence from the outer cells was masked. Fluorescence was measured using excitation wavelengths of 470 to 495 and emission of 515 to 575 in the Tetra FLIPR. The readings were made for 3 minutes in total, starting 10 seconds before the peptide was added. Maximum minimum values ​​were calculated, and plotted for each peptide concentration, and GraphPad Prism software was used to calculate EC values₅or at the inflection points of the curve, to determine the stimulation of calcium flow by the peptides.

Plasma stability test:

Materials:

Processing solution: 1 milligram / milliliter of the test article in Milli-Q water is prepared.

Extraction solution: Methanol: Acetonitrile: Water (1: 1: 1) with 0.1 percent formic acid and 400 nanograms / milliliter of

Glyburide

Plasma: Male Sprague-Dawley rat plasma (with sodium heparin), purchased from Bioreclamation LLC (Liverpool, NY).

Whole Blood: Male Sprague Dawley rat whole blood (with sodium heparin), purchased from Bioreclamation LLC (Liverpool, NY).

Lung Homogenate: Male Sprague Dawley rat lung was purchased from Bioreclamation LLC (Liverpool, NY). The lung was homogenized using a Polytron homogenizer after the addition of 5 times the volume of 1X phosphate regulated serum (PBS). The homogenate was centrifuged at 9,000 revolutions per minute (rpm) for 10 minutes at 4 ° C. The supernatant was centrifuged again at 3,000 revolutions per minute (rpm) for 30 minutes to make a transparent supernatant. Protein concentration was determined using a commercial kit (Pierce, Thermo Scientific).

Sample preparation procedure:

The test article was prepared in one of the following biological matrices: heparinized rat plasma, heparinized rat whole blood, or lung homogenate. The plasma and whole blood sample was prepared at 5,000 nanograms / milliliter by adding 5 microliters of a 1 milligram / milliliter processing solution to 995 microliters of rat or whole blood plasma. Lung homogenate samples were prepared by diluting the lung homogenate in a concentration of 1 milligram / milliliter of protein with phosphate-regulated serum (PBS), followed by the addition of 5 microliters of the processing solution to 995 microliters of diluted lung homogenate. Samples were incubated at 37 ° C with gentle agitation (approximately 65 to 75 revolutions per minute (rpm)) in an incubator with a water bath. In the times of 0 minutes, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 120 and 240 minutes, aliquots of 25 microliters of incubation samples were transferred to the 96-well plate, and the protein was immediately precipitated using 150 microliters of the extraction solution. After the incubation experiment was completed, the sample plate was centrifuged at 4,000 revolutions per minute (rpm) at 4 ° C for 10 minutes. Then, a pipetting device (Tecan Temo) was used to transfer the supernatants to another plate and add 50 microliters of water to all samples. The plaque was placed in vertex before the LC-MS analysis.

LC-MS Sample stability analysis.

HPLC: Agilent 1290 HPLC with autosampler.

Column: MAC-MOD ACE C18, 3 microns, 30 mm x 2.1 mm internal diameter.

Mobile phase A: 0.1 percent formic acid in acetonitrile. Mobile phase B: 0.1 percent formic acid in water.

Gradient Program:

Time (min) Flow (mL) Mobile phase A (%) Mobile phase B (%)

0.4 95 5

0.5 0.4 95 5

1.5

0.4

4.1

0.4

4.2

0.4

0.4

Mass spectrometer: Agilent Q-TOF 6530.

Data acquisition mode: Full scan with mass range of 100 to 1,000 m / z.

Data acquisition and analysis software: MassHunter.

Analysis of data:

Stability test: Stability half-life, (t ¹/₂), the values ​​were determined by converting the peak areas at each time point to the percentage in relation to the initial peak area (t = 0).

Remaining percentage = 100 x (peak sample area) -e- (t = 0 peak area).

The natural log of the remaining percentage values ​​was calculated and plotted against the sampling time (Microsoft Excel). The slope of this line, k, was determined by linear regression (Microsoft Excel).

Then the stability of the half-life was calculated using the formula, t ¹/₂ = 0.693 k.

Plasma stability test based on surrogate activity:

The calcium flow protocol described above was followed, with the following changes. The peptides were also incubated with 5 percent rat plasma (Bioreclamation # RATPLNAHP-M, treated with sodium heparin). Readings were taken at time points t₀ yt₂₄ hours, after incubation in a tissue culture incubator at 37 ° C. The plasma half-life of the peptide was estimated by calculating the following:

1) LN ((EC5o in to) / (EC50 in t₂4 hours)),

2) Calculate the slope of the previous value, and

3) you /₂ = 0.693 / (pending^TO2).

Using the test assay (as described above) the polypeptides of the invention exhibited efficacy and stability in accordance with Tables 2 and 3, provided below.

Table 2

Activity and Stability of Polypeptides

<td>Peptide</td><td>hAPJ Ca Flow²⁺EC₅or [nM]</td><td>Plasma stability based on surrogate activity t¹Z&gt; [min]</td>

<td>Example 1</td><td> 90.13</td><td> 30.7</td>

<td>Example 2</td><td> 132.58</td><td> 27.1</td>

<td>Example 3</td><td> 1008.19</td><td> 98.0</td>

<td>Example 4</td><td> 746.74</td><td> 50.0</td>

<td>Example 5</td><td> 21 .25</td><td> 126.2</td>

<td>Example 6</td><td> 21 .98</td><td> 1 17.2</td>

<td>Example 7</td><td> 90.85</td><td> &gt;1 000</td>

<td>Peptide</td><td>hAPJ Ca Flow²⁺EC₅₀ [nM]</td><td>Plasma stability based on surrogate activity t¹Z&gt; [min]</td>

<td>Example 8</td><td> 1 .04</td><td> 407</td>

<td>Example 9</td><td> 2.16</td><td> &gt;1 000</td>

<td>Example 1 0</td><td> 2.54</td><td> &gt;1 000</td>

<td>Example 11</td><td> 3.52</td><td> &gt;1000</td>

<td>Example 1 2</td><td> 2.07</td><td> 93.6</td>

<td>Example 1 3</td><td> 2.26</td><td> 283.6</td>

<td>Example 1 4</td><td> 8.83</td><td> 85.2</td>

<td>Example 1 5</td><td> 3.53</td><td> 180.3</td>

<td>Example 1 6</td><td> 1.43</td><td> 13.4</td>

<td>Example 1 7</td><td> 3.29</td><td> 14.1</td>

<td>Example 1 8</td><td> 1.62</td><td> 248.1</td>

<td>Example 1 9</td><td> 8.46</td><td> 28.4</td>

<td>Example 20</td><td> 173.24</td><td> 490.4</td>

<td>Example 21</td><td> 75.81</td><td> 639.7</td>

<td>Example 22</td><td> 42.03</td><td> 799.9</td>

<td>Peptide</td><td>hAPJ Ca Flow²⁺ECso [nM]</td><td>Plasma stability based on surrogate activity t¹Z&gt; [min]</td>

<td>Example 23</td><td> 52.42</td><td> &gt;1 000</td>

<td>Example 24</td><td> 32.65</td><td> 303.5</td>

<td>Example 25</td><td> 24.50</td><td> &gt;1 000</td>

<td>Example 26</td><td> 29.84</td><td> &gt;1000</td>

<td>Example 27</td><td> 65.55</td><td> &gt;1 000</td>

<td>Example 28</td><td> 5.68</td><td> 440.3</td>

<td>Example 29</td><td> 4.33</td><td> 216.6</td>

<td>Example 30</td><td> 6.05</td><td> &gt;1000</td>

<td>Example 31</td><td> 84.1 1</td><td> &gt;1 000</td>

<td>Example 32</td><td> 51 8.08</td><td> &gt;1 000</td>

<td>Example 33</td><td> 8.10</td><td> &gt;1000</td>

<td>Example 34</td><td> 3.21</td><td> 505.2</td>

<td>Example 35</td><td> 11.12</td><td> &gt;1 000</td>

<td>Example 36</td><td> 7.36</td><td> &gt;1 000</td>

<td>Example 37</td><td> 1.40</td><td> 164.3</td>

<td>Peptide</td><td>hAPJ Ca Flow²⁺EC₅₀ [nM]</td><td>Plasma stability based on surrogate activity t¹Z&gt; [min]</td>

<td>Example 38</td><td> 181 .77</td><td> &gt;1 000</td>

<td>Example 39</td><td> 8.20</td><td> 654.9</td>

<td>Example 40</td><td> 7.99</td><td> &gt;1 000</td>

<td>Example 41</td><td> 6.91</td><td> 627.1</td>

<td>Example 42</td><td> 92.77</td><td> &gt;1 000</td>

<td>Example 43</td><td> 4.14</td><td> &gt;1 000</td>

<td>Example 44</td><td> 5.94</td><td> 855.8</td>

<td>Example 45</td><td> 6.55</td><td> 167.3</td>

<td>Example 46</td><td> 3.87</td><td> 502.1</td>

<td>Example 47</td><td> 2.41</td><td> 418.9</td>

<td>Example 48</td><td> 4.1 0</td><td> 546.4</td>

<td>Example 49</td><td> 34.19</td><td> &gt;1 000</td>

<td>Example 50</td><td> 1.92</td><td> &gt;1 000</td>

<td>Example 51</td><td> 1.09</td><td> 655.9</td>

<td>Example 52</td><td> 53.88</td><td> 777.8</td>

<td>Peptide</td><td>hAPJ Ca Flow²⁺EC₅₀ [nM]</td><td>Plasma stability based on surrogate activity t¹Z&gt; [min]</td>

<td>Example 53</td><td> 1 .07</td><td> &gt;1 000</td>

<td>Example 54</td><td> 6.70</td><td> &gt;1 000</td>

<td>Example 55</td><td> 8.16</td><td> &gt;1 000</td>

<td>Example 56</td><td> 1.12</td><td> &gt;1000</td>

<td>Example 57</td><td> 2.01</td><td> &gt;1 000</td>

<td>Example 58</td><td> 13.09</td><td> &gt;1 000</td>

<td>Example 59</td><td> 10.00</td><td> &gt;1 000</td>

<td>Example 60</td><td> 8.15</td><td> &gt;1000</td>

<td>Example 61</td><td> 89.15</td><td> 259.9</td>

<td>Example 62</td><td> 3.32</td><td> &gt;1000</td>

<td>Example 63</td><td> 3.1 8</td><td> 703.9</td>

<td>Example 64</td><td> 6.3</td><td> 455</td>

<td>Example 65</td><td> 1.1</td><td> 596</td>

<td>Example 66</td><td> 589</td><td> 596</td>

<td>Peptide</td><td>hAPJ Ca Flow²⁺EC₅₀ [nM]</td><td>Plasma stability based on surrogate activity t¹Z&gt; [min]</td>

<td>Example comparative: Pyr1 -pelpel- 13</td><td> 1.79</td><td> 5.0</td>

Table 3

Correlation between the plasma stability test and the plasma stability test based on the surrogate activity

<td></td><td>Stability in</td><td>Plasma stability</td>

<td>Peptide</td><td>plasma t¹Z&gt;</td><td>activity based</td>

<td></td><td>[min]</td><td>surrogate t¹Z&gt; [min]</td>

<td>Example 8</td><td> 163</td><td> 407</td>

<td>Example 1 2</td><td> 53.9</td><td> 96.16</td>

<td>Example 1 3</td><td> 183</td><td> 283.6</td>

<td>Example 1 4</td><td> 63</td><td> 85.2</td>

<td>Example 1 6</td><td> 10.2</td><td> 13.4</td>

<td>Example 1 7</td><td> 2.3</td><td> 14.1</td>

<td>Peptide</td><td>Stability in plasma t¹Z&gt; [min]</td><td>Plasma stability activity based surrogate t¹Z&gt; [min]</td>

<td>Example 1 8</td><td> 220</td><td> 248.1</td>

<td>Pyr-1 -Apelina 1 3</td><td> 6.6</td><td> 5.0</td>

The polypeptide of the present invention may have an APJ receptor potency similar to that of apeline-13 or pyr1-apeline-13. In one embodiment, the polypeptide of the present invention has an EC₅₀ less than 100 nM. In another embodiment, the polypeptide of the invention has an EC₅or less than 50 nM, preferably less than 25 nM, and most preferably less than 15 nM. In yet another embodiment, the polypeptide of the present invention has an EC₅₀ less than 10 nM.

The polypeptide of the present invention may have a plasma stability superior to that of the apelina-13 or pyr-1apelina-13. In one embodiment, the improvement in plasma stability is at least 2 times. In one embodiment, the polypeptide of the invention has a plasma stability of at least 30 minutes. In another embodiment, the polypeptide of the invention has a plasma stability of at least 10 minutes, of at least 40 minutes, and in a very preferable manner of at least minutes.

The polypeptide of the present invention can be administered either simultaneously with, or before or after, one or more different therapeutic agents. The polypeptide of the present invention can be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents.

In one embodiment, the invention provides a product comprising a polypeptide of any of formulas I to IX, or an amide, an ester or a salt thereof, and at least one other therapeutic agent, as a combined preparation for simultaneous use, separate, or in sequence, in therapy. In one embodiment, therapy is the treatment of a disease or condition that responds to the activation of the APJ receptor.

Products provided as a combined preparation include a composition comprising a polypeptide of any of formulas I to IX, or an amide, an ester or a salt thereof, and the other therapeutic agents together in the same pharmaceutical composition, or a polypeptide of any of formulas I to IX, or an amide, an ester or a salt thereof, and the other therapeutic agents in a separate form, for example, in the form of a kit.

In one embodiment, the invention provides a pharmaceutical composition, which comprises a polypeptide of any of formulas I to IX, or an amide, an ester or a salt thereof, and other therapeutic agents. Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, as described above.

In one embodiment, the invention provides a kit, which comprises two or more separate pharmaceutical compositions, at least one of which contains a polypeptide of any of formulas I 'and I to IX, or an amide, an ester or a salt of the same. In one embodiment, the kit comprises elements for containing these compositions separately, such as a container, a divided bottle, or a divided sheet package. An example of this kit is a bubble pack, as is typically used for the packaging of tablets, capsules and the like.

The kit of the invention can be used to administer different dosage forms, for example, orally and parenterally, to administer the separate compositions at different dosage ranges, or to titrate the separate compositions against each other. To aid compliance, the kit of the invention typically comprises instructions for administration.

In the combination therapies of the invention, the compound of the invention and the other therapeutic agent can be made and / or formulated by the same or by different manufacturers. Moreover, the compound of the invention and the other therapeutic agent can be combined in a combination therapy: (i) before releasing the combination product to doctors (for example, in the case of a kit comprising the compound of the invention and the other therapeutic agent); (ii) by the doctors themselves (or under the guidance of the doctor) shortly before administration; (iii) in the patients themselves, for example, during the sequential administration of a polypeptide of the invention and the other therapeutic agent.

In accordance with the foregoing, the invention provides the use of a polypeptide of any of formulas I to IX, or of an amide, an ester or a salt thereof, for the treatment of a disease or condition that responds to receptor agonism of APJ, wherein the medicament is prepared for administration with another therapeutic agent. The invention also provides the use of another therapeutic agent for the treatment of a disease or condition that responds to apelin receptor agonism, wherein the medicament is administered with a polypeptide of any of formulas I to IX, or with an amide, an ester or a salt thereof.

The invention also provides a polypeptide of any of formulas I to IX, or a pharmaceutically acceptable salt thereof, for use in a method for the treatment of a disease or condition that responds to agonism of the APJ receptor, wherein the polypeptide of any of formulas I to IX, or an amide, an ester or a salt thereof, is prepared for administration with another therapeutic agent. The invention also provides another therapeutic agent for use in a method for the treatment of a disease or condition that responds to agonism of the APJ receptor, wherein the other therapeutic agent is prepared for administration with a polypeptide of any of formulas I to IX, or with an amide, an ester or a salt thereof. The invention also provides a polypeptide of any of formulas I to IX, or an amide, an ester or a salt thereof, for use in a method for the treatment of a disease or condition that responds to APJ receptor agonism, in wherein the polypeptide of any of formulas I to IX, or an amide, an ester or a salt thereof, is administered with another therapeutic agent. The invention also provides another therapeutic agent for use in a method for the treatment of a disease or condition that responds to agonism of the APJ receptor, wherein the other therapeutic agent is administered with a polypeptide of any of formulas I to IX, or with an amide, an ester or a salt thereof.

The invention also provides the use of a polypeptide of any of formulas I to IX, or of an amide, an ester or a salt thereof, for the treatment of a disease or condition that responds to agonism of the APJ receptor, wherein The patient has been previously treated (for example, within 24 hours) with another therapeutic agent. The invention also provides the use of another therapeutic agent for the treatment of a disease or condition that responds to agonism of the APJ receptor, wherein the patient has been previously treated (for example, within 24 hours) with a polypeptide of any of formulas I to IX, or with an amide, an ester or a salt thereof.

In one embodiment, the other therapeutic agent is selected from inotropes, beta-adrenergic receptor blockers, HMG-Co-A-reductase inhibitors, angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, calcium channel blockers (CCB), endothelin antagonists, renin inhibitors, diuretics, ApoA-1 mimetics, anti-diabetic agents, obesity reducing agents, aldosterone receptor blockers, endothelin receptor blockers, aldosterone synthase (ASI) inhibitors, a CETP inhibitor, anti-coagulants, relaxin, BNP (nesiritide), and an NEP inhibitor.

The term "in combination with" a second agent or treatment includes co-administration of the polypeptide of the invention (for example, a polypeptide according to any of formulas I to IX, or a polypeptide described otherwise herein) with the second agent or treatment; firstly the administration of the compound of the invention, followed by the second agent or treatment, and firstly the administration of the second agent or treatment, followed by the compound of the invention.

The term "second agent" includes any agent that is known in the art to treat, prevent, or reduce the symptoms of a disease or disorder described herein, for example, a disorder or disease that responds to receptor activation. of APJ, such as, for example, acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, cardiovascular ischemic diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral artery disease, strokes, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, lesions burns (including sunburn), and preeclampsia.

Examples of the second agents include inotropes, beta-adrenergic receptor blockers, HMG-Co-A-reductase inhibitors, angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, channel blockers. calcium (CCB), endothelin antagonists, renin inhibitors, diuretics, ApoA-1 mimetics, anti-diabetic agents, reducing agents

<td>the obesity,</td><td>blockers of</td><td>the</td><td>receivers</td><td>from</td><td>aldosterone,</td>

<td>blockers</td><td>of the recipients</td><td>from</td><td>endothelin,</td><td colspan="2">inhibitors of</td>

<td>synthase of</td><td>aldosterone (ASI),</td><td>a</td><td>inhibitor</td><td>from</td><td>CETP, anti</td>

coagulants, relaxin, BNP (nesiritide) and / or an NEP inhibitor.

Inotropes, as used herein, include, for example, dobutamine, isoproterenol, milrinone, amirinone, levosimendan, epinephrine, norepinephrine, isoproterenol and digoxin.

Beta-adrenergic receptor blockers, as used herein, include, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, metoprolol, nadolol, propranolol, sotalol and timolol.

Anti-coagulants, as used herein, include Dalteparin, Danaparoid, Enoxaparin, Heparin, Tinzaparin,

Warfarin

The term "HMG-Co-A-reductase inhibitor" (also referred to as beta-hydroxy-beta-methyl-glutaryl-coenzyme-A-reductase inhibitors) includes active agents that can be used to lower lipid levels, including blood cholesterol. Examples include atorvastatin, cerivastatin, compactin, dalvastatin, dihydrocompactin, fluindostatin, fluvastatin, lovastatin, pitavastatin, mevastatin, pravastatin, rosuvastatin, rivastatin, simvastatin, and velostatin, or pharmaceutically acceptable salts thereof.

The term "ACE inhibitor" (also referred to as angiotensin-converting enzyme inhibitors) includes molecules that disrupt the enzymatic degradation of angiotensin I to angiotensin II. These compounds can be used for the regulation of blood pressure and for the treatment of congestive heart failure. Examples include alacepril, benazepril, benazeprilat, captopril, ceronapril, cilazapril, delapril, enalapril, enaprilat, fosinopril, imidapril,

100 lisinopril, moexipril, moveltopril, perindopril, quinapril, ramipril, spirapril, temocapril, and trandolapril, or pharmaceutically acceptable salts thereof.

The term "endothelin antagonist" includes bosentan (see European Patent Number EP 526708 A), tezosentan (see International Publication Number WO 96/19459), or pharmaceutically acceptable salts thereof.

The term "renin inhibitor" includes ditequirene (chemical name: [1 S- [1 fl *, 2fl *, 4fl * (1 fl *, 2fl *)]] - 1 - [(1,1-dimetiI-ethoxy) -carbony] L-prolyl-L-phenylalanyl-N- [2-hydroxy-5-methyl-1 - (2-methyl-propyl) -4 - [[[2-methyl-1 - [[(2-pyridyl- methyl) -amino] -carbonyl] -butyl] -amino] -carbonyl] hexyl] -A / -alpha-methyl-L-histidinamide); terlaquirene (chemical name: [P? - (/^:? *, S *)] - N- (4-morpholinyl-carbonyl) -1-phenylalanyl-N- [1 - (cyclohexylmethyl) -2-hydroxy-3- (1-methyl-ethoxy) -3-oxo-propyl ] -S-methyl -L-cis teínaamide); Alisquirene (chemical name: (2S, 4S, 5S, 7S) -5-amino-A / - (2carbamoyl-2,2-dimethyl-ethyl) -4-hydroxy-7 - {[4-methoxy-3- (3 -methoxypropoxy) -phenyl] -methyl} -8-methyl-2- (propan-2-yl) -nonanamide), and zanquirene (chemical name: [1 S- [1 R * [R * (R *)], 2 S *, 3 R *]] - N- [1 (cyclohexyl-methyl) -2,3-dihydroxy-5-methyl-hexyl] - alpha - [[2 - [[(4-methyl-1 piperazinyl) -sulfonyl] -methyl] -1-oxo-3-phenyl-propyl] -amino] -4-thiazolpropanamide), or hydrochloride salts thereof , or SPP630, SPP635 and SPP800 as developed by Speedel, or RO 661 132 and RO 66-1 168 of formulas (A) and (B):

101

<img file="CU20140097A7_D0004.tif" />

<img file="CU20140097A7_D0005.tif" />

í's

<img file="CU20140097A7_D0006.tif" />

(B) or pharmaceutically acceptable salts thereof.

The term "alisquirene", if not defined in a specific way, should be understood both as the free base and as a salt thereof, especially a pharmaceutically acceptable salt thereof, more preferably a hemi-fumarate salt thereof.

The term "a calcium channel blocker (CCB)" includes dihydro-pyridines (DHPs), and non-dihydro-pyridines (non-DHPs) (for example, calcium channel blockers (CCBs) type diltiazem and verapamil type ). Examples include amlodipine, Bepridil, Diltiazem, felodipine, riosidine, isradipine, lacidipine, nicardipine, nifedipine, niguldipine, niludipine, nimodipine, nisoldipine, nitrendipine, Verapamil and nivaldipine-and is not di-non-di (non-representative) DHP) selected from the group consisting of flunarizine, prenylamine, diltiazem, fendiline, gallopamil, mibefradil, anipamil, thiapamil and verapamil, or the pharmaceutically acceptable salts thereof. Calcium channel blockers (CCBs) can be used as anti-hypertensive, anti-angina, or anti-arrhythmic drugs.

The term "diuretic" includes thiazide derivatives (for example,

102 chlorothiazide, hydrochlorothiazide, methylclothiazide, and chlorothalidone).

The term "ApoA-1 mimetic" includes D4F peptides (for example, of the formula DWFKAFYDKVAEKFKEAF) (SEQ ID NO: 7).

It is understood that an angiotensin II receptor antagonist or a pharmaceutically acceptable salt thereof is an active ingredient that binds to the ΑΉ receptor subtype of the angiotensin II receptor, but does not result in receptor activation. As a consequence of the inhibition of the AT receptor₁₅these antagonists can be used, for example, as antihypertensives, or for the treatment of congestive heart failure.

The class of ATÚ receptor antagonists comprises compounds that have different structural characteristics, and non-peptides are essentially preferred. For example, mention may be made of the compounds that are selected from the group consisting of waltz, losarian, candesartan, eprosartan, irbesartan, saprisartan, tasosartan, telmisartan, the compound with the designation E-1477 of the following formula:

<img file="CU20140097A7_D0007.tif" />

CQOH

103 the compound with the designation SC-52458 of the following formula:

<img file="CU20140097A7_D0008.tif" />

and the compound with the designation ZD-8731 of the following formula:

<img file="CU20140097A7_D0009.tif" />

or, in each case, a pharmaceutically acceptable salt thereof.

ΑΤ receptor antagonists_ή Preferred are candesartan, eprosartan, irbesartan, losarían, telmisartan, waltz. Also preferred are agents that have been commercialized, more preferably waltz, or a pharmaceutically acceptable salt thereof.

104

The term "anti-diabetic agent" includes the secretion of insulin enhancers that promote insulin secretion from pancreatic β-cells. Examples include biguanide derivatives (for example, metformin), sulfonylureas (SU) (for example, tolbutamide, chlorpropamide, tolazamide, acetohexamide, 4-chloro-A / - [(1-pyrrolidinyl-amino) -carbonyl] -benzensulfonamide (glycopyramide) , glibenclamide (glyburide), gliclazide, 1-butyl-3-methanylylurea, carbutamide, glibonuride, glipizide, gliquidone, glisoxepide, glibutiazole, glibuzol, glihexamide, glimidine, glipinamide, fenbutamide, and tolyl cytamide or pharmaceutically acceptable salts thereof. Other examples include phenylalanine derivatives (for example, nateglinide [A / - (í / 'ans-4-isopropyl-cyclohexylcarbonyl) -D-phenylalanine] (see European Patent Numbers EP 196222 and EP 526171) of the formula:

K ^ = 0

H-O repagUnida [(S) -2-ethoxy-4- {2 - [[3-methyl-1 - [2- (1 -piperidinyl) phenyl] -butyl] -amino] -2-oxo-ethyl} -benzoic] (see European Patents Numbers EP 589874, EP 147850 A2, in particular Example 11 on page 61, and EP 207331 A1); (2S) -2-benzyl-3- (c / s-hexahydro-2-isoindolinyl-carbonyl) -propionate dihydrate (for example, mitiglinide (see European Patent Number EP 507534)); and glimepiride (see European Patent Number EP 31058).

<img file="CU20140097A7_D0010.tif" />

<img file="CU20140097A7_D0011.tif" />

105

Other examples of second agents with which the peptide and polypeptide of the invention can be used in combination include DPP-IV inhibitors, GLP-1, and GLP-1 agonists.

DPP-IV is responsible for inactivating GLP-1. More particularly, DPP-IV generates an antagonist of GLP-1 receptors, and thus reduces the physiological response to GLP-1. GLP-1 is a major stimulant of pancreatic insulin secretion and has direct beneficial effects on glucose elimination.

The DPP-IV inhibitor (dipeptidyl peptidase IV) may be peptide or, preferably, non-peptide. DPP-IV inhibitors are disclosed in each case generically and specifically, for example, in International Publication Number WO 98/19998, DE 196 16 486 A1, and in International Publications Numbers WO 00/34241 and WO 95 / 15309, in each particular case in the claims of the compound, and the final products of the processing examples, the subject matter of the final products, Pharmaceutical preparations and claims are incorporated into the present application by reference to these publications. Compounds that are specifically disclosed in Example 3 of International Publication Number WO 98/19998 and in Example 1 of International Publication Number WO 00/34241, respectively, are preferred.

GLP-1 (glucagon-1 type peptide) is an insulinotropic protein that is described, for example, by WE Schmidt et al., In

106

Diabetology, 28, 1985, 704-707, and in the United States Patent

United States of America Number US 5,705,483.

The term "GLP-1 agonists" includes variants and analogs of GLP-1 (7-36) NH₂ which are disclosed in particular in United States Patents US Numbers

5,120,712, US 5.1 18666, US 5,512,549, in the Publication

International Number WO 91/1 1457, and by C. Orskov et al. In J. Biol. Chem. 264 (1989) 12826. Other examples include GLP1 (7-37), in which compound, the carboxy-terminal amide functionality of Arg³⁶ is displaced with Gly in 37^to GLP-1 molecule position (7-36) NH₂, and variants and analogs thereof, including GLN⁹-GLP-1 (7-37), D-GLN⁹-GLP-1 (7-37), acetyl LYS⁹-GLP-1 (7-37), LYS¹⁸-GLP-1 (7-37) and, in particular, GLP-1 (737) OH, VAL⁸-GLP-1 (7-37), GLY⁸-GLP-1 (7-37), TH R⁸-G LP-1 (7-37), MET⁸-GLP-1 (7-37), and 4-imidazopropionyl-GLP-1. A special preference is also given to the exendin-4 LPG agonist analog, described by Greig et al., In Diabetologia 1999, 42, 45-50.

Also included in the definition of "anti-diabetic agent" is the sensitivity to insulin enhancers that restore impaired insulin receptor function to reduce insulin resistance and, consequently, to improve insulin sensitivity. . Examples include the hypoglycemic derivatives of thiazolidinedione (for example, glitazone, (S) ((3,4-dihydro-2- (phenyl-methyl) -2H-1-benzopyran-6-yl) -methyl-thiazolidine-2,4dione ( englitazone), 5 - {[4- (3- (5-methyl-2-phenyl-4-oxazolyl) -1-oxo-propyl) 107 phenyl] -methyl} -thiazolidine-2,4-dione (darglitazone), 5 - {[4- (1-methylcyclohexyl) -methoxy) -phenyl] -methyl} -thiazolidine-2,4-dione (ciglitazone), 5 {[4- (2- (1-indolyl) -ethoxy) -phenyl ] -methyl} -thiazolidine-2,4-dione (DRF21 89), 5- {4- [2- (5-methyl-2-phenyl-4-oxazolyl) -ethoxy)] - benzyl} -thiazolidine-2,4-dione (BM-13.1246), 5- (2-naphthyl-sulfon L) -t¡azol¡d¡na-2,4-dione (AY-31637), bis- {4 - [(2,4-dioxo-5-thiazolidinyl) -methyl] -phenyl} -methane (YM268 ), 5- {4- [2 (5-methyl-2-phenyl-4-oxazolyl) -2-hydroxy-ethoxy] -benzyl} -thiazolidine-2,4dione (AD-5075), 5- [4- ( 1-phenyl-1-cyclopropan-carbonyl-amino) -bericyl] thiazolidine-2,4-dione (DN-108) 5 - {[4- (2- (2,3-dihydro-indole-1-yl)) - ethoxy) 10 phenyl] -methyl} -thiazolidine-2,4-dione, 5- [3- (4-Chloro-phenyl]) - 2-propynyl] -5phenyl-sulfonyl) -thiazolidin a-2,4-dione, 5- [3- (4-chloro-phenyl]) - 2-propynyl ] -5 (4-fluoro-phenyl-sulfonyl) -thiazolidine-2,4-dione, 5- {[4- (2- (methyl-2-pyridinyl-amino) -ethoxy) -phenyl] -methyl} -thiazolidine-2 , 4-dione (rosiglitazone), 5 - {[4- (2- (5-ethyl-2-pyridyl) -ethoxy) -phenyl] -methyl} -thiazoIidine15 2,4-dione (pioglitazone), 5 - {[ 4 - ((3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-be nzopyran-2-i) - methoxy) -phenyl] -methyl} -thiazo lid i na- 2,4dione (troglitazone), 5- [6- (2-Fluoro-benzyloxy) -naphthalen-2-yl-methyl] thiazolidin a-2,4-dione (MCC555), 5 - {[2- (2-naphthyl) -benzoxazol-5-yl ] methyl} -thiazolidine-2,4-dione (T-174), and 5- (2,4-di oxo -1 i azo I idi n-5 - i I20 methyl) -2-methox¡-N- ( 4-Trifluoro-methyl-benzyl) -benzamide (KRP297)).

Other anti-diabetic agents include insulin signaling path modulators, such as tyrosine protein phosphatase inhibitors (PTPases), anti-diabetic non-small molecule mimetic compounds and glutamine-fructose-6-phosphate amido25 transferase inhibitors (GFAT); compounds that

108 have an influence on the production of poorly regulated hepatic glucose, such as glucose-6-phosphatase inhibitors (G6Pase), fructose-1,6-bisphosphatase inhibitors (F-1,6-Bpase), glycogen phosphorylase (GP) inhibitors , glucagon receptor antagonists and phosphoenolpyruvate carboxykinase inhibitors (PEPCK); pyruvate dehydrogenase kinase (PDHK) inhibitors; gastric emptying inhibitors; insulin; GSK-3 inhibitors; retinoid X receptor agonists (RXR); Beta-3 AR agonists; decoupling protein agonists (PCUs); PPAR agonists and not glitazone type; PPARa / PPARy double agonists; anti-diabetic compounds containing vanadium; incretin hormones, such as glucagon-1 type peptide (GLP-1), and GLP-1 agonists; beta cell imidazoline receptor antagonists; miglitol; antagonists to₂-adrenergic; and pharmaceutically acceptable salts thereof.

In one embodiment, the invention provides a combination, in particular a pharmaceutical combination, which comprises a therapeutically effective amount of the polypeptide according to the definition of any of formulas I to IX, or of an amide, an ester or a salt of the same, and one or more therapeutically active agents selected from β-adrenergic receptor blockers, such as acebutolol, atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, sotalol and timolol; angiotensin II receptor antagonists such as AT1 blockers; anti-diabetic agents, such as DPPIVs inhibitor (for

109 example, vildagliptin), and GLP1 peptide agonist.

The term "obesity reducing agent" includes lipase inhibitors (for example, orlistate), and appetite suppressants (for example, sibutramine and phentermine).

It is understood that an aldosterone synthase inhibitor or a pharmaceutically acceptable salt thereof is an active ingredient that has the property to inhibit the production of aldosterone. Aldosterone synthase (CYP11B2) is a mitochondrial cytochrome P450 enzyme that catalyzes the last step of aldosterone production in the adrenal cortex, that is, the conversion of 11-deoxycorticosterone to aldosterone. Inhibition of aldosterone production with so-called aldosterone synthase inhibitors is known as a successful variant for the treatment of hypokalemia, hypertension, congestive heart failure, atrial fibrillation, or renal failure. This activity of aldosterone synthase inhibition is readily determined by those skilled in the art according to conventional assays (for example, United States Patent No. US 2007/0049616).

The class of aldosterone synthase inhibitors comprises both steroidal and non-steroidal aldosterone synthase inhibitors, and the latter are more preferred.

Preference is given to commercially available aldosterone synthase inhibitors or aldosterone synthase inhibitors that have been approved by the authorities

110 of health.

The class of aldosterone synthase inhibitors comprises compounds that have different structural characteristics. An example of a non-steroidal aldosterone synthase inhibitor is the (+) - enantiomer of fadrozole hydrochloride (United States Patents Numbers 4617307 and 4889861) of the formula:

<img file="CU20140097A7_D0012.tif" />

or, if appropriate, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in combination are the compounds and analogs generically and specifically disclosed, for example, in United States Patent No. US2007 / 0049616, in particular in the claims of the compound, and the products end of the processing examples, the subject matter of the final products, Pharmaceutical preparations and claims are incorporated into the present application by reference to this publication. Suitable suitable aldosterone synthase inhibitors for use in the present invention include, without limitation, 4- (6,7-dihydro-5H-pyrrolo- [1,2-c] -imidazol-5i I) -3-m et i I - be η zo nitri I o; 5- (2111 Chloro-4-cyano-phenyl) -6,7-dihydro-5H-pyrrolo- [1,2-c] -imidazole-5-carboxylic acid (4-methoxy-benzyl) -methyl-amide; 4'-fluoro-6- (6,7,8,9-tetrahydro-5 / - / - imidazo- [1,5-a] -azepin5-i I) - bi f in i I - 3-c arbo ni tri lo; 5- (4-Cyano-2-methoxyphenyl) -6,7-dihydro-5/7-pyrrolo- [1,2-c] -imidazol-5-carboxylic acid butyl ester; 4- (6,7-dih id ro-5 H- pyrro I o- [1,2-c] -imidazol-5-yl) -2-methoxy-benzonitrile; 5- (2-Chloro-4-cyano-phenyl) -6,7-dihydro-5H-pyrrolo [1,2-c] -imidazol-5-carboxylic acid 4-fluorobenzyl ester; 5- (4-Cyano-2-trifluoro-methoxy-phenyl) -6,7-dihydro-5H-pyrrolo- [1,2-c] -imidazole-5-carboxylic acid methyl ester; 5- (4-Cyano-2-methoxyphenyl) -6,7-dihydro-5 / - / - pyrrolo- [1,2-c] -imidazol-5-carboxylic acid 2-isopropoxy-ethyl ester; 4- (6,7-dihydro-5/7-pyrrolo- [1,2-c] -imidazol-5-yl) -2-methyl-benzonitrile; 4- (6,7-dihydro-5/7-pyrrolo- [1,2-c] -imidazol-5-yl) -3-fluoro-benzonitrile; 4- (6,7-dihydro-5H-pyrrolo- [1,2-c] -imidazol-5-yl) -2-methoxy-benzonitrile; 3-fluoro4- (7-methylene-6,7-dihydro-5/7-pyrrolo- [1,2-c] -imidazol-5-yl) -benzonitrile; c / 's-3-fluoro-4- [7- (4-fluoro-benzyl) -5,6,7,8-tetrahydro-imidazo- [1,5-a] pyridin-5-yl] -benzonitrile; 4'-fluoro-6- (9-methyl-6,7,8,9-tetrahydro-5Himidazo- [1,5-a] -azepin-5-yl) -biphenyl-3-carbonitrile; 4'-fluoro-6- (9-methyl6,7,8,9-tetrahydro-5H-imidazo- [1,5-a] -azepin-5-yl) -biphenyl-3carbonitrile or, in each case, the enantiomer (R) or (S) thereof; or if appropriate, a pharmaceutically acceptable salt thereof.

The term aldosterone synthase inhibitors also includes the compounds and analogs that are disclosed in the

International Publications Numbers W02008 / 076860, W02008 /

076336, W02008 / 076862, W02008 / 027284, W02004 / 046145,

112

W02004 / 01 491 4, and W02001 / 076574.

Additionally, aldosterone synthase inhibitors also include the compounds and analogs that are disclosed in United States Patent Applications

Numbers US2007 / 0225232, US2007 / 0208035, US2008 / 031 8978,

US2008 / 0076794, US2009 / 001 2068, US20090048241 and in the

TCP International Requests Numbers W02006 / 005726,

W02006 / 1 28853, W020061 28851, W02006 / 1 28852, W02007065942,

W02007 / 1 16099, W02007 / 1 1 6908, W02008 / 1 1 9744, and in European Patent Application Number EP 1886695. Suitable preferred aldosterone synthase inhibitors for use in the present invention include, without limitation, 8- (4-fluoro-phenyl) -5,6-dihydro-8H-imidazo- [5,1 -c] [1,4] -oxazine; 4- (5,6-dihydro-8H-imid scourged, 1-c] [1,4] -oxazin-8-yl) -2-fluoro-benzonitrile; 4- (5,6-dihydro-8Himidaz o- [5,1 -c] [1,4] -oxazin-8-yl) -2,6-difluoro-benzonitrile; 4- (5,6-dihydro-8H-imidazo- [5,1 -c] [1,4] -oxazin-8-yl) -2-methoxy-benzonitrile; 3 (5,6-dihydro-8 H-imid azo- [5,1-c] [1,4] -oxaz in-8-yl) -benzonityl; 4- (5,6-dihydro-8H-imidazo- [5,1 -c] [1,4] -oxazin-8-yl) -phthalonitrile; 4- (8- (4-c ¡write down I) - 5,6-dihydro-8 / 7- imid azo- [5,1 -c] [1,4] - oxaz i n-8-i ) -benzonitrile; 4 (5,6-dihyd ro-8/7-imid azo- [5,1-c] [1,4] -oxaz in-8-yl) -benzonityl; 4- (5,6dihydro-8/7-imidazo- [5,1 -c] [1,4] -oxazin-8-yl) -naphthalen-1-carbonitrile; 8 [4- (1 H-tetrazol-5-yl) -phenyl 1 -5,6-dihydro-8/7-imidazo- [5,1 -c] [1,4] oxazine, as developed by Speedel or , in each case, the (R) or (S) enantiomer thereof; or if appropriate, a pharmaceutically acceptable salt thereof.

113

The aldosterone synthase inhibitors useful in the combination are the compounds and analogs generically and specifically disclosed, for example, in International Publications Nos. WO 2009/156462 and WO 2010/130796, in particular in the claims of the compound, and the final products of the processing examples, the subject matter of the final products, the pharmaceutical preparations and the claims are incorporated by reference. Preferred suitable aldosterone synthase inhibitors for the combination of the present invention include: 3- (6-Fluoro-3-methyl-2p i rid i n-3-i I-1 H- i nd ol -1 - i I- metí I) -benzon itri lo, 1 - (4-methan hydrochloride -sulfonylb enci I) -3-m eti I - 2-pi ri di n-3 - i I -1 / - / - i nd ol, 2- (5-benzyloxy-pyridin-3-yl) -6chloro-1 -methyl-1 H-indole, 5- (3-cyano-1-methyl-1 Hindol-2-yl) -nicotinic acid ethyl ester, A / - [5- (6-chloro-3-cyano-1 -methyl-1H-indole-2-yl) pyridin-3-yl-methyl] -ethan-sulfonamide, 5- (6-chloro-3-cyano-1-methyl-1 Hi n I - 2-i I) - pi r id i n-3 - i I - this r of pyrrolidin-1-sulfonic acid, A / -methyl-A / [5- (1-methyl-1H-indol-2-yl) -pyridin-3-yl-methyl] -methanesulfonamide, 6-chloro-1-methyl-l-2- {5- [ (2-Pyrrolid in-1-yl-ethyl-amino) -methyl] -pyridin-3-yl-1 Hindol-3-carbonitrile, 6-chloro-2- [5- (4-methanesulfonyl-piperazin-1 - ilmet il) -pyridin-3-yl] -1-methyl-1 H-indole-3-carbonitrile, 6-cl gold-1-methyl -2 {5 - [(1-methyl-piperidin-4-yl- amino) -methyl] -pyridin-3-yl} -1H-indole-3carbonitrile, [5- (6-chloro-3-cyano-1-methyl-1 H-indole-2-yl) -pyridin-3-ylmethylj -amide of morpholin-4-carboxylic acid, A / - [5- (6-Chloro-1-methyl1 H -i nd ol-2-i I) -pi rid i η-3-il-m eti l] -etan-sulfonamide, C, C, C- trifluoro-N [5- (1-methyl-1 / - / - indol-2-yl) -pyridin-3-yl-methyl] -methanesulfonamide, N- [5114 sulfonamide sulfonamide sulfonamide (3-chloro-4- cyano-phenyl) -pyridin-3-yl] -4-trifluoro-methyl-benzenesulfonamide, N- [5- (3-Chloro-4-cyano-phenyl) -pyridin-3-yl] -1-phenyl-methane- (5- (3-chloro-4-cyano-phenyl) -pyridin-3-yl ) -butan-1N- (1 - (5- (4-cyano-3-methoxy-phenyl) -pyridin-3-yl) -ethyl) -ethanN - ((5- (3-chloro-4-cyano-phenyl) ) -pyridin-3-yl) (cyclopropyl) methyl) -ethan-sulfonamide, N - (ci cl op rop i I - (5- (1 Hi nd ol -5-i I) -pir id in - 3-i I) methyl) -ethan-sulfonamide, N- (cyclopropyl- (5-naphthalen-1-yl-pyridin-3-yl) methyl) -ethan-sulfon amide, Ethan-sulfonic acid [5- (6-chloro-1 -methyl 1-1 H-pyrrolo- [2,3-b] -pi ridi n2-yl) -pyridin-3-yl-methyl] -amide, and {[5- (3-Chloro-4-cyano-phenyl) -pyridi-3-yl] -cyclopropyl-methyl} -ethyl amide of ethanesulfonic acid.

The term "endothelin receptor blocker" includes bosentan and ambrisentan.

The term "CETP inhibitor" refers to a compound that inhibits cholesterol-mediated transport of cholesteryl ester transfer (CETP) of various cholesteryl esters and triglycerides from HDL to LDL and VLDL. This cholesteryl ester transfer protein (CETP) inhibition activity is actually determined by those skilled in the art according to conventional assays (for example, United States Patent No. 6,140,343). Examples include the compounds disclosed in United States Patents Nos. 6,140,343 and 6,197,786 (eg, ethyl ester of acid [2R, 4S] 4 - [(3,5-bis-trifluoro- methylbenci I) -methoxy-carbon i l-am i no] -2-ethyl-6-trif luoro-methyl I-3,4-dih id ro-2H115 quinolin-1-carboxylic (torcetrapib); the compounds given to be disclosed in the United States Patent

Number 6,723,752 (for example, (2R) -3 - {[3- (4-chloro-3-ethyl-phenoxy) phenyl] - [[3- (1,1,2,2-tetrafluoro-ethoxy) -phenyl] -methyl] -amino} -1,1,1-trifluoro2-propanol); the compounds that are disclosed in the Application for

United States Patent with Serial Number

10 / 807,838; polypeptide derivatives that are disclosed in the

United States Patent Number 5,512,548;

rosenonolactone derivatives and phosphate-containing cholesteryl ester analogs disclosed in J. Antibiot., 49 (8): 815-816 (1996), and Bioorg. Med. Chem. Lett .; 6: 1951-1954 (1996), respectively. Additionally, cholesteryl ester transfer protein (CETP) inhibitors also include those disclosed in International Publications Numbers W02000 / 017165, W02005 / 095409, W02005 / 097806, and in the

International Publications Numbers WO 2007/128568, W02008 /

009435, WO 2009/059943 and W02009 / 071 509.

The term "NEP inhibitor" refers to a compound that inhibits neutral endopeptidase (NEP) EC 3.4.24.11. Examples include Candoxatril, Candoxatrilate, Dexecadotril, Ecadotril, Racecadotril, Sampatrilate, Fasidotril, Omapatrilate, Gemopatrilate, Daglutril, SCH-42495, SCH-32615, UK-447841, AVE-0848, PL-37, and ethyl-ester (2R, 4S) -5-Biphenyl-4-yl-4- (3-carboxy-propionylamino) -2-methyl-pentanoic acid, or a pharmaceutically acceptable salt thereof. Neutral endopeptidase (NEP) inhibitors

116 also include dipeptide derivatives substituted by phosphono / biaryl, as disclosed in US Pat. No. 5,155,100. Neutral endopeptidase (NEP) inhibitors also include the Nmercaptoacyl-phenylalanine derivative, as disclosed in the Application

TCP International Number WO 2003/104200.

Neutral endopeptidase (NEP) inhibitors also include double acting agents against hypertension, as disclosed in the International Applications of TCP Numbers

WO 2008/133896, WO 2009/035543 or WO 2009/134741. Other examples include the compounds that are disclosed in the

United States Patent Applications

Numbers 12 / 788,794; 12 / 788,766 and 12 / 947,029. Neutral endopeptidase (NEP) inhibitors also include the compounds disclosed in International Publications Numbers WO

2010/136474, WO 2010/136493, WO 201 06/0171, and in

Provisional US Patent Applications for

North America Numbers 61/414171 and 61/414163.

In one embodiment, the invention provides a method for activating the APJ receptor in a subject, wherein the method comprises administering to the subject, a therapeutically effective amount of the polypeptide according to the definition of any of formulas I to IX, or of an amide, an ester or a salt thereof.

In one embodiment, the invention provides a method for the treatment of a disorder or a disease that responds to the

117 activation of the APJ receptor, in a subject, wherein the method comprises administering to the subject, a therapeutically effective amount of the polypeptide according to the definition of any of formulas I to IX, or of an amide, an ester or a salt of the same.

In one embodiment, the invention provides a method for the treatment of a disorder or a disease that responds to the activation (agonism) of the APJ receptor, in a subject, wherein the disorder or disease is selected from heart failure. acute decompensated (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, Ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral artery disease, strokes, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including burn) of sun), and preeclampsia.

In one embodiment, the invention provides a polypeptide according to the definition of any of formulas I to IX, for use as a medicament.

In one embodiment, the invention provides the use of a polypeptide according to the definition of any of formulas I to IX, or of an amide, an ester or a salt thereof, in the preparation of a medicament for the treatment of a disorder or

118 of a disease that responds to the activation of the APJ receptor. In another embodiment, the invention provides the use of a polypeptide according to the definition of any of formulas I to IX, or of an amide, an ester or a salt thereof, in the preparation of a medicament for the treatment of a disorder or disease that responds to receptor activation of

APJ, wherein this disorder or disease is selected in particular from acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral artery disease, strokes, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn), and preeclampsia. Exemplification of the invention:

Synthesis of peptides and polypeptides:

<td>Abbreviation</td><td>Definition</td>

<td>AA</td><td>Amino acid</td>

<td>Ac</td><td>Acetyl</td>

<td>Acm</td><td>Acetamidomethyl</td>

119

<td>Abbreviation</td><td>Definition</td>

<td>ACN</td><td>Acetonitrile</td>

<td>AcOH</td><td>Acetic acid</td>

<td>Ac₂OR</td><td>Acetic anhydride</td>

<td>A.M</td><td>Amino-methyl</td>

<td>BAL</td><td>Amide linker of the base structure</td>

<td>BSA</td><td>Bovine serum albumin</td>

<td>Boc</td><td>tert-butoxycarbonyl</td>

<td>DCM</td><td>Dichloromethane</td>

<td>IT GAVE</td><td>Λ /, Λ / '- di-isopropyl-carbodi-imide</td>

<td>DIPEA</td><td>Λ /, Λ / '- di -isopropyl -ethyl -ami na</td>

<td>Dma</td><td>Λ /, Λ / '- dimethyl acetamide</td>

<td>DMF</td><td>Λ /, Λ / '- dimethyl formamide</td>

<td>DMSO</td><td>Dimethyl sulfoxide</td>

<td>DVB</td><td>Divinyl Benzene</td>

<td>EDT</td><td>Etanditiol</td>

<td>FA</td><td>Formic acid</td>

120

<td>Abbreviation</td><td>Definition</td>

<td>Fmoc</td><td>9-Fl uo ren i I-methyl oxy-carbon i lo</td>

<td>HEY YOU</td><td>2- (1H-9-azabenzotriazole-) hexafluorophosphate 1-yl) -1,1,3,3-tetramethyl-uronium</td>

<td>HBSS</td><td>Hank regulated salt solution</td>

<td>HCTU</td><td>2- (6-Chloro-1H-benzo- hexafluorophosphate) triazol-yl) -1,1,3,3-tetramethyl-uronium</td>

<td>HEPES</td><td>4- (2-hydroxy-eti l) -1 - piperazin-ethan- sulfonic</td>

<td>Hfip</td><td>Hexafluoroisopropanol</td>

<td>HOAt</td><td>1-hydroxy-7-azabenzotriazole</td>

<td>HPLC</td><td>High performance liquid chromatography</td>

<td>iv</td><td>(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene) -3- m eti I - bu ti I o</td>

<td>LN</td><td>Logarithmus naturali (natural logarithm)</td>

<td>MeOH</td><td>Methanol</td>

<td>MS</td><td>Mass spectrometry</td>

<td>Nal</td><td>2-Naphthalanine</td>

121

<td>Abbreviation</td><td>Definition</td>

<td>Nle</td><td>Norleucine</td>

<td>NMP</td><td>N-methyl pyrrolidine</td>

<td>Pure oxime</td><td>2-cyano-2- (hydroxy-imino) -ethyl acetate</td>

<td>Pbf</td><td>2,2,4,6,7-Pentamethyl-dihydro-benzo-furan-5- sulfonyl</td>

<td>PE</td><td>Pyroglutamate</td>

<td>PG</td><td>Protective group</td>

<td>Ph</td><td>Phenyl</td>

<td>$</td><td>Polystyrene</td>

<td>POL</td><td>Polymer support</td>

<td>rt</td><td>Room temperature</td>

<td>SPPS</td><td>Synthesis of solid phase peptides</td>

<td>íBuOH</td><td>Terbutanol</td>

<td>TFA</td><td>Trifluoroacetic acid</td>

<td>THF</td><td>Tetrahydrofuran</td>

<td>TIS</td><td>T ri-isopropyl silane</td>

122

<td>Abbreviation</td><td>Definition</td>

<td>To go</td><td>Holding time</td>

<td>Trt</td><td>T ritilo</td>

<td>UPLC</td><td>Ultra-high liquid chromatography performance</td>

<td>UV</td><td>Ultraviolet</td>

The peptides were synthesized by conventional solid phase Fmoc chemistry. The peptides were assembled in the Prelude peptide synthesizer^MR (Protein Technologies, Inc., Tucson, USA). Peptides with a free carboxylic acid on the C terminus were synthesized from 2-chloro-tritiio-PS chloride resin (ABCR, Karlsruhe, Germany). Peptides with an unsubstituted carboxamide on the C-terminus were synthesized from the Fink-protected Rink-AmidaAM-PS resin (Merck, Darmstadt, Germany). Peptides with an N-mono-substituted carboxamide on the C terminus were synthesized from the BAL-AM-PS resin loaded with amines (EMC Microcollections, Tübingen, Germany).

The peptides were purified by reverse phase preparation HPLC. The following columns were used:

• Waters SunFire Prep C18 OBD column, 5 microns, 30 x 100 mm, Part No. 186002572 (one column or two columns

123

<td>serially). •</td><td>Waters SunFire column</td><td>Prep</td><td>C18 OBD,</td><td> 5</td><td>you love</td><td>30 x</td><td> 150</td>

<td>millimeters,</td><td>Part No. 186002797.</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> •</td><td>Waters Atlantls column</td><td>Prep</td><td>OBD T3,</td><td> 5</td><td>you love</td><td>30 x</td><td> 150</td>

<td>millimeters,</td><td>Part No. 186003703.</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> •</td><td>Waters XBridge column</td><td>Prep</td><td>C8 OBD,</td><td> 5</td><td>you love</td><td>30 x</td><td> 150</td>

<td>millimeters,</td><td>Part No. 186003083</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> •</td><td>Machery-Nagel Nucleosil '</td><td>⁸ 100</td><td>-5 C18, 5</td><td></td><td>you love</td><td>250 X</td><td> 40</td>

mm, Part No. 715340.400.

The mobile phases consisted of eluent A (0.1 percent trifluoroacetic acid (TFA) in H₂O), and eluent B (acetonitrile (ACN)). Gradients were designed based on the specific requirements of the separation problem. The pure products were lyophilized from ACN / H₂OR.

The products were analyzed by analytical HPLC using UV detection at λ = 214 nanometers (Column: Bischoff

UHC-640, 53 x 4.0 mm, ProntoSil 120-3-C18-H, 3 microns,

Part No. 0604F185PS030). The mobile phases consisted of eluent A ( trifluoroacetic acid (TFA) at 0.07 percent in H₂O), and eluent B (0.1 percent trifluoroacetic acid (TFA) in acetonitrile (ACN)). The additional characterization of the products was done by UPLC-MS (Column: Waters Acquity UPLC® BEH C18, 1.7 microns, 2.1 x 50 mm, Part No. 186002350), using electrospray ionization.

The peptides that are exemplified in Table 4 are

124 synthesized using the general procedures described below. The unsubstituted N or C terms are indicated by italics H- or -OH, respectively.

Table 4

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td>Example 1</td><td>pE-RPRLKHFGP- Nle-D-phenethyl-amine</td><td> 8</td><td>Lactam K⁶-D¹²</td>

<td>Example 2</td><td>pE-RPRLKHFGP- Nle-E-Phenethyl-Amine</td><td> 9</td><td>Lactam K⁶-AND¹²</td>

<td>Example 3</td><td>pE-RPRL-Orn-HFGP- Nle-D-phenethyl-amine</td><td> 10</td><td>Lactam O⁶-D¹²</td>

<td>Example 4</td><td>pE-RPRL-Dab-HFGP- Nle-D-phenethyl-amine</td><td> 1 1</td><td>Lactam Dab⁶-D¹²</td>

<td>Example 5</td><td>pE-RPRLKFKGP- Nle-F</td><td> 12</td><td>Lactam K⁶- term C</td>

<td>Example 6</td><td>pE-RPRLKFKGP- Nle-f</td><td> 13</td><td>Lactam K⁶- term C</td>

<td>Example 7</td><td>QRPRLCFKGP-Nle- CFGG</td><td> 14</td><td>Lactam term N- term C,</td>

125

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td></td><td></td><td></td><td>Disulfide C⁶-C¹²</td>

<td>Example 8</td><td>pE-RPRLCHKGP- Nle-CFO / 7</td><td> 15</td><td>Disulfide C⁶-C¹²</td>

<td>Example 9</td><td>pE-RPRLC-Aib-KGP- Nle-CF-OH</td><td> 16</td><td>Disulfide C⁶-C¹²</td>

<td>Example 1 0</td><td>pE-RPRLC-Aib-KGP- Nle-Cf-OH</td><td> 17</td><td>Disulfide C⁶-C¹²</td>

<td>Example 11</td><td>H-Isn-RPRLC-Aib-KG- P-Nle-Cf-OH</td><td> 18</td><td>Disulfide C⁶-C¹²</td>

<td>Example 1 2</td><td>pE-RPRLCHKGP- Nle-C-phenethyl-amine</td><td> 19</td><td>Disulfide C⁶-C¹²</td>

<td>Example 1 3</td><td>pE-RPRLCHKGP- Nle-Cf-OH</td><td> 20</td><td>Disulfide C⁶-C¹²</td>

<td>Example 1 4</td><td>pE-RPR-Cha-CHKGP- Cha-CF-OH</td><td> 21</td><td>Disulfide C⁶-C¹²</td>

<td>Example 1 5</td><td>pE-RPRLCFKGP- Nle-CF-OH</td><td> 22</td><td>Disulfide C⁶-C¹²</td>

126

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td>Example 1 6</td><td>HRPRLCHKGP-Nle- CF-OH</td><td> 23</td><td>Disulfide C⁵-C¹¹</td>

<td>Example 1 7</td><td>HRRPRLCHKGP- Nle-CF-OH</td><td> 24</td><td>Disulfide C⁶-C¹²</td>

<td>Example 1 8</td><td>H-Isn-RPRLCHKGP- Nle-CFO / 7</td><td> 25</td><td>Disulfide C⁶-C¹²</td>

<td>Example 1 9</td><td>pE-RPRLCHFGP- Nle-C-phenethyl-amine</td><td> 26</td><td>Disulfide C⁶-C¹²</td>

<td>Example 20</td><td>pE-RPRLCHK-Aib-P- Nle-CF-OH</td><td> 27</td><td>Disulfide C⁶-C¹²</td>

<td>Example 21</td><td>pE-RPRLCH- (4-NH- lsn) -GP-Nle-CF-OH</td><td> 28</td><td>Disulfide C⁶-C¹²</td>

<td>Example 22</td><td>pE-RPRLCHKGP- Nle-CK (Palmitoil) -OH</td><td> 29</td><td>Disulfide C⁶-C¹²</td>

<td>Example 23</td><td>pE-RPRLCK (Palmitoil) - KGP-Nle-CF-OH</td><td> 30</td><td>Disulfide C⁶-C¹²</td>

<td>Example 24</td><td>Palmitoil-020c-QRPR- LCHKGP-Nle-CF-OH</td><td> 31</td><td>Disulfide C⁶-C¹²</td>

127

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td>Example 25</td><td>Lauroil-O2Oc-QRPRL- CHKGP-Nle-CF-OH</td><td> 32</td><td>Disulfide C⁶-C¹²</td>

<td>Example 26</td><td>pE-RPRLCHKGP- Nle-CK (Lauroyl) -OH</td><td> 33</td><td>Disulfide C⁶-C¹²</td>

<td>Example 27</td><td>pE-RPRLCK (Lauroil) - KGP-Nle-CF-OH</td><td> 34</td><td>Disulfide C⁶-C¹²</td>

<td>Example 28</td><td>pE-RPCLCCKGP- Nle-CF-OH</td><td> 35</td><td>Disulfides C⁶-C¹², C⁴-C⁷</td>

<td>Example 29</td><td>pE-RCRLCCKGP- Nle-CF-OH</td><td> 36</td><td>Disulfides C⁶-C¹², C³-C⁷</td>

<td>Example 30</td><td>pE-rPRLCHKGP- Nle-CF-OH</td><td> 37</td><td>Disulfide C⁶-C¹²</td>

<td>Example 31</td><td>pE-FPRLCHKGP- Nle-CF-OH</td><td> 38</td><td>Disulfide C⁶-C¹²</td>

<td>Example 32</td><td>pE-EPRLCHKGP- Nle-CF-OH</td><td> 39</td><td>Disulfide C⁶-C¹²</td>

<td>Example 33</td><td>pE-RpRLCHKGP- Nle-CF-OH</td><td> 40</td><td>Disulfide C⁶-C¹²</td>

128

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td>Example 34</td><td>pE-RKRLCHKGP- Nle-CF-OH</td><td> 41</td><td>Disulfide C⁶-C¹²</td>

<td>Example 35</td><td>pE-RDRLCHKGP- Nle-CF-OH</td><td> 42</td><td>Disulfide C⁶-C¹²</td>

<td>Example 36</td><td>pE-RPFLCHKGP- Nle-CFO / 7</td><td> 43</td><td>Disulfide C⁶-C¹²</td>

<td>Example 37</td><td>pE-RPRKCHKGP- Nle-CF-OH</td><td> 44</td><td>Disulfide C⁶-C¹²</td>

<td>Example 38</td><td>pE-RPRLCHEGP- Nle-CF-OH</td><td> 45</td><td>Disulfide C⁶-C¹²</td>

<td>Example 39</td><td>pE-RPRLCHKDP- Nle-CF-OH</td><td> 46</td><td>Disulfide C⁶-C¹²</td>

<td>Example 40</td><td>pE-RPELCHKGP- Nle-CF-OH</td><td> 47</td><td>Disulfide C⁶-C¹²</td>

<td>Example 41</td><td>pE-RPR- (4-PhF) -CHK- GP-Nle-CF-OH</td><td> 48</td><td>Disulfide C⁶-C¹²</td>

<td>Example 42</td><td>pE-RPRDCHKGP- Nle-CF-OH</td><td> 49</td><td>Disulfide C⁶-C¹²</td>

129

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td>Example 43</td><td>pE-RPRLCEKGP- Nle-CF-OH</td><td> 50</td><td>Disulfide C⁶-C¹²</td>

<td>Example 44</td><td>pE-RPRLCHKLP- Nle-CF-OH</td><td> 51</td><td>Disulfide C⁶-C¹²</td>

<td>Example 45</td><td>pE-RPRLCHKRP- Nle-CFO / 7</td><td> 52</td><td>Disulfide C⁶-C¹²</td>

<td>Example 46</td><td>pE-RPRLCHKG- (Pipecolic acid) -Nle-C- F-OH</td><td> 53</td><td>Disulfide C⁶-C¹²</td>

<td>Example 47</td><td>pE-RPRLCHKGP- (3- PyA) -CF-OH</td><td> 54</td><td>Disulfide C⁶-C¹²</td>

<td>Example 48</td><td>pE-RPRLCHKGP- Nle-CHO / 7</td><td> 55</td><td>Disulfide C⁶-C¹²</td>

<td>Example 49</td><td>pE-RPRLCHKGP- Nle-CE-OH</td><td> 56</td><td>Disulfide C⁶-C¹²</td>

<td>Example 50</td><td>pE-RPRLCHKGP- Nle-C-OH</td><td> 57</td><td>Disulfide C⁶-C¹²</td>

130

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td>Example 51</td><td>pE-RPRLCHKGP- Nle-hC-F-OH</td><td> 58</td><td>Disulfide C⁶-C¹²</td>

<td>Example 52</td><td>pE-RPRL- / 7C-HKGP- Nle-hC-F-OH</td><td> 59</td><td>Disulfide C⁶-C¹²</td>

<td>Example 53</td><td>pE-RPRLcHKGP- Nle-CF-OH</td><td> 60</td><td>Disulfide C⁶-C¹²</td>

<td>Example 54</td><td>pE-RPRLCHKGP- Nle- (D- / 7C) -F-OH</td><td> 61</td><td>Disulfide O⁶-C¹²</td>

<td>Example 55</td><td>pE-RPRL- (D - /? C) -HKG- P-Nle- (D- / 7C) -F-OH</td><td> 62</td><td>Disulfide C⁶-C¹²</td>

<td>Example 56</td><td>pE-RPRLCHKGP- Nle-cF-OH</td><td> 63</td><td>Disulfide C⁶-C¹²</td>

<td>Example 57</td><td>pE-RPRLcHKGP- Nle-cF-OH</td><td> 64</td><td>Disulfide C⁶-C¹²</td>

<td>Example 58</td><td>Miristoil-O2Oc-O2Oc-QR- PRLCHKGP-Nle-Cf- OH</td><td> 65</td><td>Disulfide O⁶-C¹²</td>

131

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td>Example 59</td><td>Miristoil-O2Oc-O2Oc- O2Oc-QRPRLCHKG- P-Nle-Cf-OH</td><td> 66</td><td>Disulfide C⁶-C¹²</td>

<td>Example 60</td><td>Miristoil-O2Oc-O2Oc- O2Oc-O2Oc-QRPRLC- HKGP-Nle-Cf-OH</td><td> 67</td><td>Disulfide C⁶-C¹²</td>

<td>Example 61</td><td>pE-RPRLCH- K (Miristoil) -GP-Nle-CF- OH</td><td> 68</td><td>Disulfide C⁶-C¹²</td>

<td>Example 62</td><td>pE-RPRLCHKGP- Nle-CFA / H₂</td><td> 69</td><td>Disulfide C⁶-C¹²</td>

<td>Example 63</td><td>pE-RPRLCHKGP- Nle-CA / H₂</td><td> 70</td><td>Disulfide C⁶-C¹²</td>

<td>Example 64</td><td>pE-RPRLCHKGP- Nle-CF-OH</td><td> 71</td><td>-s-ch₂- C (= O) CH₂-S- [C⁶- C¹²]</td>

<td>Example 65</td><td>pE-RPRLCHKGP- Nle-CF-OH</td><td> 72</td><td>Monosulfide C⁶- C¹²</td>

132

<td>Example</td><td>Sequence</td><td>I KNOW THAT ID NO:</td><td>Ring Type</td>

<td>Example 66</td><td>pE-RPRLCHKGP- Nle-CF-OH</td><td> 73</td><td>-S-CH₂-C (= Z) - CH₂-S- [C⁶-C¹²]</td>

Analytical methods

1) HPLC - Analytical method A • Column; Bischoff UHC-640 (53 x 4.0 mm) with

ProntoSII 120-3-C18-H, 3 microns; Part Number: 0604F185PS030.

• Eluent A: 0.07 percent trifluoroacetic acid (TFA) in water / Eluent B: 0.1 percent trifluoroacetic acid (TFA) in acetonitrile (ACN).

• Flow: 1.5 milliliters / minute.

• Temperature: 40 ° C.

• Gradient:

<td>Time [min]</td><td>TO [%]</td><td>B [%]</td>

<td> 0.0</td><td> 90</td><td> 1 0</td>

<td> 9.5</td><td> 0</td><td> 100</td>

<td> 12.0</td><td> 0</td><td> 100</td>

<td> 12.2</td><td> 90</td><td> 10</td>

133

2a) UPLC-MS - Analytical Method B • Waters Acquity UPLC® BEH C18, 1.7 microns, 2.1 x 50 mm; Part Number: 186002350.

• Eluent A: 0.1 percent trifluoroacetic acid (TFA) in water; Eluent B: 0.1 percent trifluoroacetic acid (TFA) in acetonitrile (ACN).

• Flow: 0.7 milliliters / minute.

• Temperature: 40 ° C.

• Gradient:

<td>Time [min]</td><td>TO [%]</td><td>B [%]</td>

<td> 0 .0</td><td> 80</td><td> 20</td>

<td> 1.0</td><td> 75</td><td> 25</td>

<td> 4.2</td><td> 10</td><td> 90</td>

<td> 4.3</td><td> 0</td><td> 100</td>

<td> 4.6</td><td> 80</td><td> 20</td>

2b) UPLC-MS - Analytical Method C • Waters Acquity UPLC® BEH C18, 1.7 microns, 2.1 x 50 mm; Part Number: 186002350.

• Eluent A: 0.1 percent trifluoroacetic acid (TFA) in water; Eluent B: 0.1 percent trifluoroacetic acid (TFA) in

134 acetonitrile (ACN).

• Flow: 0.7 milliliters / minute.

• Temperature: 40 ° C.

• Gradient:

<td>Time [min]</td><td>TO [%]</td><td>B [%]</td>

<td> 0.0</td><td> 99</td><td> 1</td>

<td> 1.0</td><td> 97</td><td> 3</td>

<td> 3.5</td><td> 50</td><td> 50</td>

<td> 4.0</td><td> 10</td><td> 90</td>

<td> 4.3</td><td> 0</td><td> 100</td>

<td> 4.6</td><td> 80</td><td> 20</td>

The analytical data for the peptides of Examples 1 to 63 are summarized in Table 5, and were generated using the analytical methods described above.

Table 5

<td></td><td colspan="2">HPLC</td><td colspan="5">Mass spectrometry</td>

<td></td><td>tR</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td>

<td>Peptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>[min]</td><td>do</td><td>(measured)</td><td>(measured)</td><td>do</td><td>(cale.)</td><td>(cale.)</td>

135

<td></td><td colspan="2">HPLC</td><td colspan="3">Spectrometry c</td><td colspan="2">you masses</td>

<td>Peptide</td><td>To go</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td>

<td></td><td>[min]</td><td>do</td><td>(measured)</td><td>(measured)</td><td>do</td><td>(cale.)</td><td>(cale.)</td>

<td>Example 1</td><td> 4.16</td><td>TO</td><td> 766.3</td><td> 511.2</td><td>C</td><td> 766.4</td><td> 511.3</td>

<td>Example 2</td><td> 4.18</td><td>TO</td><td> 773.5</td><td> 515.8</td><td>C</td><td> 773.4</td><td> 516.0</td>

<td>Example 3</td><td> 4.14</td><td>TO</td><td></td><td> 506.6</td><td>c</td><td> 759.4</td><td> 506.6</td>

<td>Example 4</td><td> 4.15</td><td>TO</td><td> 752.4</td><td> 501.9</td><td>c</td><td> 752.4</td><td> 501.9</td>

<td>Example 5</td><td> 3.70</td><td>TO</td><td></td><td> 484.5</td><td>c</td><td> 726.4</td><td> 484.6</td>

<td>Example 6</td><td> 3.84</td><td>TO</td><td></td><td> 484.5</td><td>c</td><td> 726.4</td><td> 484.6</td>

<td>Example 7</td><td> 3.85</td><td>TO</td><td></td><td> 553.6</td><td>c</td><td> 829.9</td><td> 553.6</td>

<td>Example 8</td><td> 3.43</td><td>TO</td><td> 768.1</td><td> 512.4</td><td>c</td><td> 768.4</td><td> 512.6</td>

<td>Example 9</td><td> 3.77</td><td>TO</td><td></td><td> 495.2</td><td>c</td><td> 742.4</td><td> 495.3</td>

<td>Example 10</td><td> 3.74</td><td>TO</td><td> 742.5</td><td> 495.1</td><td>c</td><td> 742.4</td><td> 495.3</td>

<td>Example 11</td><td> 3.61</td><td>TO</td><td> 742.9</td><td> 495.2</td><td>c</td><td> 742.4</td><td> 495.3</td>

<td>Example 12</td><td> 3.62</td><td>TO</td><td></td><td> 497.8</td><td>c</td><td> 746.4</td><td> 497.9</td>

<td>Example 13</td><td> 3.49</td><td>TO</td><td> 768.3</td><td> 512.5</td><td>c</td><td> 768.4</td><td> 512.6</td>

<td>Example 14</td><td> 4.14</td><td>TO</td><td> 808.5</td><td> 539.2</td><td>c</td><td> 808.4</td><td> 539.3</td>

136

<td></td><td colspan="2">HPLC</td><td colspan="5">Mass spectrometry</td>

<td>Peptide</td><td>To go</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td>

<td></td><td>[min]</td><td>do</td><td>(measured)</td><td>(measured)</td><td>do</td><td>(cale.)</td><td>(cale.)</td>

<td>Example 15</td><td> 3.99</td><td>TO</td><td> 773.4</td><td> 515.8</td><td>C</td><td> 773.4</td><td> 515.9</td>

<td>Example 16</td><td> 3.36</td><td>TO</td><td></td><td> 475.5</td><td>C</td><td> 712.9</td><td> 475.6</td>

<td>Example 17</td><td> 3.28</td><td>TO</td><td></td><td> 527.5</td><td>c</td><td> 790.9</td><td> 527.6</td>

<td>Example 18</td><td> 3.36</td><td>TO</td><td></td><td> 512.5</td><td>c</td><td> 768.4</td><td> 512.6</td>

<td>Example 19</td><td> 4.38</td><td>TO</td><td> 756.0</td><td> 504.2</td><td>c</td><td> 755.9</td><td> 504.3</td>

<td>Example 20</td><td> 3.17</td><td>TO</td><td> 782.6</td><td> 522.0</td><td>c</td><td> 782.4</td><td> 521.9</td>

<td>Example 21</td><td> 3.45</td><td>TO</td><td></td><td> 512.0</td><td>c</td><td> 767.4</td><td> 511.9</td>

<td>Example 22</td><td> 6.12</td><td>TO</td><td></td><td> 585.6</td><td>B</td><td> 878.0</td><td> 585.7</td>

<td>Example 23</td><td> 6.46</td><td>TO</td><td> 883.2</td><td> 588.9</td><td>B</td><td> 883.0</td><td> 589.0</td>

<td>Example 24</td><td> 5.20</td><td>TO</td><td></td><td> 646.0</td><td>B</td><td> 968.6</td><td> 646.0</td>

<td>Example 25</td><td> 5.18</td><td>TO</td><td></td><td> 627.3</td><td>B</td><td> 940.5</td><td> 627.3</td>

<td>Example 26</td><td> 5.11</td><td>TO</td><td></td><td> 567.0</td><td>B</td><td> 850.0</td><td> 567.0</td>

<td>Example 27</td><td> 5.44</td><td>TO</td><td></td><td> 570.0</td><td>B</td><td> 855.0</td><td> 570.3</td>

<td>Example 28</td><td> 4.16</td><td>TO</td><td> 723.7</td><td></td><td>C</td><td> 723.8</td><td> 482.9</td>

137

<td></td><td colspan="2">HPLC</td><td colspan="5">Mass spectrometry</td>

<td>Peptide</td><td>To go</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td>

<td></td><td>[min]</td><td>do</td><td>(measured)</td><td>(measured)</td><td>do</td><td>(cale.)</td><td>(cale.)</td>

<td>Example 29</td><td> 3.85</td><td>TO</td><td> 753.0</td><td> 502.5</td><td>C</td><td> 753.3</td><td> 502.6</td>

<td>Example 30</td><td> 3.39</td><td>TO</td><td></td><td> 512.5</td><td>C</td><td> 768.4</td><td> 512.6</td>

<td>Example 31</td><td> 4.08</td><td>TO</td><td> 763.8</td><td> 509.4</td><td>c</td><td> 763.9</td><td> 509.6</td>

<td>Example 32</td><td> 3.59</td><td>TO</td><td> 754.8</td><td> 503.6</td><td>c</td><td> 754.9</td><td> 503.6</td>

<td>Example 33</td><td> 3.36</td><td>TO</td><td></td><td> 512.5</td><td>c</td><td> 768.4</td><td> 512.6</td>

<td>Example 34</td><td> 3.14</td><td>TO</td><td></td><td> 522.8</td><td>c</td><td> 783.9</td><td> 522.9</td>

<td>Example 35</td><td> 3.36</td><td>TO</td><td></td><td> 518.5</td><td>c</td><td> 777.4</td><td> 518.6</td>

<td>Example 36</td><td> 3.91</td><td>TO</td><td> 763.8</td><td> 509.4</td><td>c</td><td> 763.9</td><td> 509.6</td>

<td>Example 37</td><td> 3.05</td><td>TO</td><td></td><td> 517.5</td><td>c</td><td> 775.9</td><td> 517.6</td>

<td>Example 38</td><td> 3.67</td><td>TO</td><td> 768.7</td><td> 512.8</td><td>c</td><td> 768.9</td><td> 512.9</td>

<td>Example 39</td><td> 3.47</td><td>TO</td><td></td><td> 531.7</td><td>c</td><td> 797.4</td><td> 531.9</td>

<td>Example 40</td><td> 3.60</td><td>TO</td><td> 754.9</td><td> 503.6</td><td>c</td><td> 754.9</td><td> 503.6</td>

<td>Example 41</td><td> 3.91</td><td>TO</td><td></td><td> 549.1</td><td>c</td><td> 823.4</td><td> 549.3</td>

<td>Example 42</td><td> 3.10</td><td>TO</td><td> 769.2</td><td> 513.1</td><td>c</td><td> 769.4</td><td> 513.2</td>

138

<td></td><td colspan="2">HPLC</td><td colspan="5">Mass spectrometry</td>

<td>Peptide</td><td>To go</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td>

<td></td><td>[min]</td><td>do</td><td>(measured)</td><td>(measured)</td><td>do</td><td>(cale.)</td><td>(cale.)</td>

<td>Example 43</td><td> 3.58</td><td>TO</td><td> 764.2</td><td> 509.7</td><td>C</td><td> 764.4</td><td> 509.9</td>

<td>Example 44</td><td> 3.82</td><td>TO</td><td></td><td> 531.1</td><td>C</td><td> 796.4</td><td> 531.3</td>

<td>Example 45</td><td> 3.16</td><td>TO</td><td></td><td> 545.5</td><td>c</td><td> 817.9</td><td> 545.6</td>

<td>Example 46</td><td> 3.54</td><td>TO</td><td></td><td> 517.1</td><td>c</td><td> 775.4</td><td> 517.3</td>

<td>Example 47</td><td> 2.53</td><td>TO</td><td></td><td> 524.1</td><td>c</td><td> 785.9</td><td> 524.3</td>

<td>Example 48</td><td> 2.49</td><td>TO</td><td></td><td> 509.2</td><td>c</td><td> 763.4</td><td> 509.3</td>

<td>Example 49</td><td> 2.73</td><td>TO</td><td> 759.3</td><td> 506.5</td><td>c</td><td> 759.4</td><td> 506.6</td>

<td>Example 50</td><td> 2.72</td><td>TO</td><td> 694.5</td><td></td><td>c</td><td> 694.8</td><td> 463.6</td>

<td>Example 51</td><td> 3.38</td><td>TO</td><td></td><td> 517.1</td><td>c</td><td> 775.4</td><td> 517.3</td>

<td>Example 52</td><td> 3.45</td><td>TO</td><td></td><td> 521.9</td><td>c</td><td> 782.4</td><td> 521.9</td>

<td>Example 53</td><td> 3.52</td><td>TO</td><td> 768.4</td><td> 512.5</td><td>c</td><td> 768.4</td><td> 512.6</td>

<td>Example 54</td><td> 3.43</td><td>TO</td><td> 775.3</td><td> 517.1</td><td>c</td><td> 775.4</td><td> 517.3</td>

<td>Example 55</td><td> 3.83</td><td>TO</td><td> 782.3</td><td> 521.8</td><td>c</td><td> 782.4</td><td> 521.9</td>

<td>Example 56</td><td> 3.42</td><td>TO</td><td> 768.1</td><td> 512.4</td><td>c</td><td> 768.4</td><td> 512.6</td>

139

<td></td><td colspan="2">HPLC</td><td colspan="5">Mass spectrometry</td>

<td>Peptide</td><td>To go</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td><td>Me to-</td><td>[M + 2H]²⁺</td><td>[M + 3H]³⁺</td>

<td></td><td>[min]</td><td>do</td><td>(measured)</td><td>(measured)</td><td>do</td><td>(cale.)</td><td>(cale.)</td>

<td>Example 57</td><td> 3.66</td><td>TO</td><td> 768.3</td><td> 512.4</td><td>C</td><td> 768.4</td><td> 512.6</td>

<td>Example 58</td><td> 5.68</td><td>TO</td><td></td><td> 685.0</td><td>B</td><td> 1027.1</td><td> 685.0</td>

<td>Example 59</td><td> 5.58</td><td>TO</td><td></td><td> 733.4</td><td>B</td><td> 1099.6</td><td> 733.4</td>

<td>Example 60</td><td> 5.55</td><td>TO</td><td></td><td> 781.8</td><td>B</td><td> 1172.1</td><td> 781.8</td>

<td>Example 61</td><td> 3.18</td><td>D</td><td> 874.5</td><td> 582.9</td><td>D</td><td> 873.5</td><td> 585.7</td>

<td>Example 62</td><td> 3.22</td><td>TO</td><td></td><td> 512.3</td><td>C</td><td> 767.9</td><td> 512.3</td>

<td>Example 63</td><td> 2.71</td><td>TO</td><td> 694.3</td><td> 463.1</td><td>C</td><td> 694.4</td><td> 463.2</td>

General Synthesis Procedures

1) Load of the first amino acid on the chloride resin of

2-Chloro-Trityl and Fmoc Removal

The 2-chloro-trityl chloride resin (1 equivalent, 1.0 to 1.6 mmol / gram) was washed thoroughly with dichloromethane (DCM). The desired amino acid (typically 0.5 to 2 equivalents in relation to the resin, considering a load of 1.6 millimoles / gram) was dissolved in dichloromethane (DCM) (approximately 10 milliliters per gram of resin), and di-isopropyl-ethyl amine (DIPEA) (4 equivalents in relation to the resin,

140 considering a load of 1.6 millimoles / gram). The solution was added to the resin, and the suspension was stirred at room temperature for 19 hours. The resin was drained, and then washed thoroughly in sequence with dichloromethane (DCM) / MeOH / DIPEA (17: 2: 1), dichloromethane (DCM), DMA, dichloromethane (DCM).

For Fmoc removal and load determination, the resin was repeatedly stirred with piperidine / DMA (1: 4) or with 4-methyl-piperidine / DMA (1: 4) (12 x 10 milliliters per gram of the initial resin) , and washed with DMA (10 milliliters, 2 times per gram of the initial resin). The combined solutions were diluted with methanol to a volume V of 250 milliliters per gram of the initial resin. An aliquot of 2 milliliters (V_to) of this solution was further diluted to 250 milliliters (V,) with methanol. Absorption

UV was measured at 299.8 nanometers against a MeOH reference, giving absorption A. The resin was washed thoroughly in sequence with DMA, dichloromethane (DCM), DMA, dichloromethane (DCM), and dried under high vacuum. at 40 ° C, providing m grams of resin.

The resin load is calculated according to the formula:

Load [moles / gram] = (W x V_t χ V) ld χ ε x V_to xm) (with d: cuvette width; ε = 7800 L mol¹ cm¹)

2) Synthesis of solid phase peptides in the Prelude synthesizer^Mfi

2a) Synthesis cycle A

The resin was washed with DMA. Fmoc was removed by repetitive treatment with 4-methyl-piperidine / DMA (1: 4). The resin is

141 washed with DMA. Coupling was done by the addition of Fmoc-amino acid (3 equivalents; 0.2 M solution in NMP), HCTU (3 equivalents; 0.3 M solution in NMP), and di-isopropyl ethyl amine (DIPEA) (3.3 equivalents; solution 0.66 M in NMP), followed by mixing the suspension with nitrogen at room temperature for typically 15 minutes to 4 hours, depending on the specific requirements. After washing with DMA, the coupling step was typically repeated 1 to 3 times, depending on the specific requirements. After washing with DMA, the capping was carried out by adding a mixture of Ac₂O / pyridine / DMA (1: 1: 8), and the subsequent mixture of the suspension at room temperature. The resin was washed with DMA.

2b) Synthesis cycle B

The resin was washed with DMA. Fmoc was removed by repetitive treatment with piperidine / DMA (1: 4). The resin was washed with DMA. Coupling was done by adding Fmocamino acid (3 equivalents; 0.3 M solution in NMP), HCTU (3 equivalents; 0.3 M solution in NMP), and di-isopropyl ethyl amine (DIPEA) (4.5 equivalents; 0.9 M solution in NMP), followed by mixing the suspension with nitrogen at room temperature for typically 15 minutes to 4 hours, depending on the specific requirements. After washing with DMA, the coupling step was typically repeated 1 to 3 times, depending on the specific requirements. After washing with DMA, the capping was carried out by adding a mixture of Ac₂O / pyridine /

142

DMA (1: 1: 8), and the subsequent mixing of the suspension at room temperature. The resin was washed with DMA.

2c) Synthesis cycle C

The resin was washed with DMA. Fmoc was removed by repetitive treatment with piperidine / DMA (1: 4). The resin was washed with DMA. Coupling was done by adding Fmocamino acid (3 equivalents; 0.3 M solution in NMP), HCTU (3 equivalents; 0.3 M solution in NMP), and di-isopropyl ethyl amine (DIPEA) (6 equivalents; 0.9 M solution in NMP), followed by mixing the suspension with nitrogen at room temperature for typically 15 minutes to 4 hours, depending on the specific requirements. After washing with DMA, the coupling step was typically repeated 1 to 3 times, depending on the specific requirements. After washing with DMA, the capping was carried out by adding a mixture of Ac₂O / pyridine / DMA (1: 1: 8), and the subsequent mixture of the suspension at room temperature. The resin was washed with DMA.

2d) Synthesis cycle D

The resin was washed with DMA. Fmoc was removed by repetitive treatment with 4-methyl-piperidine / DMA (1: 4). The resin was washed with DMA. The coupling was done by adding a mixture of the Fmoc-amino acid and Pure Oxima (3 equivalents of each; 0.2 M of both in NMP), and DIC (3 equivalents; solution 0.3 M in NMP), followed by the mixture of the suspension with nitrogen at room temperature for typically 15 minutes to 4 hours,

143 Depending on the specific requirements. After washing with DMA, the coupling step was typically repeated 1 to 3 times, depending on the specific requirements. After washing with DMA, the capping was carried out by adding a mixture of Ac₂O / pyridine / DMA (1: 1: 8), and the subsequent mixture of the suspension at room temperature. The resin was washed with DMA.

3) Dissociation from the resin with or without the concomitant removal of the protective groups

3a) Dissociation method A

The resin (0.1 millimoles) was stirred at room temperature for 2 hours with 95 percent aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (3 milliliters). The dissociation solution was filtered, and fresh solution (3 milliliters) was added. The suspension was stirred at room temperature for 1 hour, then the dissociation solution was filtered. Fresh solution (3 milliliters) was added, and the suspension was stirred at room temperature for 1 hour. The dissociation solution was filtered. The combined dissociation solutions were slowly poured onto a mixture of cold heptane / diethyl ether (1: 1) (35 milliliters), giving a precipitate. The suspension was centrifuged and the supernatant poured. The residue was washed with cold heptane / diethyl ether (1: 1) (10 milliliters), the suspension was centrifuged, and the supernatant was poured. The solid was dried in a high vacuum.

3b) Dissociation method B

The resin (0.1 mmol) was treated with 95 percent acid

144 aqueous trifluoroacetic acid (TFA) / EDT (4: 1) (0.75 milliliters), and the suspension was stirred at room temperature for 1 hour. A mixture of 95 percent aqueous trifluoroacetic acid (TFA) (2.18 milliliters), and TES (75 microliters) was added, and stirring was resumed at room temperature for 1 hour. The dissociation solution was then filtered, 95 percent aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (3 milliliters) was added to the resin, and the suspension was stirred at room temperature for 1 hour. The dissociation solution was filtered and collected, and fresh solution (3 milliliters) was added. The suspension was stirred at room temperature for 1 hour, then the dissociation solution was filtered. The combined dissociation solutions were poured onto cold heptane / diethyl ether (1: 1) (35 milliliters). The precipitate formed in this way was allowed to settle, centrifuged, and then the supernatant was carefully poured. The precipitate was washed once with cold heptane / diethyl ether (1: 1) (10 milliliters), the suspension was centrifuged, and the supernatant was poured. The residue was dried under high vacuum.

3c) Dissociation method C

HFIP / DCM (30:70) (5 milliliters) was added to the resin (0.1 millimoles), and the suspension was stirred at room temperature for 1.5 hours. The dissociation solution was filtered and collected, and fresh HFIP / DCM (30:70) (5 milliliters) were added. The suspension was stirred at room temperature for 30 minutes. The dissociation solution was filtered and collected. The resin was washed with dichloro145 methane (DCM) (5 milliliters, 2 times), which was also collected.

The combined dissociation and washing solutions were concentrated to dryness under high vacuum. The residue was lyophilized from tBuOH / H₂Or (1: 1).

4) Cycling methods

4a) Cyclization method A (Disulfide formation)

The completely unprotected linear precursor peptide was dissolved in H₂0 / dimethyl sulfoxide (DMSO) (9: 1) or (4: 1), to typically give a concentration of 1 to 15 milligrams / milliliter. The reaction mixture was then stirred at room temperature for typically 40 hours, depending on the requirements, and then concentrated to dryness in a high vacuum.

4b) Cyclization method B (Disulfide formation)

The completely unprotected linear precursor peptide (1 equivalent) was dissolved in H₂Or to typically give a concentration of 10 milligrams / milliliter. A solution of l was added₂ 50 mM in AcOH (1.2 equivalents) in one portion to the stirred solution, and the reaction was stirred for 10 minutes at room temperature, 0.5 M ascorbic acid in H was added₂O (1.5 equivalents) to turn off excess l₂. The solution was concentrated to near dryness in vacuo.

<td>4c) Method</td><td>of cyclisation C</td><td colspan="2">(Selective formation of two</td>

<td>disulfides)</td><td></td><td></td><td></td>

<td>Peptide</td><td>linear precursor</td><td>partially protected</td><td> (1</td>

<td>equivalent) (two</td><td>cysteines were</td><td>protected with Acm and</td><td>two</td>

146 cysteines were left unprotected) dissolved in AcOH / H₂Or (4: 1), to typically give a concentration of 1 milligram / milliliter. L was added₂ 50 mM in AcOH (2 equivalents), and the reaction mixture was stirred at room temperature for 1 hour. Added more l₂fifty mM in AcOH (10 equivalents) in portions for 4 hours. After 21 hours, the reaction mixture was concentrated to near dryness in vacuo, and 1M ascorbic acid in H was added.₂Or in excess to turn off the l₂ without reacting

4d) Cycling method D (Lactam formation between the side chains)

The completely unprotected linear precursor peptide (1 equivalent), and HATU (1.5 equivalents), were dissolved in NMP (peptide concentration: typically 1 millimol / liter). Diisopropyl ethyl amine (DIPEA) (3 equivalents) was added, and the solution was stirred at room temperature for 90 minutes. The reaction mixture was concentrated to dryness in vacuo.

4e) Cyclization method E (Lactam formation between the side chain and the term C)

A solution of the peptide (1 equivalent), HATU (1.3 equivalents), and HOAt (1.3 equivalents) in N, N-dimethylformamide (DMF) (peptide concentration: 2.6 mmol / liter), was treated with 2.6- lutidine (20 equivalents), and the reaction was stirred at room temperature for 2 hours. The reaction mixture was concentrated to dryness in vacuo.

In the following, the synthesis of the examples are described

147 representative.

Example 1 Synthesis of pE-RPRLKHFGP-Nle-D-phenethylamine (SEQ ID NO: 8) (lactam K⁶-D¹²). The following Example 1 discloses SEQ ID NO: 8.

SPPS

1, Dissociation / Removal of PG

two. Purification (Fenetf / o (1b)

1. Cyclization

two. Purification

Example 1 • Preparation of Intermediary • (Linear Peptide Assembly)

Phenethyl amine-BAL-PS resin (167 milligrams, 0.100 millimoles) was subjected to solid phase peptide synthesis in the Prelude peptide synthesizer^MR. The coupling was carried out

148 as follows:

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 1</td><td>D (tBu)</td><td>2 x 4 h</td><td>C</td>

<td> 2</td><td>Nle</td><td>1 x 3 h</td><td>C</td>

<td> 3</td><td>P</td><td>2 x 45 min</td><td>c</td>

<td> 4</td><td>G</td><td>2 x 90 min</td><td>c</td>

<td> 5</td><td>F</td><td>1 x 3 h</td><td>c</td>

<td> 6</td><td>H (Trt)</td><td>2 x 45 min</td><td>c</td>

<td> 7</td><td>K (Boc)</td><td>2 x 4 h</td><td>c</td>

<td> 8</td><td>L</td><td>4 x 1 h</td><td>c</td>

<td> 9</td><td>R (Pbf)</td><td>4 x 1 h</td><td>c</td>

<td> 10</td><td>P</td><td>2 x 90 min</td><td>c</td>

<td> 1 1</td><td>R (Pbf)</td><td>4 x 1 h</td><td>c</td>

149

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 12</td><td>PE</td><td>2 x 90 min</td><td>C</td>

• Preparation of Intermediate Ib • (Dissociation from the resin with the concomitant removal of the protective group and then purification)

A mixture of 95 percent aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (2 milliliters) was added to intermediate 1a (0.1 millimoles), and the suspension was stirred at room temperature for 2.5 hours. The dissociation solution was filtered, and fresh dissociation solution (2 milliliters) was added. The suspension was stirred at room temperature for 45 minutes, and then the dissociation solution was filtered. Fresh solution (2 milliliters) was added, and the suspension was stirred at room temperature for 45 minutes. The dissociation solution was filtered, and the resin was washed with 95 percent aqueous trifluoroacetic acid (TFA) (1 milliliter). The combined dissociation solutions were poured onto a mixture of cold heptane / diethyl ether (1: 1) (35 milliliters), giving a precipitate. The suspension was centrifuged and the supernatant poured. The residue was washed with cold heptane / diethyl ether (1: 1) (20 milliliters), the suspension was centrifuged and the supernatant was poured. The solid was dried in a high vacuum. The crude product was purified by HPLC

150 of preparation and lyophilized from ACN / H₂Or, to provide intermediate 1b as a white solid in two batches of different qualities: Lot A (35.9 milligrams (98 percent purity), 0.018 millimoles), and Lot B (52.9 milligrams (80 percent purity), 0.021 millimoles).

• Preparation of Example 1 • (delation and purification)

Both batches from the previous step were treated separately following the same protocol:

Lot A: A solution of the peptide (35.9 milligrams (98 percent purity), 0.018 millimoles), and HATU (10.0 milligrams, 0.026 millimoles) in NMP (18 milliliters), and di-isopropyl-ethyl-amine (DIPEA) ( 9.2 microliters, 0.053 mmol), was stirred at room temperature for 2 hours.

Lot B: A solution of the peptide (52.9 milligrams (80 percent purity), 0.021 millimoles), and HATU (14.5 milligrams, 0.038 millimoles) in NMP (26 milliliters), and di-isopropyl-ethyl-amine (DIPEA) ( 13.0 microliters, 0.076 mmol), was stirred at room temperature for 2 hours.

Each batch was concentrated to dryness under vacuum. The product was isolated by preparation HPLC. The pure fractions of both purifications were combined and lyophilized from ACN / H₂Or to give Example 1 as a white solid (52.0 milligrams, 0.025 millimoles).

The pure product was analyzed by analytical HPLC (Method

151 analytical A: t_R = 4.16 minutes), and UPLC-MS (Analytical Method C; measured: [M + 3]³⁺= 511.2; calculated: [M + 3]³⁺= 511.3).

Examples Synthesis pE-RPRLKFKGP-Nle-F (SEQ ID NO:

12) (Lactam K⁶-term C). The following Example 5 discloses SEQ ID NO: 12)

GÉPOL

<img file="CU20140097A7_D0013.tif" />

1, Removal of ivDde

two. RFIP dissociation

-K- F ~ K (Boc&gt; -G 'P-Nle-F-OH

Ctdation

<img file="CU20140097A7_D0014.tif" />

(5c) .pR (Pbf&gt; LKFK (Boc&gt; GP »Nte-F

1, PG Removal

two. Purification

152 • Preparation of Intermediary 5a • (Loading of 2-chloro-trityl chloride resin with Fmoc-F-OH, removal of Fmoc, and determination of resin loading)

The 2-chloro-trityl chloride resin (10.0 grams, 16.0 millimoles) was reacted with a solution of Fmoc-F-OH (6.24 grams, 32.0 millimoles) in dichloromethane (DCM) (100 milliliters), and di -isopropyl-ethyl-amine (DIPEA) (11.2 milliliters, 64.0 millimoles), in analogy to the general procedure described above, to give Intermediary 5a (12.8 grams, load = 0.79 millimoles / gram).

• Preparation of Intermediary 5b • (Linear Peptide Assembly)

Intermediate 5a (0.100 mmol) was subjected to solid phase peptide synthesis in the Prelude peptide synthesizer^MR. The coupling was carried out as follows:

<td rowspan="2">Couple I lie</td><td rowspan="2">AA</td><td colspan="2">Number of links</td><td rowspan="2">Cycle synthesis</td>

<td>x Time of</td><td>reaction</td>

<td> 2</td><td>Nle</td><td>2 x 90</td><td>min</td><td>B</td>

<td> 3</td><td>P</td><td>2 x 30</td><td>min</td><td>B</td>

<td> 4</td><td>G</td><td>2 x 90</td><td>min</td><td>B</td>

<td> 5</td><td>K (ivDde)</td><td>2 x 30</td><td>min</td><td>B</td>

<td> 6</td><td>F</td><td>2 x 30</td><td>min</td><td>B</td>

153

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 7</td><td>K (Boc)</td><td>4 x 1 h</td><td>B</td>

<td> 8</td><td>L</td><td>2 x 30 min</td><td>B</td>

<td> 9</td><td>R (Pbf)</td><td>4 x 1 h</td><td>B</td>

<td> 1 0</td><td>P</td><td>2 x 90 min</td><td>B</td>

<td> 1 1</td><td>R (Pbf)</td><td>4 x 1 h</td><td>B</td>

<td> 1 2</td><td>PE</td><td>2 x 90 min</td><td>B</td>

• Preparation of Intermediary 5c • (Removal of ivDde and dissociation from the resin)

Intermediate 5b (0.100 millimoles) was treated six times for 10 minutes with a solution of hydrazine monohydrate (0.081 milliliters, 1.67 mmol) in N, N-dimethyl-acetamide (4 milliliters). The resin was then treated three times for 20 minutes with a solution of hydrazine monohydrate (0.081 milliliters, 1.67 millimoles) in tetrahydrofuran (THF) (4 milliliters). The resin was washed with dichloromethane (DCM) (3 times). HFIP / DCM (30:70) (5 milliliters) was added to the resin (0.100 millimoles), and the suspension was stirred at room temperature for 1.5 hours. The dissociation solution was filtered and fresh HFIP / DCM (30:70) (5 milliliters) were added. The

154 suspension was stirred at room temperature for 30 minutes. The dissociation solution was filtered. The resin was washed with dichloromethane (DCM) (5 milliliters, 2 times). The combined dissociation and washing solutions were concentrated to dryness under vacuum. The residue was lyophilized from tBuOH / H₂Or (1: 1), to give Intermediary 5c (187 milligrams, 0.090 millimoles).

• Preparation of Example 5 • (Cycling and removal of protective groups)

A solution of Intermediary 5c (187 milligrams, 0.090 millimoles), HATU (44.6 milligrams, 0.117 millimoles), and HOAt (16.0 milligrams, 0.117 millimoles) in Ν, Ν-dimethylformamide (DMF) (35 milliliters), was treated with 2,6-lutidine (0.210 milliliters, 1.80 mmol), and the reaction was stirred at room temperature for 2 hours. The reaction mixture was concentrated to dryness in vacuo. The residue was dissolved in 95 percent aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (5 milliliters), and the solution was stirred at room temperature for 2.5 hours. The dissociation solution was poured onto cold heptane / diethyl ether (1: 1) (30 milliliters), giving a precipitate. The suspension was centrifuged and the supernatant poured. The residue was washed with cold heptane / diethyl ether (1: 1) (10 milliliters), the suspension was centrifuged and the supernatant was poured. The washing step was repeated once. The residue was dried under high vacuum. The product was isolated by preparation HPLC, and lyophilized from ACN / H₂Or, to provide Example 5 as a white solid (41.4 milligrams,

155

0.023 millimoles).

The pure product was analyzed by analytical HPLC (Analytical method A: t_R = 3.70 minutes), and UPLC-MS (Analytical Method C; measured: [M + 3]³⁺= 484.5; calculated: [M + 3]³⁺= 484.6).

Example 7 Synthesis of QRPRLCFKGP-Nle-CFGG (SEQ ID NO: 14) (Lactam term N - term C). The following Example 7 discloses SEQ ID NO: 14.

156

CÍ-POL

Resin loading

HGO-POL

1. SPPS

two. Fmoc Removal

HQ (Trt) -R (Pbf) -PR (Pbf&gt; LC (Trt&gt; -FK (Boc&gt; GP-Nte-C (Trt&gt; FGG ~ GPClL W

Dissociation with HFIP

H-Gfm) -R (Pbf) -PR (Pbf&gt; LC (T &lt;FK (BQC&gt; GP-Nte-C (Trt) -FGG-OH

Cídaclón

f) ~ PR (PbfK “C {Trt&gt; F &lt;(Boc&gt; G ~ P-Nle-C (Trt&gt; FG \ *&gt;

(7d)

1. PG removal

2, Purification

QRP ~ RL ~ C-'F »K'GP-Nte-C''F ~ GG

1. delation

Purification

GRPRLCFKGP-Nle-CFGG

Example 7

Intermediate Preparation 7a

157 • (Loading of 2-chloro-trityl chloride resin with Fmoc-GlyOH, Fmoc removal, and determination of resin loading)

The 2-chloro-trityl chloride resin (2.00 grams, 3.20 millimoles) was reacted with a solution of Fmoc-Gly-OH (0.476 grams, 1.60 millimoles) in dichloromethane (DCM) (20 milliliters), and di -isopropyl-ethyl-amine (DIPEA) (2.24 milliliters, 12.8 millimoles), in analogy to the general procedure described above, to give Intermediary 7a (2.22 grams; load = 0.68 millimoles / gram).

• Preparation of Intermediary 7b • (Fmoc linear peptide removal assembly)

Intermediary 7a (147 milligrams, 0.100 millimoles) was subjected to solid phase peptide synthesis in the Prelude peptide synthesizer^MR. The coupling was carried out as follows:

<td rowspan="2">Couple I lie</td><td rowspan="2">AA</td><td colspan="2">Number of links</td><td rowspan="2">Cycle synthesis</td>

<td>x Time of</td><td>reaction</td>

<td> 1</td><td>G</td><td>2 x 30</td><td>min</td><td>B</td>

<td> 2</td><td>F</td><td>2 x 30</td><td>min</td><td>B</td>

<td> 3</td><td>C (Trt)</td><td>2 x 30</td><td>min</td><td>B</td>

<td> 4</td><td>Nle</td><td>2 x 90</td><td>min</td><td>B</td>

<td> 5</td><td>P</td><td>2 x 30</td><td>min</td><td>B</td>

158

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 6</td><td>G</td><td>2 x 90 min</td><td>B</td>

<td> 7</td><td>K (Boc)</td><td>2 x 30 min</td><td>B</td>

<td> 8</td><td>F</td><td>2 x 30 min</td><td>B</td>

<td> 9</td><td>C (Trt)</td><td>2 x 30 min</td><td>B</td>

<td> 10</td><td>L</td><td>2 x 30 min</td><td>B</td>

<td> 1 1</td><td>R (Pbf)</td><td>4 x 1 h</td><td>B</td>

<td> 1 2</td><td>P</td><td>2 x 90 min</td><td>B</td>

<td> 13</td><td>R (Pbf)</td><td>4 x 1 h</td><td>B</td>

<td> 14</td><td>Q (Trt)</td><td>2 x 90 min</td><td>B</td>

After assembly of the peptide, the Fmoc was removed by repetitive treatment with piperidine / DMA (1: 4). The resin was washed with DMA, to provide intermediate 7b (0.100 millimoles).

• Preparation of Intermediary 7c • (Dissociation of HFIP from resin)

HFIP / DCM (30:70) (3 milliliters) was added to the intermediary

159

7b (0.100 mmol), and the suspension was stirred at room temperature for 1.5 hours. The dissociation solution was filtered, and fresh HFIP / DCM (30:70) (3 milliliters) were added. The suspension was stirred at room temperature for 30 minutes. The dissociation solution was filtered. The resin was washed with dichloromethane (DCM) (3 milliliters, 2 times). The combined dissociation and washing solutions were concentrated to dryness under vacuum. The residue was lyophilized from tBuOH / H₂Or (1: 1), to give Intermediary 7c (203 milligrams, 0.067 millimoles).

• Preparation of Intermediary 7d • (Cycle of the base structure)

A solution of Intermediary 7c (203 milligrams, 0.067 millimoles), HATU (33.3 milligrams, 0.088 millimoles), and HOAt (11.9 milligrams, 0.088 millimoles) in Ν, Ν-dimethylformamide (DMF) (40 milliliters), was treated with 2,6-lutidine (0.157 milliliters, 1.35 millimoles), and the reaction was stirred at room temperature for 2 hours. The reaction mixture was concentrated to dryness in vacuo, to provide intermediate 7d (0.067 mmol).

• Preparation of Intermediary 7e • (Removal of protective groups, and then purification)

A mixture of 95 percent aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (3 milliliters), was added to intermediate 7d (0.067 millimoles), and the suspension was stirred at room temperature for 2.5 hours The solution was poured onto a mixture of cold heptane / diethyl ether (1: 1) (30 milliliters), giving a precipitate.

160

The suspension was centrifuged and the supernatant poured. The residue was washed with cold heptane / diethyl ether (1: 1) (10 milliliters), the suspension was centrifuged and the supernatant was poured. The washing step was repeated once. The solid was dried in a high vacuum. The crude product was purified by preparation HPLC, and lyophilized from ACN / H₂Or, to provide intermediate 7e as a white solid (33.6 milligrams, 0.017 millimoles).

• Preparation of Example 7 • (Cycling and purification)

Intermediary 7e (33.6 milligrams, 0.017 millimoles) dissolved in H₂O / dimethyl sulfoxide (DMSO) (9: 1) (30 milliliters). The reaction mixture was stirred at room temperature for 40 hours; then it was concentrated to dryness under vacuum. The crude product was purified by preparation HPLC, and lyophilized from ACN / H₂Or, to provide Example 7 as a white solid (21.0 milligrams; 0.010 millimoles).

The pure product was analyzed by analytical HPLC (Analytical method A: t_R = 3.85 minutes), and UPLC-MS (Analytical Method C; measured: [M + 3]³⁺= 553.6; calculated: [M + 3]³⁺=553.6).

Example 8 Synthesis of pE-RPRLCHKGP-Nle-CF-OH (SEQ ID NO: 15) (disulfide C⁶-C¹²). The following Example 8 discloses SEQ ID NO: 15.

161

CI-POL

Resin loading

HFO-POL

SPPS pE-R (Pbf&gt; PR (Pbf&gt; LC (Trt&gt; H (Trt&gt; K (8oc&gt; -GP-Nte-C (Trt) -F-ai

Dissociation / Removal of PG pE-RPRL &lt;PR-K ~ G ~ P-Nie ~ C ~ F-OW

1, Cídadón

two. Purification pE'RPRLC&gt; HK «GP-Nie-OF&gt; OW

Example 8 • Preparation of Intermediary 8a • (Loading of 2-chloro-trityl chloride resin with Fmoc-f-OH, removal of Fmoc, and determination of resin loading)

The 2-chloro-trityl chloride resin (40.0 grams, 64.0 mmol) was washed with dichloromethane (DCM) (3 times). A solution of Fmoc-F-OH (24.8 grams, 64.0 mmol) in dichloromethane (DCM) (400 milliliters), and di-isopropyl-ethyl amine (DIPEA) (44.7 milliliters, 256 millimoles) was added, and the suspension was stirred for 22 hours to

162 room temperature. The resin was washed thoroughly with dichloromethane (DCM) / MeOH / DIPEA (17: 2: 1) (3 times), dichloromethane (DCM) (3 times), DMA (3 times), dichloromethane (DCM) ( 3 times).

The resin was then treated four times for 10 minutes with a mixture of piperidine / DMA (1: 4) (400 milliliters), followed by washing with DMA (180 milliliters, 2 times). Piperidine / DMA solutions and DMA wash solutions were collected for the determination of resin loading. 1 milliliter of the combined solutions was diluted to 500 milliliters with methanol, and UV absorption was measured at 299.8 nanometers as A = 0.368. This corresponds to an Fmoc amount of 46.2 millimoles.

The resin was washed thoroughly with dichloromethane (DCM) (3 times), DMA (3 times), dichloromethane (DCM) (3 times), and dried under vacuum, to give Intermediary 8a (50.7 grams; load = 0.91 millimoles / gram).

• Preparation of Intermediate 8b • (Linear Peptide Assembly)

Intermediary 8a (2.64 grams, 2.40 mmol) was subjected to solid phase peptide synthesis in the Prelude peptide synthesizer^MR. The coupling was carried out as follows:

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 1</td><td>C (Trt)</td><td>2 x 30 min</td><td>D</td>

163

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 2</td><td>Nle</td><td>2 x 15 min</td><td>TO</td>

<td> 3</td><td>P</td><td>2 x 15 min</td><td>TO</td>

<td> 4</td><td>G</td><td>2 x 30 min</td><td>TO</td>

<td> 5</td><td>K (Boc)</td><td>2 x 15 min</td><td>TO</td>

<td> 6</td><td>H (Trt)</td><td>2 x 15 min</td><td>TO</td>

<td> 7</td><td>C (Trt)</td><td>2 x 60 min</td><td>D</td>

<td> 8</td><td>L</td><td>2 x 15 min</td><td>TO</td>

<td> 9</td><td>R (Pbf)</td><td>4 x 1 h</td><td>TO</td>

<td> 10</td><td>P</td><td>2 x 15 min</td><td>TO</td>

<td> 1 1</td><td>R (Pbf)</td><td>4 x 1 h</td><td>TO</td>

<td> 12</td><td>PE</td><td>2 x 15 min</td><td>TO</td>

• Preparation of Intermediary 8c • (Dissociation from the resin with the concomitant removal of the protective group)

Intermediary 8b (2.40 mmol) was washed thoroughly with dichloromethane (DCM) (4 times). A mixture of 95 was added

164 percent aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (50 milliliters), and the suspension was stirred at room temperature for 1 hour. The dissociation solution was filtered, and fresh dissociation solution (35 milliliters) was added. The suspension was stirred at room temperature for 1 hour, and then the dissociation solution was filtered. Fresh solution (35 milliliters) was added, and the suspension was stirred at room temperature for 1 hour. The dissociation solution was filtered. The combined dissociation solutions were slowly poured onto a stirred mixture of cold heptane / diethyl ether (1: 1) (500 milliliters), giving a precipitate. The suspension was stirred at room temperature for 2 hours, and then the precipitate was allowed to settle. The supernatant was suctioned with a frit. The residue was washed with cold heptane / diethyl ether (1: 1) (100 milliliters, 2 times), and the supernatant was suctioned with a frit. The solid was dried in a high vacuum, to provide intermediate 8c as a grayish solid (3.75 grams, 1.88 mmol).

• Preparation of Example 8 • (Cycling and purification)

Intermediary 8c (3.75 grams, 1.88 mmol) dissolved in H₂Or (375 milliliters). A solution of l was added₂ 50 mM in AcOH (45.1 milliliters, 2.26 mmol) in one portion to the stirred solution, and the solution was stirred for 10 minutes at room temperature. 0.5 M ascorbic acid in H was added₂Or (5.64 milliliters, 2.82 millimoles), to quench excess l₂. The solution was concentrated to almost

165 dryness. The reaction was carried out in two portions: scale of 0.188 millimoles and scale of 1.69 millimoles. The raw products were combined for purification. The crude product was purified by preparation HPLC and lyophilized from ACN / H₂Or, to provide Example 8, as a white solid (1.53 grams, 0.767 mmol).

The pure product was analyzed by analytical HPLC (Analytical Method C: t_R = 3.43 minutes), and UPLC-MS (Analytical Method B; measured: [M + 3]³⁺= 512.4; calculated: [M + 3]³⁺= 512.6).

Alternatively, the crude polypeptide of Example 8 was dissolved in water (500 milliliters of water / millimole of polypeptide), and converted to the acetate salt with the help of an ion exchange resin (i.e., Amberlite IRA -67 (acetate form) (200 grams / millimol of polypeptide), and purified by preparation HPLC (C8 modified reverse phase silica gel from Daisogel, gradient: ACN / H₂Or: 3 percent acetonitrile (ACN) and 97 percent of [0.3 percent acetic acid / water mixture] up to 12 percent acetonitrile (ACN) and 88 percent of [0.3 acetic acid mixture per percent / water]), and lyophilized, to provide an acetate salt of Example 8 as a white solid (60 to 100 percent yield).

The stoichiometry of the salt was evaluated based on the analysis of the acetic acid content (ion chromatography) and the water content, and it was determined to be in the range between 1: 3 and 1: 4 (polypeptide: acetate).

166

Example 26 Synthesis of pE-RPRLCHKGP-Nle-CK (Lauroyl) -OH (SEQ ID NO: 33) (disulfide C⁶-C¹²). The following Example 26 discloses SEQ ID NO: 33.

CM »OL

Resin loading

I

SPPS

T pE-R (Pbf&gt; PR (R&gt; f&gt; L “C (Trt&gt; H {Trt&gt; K (Boc&gt; GP-Nle * C &lt;Trt&gt; K (ívDde&gt; aPOL (26b)

1. IvDde removal

2, Coupling of tauric acid pE - R (Pbf&gt; P ~ R (PbffLG (Trt) -H (TRpK (8oc&gt; G - P-Nle - C {Trt&gt; K (Lauroí ^ T&gt; POL (26o) t Dissociation / Removal of PG 1 2 .. Purification jEjE-RPRLCHKGP-Nte-C-KÍLauroí ^ -QW (26d)

1. Cyclization

2, Purification

<img file="CU20140097A7_D0015.tif" />

pE ^ PR ^ L ^ HK ~ GP ^ Nte ~ C &lt;tauroOcfQH

Example 26

167 • Preparation of Intermediate 26a • (Loading of 2-chloro-trityl chloride resin with FmocK (ivDde) ​​-OH, removal of Fmoc, and determination of resin loading)

The 2-chloro-trityl chloride resin (1.00 grams, 1.60 millimoles) was reacted with a solution of Fmoc-K (ivDde) ​​-OH (1.84 grams, 3.20 millimoles) in dichloromethane (DCM) (10 milliliters) , and di-isopropyl-ethyl-amine (DIPEA) (1.12 milliliters, 6.40 millimoles), in analogy to the general procedure described above, to give Intermediary 26a (1.39 grams; load = 0.75 millimoles / gram).

• Preparation of Intermediate 26b • (Linear Peptide Assembly)

Intermediary 26a (134 milligrams, 0.100 millimoles) was subjected to solid phase peptide synthesis in the Prelude peptide synthesizer^MR. The coupling was carried out as follows:

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 1</td><td>C (Trt)</td><td>2 x 30 min</td><td>B</td>

<td> 2</td><td>Nle</td><td>2 x 30 min</td><td>B</td>

168

<td rowspan="2">Couple I lie</td><td rowspan="2">AA</td><td colspan="2">Number of links</td><td rowspan="2">Cycle synthesis</td>

<td>x Time</td><td>of reaction</td>

<td> 3</td><td>P</td><td>2 x</td><td>30 min</td><td>B</td>

<td> 4</td><td>G</td><td>2 x</td><td>90 min</td><td>B</td>

<td> 5</td><td>K (Boc)</td><td>2 x</td><td>30 min</td><td>B</td>

<td> 6</td><td>H (Trt)</td><td>2 x</td><td>30 min</td><td>B</td>

<td> 7</td><td>C (Trt)</td><td>2 x</td><td>30 min</td><td>B</td>

<td> 8</td><td>L</td><td>2 x</td><td>30 min</td><td>B</td>

<td> 9</td><td>R (Pbf)</td><td> 4</td><td>x 1 h</td><td>B</td>

<td> 10</td><td>P</td><td>2 x</td><td>90 min</td><td>B</td>

<td> 1 1</td><td>R (Pbf)</td><td> 4</td><td>x 1 h</td><td>B</td>

<td> 12</td><td>PE</td><td>2 x</td><td>90 min</td><td>B</td>

• Preparation of Intermediary 26c • (Removal of ivDde and coupling of lauric acid)

Intermediary 26b (0.100 mmol) was washed with tetrahydrofuran (THF) (3 times). A solution of hydrazine monohydrate (0.245 milliliters, 5.00 millimoles) in tetrahydrofuran (THF) was added

169 (12 milliliters), and the suspension was stirred at room temperature for 1 hour. The solution was filtered. This step was done twice more. The resin was washed with DMA (3 times), dichloromethane (DCM) (3 times), DMA (2 times), dichloromethane (DCM) (5 times), and DMA (3 times). Lauric acid (100 milligrams, 0.500 millimoles) and HCTU (207 milligrams, 0.500 millimoles) were dissolved in NMP (3 milliliters) and di-isopropyl-ethyl-amine (DIPEA) (0.087 milliliters, 0.500 millimoles). After 5 minutes of activation, the solution was added to the resin, and the suspension was stirred at room temperature for 16 hours. The reaction mixture was filtered, and the resin was washed with DMA (3 times), dichloromethane (DCM) (3 times), DMA (3 times), dichloromethane (DCM) (5 times), to provide the intermediary

26c.

• Preparation of Intermediary 26d • (Dissociation from the resin with the concomitant removal of the protective group, and then purification)

A mixture of 95 percent aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (3 milliliters), was added to intermediate 26c (0.100 millimoles), and the suspension was stirred at room temperature for 2 hours. The dissociation solution was filtered, and fresh dissociation solution (3 milliliters) was added. The suspension was stirred at room temperature for 1 hour, then the dissociation solution was filtered. Fresh solution (3 milliliters) was added, and the suspension was stirred at room temperature for 1 hour. The dissociation solution was filtered. Dissociation solutions

170 combined were poured onto a mixture of cold heptane / diethyl ether (1: 1) (35 milliliters), giving a precipitate. The suspension was centrifuged and the supernatant poured. The residue was washed with cold heptane / diethyl ether (1: 1) (10 milliliters), the suspension was centrifuged, and the supernatant was poured. The solid was dried in a high vacuum.

The crude product was purified by preparation HPLC and lyophilized from ACN / H₂Or, to provide intermediate 26d as a white solid (74.1 milligrams, 0.034 millimoles).

• Preparation of Example 26 • (Cycling and purification)

Intermediary 2d (74.1 milligrams, 0.034 millimoles) dissolved in H₂0 / dimethyl sulfoxide (DMSO) (9: 1) (74 milliliters). The reaction mixture was stirred at room temperature for 40 hours; then it was concentrated to dryness under vacuum. The crude product was purified by preparation HPLC and lyophilized from ACN / H₂Or, to provide Example 26 as a white solid (60.0 milligrams; 0.028 millimoles).

The pure product was analyzed by analytical HPLC (Analytical method A: t_R = 5.11 minutes), and UPLC-MS (Analytical Method B; measured: [M + 3]³⁺ = 567.0; calculated: [M + 3]³⁺ = 567.0).

Example 28 Synthesis of pE-RPCLCCKGP-Nle-C-FOH (SEQ ID NO: 35) (C disulfides⁴-C⁷ and C⁶-C¹²). The following Example 28 discloses SEQ ID NO: 35.

171

C¡ressrsa

SPPS pE-R (Pbf&gt; PC (Acm&gt; L ^ C (W&gt; -C (Acm&gt; K (Boc) -G ~ P-Nte-C (Trt) -F-0 ~ P0L

Dissociation / Partial Removal of PG

1, CIciaáán

2, Purification

i .........] Ί i pE-RPCtCCK-G'P-Nte-CF &lt;

Example 23 • Preparation of Intermediate 28a • (Loading of 2-chloro-trityl chloride resin with Fmoc-F-OH, removal of Fmoc, and determination of resin loading)

The 2-chloro-trityl chloride resin (10.0 grams, 16.0 millimoles) was reacted with a solution of Fmoc-F-OH (6.20 grams, 16.0 millimoles) in dichloromethane (DCM) (100 milliliters) and di- isopropyl-ethyl-amine (DIPEA) (11.2 milliliters, 64.0 millimoles), in analogy to the general procedure described above, to give the

172

Intermediary 28a (11.6 grams, load = 0.87 millimoles / gram).

• Preparation of Intermediate 28b • (Linear Peptide Assembly)

Intermediary 28a (115 milligrams, 0.100 millimoles) underwent solid phase peptide synthesis in the Prelude peptide synthesizer^MR. The coupling was carried out as follows:

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 1</td><td>C (Trt)</td><td>2 x 15 min</td><td>B</td>

<td> 2</td><td>Nle</td><td>2 x 15 min</td><td>B</td>

<td> 3</td><td>P</td><td>2 x 15 min</td><td>B</td>

<td> 4</td><td>G</td><td>2 x 90 min</td><td>B</td>

<td> 5</td><td>K (Boc)</td><td>2 x 1 5 min</td><td>B</td>

<td> 6</td><td>C (Acm)</td><td>2 x 15 min</td><td>B</td>

<td> 7</td><td>C (Trt)</td><td>2 x 15 min</td><td>B</td>

<td> 8</td><td>L</td><td>2 x 15 min</td><td>B</td>

<td> 9</td><td>C (Acm)</td><td>2 x 15 min</td><td>B</td>

<td> 10</td><td>P</td><td>2 x 15 min</td><td>B</td>

<td> 1 1</td><td>R (Pbf)</td><td>4 x 1 h</td><td>B</td>

173

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 12</td><td>PE</td><td>2 x 15 min</td><td>B</td>

• Preparation of Intermediary 28c • (Dissociation from the resin with the concomitant partial removal of the protective group)

Intermediary 28b (0.100 millimoles)) was washed thoroughly with dichloromethane (DCM) (4 times). A mixture of 95 percent aqueous trifluoroacetic acid (TFA) / EDT (4: 1) (0.750 milliliters) was added, and the suspension was stirred at room temperature for 1 hour. A mixture of trifluoroacetic acid (TFA) / H was added₂Or (95: 5) (2.18 milliliters) and TES (75 microliters) to the suspension, and stirring was continued at room temperature for 1 hour. The dissociation solution was filtered, and a 95 percent mixture of aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (3 milliliters) was added to the resin. The suspension was stirred at room temperature for 1 hour, and the dissociation solution was filtered. Fresh solution (3 milliliters) was added, and the suspension was stirred at room temperature for 1 hour. The dissociation solution was filtered. The combined dissociation solutions were poured onto cold heptane / diethyl ether (1: 1) (35 milliliters), giving a precipitate. The suspension was centrifuged and the supernatant poured. The residue was washed with cold heptane / diethyl ether (1: 1) (10 milliliters), the suspension

174 It was centrifuged, and the supernatant was poured. The washing step was repeated once. The residue was dried under high vacuum. The crude product was purified by preparation HPLC, and lyophilized from ACN / H₂Or, to provide intermediate 28c as a white solid (51.1 milligrams, 0.028 millimoles).

• Preparation of Example 28 • (Formation of two disulfides in a container)

Intermediary 28c (51.1 milligrams, 0.028 mmol) was dissolved in AcOH (48 milliliters) and H₂Or (12 milliliters). A solution of l was added₂ 50 mM in AcOH (1.12 milliliters, 56 micromoles), and the yellow solution was stirred at room temperature. Added more l₂fifty mM in AcOH (5.61 milliliters, 0.281 millimoles) in portions for 4 hours. After 21 hours, the reaction mixture was concentrated to 2 milliliters in vacuo, and 1M ascorbic acid in H was added.₂Or (6 milliliters), to turn off excess l₂. The product was isolated by preparation HPLC, and lyophilized from ACN / H₂Or, to provide Example 28, as a white solid (19.3 milligrams, 0.012 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A: t_R = 4.16 minutes), and UPLC-MS (Analytical Method C; measured: [M + 2]²⁺= 723.7; calculated: [M + 2]²⁺= 723.8).

Example 62 Synthesis of pE-RPRLCHKGP-Nle-C-FNH₂ (SEQ ID NO: 69) (disulfide C⁶-C¹²). The following Example 62 discloses SEQ ID NO: 69.

175

SPPS

Dissociation / Removal of PG pE-RP ^ LCHKGP-Nie-CF-ÍVHs »(62b)

1, Cycling

2, Purification

-PRLCHKGP-Nte-CF-Á^FH_;

Example 62 • Preparation of Intermediary 62a • (Linear Peptide Assembly)

The Fink-protected Rink-Amida-AM-PS resin (217 milligrams, 0.100 millimoles) was subjected to solid phase peptide synthesis in the Prelude peptide synthesizer^MR. The coupling was carried out as follows:

<td>Couple I lie</td><td>AA</td><td>Number of links x Reaction time</td><td>Cycle synthesis</td>

<td> 1</td><td>F</td><td>2 X 15 min</td><td>TO</td>

176

<td rowspan="2">Couple I lie</td><td rowspan="2">AA</td><td colspan="2">Number of links</td><td rowspan="2">Cycle synthesis</td>

<td>x Time of</td><td>reaction</td>

<td> 2</td><td>C (Trt)</td><td>2 X 30</td><td>min</td><td>D</td>

<td> 3</td><td>Nle</td><td>2X15</td><td>min</td><td>TO</td>

<td> 4</td><td>P</td><td>2X15</td><td>min</td><td>TO</td>

<td> 5</td><td>G</td><td>2 x 30</td><td>min</td><td>TO</td>

<td> 6</td><td>K (Boc)</td><td>2X15</td><td>min</td><td>TO</td>

<td> 7</td><td>H (Trt)</td><td>2X15</td><td>min</td><td>TO</td>

<td> 8</td><td>C (Trt)</td><td>2 x 1</td><td>h</td><td>D</td>

<td> 9</td><td>L</td><td>2X15</td><td>min</td><td>TO</td>

<td> 10</td><td>R (Pbf)</td><td>4 x 1</td><td>h</td><td>TO</td>

<td> 1 1</td><td>P</td><td>2X15</td><td>min</td><td>TO</td>

<td> 12</td><td>R (Pbf)</td><td>4 x 1</td><td>h</td><td>TO</td>

<td> 13</td><td>pE</td><td>2X15</td><td>min</td><td>TO</td>

• Preparation of Intermediary 62b (Dissociation from resin with removal

177 concomitant of the protective group)

A mixture of 95 percent aqueous trifluoroacetic acid (TFA) / EDT / TES (95: 2.5: 2.5) (3 milliliters) was added to intermediate 62a (0.1 millimoles), and the suspension was stirred at room temperature for 1.5 hours. The dissociation solution was filtered, and fresh dissociation solution (2 milliliters) was added. The suspension was stirred at room temperature for 45 minutes, and then the dissociation solution was filtered. Fresh solution (2 milliliters) was added, and the suspension was stirred at room temperature for 45 minutes. The combined dissociation solutions were poured onto a mixture of cold heptane / diethyl ether (1: 1) (35 milliliters), giving a precipitate. The suspension was centrifuged and the supernatant poured. The residue was washed with cold heptane / diethyl ether (1: 1) (10 milliliters), the suspension was centrifuged, and the supernatant was poured. The solid was dried in a high vacuum. The crude product of Intermediary 62b was used in the next step without purification.

• Preparation of Example 62 • (Cycling and purification)

Intermediary 62b (0.100 millimoles) dissolved in H₂Or (20 milliliters). A solution of l was added₂ 50 mM in AcOH (2.4 milliliters, 0.120 mmol) in one portion to the stirred solution, and the solution was stirred for 30 minutes at room temperature. 0.5 M ascorbic acid in H was added₂Or (0.30 milliliters, 0.300 millimoles), to turn off excess l₂. The solution was concentrated to almost dryness. The crude product was purified by HPLC of

178 preparation, and lyophilized from ACN / H₂Or, to provide Example 62, as a white solid (50.5 milligrams, 0.025 millimoles).

The pure product was analyzed by analytical HPLC (Analytical Method C: t_R = 3.22 minutes), and UPLC-MS (Analytical Method C; measured: [M + 3]³⁺= 512.3; calculated: [M + 3]³⁺= 512.3).

The other examples were synthesized in analogy:

• Examples 2 to 4 were synthesized in analogy to Example 1.

• Example 6 was synthesized in analogy to Example 5.

• Examples 9 to 25, and 27 were synthesized in analogy to

Example 26

• Example 29 was synthesized in analogy to Example 28.

• Examples 30 to 61 were synthesized in analogy to Example 8.

• Example 63 was synthesized in analogy to Example 62.

Example 64: pE-RPRLCHKGP-Nle-CF-OH (SEQ ID

NO: 71) with a -S-CH link₂-C (O) CH₂-S- between the cysteines in positions 6 and 12 [C⁶-C¹²]

Analytical method

Method D (HRMS);

Eluent A: water + 0.05 percent formic acid + 3.75 mM ammonium acetate, Eluent B: Acetonitrile + 0.04 percent formic acid.

Gradient: From 2 to 98 percent of B in 4.4 minutes - Flow of 1.0 milliliter / minute. Column: Acquity CSH 1.7 microns, 2.1 * 50 mm, 50 ° C.

179

<img file="CU20140097A7_D0016.tif" />

<img file="CU20140097A7_D0017.tif" />

Example 8 ((S) -2 - ((3S, 6R, 11R, 14S, 17S, 25aS) -14 - ((1Himidazol-5-yl) -methyl) -17- (4-amino-butyl) - 3-butyl-11 - ((S) -2 - ((S) -5guanidino-2 - ((S) -1 - ((S) -5-guanidino-2 - ((S) -5-oxo-pyrrolidin -2carboxamido) -pentanoyl) -pyrrolidin-2-carboxamido) -pentanamido) -4m eti l-pe ntanam gone) -1,4,1 2,15,1 8,21 -hexaoxodocosahidro-1 H-pirrolo [2,1 -j] [1,2,5,8,11,14,1 7,20] -dithiahexa-azacycllotricosin-6180 carboxamido) -3-phenyl-propanoic acid) (12 milligrams, 6.76 micromoles) was dissolved in 50 mM sodium phosphate regulator, pH 6.5 (1.5 milliliters), to which TCEP HCI ((f / 7's- (2-carboxy-ethyl) phosphine) was added (2.91 milligrams, 10.13 micromoles ), at room temperature This reaction mixture was stirred for 1 hour at room temperature To the above solution was added 1,3-dichloro-propan-2-one (4.29 milligrams, 0.034 mmol), at room temperature, which was stirred for 30 minutes at room temperature. RP-HPLC eluting from 15 to 60 percent of MeCN / water with 0.1 percent trifluoroacetic acid (TFA), gave Example 64 ((S) -2 - ((3S, 6R, 14R, 17S, 20S, 28aS) -17 - ((1Himidazol-5-yl) -methyl) -20- (4-amino-butyl) -3-butyl-14 - ((S) -2 - ((S) -5guanidino-2 - ((S) -1 - ((S) -5-guanidino-2 - ((S) -5-oxo- pyrrolidin-2-carboxamido) -pentanoyl) -pyrrolidin-2-carboxamido) -pentanamido) -4-methyl-pentana mido) -1,4,10,1 5.1 8,21,24-heptaoxo-hexacosahidropyrol- [2,1 - i] [1,23,4,7,1 0,1 3,1,1 9] -dithiahexa -azacyclohexacosin-6-carboxamido) -3-phenyl-propanoic acid) (6 milligrams, 2.93 micromoles, 43.4 percent yield). HRMS (method D) [M + 1]; 1590.791 1 (observed), 1590.7912 (expected). Holding time; 3.08 minutes

Example 65: pE-RPRLCHKGP-Nle-CF-OH (SEQ ID

NO: 72) with a monosulfide bond between the 2 cysteines at positions 6 and 12 [C⁶-C¹²]

181

<img file="CU20140097A7_D0018.tif" />

<img file="CU20140097A7_D0019.tif" />

<img file="CU20140097A7_D0020.tif" />

The mixture of Example 8 ((S) -2 - ((3S, 6R, 11 R, 14S, 17S, 25aS) -14 - ((1 H-imidazol-5-yl) -methyl) -1 7- (4 -amino-butyl) -3-butyl-11 - ((S) 2 - ((S) -5-guanidino-2 - ((S) -1 - ((S) -5-guanidino-2 - ((S ) -5-oxo-pyrrolidin-2-carboxamido) -pentanoyl) -pyrrolidin-2-carboxamido) -pentanamido) -4meti l-pe ntanam gone) -1,4,12,15,1 8,21 -hexaoxodocosahydro-1 H -pirro lo [2,1 -j] [1,2,5,8,11,14,1 7,20] -dithiahexa-azaciclotricosin-6carboxamido) -3-phenyl-propanoic acid, salts of trifluoroacetic acid (TF A )) (30 milligrams, 0.15 millimoles), and N, N, N ', N', N, N-hexamethyl182 phosphinatriamine (12.3 milligrams, 0.75 mmol) in phosphate regulated serum regulator, pH 9.2 (1 milliliter), was stirred at room temperature for 3 days. The reaction mixture was purified by preparation HPLC (Sunfire C18, 0.1 percent trifluoroacetic acid (TFA) in water / MeCN) twice, and the product fraction was lyophilized to a white powder (Example 65: 4 milligrams, 13.4 percent). [M + 2H] 2+ (calculated) =

752.88, [M + 2H] 2+ (measured) = 752.40, [M + 3H] 3+ (calculated) = 502.26, [M + 3H] 3+ (measured) = 501.94. HPLC (analytical method C), Tr min =

6.93.

Example 66: pE-RPRLCHKGP-Nle-CF-OH (SEQ ID

NO: 73) with a -S-CH link₂-C (= Z) -CH₂-S- between the 2 cysteines in positions 6 and 12 [C⁶-C¹²], and Z is:

OR

<img file="CU20140097A7_D0021.tif" />

Analytical method

Method D (HRMS);

Eluent A: water + 0.05 percent formic acid + 3.75 mM ammonium acetate, Eluent B: Acetonitrile + 0.04 percent formic acid.

183

Gradient: From 2 to 98 percent of B in 4.4 minutes Flow of 1.0 milliliter / minute. Column: Acquity CSH 1.7 microns, 2.1 * 50 mm, 50 ° C.

OH

HN ^

H₂N NH H₂N NHF ^ N

V r _s^H

NH '

NH

Ά^Ν or

r'i l

J CU '·' - \ O. NH

Η - Η II - H

Or γ or

NHo

OR

<img file="CU20140097A7_D0022.tif" />

<img file="CU20140097A7_D0023.tif" />

OR

CF3COOH

O and

HN .O

<img file="CU20140097A7_D0024.tif" />

<img file="CU20140097A7_D0025.tif" />

NH

O 4 O

<img file="CU20140097A7_D0026.tif" />

OR

<img file="CU20140097A7_D0027.tif" />

184

To a solution of Example 64 (acid (S) -2 - ((3S, 6R, 1 4R, 1 7S, 20S, 28aS) -1 7 - ((1 H-imid azol-5-yl) -met il) -20- (4-amino-butyl) -3-butyl-14 ((S) -2 - ((S) -5-guanidino-2 - ((S) -1 - ((S) -5-guanidino- 2 - ((S) -5-oxopyrrolidin-2-carboxamido) -pentanoyl) -pyrrolidin-2-carboxamido) pentanamido) -4-methyl-pentanamido) -1,4,10,15,18,21,24-heptaoxohexacosahydro -pyrrolo- [2,1 - i] [1,23,4,7,1 0,1 3,1 6,1 9] -dithiahexaazacyclohexacosin-6-carboxamido) -3-phenyl-propanoic acid) (11.5 milligrams, 5.62 micromoles), and the compound of (S) -1- (aminooxy) -1 9-carboxy-2,7,1 6,21-tetraoxo-9,1 2-dioxa-3,6,1 5,20-tetra- azaoctatriacontan-38-oic with 2,2,2-trifluoroacetic acid (1: 1) (9.19 milligrams, 0.011 millimoles) in 100nM sodium phosphate regulator, pH 6.0 (1 milliliter), aniline (2,051 microliters, 0.022 mmol), at room temperature. The addition of dimethyl sulfoxide (DMSO) (50 microliters) gave a homogeneous solution. This reaction mixture was stirred at room temperature for 2 hours. RP-HPLC eluting with 15 to 60 percent MeCN / water with 0.1 percent trifluoroacetic acid (TFA), gave Example 66 (1 - ((Z) ((3S, 6R, 14R, 17S, 20S, 28aS) 17 - ((1H-imidazol-5-yl) -methyl) -20- (4-amino-butyl) acid ) -3-butyl-6 - ((S) -1-carboxy-2-phenyl-ethyl-carbamoyl) -14 - ((S) -2 - ((S) -5-guanidino-2 - ((S) -1 ((S) -5-guanidino-2 - ((S) -5-oxo-pyrrolidin-2-carboxamido) -pentanoyl) pyrrolidin-2-carboxamido) -pentanamide) -4-methyl-pentanamide) 1,4,15 , 18,21,24-hexaoxodocosahidro-pyrrolo- [2,1i] [1,23,4,7,10,13,16,19] -dithiahexa-azacyclohexacosin-10 (1H, 9H, 11H) iliden) -amino -oxy) -19-carboxy-2,7,16,21 - tetraoxo-9,1 2-di oxa185

3,6,1 5,20-tetra-aza-octatriacontan-38-oico) (4.5 milligrams, 1,646 micromoles, 29.3 percent yield). HRMS (method D) [(M + 3) / 3]; 759.7487 (observed), 759.7462 (expected). Holding time; 4.12 minutes

Synthesis of the compound of acid (S) -1 - (amino-oxy) -l 9-carboxy-2,7,1 6,

-tetraoxo-9.1 2-di oxa-3.6.1 5.20-tetra-aza-octatriacontan-38-oic with 2,2,2-trifluoroacetic acid (TFA) (1: 1):

<img file="CU20140097A7_D0028.tif" />

<img file="CU20140097A7_D0029.tif" />

H of 2-CI Trt.

The 1-chloro-tritiium chloride resin (1.55 millimoles / gram) (0.500 grams, 0.775 millimoles) in a 100 milliliter glass vessel swelled in dichloromethane (DCM) (20 milliliters) for 30 minutes, and was drained. To the resin was added a suspension of 2- (amino-oxy) -acetic acid (0.338 grams, 3.10 millimoles), and di-isopropyl-ethyl-amine (DIPEA) (1,354 milliliters, 7.75 millimoles) hemohydrochloride in NMP (7 milliliters) / DCM (4 milliliters), which was stirred for 5 hours. The solvent was drained. The resin was rinsed with dichloromethane (DCM) / MeOH / DIPEA (17/2/1, 40 milliliters), DCM (50 milliliters), NMP (50 milliliters) DCM (50 milliliters), respectively.

The resulting resin was dried with KOH / NaOH overnight.

2 * C | Tri v

XA, «3

186

The resin (0.775 mmol) in a 100 milliliter glass vessel was swollen in dichloromethane (DCM) (20 milliliters) for 30 minutes, and drained. To a suspension of (9H-fluoren-9-yl) -methyl 2-aminoethyl-carbamate hydrochloride (0.081 grams, 0.775 mmol), HOAt (0.422 grams, 3.10 mmol), and di-isopropyl-ethylamine (DIPEA) ( 1,354 milliliters, 7.75 millimoles) in NMP (8 milliliters), HBTU (1,176 grams, 3.10 millimoles) in NMP (2.5 milliliters) was added, which was stirred for 2 hours at room temperature. The solvent was drained. The resin was rinsed with NMP (10 milliliters), and DCM (10 milliliters), respectively. The resulting resin was dried overnight.

/ Ά, i

ΟγΆ

GOES

χΤ η ΐ

Ιο&gt;

.fA 'Ύ n'

H m + 3:? - C i Tft

The resin (0.775 mmol) was loaded into a reaction vessel. 10 milliliters of 20 percent PIPERIDINE / NMP was added to the resin, whose suspension was stirred at room temperature for 5 minutes. After the solvent was drained, an additional 10 milliliters of 20 percent PIPERIDINE was added /

187

NMP, and stirred for 20 minutes at room temperature. A solution of HOAt (0.316 grams, 2,325 mmol) and 1- (9H-fluoren-9-yl) -3-oxo-2,7,10-trioxa-4-azadodecan-12-oic acid (1) solution was added to the resin 0.896 grams, 2,325 millimoles) in NMP (8 milliliters), and DIC (0.362 milliliters, 2,325 millimoles) in NMP (1 milliliter) was added. The reaction mixture was stirred for 2 hours at room temperature. The resin was filtered and rinsed with NMP (10 milliliters) four times. The resulting resin was dried overnight.

<img file="CU20140097A7_D0030.tif" />

The resin (0.775 mmol) was loaded into a reaction vessel. 10 milliliters of 20 percent PIPERIDINE / NMP was added to the resin, whose suspension was stirred at room temperature for 5 minutes. After the solvent was drained, it was

188 They added an additional 10 milliliters of 20 percent PIPERIDINE / NMP, and stirred for 20 minutes at room temperature. A solution of HOAt (0.316 grams, 2.325 millimoles) and Fmoc-Glu-OtBu (0.989 grams, 2.325 millimoles) in NMP (8 milliliters) was added to the resin, DIC (0.362 milliliters, 2.325 millimoles) in NMP (2.00 milliliters) The reaction mixture was stirred for 2 hours at room temperature. The resin was filtered and rinsed with NMP (10 milliliters) four times. The resulting resin was dried overnight.

! tóa2-CI Tít

Or j

HC '' xz'Sz

O η H rasifts AGI Trt

The resin (0.775 mmol) was loaded into a reaction vessel. 10 milliliters of 20 percent PIPERIDINE / NMP (0.775 mmol) was added to the resin, whose suspension was stirred at room temperature for 5 minutes. After the solvent was drained, an additional 10 milliliters of PIPERIDINE was added to the

189 percent / NMP (0.775 mmol) (0.775 mmol), and stirred for 20 minutes at room temperature. A solution of 18-terbutoxy-18-oxo-octadecanoic acid (0.862 grams, 2.325 millimoles) and HOAt (0.316 grams, 2.325 millimoles) in NMP (8 milliliters) was added to the resin, and DIC (0.362 milliliters, 2.325 millimoles) in NMP (2.00 milliliters). The reaction mixture was stirred for 4 hours at room temperature. The resin was filtered and rinsed with NMP (10 milliliters) four times. The resulting resin was dried overnight.

.1

O., A - '' ^ Υ'νΑ ÍSSíftS 2 € l Title

ITEM

The resin (0.775 mmol) was treated with 20 milliliters of dissociation cocktail (TFA / TIPS / water = 95 / 2.5 / 2.5) for 1.5 hours at room temperature. The resin was removed by filtration, and rinsed with trifluoroacetic acid (TFA). The filtrate was concentrated in vacuo. RP-HPLC with a C18 column, eluting with 15 to 50 percent MeCN / water with 0.1 percent trifluoroacetic acid (TFA), gave the acid (S) -1- (amino-oxy) -l 9-carboxy-2,7,1 6,21 tetraoxo-9,1 2-di oxa-3,6,1 5,20-tetra-aza-octatriacontan-38-oico with

2,2,2-trifluoroacetic acid (TFA) (1: 1) (207 milligrams, 0.294 millimoles, 37.9 percent yield). HRMS (method D) [M + 1j; 704.4459 (observed), 704.4486 (expected). Holding time;

2.63 minutes

190

The polypeptides of Examples 1 to 66 can be purified and isolated as described above and / or by a combination of conventional purification techniques, such as solvent extraction, column chromatography, liquid chromatography and recrystallization. When the polypeptide isolated in the previous examples is a free compound, it can be converted to a suitable salt by the known method. Accordingly, the peptides of Examples 1 to 66 can be converted into their corresponding salt (for example, hydrochloride, hydrobromide, sulfate, phosphate, citrate, acetate, lactate or other pharmaceutical salt suitable for injection) with a proportion of po I ip ép t id o: s al in the range of 1: 1 to 1: 4. For example, the polypeptides of Examples 1 to 66 can be dissolved in water, and can be converted into a salt using ion exchange resins. Conversely, when the isolated peptide is a salt, it can be converted to the free peptide by a known method, or directly to a different salt with the help of ion exchange resins.

It has been found that the polypeptides of the following Examples have EC values₅or in the range of about 0.01 nM to about 1,100 nM for the power of the APJ receiver. It has been found that the polypeptides of the following Examples have a plasma stability greater than 2 minutes, greater than 5 minutes, greater than 10 minutes, greater than 20 minutes, greater than 50 minutes and greater than 60 minutes.

It can be seen that the polypeptides of the invention are useful

191 as agonists of the APJ receptor and, therefore, are useful in the treatment of diseases and conditions that respond to activation of the APJ receptor, such as the diseases disclosed herein.

Having described in this way the exemplary embodiments of the present invention, it should be noted by those of ordinary experience in this field that the disclosures herein are for example only, and that various other alternatives, adaptations, and modifications can be made within the scope of the present invention. In accordance with the foregoing, the present invention is not limited to the specific embodiments as illustrated therein.