GB2551945A - Novel GLP-1 receptor agonist peptides - Google Patents

Abstract

A compound of formula (I): (I) wherein Q is optionally substituted phenyl or monocyclic heteroaryl; x is an integer from 1 to 3; R1 & R2 are hydrogen or C1-6 alkyl, or form a ring; aa1 is an amino acid; G is glycinyl;T is L-threoninyl; aa2 is an amino acid; W is L-serinyl or 2,3-diaminopropionyl; aa3 is an amino acid; aa4 is an amino acid; aa5 is a linking group; Z is hydrogen, -COOR8 or -CONR9R10; R8, R9 and R10 are hydrogen or C1-6 alkyl; or a tautomeric or stereochemically isomeric form or a prodrug, salt or zwitterion thereof. The compounds of formula (I) are Glucagon-like peptide-1 (GLP-1) receptor agonist peptides and are useful in treating diseases or conditions mediated by insulin, such as diabetes mellitus type 1 or 2. The invention further relates to compositions containing said peptides and processes for their preparation.

Description

(54) Title of the Invention: Novel GLP-1 receptor agonist peptides Abstract Title: GLP-1 receptor agonist peptides

aa-_;-G-T-aa:.T.W.aa.»-a3_fc-aai-Z wherein Q is optionally substituted phenyl or monocyclic heteroaryl; x is an integer from 1 to 3; R1 & R2 are hydrogen or C1-6 alkyl, or form a ring; aa1 is an amino acid; G is glycinyl;T is L-threoninyl; aa2 is an amino acid; W is L-serinyl or 2,3-diaminopropionyl; aa3 is an amino acid; aa4 is an amino acid; aa5 is a linking group; Z is hydrogen, -COOR8 or -CONR9R10; R8, R9 and R10 are hydrogen or C1-6 alkyl; or a tautomeric or stereochemically isomeric form or a prodrug, salt or zwitterion thereof. The compounds of formula (I) are Glucagonlike peptide-1 (GLP-1) receptor agonist peptides and are useful in treating diseases or conditions mediated by insulin, such as diabetes mellitus type 1 or 2. The invention further relates to compositions containing said peptides and processes for their preparation.

1/2

Time (mmutes)

FIGURE 1

On

FIGURE 2

2/2

Log [cold competing ligand] BI

FIGURE 3

H-radioligand bound (cpm)

NOVEL GLP-1 RECEPTOR AGONIST PEPTIDES

FIELD OF THE INVENTION

The invention relates to novel GLP-1 receptor agonist peptides, to the use of said peptides in treating diseases or conditions mediated by insulin, such as diabetes mellitus type 1 or 2, to compositions containing said peptides and processes for their preparation.

BACKGROUND OF THE INVENTION

Glucagon-like peptide-1 (GLP-1) is an incretin derived from the transcription product of the proglucagon gene. The major source of GLP-1 in the body is the intestinal L cell that secretes GLP-1 as a gut hormone. The biologically active forms of GLP-1 are: GLP-1-(7-37) and GLP-1-(7-36)NH₂. Those peptides result from selective cleavage of the proglucagon molecule.

GLP-1 secretion by ileal L cells is dependent on the presence of nutrients in the lumen of the small intestine. The secretagogues (agents that cause or stimulate secretion) of this hormone include major nutrients like carbohydrate, protein and lipid. Once in the circulation, GLP-1 has a half-life of less than 2 minutes, due to rapid degradation by the enzyme dipeptidyl peptidase-4. It is a potent antihyperglycemic hormone, inducing glucosedependent stimulation of insulin secretion while suppressing glucagon secretion. Such glucose-dependent action is particularly attractive because, when the plasma glucose concentration is in the normal fasting range, GLP-1 no longer stimulates insulin to cause hypoglycemia. GLP-1 appears to restore the glucose sensitivity of pancreatic β-cells, with the mechanism possibly involving the increased expression of GLUT2 and glucokinase. GLP-1 is also known to inhibit pancreatic β-cell apoptosis and stimulate the proliferation and differentiation of insulin-secreting β-cells. In addition, GLP-1 inhibits gastric secretion and motility. This delays and protracts carbohydrate absorption and contributes to a satiating effect. The gastric motility aspect of GLP-1 function is very important and is a primary role of GLP-1.

Glucagon-like peptide 1 receptor (GLP-1 R) is a human gene which resides on chromosome 6. The protein encoded by this gene is a member of the glucagon receptor family of G protein-coupled receptors.

GLP-1 R binds specifically to the glucagon-like peptide-1 (GLP-1) and has much lower affinity for related peptides such as the gastric inhibitory polypeptide and glucagon. GLP-1 R is known to be expressed in pancreatic β-cells. Activated GLP-1R stimulates the adenylyl cyclase pathway which results in increased insulin synthesis and release of insulin. Consequently GLP-1R has been suggested as a potential target for the treatment of diabetes. GLP-1R is also expressed in the brain where it is involved in the control of appetite. The diabetic, pancreatic, and neuroprotection implications of GLP-1R are also thought to be potential therapies for treating the diabetes and energy metabolism abnormalities associated with Huntington’s disease affecting the brain and periphery. Coupling of the GLP-1 receptor to other signaling pathways has also been demonstrated and may influence the pharmacological outcome of stimulating the GLP-1 receptor with peptide or small molecule agonists.

WO 2004/067548 and WO 2011/073328 describe a series of GLP-1 peptides having a modified N-terminus. WO 2007/082264, WO 2003/033671, WO 2006/014287 and WO 2006/127948 describe a series of GLP-1 analogs. US2006004222 describes the synthesis of intermediates useful in the preparation of biphenyl amino acids used in preparing peptide receptor modulators. W02007017892, W02008062457, W02009125424 and WO2011048614 describe a series of GLP-1 peptidomimetics useful for the treatment of diabetes.

There is therefore a need to provide alternative GLP-1 modulators, agonists or partial agonists, particularly GLP-1 agonists, which may be useful for the treatment or amelioration of diseases or conditions mediated by insulin, such as diabetes mellitus type 1 or 2.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a compound of formula (I):

Q 'X aai -G-T-aas-T-W-aas-aa^-aas-Z (I) wherein Q represents phenyl or a monocyclic heteroaryl ring each of which may be optionally substituted by one or more R^q groups;

R^q represents halogen, hydroxyl, amino or Ci.₆ alkyl having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S; x represents an integer selected from 1 to 3;

R¹ and R² independently represent hydrogen or a Ci_₆ alkyl group, or together with the carbon to which they are attached join to form a C₃.₈ cycloalkyl or a heterocyclyl group; aa! represents a -NH-C(H)(R³)-CO- group;

R³ represents a-(CH2)y-COR¹¹ or-(CH2)_y-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more halogen groups or Ci.₆ alkyl groups having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S; y represents an integer selected from 1 or 2;

R¹¹ represents hydroxyl, amino or Ci._2O alkoxy having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

G represents a glycine residue;

T represents an L-threonine residue;

aa₂ represents a -NH-C(R^4a)(R^4b)-CO- group;

R^4a represents hydrogen or a Ci_₆ alkyl group having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

R^4b represents a Cmo alkyl group having an alkyl chain that optionally contains one or more heteroatoms, selected from Ο, N, or S, or a benzyl group optionally substituted by one or more Ci_₆ alkoxy groups, halogen atoms, or Ci_₆ alkyl groups having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

W is selected from L-serine and 2,3-diaminopropionic acid residues;

aa₃ represents a -NH-C(H)(R⁵)-CO- group;

R⁵ represents a-CH2-COOR¹² or-(CH2)_y-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more halogen groups or Ci_₆ alkyl groups having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

R¹² represents hydrogen or a Ci._2O alkyl group having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

aa₄ represents a -NH-C(H)(R⁶)-CO- group;

R⁶ represents a -(CH2)k-Ar¹ group or a Ci-i6 alkyl group having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S, k represents an integer selected from 1 to 5; such that when k represents an integer greater than 2, one CH₂ group may be replaced by-O-; aa₅ represents a -NH-C(H)(R⁷)- group;

R⁷ represents a -(CH2)d-Ar³ group or a Ci_i6 alkyl group having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S, and wherein the alkyl chain or heteroatom can be optionally substituted by a Ci.₆ alkyl group, a -(CH₂)d-Ar³ group or a C(O)-Ci-₆ alkyl or alkenyl group;

Ar¹ and Ar³ represents an aromatic or heteroaromatic ring system selected from phenyl, naphthyl, indolyl, pyridyl, pyrazinyl, pyrimidinyl, oxazolyl, imidazolyl, pyrazolyl, isoxazolyl, quinolinyl or isoquinolinyl which may be optionally substituted by one or more halogen, Ci_6 alkoxy, Ar² groups, or Ci_6 alkyl having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

Ar² represents an aromatic or heteroaromatic ring system selected from phenyl, naphthyl, indolyl, pyridyl, pyrazinyl, pyrimidinyl, oxazolyl, imidazolyl, pyrazolyl, isoxazolyl, quinolinyl or isoquinolinyl wherein said Ar² groups may be optionally substituted by one or more Ci._2O alkyl or Ci._2O alkoxy groups, wherein the Ci._2O alkyl or Ci._2O alkoxy groups have an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

d represents an integer selected from 1 to 5;

Z represents hydrogen, -COOR⁸ or -CONR⁹R¹⁰; and

R⁸, R⁹ and R¹⁰ independently represent hydrogen or Ci_₆ alkyl;

or a tautomeric or stereochemically isomeric form or a prodrug, salt or zwitterion thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1 and 2 describe the results obtained when testing the peptide of Example 3 in the Oral Glucose Tolerance Test (OGTT).

Figure 3 represents in vitro pharmacology data to determine the dissociation constant (Kd) for the radioligand [³H]-{H-[Aib]-E-G-T-[a-Me-Phe]-T-S-D-[(2’-ethyl-4’methoxy)Bip]-[(3,5-dimethyl)hhPhe]-NH₂}.

DETAILED DESCRIPTION OF THE INVENTION

It will be apparent to the skilled person that references herein to an amino acid include a compound represented by the general structure:

D- or (R)-a-amino acid L- or (S)-a-amino acid (for example, where R¹ = H, R² = CH3) (for example, where R¹ = H, R² = CH3) where R¹ and R² can be a natural or an un-natural side chain, including hydrogen. References herein to the term amino acid include an amino group and a carboxyl group linked to the same carbon, referred to as a carbon. The absolute S configuration at the a carbon is commonly referred to as the L or natural configuration. In one embodiment, each amino acid residue within the peptides of the invention is in the (S)-configuration.

In this application, unless the context indicates to the contrary, the term “residue” when used in connection with an amino acid (such as serine or threonine), for example in the term “serine residue” is meant the moiety derived from the amino acid after removal of a hydrogen atom and hydroxyl group from respectively the amino and carboxylic acid groups.

The term “Ci-xalkyl” as used herein as a group or part of a group refers to a linear or branched, saturated or partially unsaturated hydrocarbon group containing from 1 to x carbon atoms which may be optionally substituted. Examples of such groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4di methyl pentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl and the like. The term “Ci-xalkyl” also includes cycloalkyl which may be substituted or substituted, for example, cyclopropyl, cyclopropylmethyl and cyclobutyl. The Ci._x alkyl has an alkyl chain that optionally contains one or more optionally substituted heteroatoms selected from Ο, N or S. For example, one or more non-terminal carbons and/or one or more terminal carbons of the alkyl chain may be replaced by a heteroatom. Examples include -(CH₂)4-NH₂, (CH₂)₄NHCH₃, -(CH₂)₃OCH₃ and -(CH₂)₅OH. In particular, the alkyl chain contains one or more atoms of O.

The term “C₃.xcycloalkyl” as used herein refers to a saturated monocyclic hydrocarbon ring containing from 3 to x carbon atoms which may be substituted or unsbustituted. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl and the like.

The term “halogen” as used herein refers to fluorine, chlorine, bromine or iodine.

The term ‘Ci__xalkoxy’ as used herein as a group or part of a group refers to an -O-Ci-xalkyl group wherein Ci__xalkyl is as defined herein. Examples of such groups include methoxy, ethoxy, propoxy, butoxy, and the like. The Ci__x alkoxy has an alkyl chain that optionally contains one or more optionally substituted heteroatoms selected from Ο, N or S. For example, one or more non-terminal carbons and/or one or more terminal carbons of the alkyl chain may be replaced by a heteroatom. Examples include -OCH₂OH, -O(CH₂)₃NHCH₃and -O(CH₂)₄SH. In particular, the alkyl chain contains one or more atoms of 0.

An example of a Ci-xalkyl group or -O-Ci_xalkyl group having an alkyl chain with one or more heteroatoms is polyethylene glycol (PEG) and the invention includes PEGylated derivatives of the compounds of formula (I). The PEG chain may be linear or branched and may have a terminal alkyl ether group, for example a methyl ether group.

The term monocyclic heteroaryl ring means a monocyclic heterocyclyl group containing one or more carbon atoms, one or more hydrogen atoms and one or more heteroatoms such as nitrogen, oxygen and sulfur; the carbon and heteroatoms being interconnected to form a ring. Examples of five membered monocyclic heteroaryl rings include but are not limited to pyrrole, furan, thiophene, imidazole, furazan, oxazole, oxadiazole, oxatriazole, isoxazole, thiazole, thiadiazole, isothiazole, pyrazole, triazole and tetrazole groups. Examples of six membered monocyclic heteroaryl rings include but are not limited to pyridine, pyrazine, pyridazine, pyrimidine and triazine.

The term heterocyclyl group typically refers to a 4- to 7-membered non-aromatic heterocyclic ring containing one or more carbon atoms and one or more heteroatoms such as nitrogen, oxygen and sulfur; the carbon and heteroatoms being interconnected to form a ring. The term “non-aromatic” embraces, unless the context indicates otherwise, unsaturated ring systems without aromatic character, partially saturated and fully saturated heterocyclyl ring systems. The terms “unsaturated” and “partially saturated” refer to rings wherein the ring structure(s) contains atoms sharing more than one valence bond i.e. the ring contains at least one multiple bond e.g. a C=C, C=C or N=C bond. The term “saturated” or “fully saturated” refers to rings where there are no multiple bonds between ring atoms. The heterocyclyl groups typically contain from 1 to 3 heteroatom ring members and more usually 1 or 2 heteroatom ring members. Particular examples include morpholine, piperidine (e.g. 1piperidinyl, 2-piperidinyl, 3-piperidinyl and 4-piperidinyl), piperidone, pyrrolidine (e.g. 1pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl), pyrrolidone, oxetane, azetidine, pyran (2Hpyran or4H-pyran), dihydrothiophene, dihydropyran, dihydrofuran, dihydrothiazole, tetrahydrofuran, tetrahydrothiophene, dioxane, tetrahydropyran (e.g. 4-tetrahydro pyranyl), imidazoline, imidazolidinone, oxazoline, thiazoline, 2-pyrazoline, pyrazolidine, piperazone, piperazine, and N-alkyl piperazines such as N-methyl piperazine. In general, preferred nonaromatic heterocyclyl groups include saturated groups such as piperidine, pyrrolidine, azetidine, morpholine, piperazine and N-alkyl piperazines.

In one embodiment, Q represents phenyl or a 5 membered monocyclic heteroaryl ring each of which may be optionally substituted by one or more R^q groups. For example, Q can be selected from phenyl, imidazole and oxazole, each optionally substituted by one or more R^q groups. Typically, there will be 0, 1 or 2 substituents present, and more usually 0 or 1 substituent R^q.

Ina further embodiment, Q is selected from:

Q^d.

wherein R^q is as defined hereinbefore and n represents an integer selected from 0 or 1.

In a yet further embodiment, Q is selected from:

Q^a ; or Q^b wherein R^q is as defined hereinbefore and n represents 0 or 1.

In each of the foregoing embodiments R^q is selected from halogen, hydroxyl, amino or a Ci_₆ alkyl chain optionally containing one or more heteroatoms selected from Ο, N, or S.

More particularly, R^q can be selected from fluorine, chlorine, bromine, hydroxyl, amino and Ci-₄ alkyl chain optionally containing one or more heteroatoms selected from O and N.

In one embodiment, R^q is selected from fluorine, chlorine, hydroxyl, amino and Ci_₄ alkyl chain optionally containing one or more heteroatoms selected from O and N.

In a further embodiment, R^q is selected from fluorine, chlorine and hydroxyl.

In one embodiment, x represents 2.

In one embodiment, R¹ and R² independently represent hydrogen or a Ci_₆ alkyl group, or together with the carbon to which they are attached join to form a C₃.₈ cycloalkyl or a heterocyclyl group. The heterocyclyl group typically contains 1 or 2 heteroatom ring members, and more usually a single heteroatom ring member, selected from Ο, N and S.

In a further embodiment, R¹ and R² independently represent hydrogen or a Ci_₆ alkyl group, or together with the carbon to which they are attached join to form a cyclopropyl or cyclobutyl group or oxetanyl.

In a further embodiment, R¹ and R² independently represent hydrogen or a Ci_₄ alkyl group, or together with the carbon to which they are attached join to form a cyclopropyl or cyclobutyl group.

In a yet further embodiment, R¹ and R² independently represent hydrogen or a Ci_₆ alkyl group (e.g. a Ci_₄ alkyl group or a Ci.₃ alkyl group or a Ci_₂ alkyl group).

In a yet further embodiment, R¹ and R² both represent a Ci_₆ alkyl group (e.g. a Ci_₄ alkyl group or a Ci_₃ alkyl group or a Ci_₂ alkyl group).

In a yet further embodiment, R¹ and R² both represent methyl.

In one embodiment, R³ represents a -(CH2)2-COR¹¹ or-CH2-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more Ci_₆ alkyl or halogen groups.

In one embodiment, R¹¹ is selected from hydroxyl, amino and Cmo alkoxy.

In another embodiment, R¹¹ is selected from hydroxyl, amino and Ci_₆ alkoxy (e.g. Ci_₄ alkoxy or Ci.₃ alkoxy).

In one embodiment, R¹¹ represents hydroxyl, amino or methoxy.

In one embodiment, R³ represents a -(CH₂)₂-CO₂Me, -(CH₂)₂-CO₂H, -(CH₂)₂-CONH₂ orCH₂-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by Ci_₆ alkyl or halogen.

In a further embodiment, R³ represents a -(CH₂)₂-CO₂Me, -(CH₂)₂-CO₂H, -(CH₂)₂-CONH₂ or -CH₂-tetrazolyl group, wherein said tetrazolyl group is unsubstituted.

The tetrazolyl group can be linked to the (CH₂)_y group through either a carbon ring member of the tetrazole ring or through a nitrogen ring member. In one embodiment, the tetrazolyl group is linked to the (CH₂)_y group through a carbon ring member of the tetrazole ring. In another embodiment, the tetrazolyl group is linked to the (CH₂)_y group through a nitrogen ring member of the tetrazole ring.

In one embodiment, R^4a represents hydrogen or a Ci_₄ alkyl group; for example a Ci.₃ alkyl group such as a methyl group.

In one embodiment, R^4b represents a Ci.₉ alkyl group or a benzyl group optionally substituted by one or two substituents selected from fluorine, chlorine, Ci_₄ alkyl and Ci_₄ alkoxy.

In another embodiment, R^4b represents a C₃.₉ alkyl group or a benzyl group optionally substituted by one or two substituents selected from fluorine, chlorine, methyl, ethyl, isopropyl, methoxy, ethoxy and isopropoxy.

In another embodiment, R^4b represents a C₇.₉ alkyl group or a benzyl group optionally substituted by one or two substituents selected from fluorine, chlorine, methyl and methoxy.

In another embodiment, R^4b represents a C₈ alkyl group or a benzyl group optionally substituted by one or two fluorine atoms.

In a further embodiment, R^4b represents a benzyl group optionally substituted by one fluorine atom (such as 2-fluorobenzyl).

In one embodiment, W is an L-serine residue.

In another embodiment, W is a 2,3-diaminopropionic acid residue.

In one embodiment, R¹² represents hydrogen or a C1.12 alkyl group.

In one embodiment, R¹² represents hydrogen or a Cmo alkyl group.

In one embodiment, R¹² represents hydrogen or a C₆-12 alkyl group.

In one embodiment, R⁵ represents a -CH₂-COOH, -CH₂-COOMe, -CH₂-COO-decan-2-yl or -CH₂-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more Ci_₆ alkyl or halogen groups.

In a further embodiment, R⁵ represents a -CH₂-COOH, -CH₂-COOMe, -CH₂-COO-decan-2yl or -CH₂-tetrazolyl group, wherein said tetrazolyl group is unsubstituted.

In one embodiment, R⁶ represents a C6-15 alkyl group or a -(CH2)k-Ar¹ group, such as (CH2)_k-phenyl, wherein said phenyl group may be optionally substituted by one or more methyl groups or a further phenyl group wherein said further phenyl group may also be substituted by one or more ethyl, methoxy or-(OCH₂CH₂)₅-O-CH₃ groups.

In one embodiment, k represents an integer selected from 1 to 4.

In one embodiment, R⁶ represents a C₇ alkyl group, a -(CH₂)₃-phenyl group substituted by one or more methyl groups or a -CH₂-phenyl group optionally substituted by a further phenyl group wherein said further phenyl group may also be substituted by one or more ethyl or methoxy groups.

In one embodiment, R⁷ represents C6-15 alkyl or a -(CH2)d-Ar³ group, such as -(CH2)_dphenyl, wherein said phenyl group may be optionally substituted by one or more methyl groups or a further phenyl group wherein said further phenyl group may also be substituted by one or more ethyl or methoxy groups.

In one embodiment, when R⁷ is -(CH₂)_d-Ar³, d represents an integer selected from 1 to 4.

When R⁷ is a Ci-i₆ alkyl group, it can be, for example, selected from C₆-is alkyl groups, or from C7.14 alkyl groups, particular examples being C₈ and Ci₄ alkyl groups.

In one embodiment, R⁷ is selected from a C₈ alkyl group, a Ο_Ί4 alkyl group and a -(CH₂)₃phenyl group substituted by one or more methyl groups.

In one embodiment, R⁷ represents -(CH₂)₄-NH₂ optionally substituted on the nitrogen atom with a -C(O)-C₂-₆ alkenyl or -C(O)-C₂.₆ alkynyl group.

In one embodiment, Z represents hydrogen or-CONR⁹R¹⁰.

In a further embodiment, Z represents hydrogen or-NH₂.

In another embodiment, Z represents -NH₂.

In another embodiment, Z represents hydrogen.

In one embodiment, the compound of formula (I) is present as a PEGylated derivative.

In one embodiment, the compound of formula (I) is selected from a compound of Examples 1-24.

It will be appreciated by those skilled in the art that certain protected derivatives of compounds of formula (I), which may be made prior to a final deprotection stage, may not possess pharmacological activity as such, but may, in certain instances, be administered orally or parenterally and thereafter metabolised in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as “prodrugs”. All such prodrugs of compounds of the invention are included within the scope of the invention. Examples of pro-drug functionality suitable for the compounds of the present invention are described in Drugs of Today, Volume 19, Number 9, 1983, pp 499 - 538 and in Topics in Chemistry, Chapter 31, pp 306 - 316 and in “Design of Prodrugs” by H. Bundgaard, Elsevier, 1985, Chapter 1 (the disclosures in which documents are incorporated herein by reference). It will further be appreciated by those skilled in the art, that certain moieties, known to those skilled in the art as “pro-moieties”, for example as described by H. Bundgaard in “Design of Prodrugs” (the disclosure in which document is incorporated herein by reference) may be placed on appropriate functionalities when such functionalities are present within compounds of the invention.

Compounds of formula (I) may exist in a number of different geometric isomeric, and tautomeric forms and references to compounds of formula (I) include all such forms. The invention also includes the zwitterionic forms of the compounds of formula (I). For the avoidance of doubt, where a compound can exist in one of several geometric isomeric, tautomeric, or zwitterionic forms and only one is specifically described or shown, all others are nevertheless embraced by formula (I).

For example, in compounds of the formula (I) containing an imidazole or tetrazole group, the imidazole and tetrazole groups may each exist in several tautomeric forms. Unless the context indicates to the contrary, the illustration of one tautomeric form should be taken also to include any other possible tautomeric forms.

The present invention includes all pharmaceutically acceptable isotopically-labeled compounds of the invention, i.e. compounds of formula (I), wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

Examples of isotopes suitable for inclusion in the compounds of the invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T), carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶CI, fluorine, such as ¹⁸F, iodine, such as ¹²³l, ¹²⁵l and ¹³¹l, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulfur, such as ³⁵S.

Certain isotopically-labelled compounds of formula (I), for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The compounds of formula (I) can also have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.

The compounds of the invention can be present as salts, in particular pharmaceutically acceptable salts. Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means and suitable pharmaceutically salts will be known to the skilled person.

According to a further aspect of the invention there is provided a process for preparing a compound of formula (I) as herein defined.

Pharmaceutical Compositions

While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation).

Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising (e.g admixing) at least one compound of formula (I) (and sub-groups thereof as defined herein), together with one or more pharmaceutically acceptable excipients and optionally other therapeutic or prophylactic agents, as described herein.

The pharmaceutically acceptable excipient(s) can be selected from, for example, carriers (e.g. a solid, liquid or semi-solid carrier), adjuvants, diluents, fillers or bulking agents, granulating agents, coating agents, release-controlling agents, binding agents, disintegrants, lubricating agents, preservatives, antioxidants, buffering agents, suspending agents, thickening agents, flavouring agents, sweeteners, taste masking agents, stabilisers or any other excipients conventionally used in pharmaceutical compositions. Examples of excipients for various types of pharmaceutical compositions are set out in more detail below.

The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

Pharmaceutical compositions containing compounds of the formula (I) can be formulated in accordance with known techniques, see for example, Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

The pharmaceutical compositions can be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, sublingual, ophthalmic, otic, buccal, rectal, intra-vaginal, or transdermal administration. In one embodiment, administration is via a non-invasive route, such as intranasal, pulmonary or oral administration.

Where the compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery. The delivery can be by bolus injection, short term infusion or longer term infusion and can be via passive delivery or through the utilisation of a suitable infusion pump or syringe driver.

Pharmaceutical formulations adapted for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, co-solvents, surface active agents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels, lyophilisation protectants and combinations of agents for, inter alia, stabilising the active ingredient in a soluble form and rendering the formulation isotonic with the blood of the intended recipient. Pharmaceutical formulations for parenteral administration may also take the form of aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents (R. G. Strickly, Solubilizing Excipients in oral and injectable formulations, Pharmaceutical Research, Vol 21(2) 2004, p 201-230).

The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules, vials and prefilled syringes, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

The pharmaceutical formulation can be prepared by lyophilising a compound of formula (I), or sub-groups thereof. Lyophilisation refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilisation are therefore used herein as synonyms.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Pharmaceutical compositions of the present invention for parenteral injection can also comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as sunflower oil, safflower oil, corn oil or olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of thickening or coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include agents to adjust tonicity such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In one preferred embodiment of the invention, the pharmaceutical composition is in a form suitable for i.v. administration, for example by injection or infusion. For intravenous administration, the solution can be dosed as is, or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% dextrose), before administration.

In another preferred embodiment, the pharmaceutical composition is in a form suitable for sub-cutaneous (s.c.) administration.

Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft shell), caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches such as buccal patches.

Thus, tablet compositions can contain a unit dosage of active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, eg; lactose, sucrose, sorbitol or mannitol; and/or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as microcrystalline cellulose (MCC), methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g. swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g. stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT), buffering agents (for example phosphate or citrate buffers), and effervescent agents such as citrate/bicarbonate mixtures. Such excipients are well known and do not need to be discussed in detail here.

Tablets may be designed to release the drug either upon contact with stomach fluids (immediate release tablets) or to release in a controlled manner (controlled release tablets) over a prolonged period of time or with a specific region of the Gl tract.

Capsule formulations may be of the hard gelatin or soft gelatin variety and can contain the active component in solid, semi-solid, or liquid form. Gelatin capsules can be formed from animal gelatin or synthetic or plant derived equivalents thereof.

The solid dosage forms (eg; tablets, capsules etc.) can be coated or un-coated. Coatings may act either as a protective film (e.g. a polymer, wax or varnish) or as a mechanism for controlling drug release or for aesthetic or identification purposes. The coating (e.g. a Eudragit ™ type polymer) can be designed to release the active component at a desired location within the gastro-intestinal tract. Thus, the coating can be selected so as to degrade under certain pH conditions within the gastrointestinal tract, thereby selectively release the compound in the stomach or in the ileum, duodenum, jejenum or colon.

Instead of, or in addition to, a coating, the drug can be presented in a solid matrix comprising a release controlling agent, for example a release delaying agent which may be adapted to release the compound in a controlled manner in the gastrointestinal tract. Alternatively the drug can be presented in a polymer coating e.g. a polymethacrylate polymer coating, which may be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract. Alternatively, the matrix material or release retarding coating can take the form of an erodible polymer (e.g. a maleic anhydride polymer) which is substantially continuously eroded as the dosage form passes through the gastrointestinal tract. In another alternative, the coating can be designed to disintegrate under microbial action in the gut. As a further alternative, the active compound can be formulated in a delivery system that provides osmotic control of the release of the compound. Osmotic release and other delayed release or sustained release formulations (for example formulations based on ion exchange resins) may be prepared in accordance with methods well known to those skilled in the art.

The compound of formula (I) may be formulated with a carrier and administered in the form of nanoparticles, the increased surface area of the nanoparticles assisting their absorption.

In addition, nanoparticles offer the possibility of direct penetration into the cell. Nanoparticle drug delivery systems are described in “Nanoparticle Technology for Drug Delivery”, edited by Ram B Gupta and Uday B. Kompella, Informa Healthcare, ISBN 9781574448573, published 13^th March 2006. Nanoparticles for drug delivery are also described in J. Control. Release, 2003, 91 (1-2), 167-172, and in Sinha etal., Mol. Cancer Ther. August 1, (2006) 5, 1909.

The pharmaceutical compositions typically comprise from approximately 1% (w/w) to approximately 95% (w/w) active ingredient and from 99% (w/w) to 5% (w/w) of a pharmaceutically acceptable excipient or combination of excipients. Preferably, the compositions comprise from approximately 20% (w/w) to approximately 90%,% (w/w) active ingredient and from 80% (w/w) to 10% of a pharmaceutically acceptable excipient or combination of excipients. The pharmaceutical compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, pre-filled syringes, dragees, tablets or capsules.

The pharmaceutically acceptable excipient(s) can be selected according to the desired physical form of the formulation and can, for example, be selected from diluents (e.g solid diluents such as fillers or bulking agents; and liquid diluents such as solvents and cosolvents), disintegrants, buffering agents, lubricants, flow aids, release controlling (e.g. release retarding or delaying polymers or waxes) agents, binders, granulating agents, pigments, plasticizers, antioxidants, preservatives, flavouring agents, taste masking agents, tonicity adjusting agents and coating agents.

The skilled person will have the expertise to select the appropriate amounts of ingredients for use in the formulations. For example tablets and capsules typically contain 0-20% disintegrants, 0-5% lubricants, 0-5% flow aids and/or 0-99% (w/w) fillers/ or bulking agents (depending on drug dose). They may also contain 0-10% (w/w) polymer binders, 0-5% (w/w) antioxidants, 0-5% (w/w) pigments. Slow release tablets would in addition contain 0-99% (w/w) release-controlling (e.g. delaying) polymers (depending on dose). The film coats of the tablet or capsule typically contain 0-10% (w/w) polymers, 0-3% (w/w) pigments, and/or 0-2% (w/w) plasticizers.

Parenteral formulations typically contain 0-20% (w/w) buffers, 0-50% (w/w) cosolvents, and/or 0-99% (w/w) Water for Injection (WFI) (depending on dose and if freeze dried). Formulations for intramuscular depots may also contain 0-99% (w/w) oils.

The compounds of the invention may have a oral bioavailability and are therefore particularly suitable for oral administration, as long as their potency is generally satisfactory, and/or as long as their half-life is also generally satisfactory. Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragee cores or capsules. It is also possible for them to be incorporated into a polymer or waxy matrix that allow the active ingredients to diffuse or be released in measured amounts.

Oral bioavailability can also be increased through various methods known to those skilled in the art, such as those reviewed by Mitra et al. in Int. J. Pharmaceutics, 447, 75-93 (2013) and by Henrikson etal in Br. J. Clinical Pharmacol, doi: 10.1111/bcp.12557. Such methods include the use of absorption enhancers, emulsifying agents, surfactants, preservatives, fatty acids, bile salts and enzyme inhibitors (examples of such additives and excipients include chitosans, cyclodextrins, alkylsaccharides, deoxycholate salts and the like). Delivery technologies such as capsules, microcapsules, emulsions, microemulsions, micelles, microparticles, functionalized or coated nanoparticles or hydrogels can offer further improvements in oral bioavailability.

The compounds of the invention can also be formulated as solid dispersions. Solid dispersions are homogeneous extremely fine disperse phases of two or more solids. Solid solutions (molecularly disperse systems), one type of solid dispersion, are well known for use in pharmaceutical technology (see (Chiou and Riegelman, J. Pharm. Sci., 60, 1281 1300 (1971)) and are useful in increasing dissolution rates and increasing the bioavailability of poorly water-soluble drugs.

This invention also provides solid dosage forms comprising the solid solution described above. Solid dosage forms include tablets, capsules, chewable tablets and dispersible or effervescent tablets. Known excipients can be blended with the solid solution to provide the desired dosage form. For example, a capsule can contain the solid solution blended with (a) a disintegrant and a lubricant, or (b) a disintegrant, a lubricant and a surfactant. In addition a capsule can contain a bulking agent, such as lactose or microcrystalline cellulose. A tablet can contain the solid solution blended with at least one disintegrant, a lubricant, a surfactant, a bulking agent and a glidant. A chewable tablet can contain the solid solution blended with a bulking agent, a lubricant, and if desired an additional sweetening agent (such as an artificial sweetener), and suitable flavours. Solid solutions may also be formed by spraying solutions of drug and a suitable polymer onto the surface of inert carriers such as sugar beads (‘non-pareils’). These beads can subsequently be filled into capsules or compressed into tablets.

The pharmaceutical formulations may be presented to a patient in “patient packs” containing an entire course of treatment in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient’s supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician’s instructions.

Compositions for topical use and nasal delivery include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Additives and excipients may be added as solubilizing agents, enzyme inhibitors or absorption enhancers and include cyclodextrins, chitosans and oligosaccharides. Such compositions can be formulated in accordance with known methods and include those reviewed by Lochhead and Thorne in Adv. Drug Del. Rev. 64, 614-628 (2012) and Falcao et al. in Eur. J. Pharmaceutics and Biopharmaceutics, 88, 8-27 (2014).

Examples of formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped moldable or waxy material containing the active compound. Solutions of the active compound may also be used for rectal administration.

Compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. For administration by inhalation, the powdered formulations typically comprise the active compound together with an inert solid powdered diluent such as lactose, carrier materials, lubricants and flow agents,

Deposition of the aerosol or powder in the lungs is affected by particle properties. Particle properties such as size, shape, electrostatic charge and density can be controlled by a number production methods including spray drying, micromixing and supercritical fluid technologies. Pulmonary delivery formulations and delivery systems are well known to those skilled in the art and have been reviewed by Hickey in J. controlled release, 190, 182-188 (2014) and by Chan et al. in J. controlled release, 193, 228-240 (2014).

The compounds of the formula (I) will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity. For example, a formulation may contain from 1 nanogram to 2 grams of active ingredient, e.g. from 1 nanogram to 2 milligrams of active ingredient. Within these ranges, particular sub-ranges of compound are 0.1 milligrams to 2 grams of active ingredient (more usually from 10 milligrams to 1 gram, e.g. 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2 milligrams of active ingredient).

For oral compositions, a unit dosage form may contain from 1 milligram to 2 grams, more typically 10 milligrams to 1 gram, for example 50 milligrams to 1 gram, e.g. 100 milligrams to gram, of active compound.

For inhalation compositions, a unit dosage form may contain from 0.1 milligram to 5 milligrams, for example, 1 milligram.

For intranasal compositions, a unit dosage form may contain from 0.1 milligram to 30 milligrams, for example 10 milligrams.

The active compound will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.

Therapeutic Uses

According to a further aspect of the invention, there is provided a GLP-1 receptor agonist peptide of the invention, for use in therapy.

In particular embodiments, these compounds may be used for the following diseases or conditions mediated by insulin:

(i) prevention and/or treatment of all forms of diabetes, such as hyperglycemia, type diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reduction of HbA1 C;

(ii) delaying or preventing diabetic disease progression, such as progression in type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulin requiring type 2 diabetes, and/or delaying the progression of non-insulin requiring type 2 diabetes to insulin requiring type 2 diabetes;

(iii) improving β-cell function, such as decreasing β-cell apoptosis, increasing β-cell function and/or β-cell mass, and/or for restoring glucose sensitivity to β-cells;

(iv) prevention and/or treatment of cognitive disorders;

(v) prevention and/or treatment of eating disorders, such as obesity, e.g. by decreasing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa, and/or obesity induced by administration of an antipsychotic or a steroid; reduction of gastric motility; and/or delaying gastric emptying;

(vi) prevention and/or treatment of diabetic complications, such as neuropathy, including peripheral neuropathy; nephropathy; or retinopathy;

(vii) improving lipid parameters, such as prevention and/or treatment of dyslipidemia, lowering total serum lipids; lowering HDL; lowering small, dense LDL; lowering VLDL; lowering triglycerides; lowering cholesterol; increasing HDL; lowering plasma levels of lipoprotein a (Lp(a)) in a human; inhibiting generation of apolipoprotein a (apo(a)) in vitro and/or in vivo·, (viii) prevention and/or treatment of cardiovascular diseases, such as syndrome X; atherosclerosis; myocardial infarction; coronary heart disease; stroke, cerebral ischemia; an early cardiac or early cardiovascular disease, such as left ventricular hypertrophy; coronary artery disease; essential hypertension; acute hypertensive emergency; cardiomyopathy; heart insufficiency; exercise tolerance; chronic heart failure; arrhythmia; cardiac dysrhythmia; syncopy; atheroschlerosis; mild chronic heart failure; angina pectoris; cardiac bypass reocclusion; intermittent claudication (atheroschlerosis oblitterens); diastolic dysfunction; and/or systolic dysfunction;

(ix) prevention and/or treatment of gastrointestinal diseases, such as inflammatory bowel syndrome; small bowel syndrome, or Crohn's disease; dyspepsia; and/or gastric ulcers;

(x) prevention and/or treatment of critical illness, such as treatment of a critically ill patient, a critical illness poly-nephropathy (CIPNP) patient, and/or a potential CIPNP patient;

prevention of critical illness or development of CIPNP; prevention, treatment and/or cure of systemic inflammatory response syndrome (SIRS) in a patient; and/or for the prevention or reduction of the likelihood of a patient suffering from bacteraemia, septicaemia, and/or septic shock during hospitalisation;

(xi) prevention and/or treatment of polycystic ovary syndrome (PCOS); and/or (xii) prevention and/or treatment of central peripheral nervous disorders including neurodegeneration and/or cognition disorders such as Alzheimer’s disease, Parkinson’s disease, stroke, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and/or neuropathies.

In a particular embodiment, the indication is selected from the group consisting of (i)-(iii) and (v)-(viii), such as indications (i), (ii), and/or (iii); or indication (v), indication (vi), indication (vii), and/or indication (viii).

In another particular embodiment, the indication is (i). In a further particular embodiment the indication is (v). In a still further particular embodiment the indication is (viii).

Following demonstration of GLP1R receptor expression in the brain, the role of this receptor in the central nervous system has been intensely studied. It has been shown that activation of GLP1R and the subsequent activation of the cAMP pathway can result in neurite outgrowth in PC12 cells and SK-N-SH human neuroblastoma cells (Perry et al., J.

Pharmacol. Exp. Ther., 300, 958-966 (2002)). Further examination of the neurotrophic properties of GLP-1 and its longer-activing analog extendin-4 in cultured hippocampal neurons has demonstrated that they can completely protect cultured rat hippocampal neurons against glutamate-induced apoptosis (Perry et al., J. Pharmacol. Exp. Ther., 302, 881-888 (2002)).

In addition, intracerebroventricular administration of GLP-1 enhances associative and spatial learning through GLP1R. Consistently, GLP1R knock-out mice exhibit learning deficiency in contextual fear conditioning tests as well as enhanced seizure severity and neuronal injury after kainite administration. These phenotypes are corrected after hippocampal Glp 1r gene transfer. Conversely, overexpression of GLP1R in rat hippocampus results in improved learning and memory. GLP1R-deficient mice also have an intermediate phenotype in heterozygotes and phenotypic correction after Glplr gene transfer in hippocampal somatic cells (During et al., Nat. Med., 9, 1173-9 (2003)).

Taken together these data indicate that GLP1R in the CNS could be a new target for neuroprotective and cognitive enhancing agents. Consistent with this prediction, preclinical studies showed that exendin-4 administration in two different models of Parkinson’s disease resulted in significant arrest in the progression of, as well as in the reverse of, nigral lesions once established (Harkavyi et al., J. Neuroinflammation, 5, 19 (2008)). Exendin-4 treatment has also been shown to protect dopaminergic neurons against degeneration following treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP model of Parkinson’s disease) as well as a reduction in the associated deterioration of dopamine levels and motor functions (Li et al., Proc. Natl. Acad. Sci. U.S.A., 106, 1285-90 (2009)). In a mouse model of Alzheimer's disease (APP/PS1 mice), peripheral administration of a modified form of GLP-1 (liraglutide) prevented memory impairments in object recognition and water maze tasks. In addition, hippocampal synaptic loss and deterioration of synaptic plasticity were prevented. These improvements were associated with 40-50% reduction in amyloid plaque count in the cortex and dense-core plaque numbers (McClean et al., J. Neurosci., 31,6587-94 (2011)). Similarly, in a transient middle cerebral artery occlusion or60-minute focal cerebral ischemia stroke models, administration of exendin-4 reduced brain damage and improved functional outcome (Li et al., Proc. Natl. Acad. Sci. U.S.A., 106, 1285-90 (2009)). In a study to find an effective treatment for a genetic form of diabetes that is present in some Huntington's disease patients, the efficacy of exendin-4 was tested in a mouse model of Huntington’s disease. As well as controlling the blood sugar levels, administration of exendin-4 suppressed cellular pathology in both brain and pancreas leading to improved motor function and extended the survival time of the Huntington's disease mice. These positive outcomes were correlated with reduced accumulation of the disease causing mutant huntingtin aggregates in both islet and brain cells (Martin et al., Diabetes, 58, 318-28 (2009)).

More recently, in a limited clinical trial, treatment with exenatide resulted in clinically relevant improvements in Parkinson’s disease across motor and cognitive scores compared with the control group (Aviles-Olmos et al., J. Clin. Invest., 123, 2730-6 (2013)).

Examples

The invention is illustrated by the Examples described below. In the table below, the term “Cap” refers to the moiety Q-(CH₂)_X-NH-C(=O)-C(R¹)(R²)-C(=O)- in formula (I).

Table 1: Peptide Sequences for Examples 1-24

Sequence	N	CM X X O O	CM X X O O	CM X X O O
m CO CO	(3,5- dimethylphenyl) butylamine	(3,5- dimethylphenyl) butylamine	(3,5- dimethylphenyl) butylamine
co co	(2'-Et-4'-OMe)Bip	(2'-Et-4'-OMe)Bip	(2'-Et-4'-OMe)Bip
co CO co	Q	Q	Q
£	ω	ω	ω
H	H	H	H
CM CO co	a-Me-Phe(2-F)	a -Me-Phe(2-F)	a -Me-Phe(2-F)
H	H	H	H
0	0	0	0
co co	LJJ	0	tetrazol-5- yl-Ala
Cap	Cap 1	Cap 1	Cap 1
Example No	-	CN	CO

CM	CM	CM	CM
X	X	X	X
X	X	X	X
Ο	Ο	Ο	Ο
Ο	Ο	Ο	Ο
c ω	Φ	c ω	ε= Φ
ω c	C	Φ C	Φ C
ώ ο. Ε	Ε	ιό ο. Ε	ιή ο. Ε
co >, 15	_φ	co >, .<9	co ν
i-> JZ >,		i-> JZ >,	ϊ-> JZ >,
Μ-» Μ-»	C	Μ-» Μ-»	Μ-» Μ-»
ω =3	ο	Φ =3	Φ =3
Ε -°	C	Ε -°	Ε -°
Ώ		Ώ	Ώ
Ω_	Ω_	Ο.	Ώ_
ώ	ώ	in	ώ
ω	ω	φ	Φ
Ο	Ο	ο	Ο
Μ—»	Μ—»	+-»	Μ—»
LLI	LLI	LU	XI
_1	_1	_1	_1
CN	CN	CM	CM
Ο	Ο	Ω	D-0- Me
ω	ω	ω	ω
Η	Η	Η	Η
ι_ι_	ι_ι_	X	X
CN	CN	CM	CM
ω	ω	Φ	Φ
_c	_c	.C	_c
		X	X
ώ	ώ	φ	φ
ό	ό	σ	ό
Η	Η	Η	Η
Ο	Ο	Ο	Ο
1 m	1 m	1 CM	Φ
Ο —ξ	Ο —ξ	Ο —ξ	2
Ν <	Ν <	Ν <	1 ο
03 —	03 —	03 —
U_ >». Μ-»	U_ >». Μ-»	U_ >». Μ-»	1 1 1 1
ω	ω	Φ
Μ—»	Μ—»	Μ—»
CN		,-
Ω_	Ω_	Ω-	Ώ_
03	03	03	03
Ο	Ο	Ο	Ο
	m	(Ο	1^

CM X X Ο Ο	CM X X O O	CM X z O O	CM X X O O
(3,5- dimethylphenyl) butylamine	(3,5- dimethylphenyl) butylamine	nonylamine	(3,5- dimethylphenyl) butylamine
(2'-Et-4'-OMe)Bip	(2'-Et-4'-OMe)Bip	octyl-Gly	(3,5- dimethyl)hhPhe
Q	tetrazol -5-yl- Ala	Q	Q
DAP	ω	ω	ω
H	H	H	H
a-Me-Phe(2-F)	a-Me-Phe(2-F)	a-Me-Phe(2-F)	a-Me-Phe(2-F)
H	H	H	H
0	0	0	0
LJJ	tetrazol-5- yl-Ala	tetrazol-5- yl-Ala	tetrazol-5- yl-Ala
Cap 1	Cap 1	Cap 1	Cap 1
00	σ>	o	-

CM	CM	CM	CM
ΞΕ	ΞΕ	ΞΕ	Ζ
Ζ	Ζ	Ζ	ζ
Ο	Ο	Ο	Ο
ο	ο	ο	Ο
φ	φ
C Γ37	Ε cT		ΞΑ
Ε ι-	Ε	C φ	ε= Φ
φ ω Ε	φ ω Ε	Φ C ώ a Ε	Φ C ιή a Ε
ο ο Φ ω	ο ο Φ (Ζ)	co γ <	ω γ <
Ό —	Ό —	Μ-» Μ-»	<-> _c: >» Μ-» Μ-»
φ ω	φ ω	Φ =3	Φ =3
Μ—» Ε 02	Μ—» Ε 02	Ε -°	Ε -°
φ t±L Ο.	φ t±L ο.	Τ3	Τ3
ο.	ο.	Ο.	Ο.
ώ	ώ	ώ	co ΕΓ
φ	φ	Ο φ	Ο φ
		LU c	LU c
Ο	Ο	Q- Ο	Q- ο
		- (Ζ)	- (Ζ)
		-	-
-Λ	-Λ	-Λ ω	-Λ ω
LU 1	LU 1
Ν	Ν	Ν “	Ν “
Ω	Ω	Ω	Ω
ω	ω	ω	ω
Η	Η	Η	Η
υ_	υ_	LL	υ_
<Ε	CJ	CXI	CJ
Φ	φ	Φ	φ
_c	_c	.C	_c
CL	CL	CL	CL
Φ	Φ	Φ	Φ
ό	ό	σ	ό
Η	Η	Η	Η
Ο	Ο	Ο	Ο
1 m	1 m	1 m	1 m
i ro	Ε ro	Ε ro	-Ε φ
ο 3? Ν <	ο 3? Ν <	ο 3? Ν <	ο 3? Ν <
φ —	φ —	φ —	φ —
U_ >». Μ-»	U_ >». Μ-»	U_ >». Μ-»	U_ >». Μ-»
Φ	Φ	Φ	Φ
Μ—»	Μ—»	Μ—»	Μ—»
Τ-	Τ-	,-	Τ-
Ο.	Ο.	ο.	Ο.
Φ	Φ	φ	Φ
Ο	Ο	Ο	Ο
C\l	co		m

CM	CM	CM	CM
X	X	X	X
X	X	X	X
Ο	Ο	Ο	Ο
Ο	Ο	Ο	Ο
	φ
c φ	C φ	C φ
φ c	C	Φ C	Φ C
ιό ο. Ε	Ε	ιό ω. Ε	ιό Ω- Ε
	_φ	co >, 15	co
A· JZ >>		i-> χ: >>	ϊ-> JZ >>
ώ ό	ο	Φ ό	Φ b
Ε -°	C	Ε -°	Ε -°
Τ3		Τ3	Τ3
Ο.		Ο.	Ω.
ώ		όο	00
a?		α?	0?
	ο 1
Ο	Ο	Ο
1		1	1
Μ—»	ο	+-»	Μ—»
XI		LU	X
CM		CM	CM
0-2 can	Ο	Ω	Ω
ά ω L-1 Τ3
ω	ω	ω	ω
Η	Η	Η	Η
χ		X	X
CM,		ά	ά
φ	Ο 1	Φ	φ
_c	.C	_c
		X	X
φ	ο	φ	φ
	ο
ό		σ	σ
Η	Η	Η	Η
Ο	Ο	Ο	Ο
1 m	1 m	1 m	1 m
i to	i to	ό to	ό to
ο 3? Ν <	ο 3? Ν <	ο 3? Ν <	ο 3? Ν <
Φ —	φ —	φ —	φ —
U_ >». Μ-»	U_ >». Μ-»	U_ >». Μ-»	U_ >». Μ-»
Φ	Φ	Φ	Φ
Μ—»	Μ—»	Μ—»	Μ—»
τ-	τ-	CO
Ο.	Ο.	ο.	Ω.
Φ	Φ	φ	Φ
Ο	Ο	Ο	Ο
CO	1^	co	σ>

CM	CM	CM	CM
X	X	X	X
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Cap 2 =

Cap 6 =

CH

Cap 7 =

V^N5 a-Me-Phe(2-F) =

(3,5-dimethyl)hhPhe =

tetrazol-5-yl-Ala =

pentadecylamine = ίο

(3,5-dimethylphenyl)butylamine =

Solution phase chemical synthesis

General Experimental Procedures

Commercial reagents were used as received from the supplier, without further purification. Chromatography refers to column chromatography performed using 60-120 mesh silica gel and executed under nitrogen pressure (flash chromatography) conditions.

¹H NMR spectra were recorded at 400 MHz in deuterated solvents, as stated. Chemical shift values are expressed in parts per million, i.e. δ-values. The following abbreviations are used for the multiplicity for the NMR signals: s = singlet, br = broad, d = doublet, t = triplet, dd = doublet of doublets, q = quartet, m = multiplet.

Chemical abbreviations: HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b]pyridinium-3-oxide hexafluorophosphate, HOBt= 1-hydroxybenzotriazole, EDC = Λ/-(3dimethylaminopropyl)-/\/'-ethylcarbodiimide, TFA = 1,1,1-trifluoroacetic acid, Fmoc = fluorenylmethoxycarbonyl, DIEA = /V,/V-di/sopropylethylamine, THF = tetrahydrofuran, DIAD = di/sopropylazodicarboxylate, Cbz = carboxybenzyl, Boc = terf-butoxycarbonyl, DMF = N,Ndimethylformamide, DCA = dicyclohexylamine.

Preparative HPLC purification was performed under the following conditions: column = Xbridge C-18, 150 X 20 mm, 5 pm; flow rate = 17 mL/min, gradient = [time (min)/solvent B in A (%)]: 0.00/40, 15.00/50, 17.00/100, 20.00/40, (solvent A = 10 mM ammonium acetate in water, solvent B = acetonitrile).

Mass-directed preparative HPLC was performed under the following conditions: column = Gemini C-18, 100 x 21 mm, 5 pm; flow rate = 20 mL/min, gradient = [time (min)/solvent B in A (%)]: 0.00/20, 0.70/20, 9.00/60, 10.50/100, 12.00/100, 12.20/20, 14.5/20 (solvent A = 10 mM ammonium bicarbonate in water, solvent B = acetonitrile), mass detector = ZQ mass spectrometer with ESCi probe.

LCMS analysis was performed under the following conditions:

Method 1: Column: Phenomenex Gemini-NX C-18, 3 pm, 2.0 x 30 mm. Gradient [time (min)/solvent D in C (%)]: 0.00/2, 0.10/2, 8.40/95, 9.40/95, 9.50/2, 10.00/2 (solvent C = 1.58 g ammonium formate in 2.5 L water + 2.7 mL ammonia solution; solvent D = 2.5 L acetonitrile + 132 mL (5 %) solvent C + 2.7 mL ammonia solution). Injection volume 1 pL;

UV detection 230 to 400 nM; column temperature 45 °C; 1.5 mL/min. Instruments: Waters Alliance 2795, Waters 2996 PDA detector, Micromass ZQ.

Method 2: Column: Phenomenex Gemini-NX C-18, 3 pm, 2.0 x 30 mm. Gradient [time (min)/solvent D in C (%)]: 0.00/2, 0.10/2, 2.50/95, 3.50/95, (solvent C = 1.58 g ammonium formate in 2.5 L water + 2.7 mL ammonia solution; solvent D = 2.5 L acetonitrile + 132 mL (5 %) solvent C + 2.7 mL ammonia solution). Injection volume 1 pL; UV detection 230 to 400 nM; column temperature 45 °C; 1.5 mL/min. Instruments: Waters Alliance 2795, Waters 2996 PDA detector, Micromass ZQ.

Method 3: Column: Phenomenex Gemini-NX C-18, 3 pm, 2.0 x 30 mm. Gradient [time (min)/solvent B in A (%)]:0.00/2, 0.10/2, 8.40/95, 9.40/95, 9.50/2, 10.00/2, (solvent A = 1.58 g ammonium formate in 2.5 L water + 2.5 ml_ formic acid; solvent B = 2.5 L acetonitrile + 135 ml_ water + 2.5 ml_ formic acid). Injection volume 1 μΙ_; UV detection 230 to 400 nM; column temperature 45 °C; 1.5 mL/min. Instruments: Waters Alliance 2795, Waters 2996 PDA detector, Micromass ZQ.

Method 4: Column: Phenomenex Kinetic XB C-18, 100 A, 2.6 pm, 2.0 x 30 mm. Gradient [time (min)/solvent B in A (%)]: 0.00/5, 1.37/98, 1.60/98, 1.83/5, 2.29/5, (solvent A = 0.1% formic acid in water; solvent B = acetonitrile). Injection volume 1 pl_; UV detection 220, 240 and 254 nM; column temperature 40 °C; 2.0 mL/min and mass spectrometric detection. Method 5: Column: Phenomenex Gemini C-18, 110 A, 5 pm, 4.6 x 50 mm. Gradient [time (min)/solvent B in A (%)]: 0.00/20, 0.60/20, 6.50/98, 7.50/98, 7.60/20, 10.0/20 (solvent A = mM ammonium bicarbonate in water, pH 10; solvent B = acetonitrile). Injection volume 1 pL; UV detection 220, 240 and 254 nM; column temperature 40 °C; 1.2 mL/min and mass spectrometric detection.

Method 6: Column: BEH C-18, 1.7 pm, 2.1 x 50 mm. Gradient [time (min)/solvent B in A (%)]: 0.00/10, 2.50/90, 3.5/100, 4.5/100, (solvent A = 0.1% ammonium hydroxide in water; solvent B = 0.1% ammonium hydroxide in acetonitrile). Instrument: Waters Acquity H Class with PDA and SQ detectors.

Method 7: Column: BEH C-18, 1.7 pm, 2.1 x 50 mm. Gradient [time (min)/solvent B in A (%)]: 0.00/10, 2.50/90, 3.5/100, 4.5/100, (solvent A = 0.1% formic acid in water; solvent B = 0.1% formic acid in acetonitrile). Instrument: Waters Acquity H Class with PDA and SQ detectors.

Method 8: Column: BEH C-18, 1.7 pm, 2.1 x 50 mm. Gradient [time (min)/solvent B in A (%)]: 0.00/5, 0.40/5, 0.80/35, 1.20/55, 2.70/95, 3.30/95, 3.31/5, 4.00/5 (solvent A = 5 mM ammonium acetate in 0.1% formic acid in water; solvent B = 0.1% formic acid in acetonitrile). Instrument: Waters Acquity H Class with PDA and SQ detectors.

Method 9: Column: X-bridge C-18, 5 pm, 4.6 x 150 mm. Gradient [time (min)/solvent B in A (%)]: 0.00/10, 0.01/10, 5.00/90, 7.00/100, 11.0/100, 11.01/10, 12.00/10 (solvent A = 20 mM ammonium acetate in water; solvent B = MeOH). Instrument: Shimadzu Nexera with PDA and 2020 detectors.

Method 10: Column: Acquity BEH C-18, 1.7 pm, 2.1 x 100 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/2, 2.00/2, 7.00/50, 8.50/80, 9.50/2, 10.0/2; Solvents: solvent A = 5 mM ammonium acetate in water; solvent B = acetonitrile; Injection volume 1 pL; Detection wavelength 214 nm; Column temperature 30 °C; Flow rate 0.3 mL per min. Instruments: Waters Acquity UPLC, Waters 3100 PDA Detector, SQD.

Method 11: Column: Acquity HSS-T3, 1.8 pm, 2.1 x 100 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/2, 2.00/2, 7.00/50, 8.50/80, 9.50/2, 10.0/2; Solvents: solvent A = 0.1% TFA in water; solvent B = MeOH; Injection volume 1 μΙ_; Detection wavelength 214 nm; Column temperature 30 °C; Flow rate 0.3 mL per min. Instruments: Waters Acquity UPLC, Waters 3100 PDA Detector, SQD.

Method 12: Column: Acquity HSS-T3, 1.8 pm, 2.1 x 100 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/10, 2.00/15, 4.50/55, 6.00/90, 8.00/90, 9.00/10, 10.0/10; Solvents: solvent A = 0.1% TFA in water; solvent B = acetonitrile; Injection volume 1 pL; Detection wavelength 214 nm; Column temperature 30 °C; Flow rate 0.3 mL per min. Instruments: Waters Acquity UPLC, Waters 3100 PDA Detector, SQD.

Preparation of 2,2,5,5-tetramethyl-[1,3]dioxane-4,6-dione

A mixture of Meldrum’s acid (10.0 g, 69.4 mmol), potassium carbonate (47.9 g, 347 mmol) and methyl iodide (30.0 mL, 208 mmol) in acetonitrile (150 mL) was heated at 75 °C for 16 hours. The reaction mixture was cooled to room temperature, diluted with CH₂CI₂ (200 mL), filtered and evaporated to dryness in vacuo. Water (100 mL), EtOAc (150 mL) and hexane (150 mL) were added and the resulting mixture was stirred for 5 min. The organic layer was separated, washed with 10% aqueous solution of sodium thiosulfate (125 mL) and water (125 mL), dried over anhydrous Na₂SO₄ and solvent was removed in vacuo to afford the title compound as a white solid (9.0 g, 75%).

¹H NMR (400MHz, CDCI₃): δ: 1.67 (s, 6H), 1.77 (s, 6H)

Preparation of 7,7-dimethyl-6,8-dioxaspiro[3.5]nonane-5,9-dione

A solution of cyclobutane-1,1-dicarboxylic acid (1.0 g, 6.94 mmol) in acetic anhydride (0.8 mL, 8.33 mmol) and concentrated H₂SO₄, was cooled to 0 °C and treated with acetone (0.83 mL, 90.2 mmol) by drop wise addition. The resulting reaction mixture was stirred at 0 °C for 2 hours. Water was added to the reaction mixture and the solid formed was filtered off, washed with water (2 x 5 mL) and dried in vacuo to afford the title compound as a white solid (1.04 g, 81%), which was used without further purification.

¹H NMR (400 MHz, DMSO-cfe): δ ppm 1.62 (s, 6H), 2.04-2.13 (m, 2H), 2.62 (t, J - 8.5 Hz, 4H)

Preparation of 2-(1 -Trityl-1 H-imidazole-4-yl)-ethyl amine

Step 1: Histamine dihydrochloride (4.5 g, 22.4 mmol) and triethylamine (10.6 mL, 75.7 mmol) were dissolved in absolute MeOH (90 mL) and the resulting solution was stirred at room temperature for 10 min. A solution of trifluoroacetic acid ethyl ester (3.19 mL, 26.8 mmol) in

MeOH (7 mL) was added drop wise over 30 min at 0 °C and the resulting reaction mixture was warmed to room temperature and stirred for 3.5 hours. The mixture was then diluted with CH₂CI₂ (100 mL) and treated with triethylamine (6.82 mL, 44.8 mmol) and trityl chloride (7.5 g, 26.8 mmol; added in portions). The resulting reaction mixture was stirred overnight at room temperature then diluted with water (130 mL) and extracted with chloroform (3 x 90 mL). All organic layers were combined, dried over anhydrous Na₂SO4, and evaporated to a beige solid. The solid was triturated with hexane (100 mL) and filtered to afford 2,2,2triflouro-/V-[2-(1 -trityl-1 H-imidazol-4-yl)-ethyl]-acetamide as a white solid (8.92 g, 81%), which was used without further purification.

¹H NMR (400 MHz, CDCI₃): δ ppm 8.45 (1H, s, br), 7.43 (1H, s), 7.37 (9H, m), 7.15 (6H, m), 6.64 (1H, s), 3.67 (2H, s), 2.78 (m, 2H)

LCMS (Method 6): 3.27 min, [M+H]⁺ = 450 observed

Step 2: 2,2,2-trifluoro-/V-[2-(1 -trityl-1 /7-imidazol-4-yl)-ethyl]-acetamide (8.92 g, 19.8 mmol) was dissolved in THF (200 mL) and MeOH (240 mL). A solution of NaOH (3.96 g, 0.099 mol) in water (100 mL) was added and the resulting reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was concentrated in vacuo to afford a residue which was partitioned between chloroform (240 mL) and water (150 mL). The separated aqueous layer was back-extracted with chloroform (2 x 100 mL) and the organic extracts were combined, dried over anhydrous sodium sulfate and concentrated in vacuo to give brown oil, which was dried in vacuo to afford the title compound as a beige solid (7.01 g, quantitative), which was used without further purification.

¹H NMR (400MHz, CDCI₃): δ ppm 7.40-7.25 (10H, m), 7.15 (6H, m), 6.60 (1H, s), 2.95 (2H, t, J = 6.4 Hz), 2.67 (2H, t, J = 6.6 Hz), NH₂ not observed

Preparation of 3-{[2-(4-fluorophenyl)ethyl]amino}-2,2-dimethyl-3-oxopropanoic acid

2-(4-Fluorophenyl)ethan-1-amine (2.0 g, 14.4 mmol) and triethylamine (7.25 g, 71.9 mmol) were dissolved in toluene (30 mL) and heated to 75 °C. The resulting mixture was treated with a solution of 2,2,5,5-tetramethyl-[1,3]dioxane-4,6-dione (3.7 g, 21.6 mmol) in toluene (20 mL) by drop wise addition over 20 min. The reaction mixture was stirred at 75 °C for 3 hours then concentrated in vacuo. The residue was dissolved in chloroform (150 mL) and washed with 10% aqueous solution of citric acid (100 mL); the aqueous phase was then back-extracted with chloroform (2 x 50 mL) and all organic extracts were combined and dried over anhydrous Na₂SO₄. The solvents were then removed in vacuo, and the resulting residue was triturated with hot chloroform (50 mL) and hexanes (25 mL). The suspension was stirred at room temperature for 2 hours before the solids were filtered, washed with a mixture of chloroform and hexanes (1:1,2 x 25 ml_) and dried in vacuo to afford the title compound as a white solid (2.2 g, 61%).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.28 (1H, s), 7.16 (2H, m), 7.03 (2H, m), 6.23 (1H, s, br), 3.56 (2H, app. q, J = 6.7 Hz), 2.85 (2H, t, J = 6.9 Hz), 1.48 (6H, s), CO₂H not observed LCMS (Method 8): 1.91 min, [M+H]⁺ = 254 observed

Preparation of 3-{[2-( 1 -trityl-1 H-imidazole-4-yl)ethyl]amino}-2,2-dimethyl-3oxopropanoic acid

The title compound (8.32 g, 88%) was prepared from 2,2,5,5-tetramethyl-[1,3]dioxane-4,6dione (5.24 g, 30.4 mmol) and 2-(1 -trityl-1 /7-imidazole-4-yl)-ethyl amine (7.18 g, 20.3 mmol), according to the procedure described for 3-{[2-(4-fluorophenyl)ethyl]amino}-2,2-dimethyl-3oxopropanoic acid.

¹H NMR (400 MHz, CDCI₃): δ ppm 7.68 (2H, s, br), 7.40 (9H, m), 7.11 (6H, m), 6.81 (1H, s, br), 3.29 (2H, m), 2.62 (2H, t, J= 6.6 Hz), 1.21 (6H, s), CO₂H not observed LCMS (Method 6): 2.01 min, [M+H]⁺ = 468 observed

Preparation of 3-{[2-(4-chlorophenyl)ethyl]amino}-2,2-dimethyl-3-oxopropanoic acid

The title compound (655 mg, 69%) was prepared from 2,2,5,5-tetramethyl-[1,3]dioxane-4,6dione (0.912 g, 5.30 mmol) and 2-(4-chlorophenyl)ethan-1-amine (491 pL, 3.53 mmol), according to the procedure described for 3-{[2-(4-fluorophenyl)ethyl]amino}-2,2-dimethyl-3oxopropanoic acid, with a final purification performed by gradient flash chromatography, eluting with mixtures of CH₂CI₂, MeOH and acetic acid.

¹H NMR (400MHz, CDCI₃): δ ppm 7.21 (2H, d, J= 8.4 Hz), 7.05 (2H, d, J= 8.4 Hz), 6.17 (1H, s, br), 3.47 (2H, m), 2.76 (2H, t, J = 7.0 Hz), 1.39 (6H, s), CO₂H not observed LCMS (Method 1): 0.28 min, [M-H]’ = 268 observed

Preparation of 3-[(4-fluorobenzyl)amino]-2,2-dimethyl-3-oxopropanoic acid

The title compound (950 mg, 94%) was prepared from 2,2,5,5-tetramethyl-[1,3]dioxane-4,6dione (1.09 g, 6.34 mmol) and 4-fluorobenzylamine (485 pL, 4.22 mmol), according to the procedure described for 3-{[2-(4-fluorophenyl)ethyl]amino}-2,2-dimethyl-3-oxopropanoic acid, with a final purification performed by gradient flash chromatography, eluting with mixtures of CH₂CI₂, MeOH and acetic acid.

¹H NMR (400MHz, CDCI₃): δ ppm 7.23 (2H, m), 7.04 (2H, m), 4.98 (1H, s, br), 4.45 (2H, d, J = 5.5 Hz), 1.54 (6H, s), CO₂H not observed LCMS (Method 1): 0.29 min, [M-H]’ = 238 observed

Preparation of 3-{[2-(4-fert-butoxyphenyl)ethyl]amino}-2,2-dimethyl-3-oxopropanoic acid

The title compound (1.11 g, 90%) was prepared from 2,2,5,5-tetramethyl-[1,3]dioxane-4,6dione (1.03 g, 5.99 mmol) and 2-(4-terf-butoxyophenyl)ethan-1-amine (780 mg, 4.00 mmol), according to the procedure described for 3-{[2-(4-fluorophenyl)ethyl]amino}-2,2-dimethyl-3oxopropanoic acid, with a final purification performed by gradient flash chromatography, eluting with mixtures of CH₂CI₂, MeOH and acetic acid.

¹H NMR (400MHz, cfe-DMSO): δ ppm 7.64 (1H, m), 7.05 (2H d, J = 8.2 Hz), 6.84 (2H, m), 3.21 (2H, m), 2.64 (2H, t, J= 7.2 Hz), 1.23 (9H, s), 1.21 (6H, s), CO₂H not observed LCMS (Method 1): 1.57 min, [M+H]⁺ = 308 observed

Preparation of 1 -((2-(1 -trityl-1 H-imidazol-4-yl)ethyl)carbamoyl)cyclobutane-1 carboxylic acid

The title compound (0.90 g, 70%) was prepared from 7,7-dimethyl-6,8dioxaspiro[3.5]nonane-5,9-dione (750 mg, 4.03 mmol) and 2-(1-trityl-1/7-imidazole-4-yl)-ethyl amine (949 mg; 2.683 mmol), according to the procedure described for 3-{[2-(4fluorophenyl)ethyl]amino}-2,2-dimethyl-3-oxopropanoic acid.

¹H NMR (400 MHz; cfe-DMSO): δ ppm 7.68 (1H, m). 7.35 -7.42 (9H, m), 7.32 (1H, s), 7.08 (6H, m), 6.70 (1H, s), 3.29 (2H, m), 2.58 (2H, m), 2.30 (4H, m), 1.75 (1H, m), 1.66 (1H, m), CO₂H not observed

LCMS (Method 10): 4.87 min, [M+H]⁺ = 480 observed

Preparation of 3-{[3-(1H-imidazol-1-yl)propyl]amino}-3-oxopropanoic acid

A solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (4.14 g, 28.77 mmol) in anhydrous acetonitrile (50 mL) was treated with 3-(1/7-imidazol-1-yl)propan-1-amine (3.0 g, 24.0 mmol) and the resulting mixture was heated at 70 °C for 3 hours. The reaction mixture was cooled to room temperature. After decanting the supernatant liquid the crude gummy residue was purified by flash chromatography, eluting with a gradient of 5-25% methanol in CH₂CI₂ containing 0.5% ammonia, to afford the title compound as a gummy solid (840 mg, 17%).

¹H NMR (400 MHz; cfe-DMSO): δ ppm 8.19 (1H, s), 7.63 (1H, s, br), 7.19 (1H, s, br), 6.91 (1H, s, br), 3.97 (2H, m), 3.10 (2H, m), 3.01 (2H, m), 1.82 (2H, m), CO₂H not observed LCMS (Method 11): 1.90 min, [M+H]⁺ = 212 observed

Preparation of (S)-2-((((9H-fluoren-9-yl) methoxy) carbonyl) amino)-3-(2H-tetrazol-2-yl) propanoic acid (S)-2-amino-3-(2/7-tetrazol-2-yl) propanoic acid (0.95 g, 6.04 mmol) and Na₂CO₃(1.60 g,

15.1 mmol) were dissolved in H₂O (35 mL) and cooled to 0 °C. A solution of Fmoc-CI (1.7 g, 6.65 mmol) in 1,4-dioxane (10 mL) and water (5 mL) was added drop wise and the resulting mixture was stirred at room temperature for 16 hours. The mixture was diluted with H₂O (100 mL), acidified with 1M HCI to -pH 2-3 and extracted with EtOAc (3 x 200 mL). The organic layers were combined, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by trituration with MeOH and diethyl ether to afford the title compound as a white solid (810 mg, 39%).

¹H NMR (400 MHz, cfe-DMSO): δ ppm 13.35 (1H, s), 9.00 (1H, s), 7.90 (2H, m), 7.65 (2H, m), 7.40 (2H, m), 7.32 (2H, m), 5.11 (1H, m), 5.02 (1H, m), 4.65 (1H, m), 4.28-4.16 (3H, m), 3.17 (1H, d, J = 4.9 Hz)

LCMS (Method 8): 2.07 min, [M-H]' = 378 observed

Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-trityl-2H-tetrazol5-yl)propanoic acid

A solution of (S)-2-((((9/7-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2/7-tetrazol-5-yl)propanoic acid (430 mg, 1.13 mmol) in CH₂CI₂ (10 mL) was cooled to 0 °C and treated with triethylamine (0.5 mL, 3.39 mmol). After stirring for 5 min, trityl chloride (316 mg, 1.13 mmol) was added and the resulting mixture was stirred at 0 °C for 2 hours. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (3 x 20 mL). The organic layers were combined and washed with brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography, eluting with a solvent gradient of 2% to 8% MeOH in CH₂CI₂ to afford the title compound as a white solid (475 mg, 67 %).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.74 (2H, m), 7.54 (2H, m), 7.48-7.24 (13H, m), 7.12-7.02 (6H, m), 5.78 (1H, m), 4.85 (1H, m), 4.44-4.36 (2H, m), 4.32 (1H, m), 3.62 (2H, m), CO₂H not observed

LCMS (Method 10): 5.99 min, [M-H]' = 620 observed

Preparation of (S)-2-amino-4-(((S)-decan-2-yl)oxy)-4-oxo butanoic acid

Step 1: (S)-4-(feff-butoxy)-3-((terf-butoxycarbonyl)amino)-4-oxobutanoic acid (500 mg, 1.72 mmol) was dissolved in CH₂CI₂ (10 mL) and treated with triethylamine (0.480 mL, 3.45 mmol). The resulting mixture was stirred for 5 min at room temperature and treated with (S)decan-2-ol (300 mg, 1.90 mmol), EDO.HCI (858 mg, 4.49 mmol) and HOBt (700 mg, 5.18 mmol). The reaction mixture was stirred for 16 hours at room temperature then concentrated in vacuo. The residue was purified by flash chromatography, eluting with a gradient of 0 to 3 % EtOAc in hexane to afford 1-(teff-butyl)-4-((S)-decan-2-yl)(te/Y-butoxycarbonyl)-Laspartate as a colourless oil (186 mg, 25%).

¹H NMR (400 MHz, cfe-DMSO): δ ppm 7.20 (1H, d, J= 8.5 Hz), 4.79 (1H, m), 4.21 (1H, m), 2.68 (1H, m), 2.55 (1H, m), 1.55-1.35 (19H, m) 1.27-1.20 (13H, m) 1.15 (3H, d, J= 6.1 Hz), 0.86 (3H, m)

Step-2: A solution of 1 -((erf-butyl) 4-((S)-decan-2-yl) (terf-butoxycarbonyl)-L-aspartate (180 mg, 0.419 mmol) in CH₂CI₂ (5.0 ml_) was cooled to 0°C then treated with TFA (0.340 ml_,

4.19 mmol) and stirred for 15 min at 0 °C. The resulting mixture was warmed to room temperature, stirred for a further 16 hours then concentrated in vacuo. The residue triturated with diethyl ether and n-pentane to afford the title compound as an off-white solid (50.0 mg, 44%).

¹H NMR (400 MHz, TFA): δ ppm 4.80 (1H, m), 4.45 (1H, s), 3.14 (2H, m), 1.44 (1H, m), 1.34 (1H, m), 1.05 (18H, m), 0.59 (3H, m)

LCMS (Method 8): 1.83 min, [M+H]⁺ = 274 observed

Preparation of 3-(4'-((2,5,8,11,14-pentaoxahexadecan-16-yl)oxy)-2'-ethyl-[1,Γbiphenyl]-4-yl)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid

Step 1: A solution of methyl /V-(terf-butoxycarbonyl tyrosinate (5.0 g, 17 mmol) in CH₂CI₂ (15 ml_) and pyridine (6.8 ml_, 8.5 mmol) was cooled to -5 °C and treated with trifluoromethanesulfonic anhydride (4.8 g, 17 mmol). The resulting mixture was stirred at -5 °C for 3 hours then poured into water (50 ml_) and extracted with CH₂CI₂ (3 x 30 ml_). The organic extracts were combined, washed sequentially with 0.5 N NaOH solution (50 ml_), 1 N HCI (2 x 50 ml_), dried over Na₂SO₄and concentrated in vacuo to afford 2-((ferfbutoxycarbonyl) amino)-3-(4-(((trifluoromethyl)sulfonyl)oxy)phenyl)propanoate (6.2 g, 86%) as an orange gum.

¹H NMR (400 MHz, CDCI₃): δ ppm 7.24 (4 H, m), 5.03 (1H, m), 4.79 (1H, m), 3.75 (3 H, s),

3.19 (1 H, m), 3.06 (1H, m) 1.43 (9 H, s)

LCMS (Method 8): 2.64 min, [M+H-Boc]⁺ = 328 observed

Step 2: Bis(pinacolato)diboron (655 mg, 2.57 mmol), potassium acetate (460, 4.68 mmol), tricyclohexylphosphine (17 mg, 0.05 mmol) and palladium acetate (6.0 mg, 0.020 mmol) were dissolved acetonitrile (10.0 mL). The resulting mixture was stirred for 10 min at room temperature then treated with methyl 2-((terf-butoxycarbonyl)amino)-3-(4(((trifluoromethyl)sulfonyl)oxy)phenyl)propanoate (1.00 g, 2.34 mmol) and heated at 80 °C for 3 hours. The mixture was cooled, filtered through celite and concentrated in vacuo. The crude residue was purified by flash column chromatography, eluting with a gradient of 8 to 12% EtOAc in hexane to afford methyl 2-((ferf-butoxycarbonyl)amino)-3-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate (650 mg, 68%) as pale yellow gum.

¹H NMR (400 MHz, CDCI₃): δ ppm 7.58 (2 H, d, J=7.Q Hz), 7.15 (2 H, d, J=7.Q Hz), 4.96 (1H, d, J=7.Q Hz), 4.61 (1H, m), 3.51 (3 H, s), 3.13 (2H, m), 1.44 (9H, s), 1.36 (12H, s) LCMS (Method 8): 2.66 min, [M+H]⁺ = 406.5 observed

Step 3: A solution of 3-ethylphenol (4.00 g, 32.0 mmol) in acetic acid (20 ml_) was cooled to 0 °C, treated with bromine (5.30 g, 32.0 mmol) then warmed to room temperature and stirred for 12 hours. The reaction mixture was quenched with a saturated solution of NaHCO₃ (100 ml_), extracted with EtOAc (3 x 500 ml_), dried (Na₂SO₄) and concentrated in vacuo. The crude residue was purified by flash column chromatography, eluting with a gradient of 0 to 5% EtOAc in hexane to afford 4-bromo-3-ethylphenol (3.8 g, 58%) as a yellow gum.

¹H NMR (400 MHz, cfe-DMSO): δ ppm 9.60 (1H, s), 7.32 (1 H, d, J=8.4 Hz), 6.73 (1 H, d, J =2.8 Hz), 6.55 (1 H, dd, J= 8.6, 2.8 Hz), 2.59 (2 H, q, J= 7.2 Hz), 1.17 (3 H, t, J= 7.2 Hz) LCMS (Method 9): 6.82 min, [M+H]⁺ = 199 observed

Step 4: 4-Bromo-3-ethylphenol (1.0 g, 4.97 mmol), methyl 2-((te/Y-butoxycarbonyl)amino)-3(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate (2.01 g, 4.97 mmol) and Na₂CO₃ (3.16 g, 14.90 mmol) were dissolved in 1,4-dioxane (8 mL) and water (2 mL) at room temperature. The reaction mixture was degassed for 10 min, treated with tetrakis(triphenylphosphine)palladium(0) (0.174 g, 0.24 mmol) and stirred at 80 °C for 4 hours. The reaction mixture was cooled to room temperature, diluted with water (100 mL), extracted with EtOAc (3 x 50 mL) and dried over Na₂SO₄. The combined organic extracts were concentrated in vacuo and purified by flash column chromatography, eluting with a gradient of 0 to 20% EtOAc in hexane to afford methyl 2-((te/Y-butoxycarbonyl)amino)-3-(2'ethyl-4'-hydroxy-[1,1'-biphenyl]-4-yl)propanoate as a colourless gum (1.0 g, 51%).

¹H NMR (400 MHz, cfe-DMSO): δ ppm 9.35 (1H, s), 7.38 (1H, d, J= 8.0 Hz), 7.25 (2H, d, J = 7.6 Hz), 7.15 (2H, d, J=7.Q Hz), 6.93 (1H, d, J= 8.2 Hz), 6.70 (1H, d, J= 2.0 Hz), 6.61 (1H, dd, J= 8.2, 2.0 Hz), 4.23 (1H, m), 3.62 (3H, s), 3.01 (1H, m), 2.87 (1H, m), 2.44 (2H, m), 1.33 (9H, s), 1.02 (3H, t, J=7.4 Hz)

LCMS (Method 8): 2.40 min, [M-Boc+H]⁺ = 300 observed

Step 5: A solution of 2,5,8,11,14-pentaoxahexadecan-16-ol (1.0 g, 3.90 mmol) and pyridine (313 mg, 3.90 mmol) in CH₂CI₂ (20 mL) was cooled to 0 °C and treated with thionyl chloride (0.603 g, 5.07 mmol). The resulting mixture was heated to 50 °C for 2 hours with stirring, then cooled to room temperature and diluted with water (100 mL). The mixture was extracted with EtOAc (3 x 50 mL) and the combined extracts were dried over Na₂SO₄, concentrated in vacuo and purified by flash column chromatography, eluting with a gradient of 0 to 70% EtOAc in hexane to afford of 16-chloro-2,5,8,11,14-pentaoxahexadecane as a colourless gum (900 mg, 84%).

¹H NMR (400 MHz, cfe-DMSO): δ ppm 3.73-3.65 (4 H, m), 3.58-3.52 (14 H, m), 3.473.42 (2 H, m), 3.24 (3 H, s)

Step 6 : A mixture of methyl 2-((terf-butoxycarbonyl)amino)-3-(2'-ethyl-4'-hydroxy-[1,1'biphenyl]-4-yl)propanoate (1.0 g, 2.40 mmol), 16-chloro-2,5,8,11,14-pentaoxahexadecane (0.88 g, 3.24 mmol), K₂CO₃ (0.828 g, 6.00 mmol) and potassium iodide (0.131 g, 0.78 mmol) in DMF (20 ml_) was heated at 90 °C for 24 hours. The reaction mixture was cooled, diluted with water (100 ml_), extracted with EtOAc (3 x 50 ml_), dried over Na₂SO₄ and concentrated in vacuo. The crude residue was purified by column chromatography, eluting with a gradient of 0 to 60% EtOAc in hexane to afford methyl 3-(4'-((2,5,8,11,14pentaoxahexadecan-16-yl)oxy)-2'-ethyl-[1,1 '-biphenyl]-4-yl)-2-((terfbutoxycarbonyl)amino)propanoate (900 mg, 57 %).

¹H NMR (400 MHz, cfe-DMSO): δ ppm 7.38 (1H, d, J = 8.0 Hz), 7.27 (2H, d, J =8.0 Hz), 7.17 (2H, d, J =8.0 Hz), 7.03 (1H, d, J = 8.0 Hz), 6.88 (1H, d, J = 2.5 Hz), 6.81 (1H, dd, J= 8.4, 2.5 Hz), 4.24 (1H, m), 4.11 (2H, m), 3.75 (2H, m), 3.63 (3H, s), 3.61-3.45 (16H, m), 3.42 (2H, m), 3.21 (3H, s), 3.04 (1H, dd, J= 14.2, 4.7 Hz), 2.89 (1H, m), 1.37 (9H, s), 1.02 (3H, t, J=7.4 Hz)

LCMS (Method 9): 6.99 min, [M+NH₄]⁺ = 652 observed

Step 7: A solution of methyl 3-(4'-((2,5,8,11,14-pentaoxahexadecan-16-yl)oxy)-2'-ethyl-[1,1'biphenyl]-4-yl)-2-((terf-butoxycarbonyl)amino)propanoate (800 mg, 1.27 mmol) in THF/water (8:2, v.v, 10 ml_) was treated with lithium hydroxide (90.9 mg, 3.70 mmol) and the resulting mixture was stirred at room temperature for 12 hours. The reaction was diluted with water (100 ml_), acidified to pH 2 with 1 N HCI, extracted with EtOAc (2 x 100 ml_) then dried over Na₂SO₄ and concentrated in vacuo. The crude residue was purified by flash column chromatography, eluting with a gradient of 0 to 8% MeOH in CH₂CI₂ to afford 3-(4'((2,5,8,11,14-pentaoxahexadecan-16-yl)oxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)-2-((ferfbutoxycarbonyl)amino)propanoic acid as a yellow gum (650 mg, 88%).

¹H NMR (400 MHz, cfe-DMSO): δ ppm 7.28 (2H, d, J= 8.0 Hz), 7.16 (3H, m), 7.04 (1H, d, J = 8.2 Hz), 6.88 (1H, d, J = 2.1 Hz), 6.81 (1H, dd, J= 8.4, 2.3 Hz), 4.14 (3H, m), 3.76 (2H, m), 3.61 -3.31 (16H, m), 3.23 (3H, s), 3.07 (1H, dd, J= 13.7, 4.7 Hz), 2.85 (1H, m), 2.54 (2H, m), 1.32 (9H, s), 1.03 (3H, t, J=7.5 Hz), CO₂H not observed

LCMS (Method 9): 6.23 min, [M+NH₄]⁺ = 637 observed

Step 8: A solution of 3-(4'-((2,5,8,11,14-pentaoxahexadecan-16-yl)oxy)-2'-ethyl-[1,1'biphenyl]-4-yl)-2-((terf-butoxycarbonyl)amino)propanoic acid (650 mg, 1.04 mmol) in CH₂CI₂ (20.0 mL) was cooled to 0°C and treated with TFA (1.19 g, 10.40 mmol). The reaction mixture was stirred at room temperature for 16 hours and concentrated in vacuo to afford 3(4'-((2,5,8,11,14-pentaoxahexadecan-16-yl)oxy)-2'-ethyl-[1,1'-biphenyl]-4-yl)-247 aminopropanoic acid as a yellow gum (500 mg, 68%), which was used without further purification.

¹H NMR (400 MHz, cfe-DMSO): δ ppm 8.30 (3H, m), 7.30 (2H, d, 4=8.0 Hz), 7.24 (2H, d, 4=8.4 Hz), 7.04 (1H, d, J= 8.4 Hz), 6.90 (1H, d, 4=2.4 Hz), 6.82 (1H, dd, J= 8.4, 2.4 Hz), 4.26 (1H, m), 4.12 (2H, m), 3.76 (2H, m), 3.61 (2H, m), 3.60-3.31 (14 H, m), 3.21 (3 H, s), 3.18-3.08 (2 H, m), 2.64 (2H, m), 1.08 (3 H, t, 4=7.4 Hz)

LCMS (Method 9): 5.77 min, [M+H]⁺ = 520 observed

Step 9: A solution of 3-(4'-((2,5,8,11,14-pentaoxahexadecan-16-yl)oxy)-2'-ethyl-[1,1'biphenyl]-4-yl)-2-aminopropanoic acid (500 mg, 0.96 mmol) in dioxane (50 ml_) and water (50 ml_) was cooled to 0 °C and treated with Na₂CO₃ (306 mg, 2.88 mmol). The resulting mixture was stirred for 10 min and treated with a solution of Fmoc-CI (273 mg, 1.05 mmol) in 1,4-dioxane (10 ml_) and water (5 ml_). The reaction mixture was warmed to room temperature, stirred for 16 hours then diluted with water (100 ml_), acidified to pH 2 with 1 N HCI and extracted with EtOAc (2x 50 ml_). The combined organic extracts were dried over Na₂SO₄ and concentrated in vacuo. The crude residue was purified by flash column chromatography, eluting with a gradient of 0 to 4% MeOH in CH₂CI₂, to afford 3-(4'((2,5,8,11,14-pentaoxahexadecan-16-yl)oxy)-2'-ethyl-[1,T-biphenyl]-4-yl)-2-((((9/7-fluoren-9yl)methoxy)carbonyl)amino)propanoic acid as a yellow gum (500 mg, 77%).

¹H NMR (400 MHz, cfe-DMSO): δ ppm 7.88 (2H, d, J= 7.6 Hz), 7.67 (2H, m), 7.40 (2H, m), 7.30 (4H, m), 7.13 (2H, d, J =8.0 Hz), 6.97 (1H, d, J= 8.4 Hz), 6.85 (1H, m), 6.76 (1H, m), 4.34-4.16 (5H, m), 3.75 (2H, m), 3.61-3.48 (16H, m), 3.42 (2H, m), 3.23 (3 H, s), 3.13 (1H, m), 2.92 (1H, m), 2.41 (2 H, m), 0.94 (3 H, t, J=7.Q Hz), CO₂H not observed

LCMS (Method 9): 6.61 min, [M-H]’ = 740 observed

Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2'-ethyl-4'methoxy-[1,1'-biphenyl]-4-yl)propanoic acid

Step 1: A solution of (S)-2-((terf-butoxycarbonyl)amino)-3-(2'-ethyl-4'-methoxy-[1,Tbiphenyl]-4-yl)propanoic acid (6.5 g, 16.20 mmol) in 1,4-dioxane (5.0 mL) was cooled at 0 °C and treated with a solution of HCI in 1,4 dioxane (50 mL, 4.0 M). The reaction mixture was warmed to room temperature, stirred for 12 hours and concentrated in vacuo to afford (S)-2amino-3-(2'-ethyl-4'-methoxy-[1,T-biphenyl]-4-yl)propanoic acid (hydrochloride salt) as an off-white solid (4.0 g, 82%), which was used without further purification.

¹H NMR (400MHz, cfe-DMSO): δ ppm 8.43 (3H, s, br), 7.32 (2H, m), 7.24 (2H, m), 7.06 (1H, m), 6.88 (1H, s), 6.82 (1H, d, J=8.2 Hz), 4.24 (1H, m), 3.76 (3H, s), 3.16 (2H, m), 2.56 (2H, m), 1.05 (3H, t, J= 7.6 Hz)

LCMS (Method 9): 1.81 min, [M+H]⁺ = 300 observed

Step 2: A solution of (S)-2-amino-3-(2'-ethyl-4'-methoxy-[1,1'-biphenyl]-4-yl)propanoic acid (2.3 g, 7.60 mmol) in a mixture of THF (100 ml.) and water (20 ml_) was treated with NaHCO₃ (2.5 g, 30 mmol) and stirred at room temperature for 10 min. The resulting mixture was treated with Fmoc-O-succinimide (2.56 g, 7.6 mmol), by portion wise addition, and then stirred at room temperature for 12 hours. The reaction mixture was concentrated in vacuo to remove the organic solvents and the aqueous layer was extracted with diethyl ether (50 ml_). The organic phases were discarded and the aqueous layer was acidified to pH 4 with 0.1 N HCI. The mixture was extracted with EtOAc (3x 100 ml_) and the organic extracts were dried over Na₂SO₄ and concentrated in vacuo. The crude residue was purified by preparative HPLC to afford 3 (S)-2-((((9/7-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2'-ethyl-4'-methoxy[1,1'-biphenyl]-4-yl)propanoic acid as a white solid (3.0 g, 75%).

¹H NMR (400MHz, cfe-DMSO): δ ppm 7.88 (2H, d, J= 7.6 Hz), 7.68 (3H, m), 7.40 (2H, m), 7.33-7.27 (4H, m), 7.12 (2H, d, J = 8.0 Hz), 7.00 (1 H, d, J= 8.4 Hz), 6.84 (1H, d, J = 2.6 Hz), 6.76 (1H, dd, J= 8.4, 2.6 Hz), 4.27-4.15 (4H, m), 3.76 (3 H, s), 3.13 (1H, m), 2.92 (1H, m), 2.48 (2H, m), 0.98 (3 H, t, J =7.2 Hz), CO₂H not observed

LCMS (Method 9): 7.24 min, [M+H]⁺ = 522 observed

Preparation of (2S)-2-[(((9H-fluoren-9-yl)methoxy)carbonyl)amino]-5-(3,5dimethylphenyl)pentanoic acid

Step 1: A reaction vessel was charged with copper(l) iodide (600 mg, 3.16 mmol) and tetrakis(triphenylphosphine)palladium(0) (1.10 g, 0.952 mmol) then purged with N₂. Pyrrolidine (30 mL) was added and the resulting mixture was sparged with a stream of N₂. 3,5-dimethyliodobenzene (11.8 mL, 82.0 mmol) was then added, followed by /V-Boc-Lpropargylglycine (17.5 g, 82.2 mmol), portion wise, to control the exotherm. The resulting mixture was stirred at room temperature for two hours then poured into a saturated solution of aqueous ammonium chloride (200 mL) and extracted with EtOAc (2 x 200 mL). The combined organic extracts were washed with brine (200 mL), dried over MgSO₄ and concentrated in vacuo to obtain a dark orange gum. The crude residue was purified by flash column chromatography, eluting with mixtures of CH₂CI₂, MeOH and acetic acid to afford (2S)-2-[(terf-butoxycarbonyl)amino]-5-(3,5-dimethylphenyl)pent-4-ynoic acid (20.4 g, 79%) as an orange gum.

¹H NMR (400 MHz, CDCI₃): δ ppm 7.01 (2H, s), 6.92 (1H, s), 5.37 (1H, d, J= 8.2 Hz), 4.58 (1H, m), 2.97 (2H, m) 2.25 (6H, s), 1.44 (9H, s), CO₂H not observed LCMS (Method 2): 1.16 min, [M-H]'= 316 observed

Step 2: A solution of (2S)-2-[(terf-butoxycarbonyl)amino]-5-(3,5-dimethylphenyl)pent-4-ynoic acid (20.4 g, 64.4 mmol) in EtOAc (300 mL) was degassed and treated with activated palladium on charcoal (2.2 g, 10 wt% Pd). The mixture was thoroughly sparged, firstly with N₂, secondly with H₂ and the reaction was stirred at room temperature under an ambient pressure of H₂ gas for 16 hours, to effect partial hydrogenation of the alkyne. The mixture was sparged with N₂, filtered through celite and evaporated to an orange gum. The gum was dissolved in EtOAc (300 ml_) and the resulting solution was degassed and treated with fresh activated palladium on charcoal (2.2 g, 10 wt% Pd). The mixture was thoroughly sparged with N₂ and then H₂, and the reaction mixture was stirred at room temperature under an ambient pressure of H₂ gas for a further 16 hours to effect complete hydrogenation of the alkyne/alkene mixture. The mixture was sparged with N₂, filtered through celite and concentrated in vacuo to afford (2S)-2-[(fert-butoxycarbonyl)amino]-5-(3,5dimethylphenyl)pentanoic acid (20.0 g, 97%) as a pale yellow gum which was used without further purification.

¹H NMR (400 MHz, cfe-DMSO): δ ppm 7.07 (1H, d, J= 8.2 Hz), 6.77 (1H, s), 6.74 (2H, s), 3.87 (1H, m), 2.44 (2H, m), 2.20 (6H, s), 1.64-1.54 (4H, m), 1.35 (9H, s), CO₂H not observed LCMS (Method 2): 1.71 min, [M-H]’ = 320 observed

Step 3: (2S)-2-[(ferf-butoxycarbonyl)amino]-5-(3,5-dimethylphenyl)pentanoic acid was dissolved in a solution of HCI in 1,4-dioxane (100 ml_; 4.0 M) and the resulting mixture was stirred at room temperature for 3 hours to form a white precipitate. The mixture was evaporated to a white solid which was triturated with diethyl ether (150 ml_). The solid was filtered and washed with diethyl ether (80 ml_) then dried in vacuo to afford (1S)-1-carboxy-4(3,5-dimethylphenyl)butan-1-ammonium chloride (14.8 g, 92%) as a white solid which was used without further purification.

¹H NMR (400 MHz, cfe-DMSO): δ ppm 8.34 (3H, s, br), 6.79 (1H, s), 6.77 (2H, s), 3.87 (1H, m), 2.48 (2H, m), 2.21 (6H, s), 1.77-1.50 (4H, m), CO₂H not observed LCMS (Method 2): 0.92 min, [M-H]’ = 220 observed

Step 4: A suspension of (1S)-1-carboxy-4-(3,5-dimethylphenyl)butan-1-ammonium chloride (14.8 g, 57.4 mmol) in THF (200 mL) was basified to pH 8 with a saturated aqueous solution of sodium bicarbonate (100 mL) and diluted with water (100 mL). The resulting mixture was treated with Fmoc-O-succinimide (21.3 g, 63.3 mmol), stirred at room temperature for 16 hours and concentrated to remove THF. The suspension was acidified to pH 1 with 1 M HCI solution and extracted with EtOAc (2 x 200 mL). The combined organic extracts were washed with brine (200 mL), dried over MgSO₄, and concentrated in vacuo. The crude residue was purified by flash column chromatography, eluting with mixtures of CH₂CI₂,

MeOH and acetic acid to afford (2S)-2-[(((9/7-fluoren-9-yl)methoxy)carbonyl)amino]-5-(3,5dimethylphenyl)pentanoic acid (7.2 g, 28%) as a white solid.

¹H NMR (400 MHz, cfe-DMSO): δ ppm 7.87 (2H, d, J= 7.4 Hz), 7.70 (2H, d, J =7.4 Hz), 7.65 (1H, d, J = 8.2 Hz), 7.39 (2H, app t, J = 7.4 Hz), 7.29 (2H, m), 6.77 (1H, s), 6.75 (2H, s), 4.30-4.18 (3H, m), 3.95 (1H, m), 2.48 (2H, m), 2.19 (6H, s), 1.71-1.57 (4H, m), CO₂H not observed

LCMS (Method 1): 3.05 min, [M+H]⁺ = 444 observed

Preparation of 2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}hexadecanoic acid

2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}hexadecanoic acid (490 mg, 9%) was prepared from 2-aminohexadecanoic acid (2.95 g, 10.9 mmol) and Fmoc-O-succinimide (3.98 g, 10.9 mmol), according to the procedure described for the preparation of (2S)-2-[(((9/7-fluoren-9yl)methoxy)carbonyl)amino]-5-(3,5-dimethylphenyl)pentanoic acid.

¹H NMR (400MHz, CDCI₃): δ ppm 7.76 (2H, d, J= 7.4 Hz), 7.58 (2H, m), 7.39 (2H, app t,J = 7.2 Hz), 7.31 (2H, m), 5.21 (1H, d, J= 8.2 Hz), 4.41 (3H, m), 4.23 (1H, t, J= 6.8 Hz), 1.89 (1H, m), 1.70 (1H, m), 1.45-1.20 (24H, m), 0.87 (3H, t, J= 6.6 Hz), CO₂H not observed LCMS (Method 1): 4.19 min, [M+H]⁺ = 494.5 observed

Preparation of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(((S)-decan-2yl)oxy)-4-oxobutanoic acid (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(((S)-decan-2-yl)oxy)-4-oxobutanoic acid (500 mg, 97%) was prepared from (S)-2-amino-4-(((R)-decan-2-yl)oxy)-4-oxo butanoic acid (285 mg, 1.04 mmol) and Fmoc-O-succinimide (381 mg, 1.04 mmol), according to the procedure described for the preparation of (2S)-2-[(((9/7-fluoren-9yl)methoxy)carbonyl)amino]-5-(3,5-dimethylphenyl)pentanoic acid.

¹H NMR (400MHz, CDCI₃): δ ppm 7.73 (2H, m), 7.54 (2H, m), 7.36 (2H, m), 7.23 (2H, m), 6.30 (1H, s, br), 4.85 (1H, m), 4.53 (1H, m), 4.31 (2H, m), 4.15 (1H, m), 2.95 (2H, m), 1.50 (1H, m), 1.40 (1H, m), 1.24-1.10 (15H, m), 0.84 (3H, t, J= 7.0 Hz), CO₂H not observed LCMS (Method 1): 3.68 min, [M+H]⁺ = 496 observed

Preparation of (2S)-6-(acryloylamino)-2-((((9H-fluoren-9-yl) methoxy) carbonyl) amino)hexanoic acid

A suspension of (2S)-6-amino-2-((((9/7-fluoren-9-yl) methoxy) carbonyl) amino)hexanoic acid (0.5 g, 1.36 mmol) in aqueous NaOH (1 N, 10 mL) was treated with acryloyl chloride (0.15 mL, 2.04 mmol) by drop wise addition at 0 °C. Aqueous NaOH (2 N) was added to maintain the reaction at ~ pH 10. The resulting reaction mixture was stirred at 0 °C for 30 min. After completion, the reaction mixture was acidified with HCI (2 N) to adjust to ~ pH 2 and extracted with ethyl acetate (3x15 mL). The organic layers were combined and washed with brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography, eluting with a gradient of 3-5% methanol in CH₂CI₂ to afford the title compound as a white solid (125 mg, 22%).

¹H NMR (400 MHz; cfe-DMSO): δ ppm 12.50 (1H, s, br), 8.13 (1H, m), 7.89 (2H, m), 7.72 (2H, m), 7.63 (1H, m), 7.41 (2H, m), 7.32 (2H, m), 6.20 (1H, m), 6.05 (1H, m), 5.55 (1H, m), 4.20-4.27 (3H, m), 3.89 (1H, m), 3.11 (2H, m), 1.70 (2H, m), 1.47-1.26 (4H, m)

LCMS (Method 12): 5.65 min, [M+H]⁺ = 423 observed

Solid Phase Peptide Synthesis

General procedure for the preparation of peptides by solid phase peptide synthesis

Peptides were assembled manually on a roller-mixer by Fmoc SPPS (Solid Phase Peptide Synthesis) using polypropylene columns equipped with a filter disc. Rink Amide AM resin (0.4mmol, Novabiochem - loading 0.6 mmol/g) was swollen in CH₂CI₂ for 15 min. Removal of the resin-bound Fmoc protecting group was achieved using 20% piperidine in DMF for 5 min and 10 min. After 6 washes of the resin with DMF (30 seconds), HATU, Fmoc-protected amino acid solution and DIEA were added to the resin as a solution in DMF. Natural aminoacids were coupled using 5 molar equivalents (eq) of the Fmoc-amino acid with 4.5 eq HATU and 10 eq DIEA for 1 hour (typical concentration 1 M in DMF). Unnatural amino acids were coupled using 1.5-2 eq of the Fmoc-amino acid with 1.4-1.8 eq HATU and 5 eq of DIEA (typical concentration 0.4 M in DMF). Completion of coupling reactions was confirmed by TNBS resin test (2,4,6-trinitrobenzene sulfonic acid solution). Threonine was triple-coupled (i.e. the resin was subjected to the reaction conditions for 1 hour each time and the solution was drained and the resin was re-subjected to fresh reagents each time).

After completion of the synthesis, the resin was washed 3 times with DMF, 3 times with CH₂CI₂ and 3 times with diethylether. Cleavage from the reisn was performed using 95%

TFA with 2.5% water and 2.5% tri/sopropylsilane. After 3 hours, the resin was filtered and discarded; the filtrate was evaporated to dryness. Cold diethylether was added to precipitate the peptide and the solid was triturated. The supernatant was discarded and the process was repeated. Residual diethylether was evaporated and the peptide was obtained as an offwhite powder. Final purification was performed by preparative HPLC on a Gilson system at 230 nm using a 250 x 21.2 mm ACE 10 pm Ci₈ 300 A column at a flow rate of 15 mL/min. Peptide crudes were dissolved in 2-4 ml_ of 25-30% eluent B and loaded onto a 5 ml_ loop. Eluents were 0.1% TFA in milli-Q water (A) and 0.1% TFA in acetonitrile (B).

Table 2: Purifications and yield of peptides prepared by solid phase peptide synthesis

Example	Preparative Gradient (% B/time in min)	Crude amount (mg)	Isolated target (>95% pure; mg)	Overall yield (%)
1	40-70/50	-	65	11
2	40-70/50	200	60	10
3	40-70/50	275	42	7
4	50-80/40	280	45	11
5	2-90/60	180	70	12
6	2-90/60	200	12.6	2

7	50-80/40	220	34.4	4
8	30469	40-70/30	100	30
9	45-75/30	370	57	9
10	40-70/50	446	90	17
11	40-70/60	310	81	14
12	60-90/60	170	24	5
13	60-90/60	8.9	2

14	35-55/60	262	31	4.5
15	35-55/60	31	4.5
16	65-95/60	390	53	8
17	50-100/80	375	14.7	2.7
18	35-65/40	400	43	7.2
19	50-80/50	406	34.4	5.6
20	45-75/60	437	24.6	4

21	50-80/50	370	28.7	4.8
22	40-70/40	354	90.6	15.4
23	30-60/40	-	35.3	6

Table 3: HPLC purity determination and mass spectrometry of final peptides prepared by solid phase peptide synthesis

Peptide crudes were analysed on a Gilson HPLC system at 230 nm using a gradient over 30 5 min at 1.5 mL/min, with additional conditions as specified. Eluents were 0.1% TFA in milli-Q water (A) and 0.1% TFA in acetonitrile (B). Molecular weight was confirmed by MALDI-TOF mass spectroscopy (Voyager DE Pro) in reflector positive mode using a-cyano-4hydroxycinnamic acid as matrix.

Example	HPLC Analysis	MALDI-TOF MS
Analytical Gradient (% B/run time in min)	Retention time (min)	purity	Column id	theoretical monoisotopic mass	[M]+ or [M+H]+ observed
1	2-70/30	25.0	>95%	Vydac218TP54	1477.7	1478.9

2	2-100/30	18.3	>95%	Thermo Hypersil Gold 250x4.6 5pm	1476.7	1477.2
3	2-100/30	18.5	>95%	EXL-1211- 2546U	1487.7	1488.8
4	2-70/30	28.3	>95%	Thermo Hypersil Gold 250x4.6 5pm	1515.7	1516.3
5	2-70/30	24.2	>95%	Thermo Hypersil Gold 250x4.6 5pm	1453.7	1454.5
6	2-70/30	23.3	>95%	EXL-1211- 2546U	1487.7	1488.6
7	2-100/30	20.1	>95%	Vydac218TP54	1505.7	1506.5
8	2-70/30	24.2	>95%	Vydac218TP54	1476.7	1477.7

9	2-70/30	24.9	>95%	EXL-1211- 2546U	1511.7	1511.3
10	2-70/30	25.6	>95%	EXL-1211- 2546U	1341.8	1341.7
11	2-70/30	24.1	>95%	EXL-1211- 2546U	1410.6	1411.1
12	2-100/30	25.8	>95%	EXL-1211- 2546U	1537.8	1538.4
13	2-100/30	26.5	>95%	EXL-1211- 2546U	1537.8	1538.9
14	2-100/30	17.2	>95%	EXL-1211- 2546U	1708.8	1710.2
15	2-100/30	17.6	>95%	EXL-1211- 2546U	1708.8	1710.2

16	2-100/30	23.5	>95%	EXL-1211- 2546U	1627.9	1628.9
17	2-100/30	21.7	>95%	EXL-1211- 2546U	1331.8	1332.0
18	2-100/30	18.4	>95%	EXL-1211- 2546U	1499.7	1500.7
19	2-100/30	21.4	>95%	EXL-1211- 2546U	1531.7	1532.7
20	2-100/30	19.6	>95%	EXL-1211- 2546U	1513.7	1514.9
21	2-100/30	20.5	>95%	EXL-1211- 2546U	1501.7	1502.8
22	2-100/30	18.2	>95%	EXL-1211- 2546U	1473.7	1474.7

2-70/30

19.4 >95%

EXL-12112546U

1466.7

1467.7

Example 24

Preparation of ((3S)-3-{[( 1 S)-1 -{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4methoxyphenyl)phenyl]ethyl]carbamoyl}-3-[(2S)-2-[(2S,3/?)-2-[(2S)-2-[(2fluorophenyl)methyl]-2-[(2S,3/?)-3-hydroxy-2-{2-[(2S)-2-(2-{[2-(1H-imidazol-4yl)ethyl]carbamoyl}-2,2-dimethylacetamido)-3-(2H-1,2,3,4-tetrazol-5yl)propanamido]acetamido}butanamido]propanamido]-3-hydroxybutanamido]-3hydroxypropanamido]propanoic acid)

Step 1: lodomethane (0.10 mL) was added to a solution of (2S)-2-{[(9/7-fluoren-9ylmethoxy)carbonyl]amino}-3-(2-fluorophenyl)-2-methylpropanoic acid (0.48 g) and K₂CO₃ (160 mg) in DMF (10 mL). The resulting solution was stirred overnight at room temperature. The mixture was added to brine (50 mL) and the product extracted with EtOAc (4 x 15 mL), the organics were combined, washed with 10% citric acid (25 mL), saturated NaHCO₃ solution (25 mL), brine (5 x 25 mL), dried over MgSO₄ and the solvent removed to afford methyl (2S)-2-{[(9/7-fluoren-9-ylmethoxy)carbonyl]amino}-3-(2-fluorophenyl)-2methylpropanoate as a yellow oil (0.512 g, 97 %).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.77 (2H, d, J= 8 Hz), 7.60 (2H, m), 7.40, (2H, m), 7.30 (2H, m), 7.21 (1H,m), 6.99 (3H, m), 5.49 (1H, s, br), 4.41 (2H, s), 3.76 (3H, s), 3.50-3.27 (2H, m), 1.63 (3H, s)

LCMS (Method 4): 1.964 min, [M+H]⁺ = 434 observed

Step 2: Piperidine (2.4 mL) was added to a solution of methyl (2S)-2-{[(9/7-fluoren-9ylmethoxy)carbonyl]amino}-3-(2-fluorophenyl)-2-methylpropanoate (0.512 g) in CH₂CI₂ (20 mL) and the solution stirred overnight at room temperature. The mixture was concentrated and purified by column chromatography (hexane/EtOAc 10:1, 5:1, 3:1, 1:1 and 100% EtOAc) and preparative LCMS to afford (2S)-2-amino-3-(2-fluorophenyl)-2-methylpropanoate (0.25 g, 70%) as a yellow residue.

¹H NMR (400 MHz, CDCI₃): δ ppm 7.30-6.99 (4H, m), 3.72 (3H, s), 3.09 (1H, d, J= 13.4 Hz), 2.93 (1H, d, J= 13.4 Hz), 1.39 (3H, s), NH₂ not observed LCMS (Method 4): 1.019 min, [M+H]⁺ = 212 observed

Step 3: DIEA (0.62 mL, 3.6 mmol) was added to a solution of (2S)-2-amino-3-(2fluorophenyl)-2-methylpropanoate (0.377 g, 1.78 mmol), Cbz-Thr(/Bu)-OH.DCA (0.876 g, 1.75 mmol), HATU (1.01 g, 2.7 mmol) in DMF (15 mL) and the resulting solution stirred at room temperature for 1 hour. The mixture was added to brine (100 mL) and the product extracted with EtOAc (4 x 50 mL). The organics were combined, washed with 10% citric acid solution (100 mL), saturated NaHCO₃ solution (100 mL), brine (5 x 100 mL), dried over MgSO₄ and the solvent removed to afford methyl (2S)-2-[(2S,3R)-2{[(benzyloxy)carbonyl]amino}-3-(terf-butoxy)butanamido]-3-(2-fluorophenyl)-2methylpropanoate as an orange oil (0.748 g, 83%).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.89 (1H, s, br), 7.39-6.97 (9H, m), 5.95 (1H, s, br),

5.13 (2H, m), 4.17-4.10 (2H, m), 3.71 (3H, s), 3.46 (1H, d, J= 14 Hz), 3.18 (1H, d, J= 14 Hz), 1.68 (3H, s), 1.24 (9H, s) and 1.06 (1H, d, J= 4 Hz)

LCMS (Method 5): 8.40 min, [M+H]⁺ = 503 and [M-H]’ = 501 observed

Step 4: Lithium hydroxide (161 mg, 6.8 mmol) was added to a solution of methyl (2S)-2[(2S,3R)-2-{[(benzyloxy)carbonyl]amino}-3-(terf-butoxy)butanamido]-3-(2-fluorophenyl)-2methylpropanoate (0.846 g, 1.68 mmol) in THF (15 mL) and water (15 mL). The resulting solution was stirred overnight at room temperature. The solution was concentrated to remove the THF and the aqueous acidified with 10% citric acid solution. The product was extracted with EtOAc (5 x 50 mL) and washed with water (50 mL), dried over MgSO₄ and the solvent removed to give a clear oil. NMR of this material shows the product to be the major component of the mixture but approx -10% SM remains.

The residue was treated as before using LiOH (161 mg, 6.8 mmol) and THF (15 mL) and water (15 mL) and stirred overnight at room temperature. The THF was removed under vacuum and the residue acidified with 10% citric acid solution, the product extracted with EtOAc (5 x 50 mL), washed with water (3 x 40 mL), dried over MgSO₄ and the solvent removed to give (2S)-2-[(2S,3R)-2-{[(benzyloxy)carbonyl]amino}-3-(teff-butoxy)butanamidoj3-(2-fluorophenyl)-2-methylpropanoic acid (0.75 g, 91%) as a clear residue.

¹H NMR (400 MHz, CDCI₃): δ ppm 7.75 (1H, s), 7.41-6.90 (9H, m), 5.95 (1H, s), 5.205.06 (2H ,m), 4.25 - 4.07 (2H, m), 3.47 (1H, d), 3.22 (1H, d), 1.68 (3H, s) and 1.31 - 0.91 (13H, m)

LCMS (Method 4): 1.84 min, [M+H]⁺ = 489 and [M-H] = 487 observed

Step 5: DIEA (1.6 mL) was added to a solution of (2S)-2-[(2S,3R)-2{[(benzyloxy)carbonyl]amino}-3-(terf-butoxy)butanamido]-3-(2-fluorophenyl)-2methylpropanoic acid (0.747 g, 1.529 mmol) and H-Thr(fBu)OMe.HCI (1.72 g) in DMF (20 mL). The resulting solution was stirred for 10 min and HATU (0.88 g) was added. The resulting solution was stirred overnight at room temperature. The mixture was added to brine (100 mL) and the product extracted with EtOAc (5 x 50 mL). The organics were combined, washed with 10% citric acid (100 mL), saturated NaHCO₃ solution (100 mL), brine (5 x 100 mL), dried over MgSO₄ and the solvent removed to give a clear residue. Purification by column chromatography (Hexane/EtOAc 10:1, 5:1 and 3:1) gave methyl (2S,3R)-2-[(2S)-2-[(2S,3R)-2-{[(benzyloxy)carbonyl]amino}-3-(ferfbutoxy)butanamido]-3-(2-fluorophenyl)-2-methylpropanamido]-3-(ferf-butoxy)butanoate as a clear oil (0.894 g, 89 %).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.56 (1H, s, br), 7.35-7.18 (7H, m), 7.05-6.99 (2H, m), 6.92 (1H, d, J = 8.8 Hz), 5.95 (1H, d, J= 4.8 Hz), 5.16-5.06 (2H, m), 4.49 (1H, dd, J= 9.2 Hz, J=2 Hz), 4.21 -4.10 (3H, m), 3.68 (3H, s), 3.45 (1H, d, J=14 Hz), 3.30 (1H, d, J= 14 Hz), 1.24 (9H,s), 1.61 (3H, m), 1.13 (3H, d, J = 6.4 Hz), 1.09 - 1.07 (12H, m)

LCMS (Method 4): 2.11 min, masses observed [M+H]⁺ = 660 observed

Step 6: Lithium hydroxide (0.131 g, 5.4 mmol) was added to a solution of methyl (2S,3R)-2[(2S)-2-[(2S,3R)-2-{[(benzyloxy)carbonyl]amino}-3-(terf-butoxy)butanamido]-3-(2fluorophenyl)-2-methylpropanamido]-3-(tert-butoxy)butanoate (0.894 g, 1.35 mmol) in THF (20 mL) and water (20 mL). The resulting solution was stirred overnight at room temperature. The THF was removed under vacuum and the solution acidified with 10% citric acid. The product was extracted with EtOAc (5 x 50 mL), washed with water (2 x 100 mL), dried and concentrated to give (2S,3R)-2-[(2S)-2-[(2S,3R)-2-{[(benzyloxy)carbonyl]amino}-3(ferf-butoxy)butanamido]-3-(2-fluorophenyl)-2-methylpropanamido]-3-(tert-butoxy)butanoic acid as a clear glassy solid (0.842 g, 96%).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.52 (1H, s, br) 7.38-7.14 (7H, m), 7.07-6.93 (3H, m), 5.85 (1H, d, J = 5.6 Hz), 5.17-5.09 (2H, m), 4.39-4.10 (4H, m), 3.29 (1H, d, J= 14 Hz), 3.20 (1H, d, J= 14 Hz), 1.29-1.22 (21H, m), 1.11 (3H, d, J=6 Hz) and 1.01 (3H, d, J = 6.4 Hz), CO₂H not observed

LCMS (Method 4): 1.99 min, [M+H]⁺ 646 and [M-H]’ = 644 observed

Step 7: HATU (0.744 g) was added to a solution of (2S,3R)-2-[(2S)-2-[(2S,3R)-2{[(benzyloxy)carbonyl]amino}-3-(terf-butoxy)butanamido]-3-(2-fluorophenyl)-2methylpropanamido]-3-(tert-butoxy)butanoic acid (0.842 g), H-Ser((Bu)OMe.HCI (1.38 g) and DIEA (1.4 mL) in DMF (20 mL) and the solution was stirred at room temperature for 1 hour. The mixture was added to brine (100 mL) and the product extracted with EtOAc (5 x 50 mL). The organics were combined washed with 10% citric acid (2 x 100 mL), saturated NaHCO₃ (100 mL), brine (5 x 100 mL), dried over MgSO₄ and the solvent removed to give a yellow oil. Purification by column chromatography (hexane/EtOAc, 10:1, 5:1, 3:1 and 1:1) gave methyl (2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)-2-{[(benzyloxy)carbonyl]amino}-3-(ferfbutoxy)butanamido]-3-(2-fluorophenyl)-2-methylpropanamido]-3-(ferf-butoxy)butanamido]-3(ferf-butoxy)propanoate as a clear glassy solid (0.62 g, 59%).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.89 (1H, d), 7.70 (1H, s), 7.40-6.91 (10H, m), 5.94 (1H, s, br), 5.20 - 5.07 (2H, m), 4.70 (1H, m), 4.3 (1H, m), 4.22 - 4.08 (3H, m), 3.82 (1H, m), 3.70 (3H, s), 3.55 (1H, m), 3.40 (1H ,d), 3.20 (1H, d), 1.65 (3H, s), 1.45-1.00 (33H, m)

LCMS (Method 4): 2.20 min, [M-H]’ = 801 observed.

Step 8: A solution of methyl (2S)-2-[(2S,3/?)-2-[(2S)-2-[(2S,3/?)-2{[(benzyloxy)carbonyl]amino}-3-(terf-butoxy)butanamido]-3-(2-fluorophenyl)-2methylpropanamido]-3-(teff-butoxy)butanamido]-3-(te/Y-butoxy)propanoate (0.619 g, 0.772 mmol) in MeOH (50 mL) was treated with palladium on activated charcoal (0.6 g, 10% Pd by weight) and stirred under 30 bar of hydrogen gas in an autoclave for 1 hour at room temperature. The catalyst was filtered off, washed with MeOH and the solution concentrated to give methyl (2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)-2-amino-3-(te/Y-butoxy)butanamido]-2-[(2fluorophenyl)methyl]propanamido]-3-(teff-butoxy)butanamido]-3-(te/Y-butoxy)propanoate as a clear oil (0.523 g, 100%).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.91 (1H, d), 7.31-7.18 (2H, m), 7.09-6.97 (2H, m), 4.69 (1H, m), 4.33 - 4.09 (3H, m), 3.82 (1H, m), 3.70 (3H, s), 3.60 - 3.48 (3H, m), 3.35 3.00 (3H, m), 1.70 - 1.50 (5H, m), 1.30 (9H, s), 1.20 - 0.90 (24H, m)

LCMS (Method 4): 1.59 min, [M]⁺ = 669 observed

Step 9: HATU (0.44 g) was added to a solution of methyl (2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)2-amino-3-(teff-butoxy)butanamido]-2-[(2-fluorophenyl)methyl]propanamido]-3-(te/Ybutoxy)butanamido]-3-(terf-butoxy)propanoate (0.52 g), CBz-Gly-OH (0.18 g) and DIEA (0.28 mL) in DMF (20 mL). The resulting solution was stirred overnight at room temperature. The mixture was added to brine (50 mL) and the product extracted with EtOAc (5 x 50 mL). The organics were combined, washed with 10% citric acid (50 mL), saturated NaHCO₃ (50 mL), brine (5 x 50 mL), dried over MgSO₄ and the solvent removed to give an orange oil. Purification by column chromatography (hexane/EtOAc, 3:1 then 1:1) gave methyl (2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)-2-(2-{[(benzyloxy)carbonyl]amino}acetamido)-3(tert-butoxy)butanamido]-3-(2-fluorophenyl)-2-methylpropanamido]-3-(tertbutoxy)butanamido]-3-(tert-butoxy)propanoate as a pale yellow solid (0.50 g, 75%).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.90 (1H, d), 7.70 (1H, s), 7.39-6.91 (10H, m), 5.49 (1H, s), 5.14 (2H ,s), 4.69 (1H, m), 4.31 -4.12 (4H, m), 4.05-3.79 (3H, m), 3.70 (3H, s), 3.56 (1H, m), 3.44 (1H, d), 3.27 (1H, d), 1.52 (3H, s) and 1.33-0.99 (34H, m)

LCMS (Method 4): 2.10 min, [M]⁺ = 860 observed

Step 10: LiOH (17 mg) was added to a stirred solution of methyl (2S)-2-[(2S,3R)-2-[(2S)-2[(2S,3R)-2-(2-{[(benzyloxy)carbonyl]amino}acetamido)-3-(tert-butoxy)butanamido]-3-(2fluorophenyl)-2-methylpropanamido]-3-(tert-butoxy)butanamido]-3-(tert-butoxy)propanoate (0.153 g) in THF (6 mL) and water (6 mL). The resulting solution was stirred for 3 hours at room temperature. The THF was removed in vacuo and the residue acidified, the product extracted with EtOAc (4 x 20 mL), washed with brine (40 mL), dried over MgSO₄ and the solvent removed to give a white solid. Purification by mass-directed preparative HPLC afforded (2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)-2-(2-{[(benzyloxy)carbonyl]amino}acetamido)-3(teff-butoxy)butanamido]-3-(2-fluorophenyl)-2-methylpropanamido]-3-(te/Ybutoxy)butanamido]-3-(terf-butoxy)propanoic acid as a white solid (97.6 mg, 65 %) ¹H NMR (400 MHz, cfe-DMSO): δ ppm 11.90 (1H, s, br), 7.87-7.74 (2H, m), 7.64 (1H, d), 7.39-7.20 (7H, m), 7.11 -6.99 (2H, m), 5.06 (2H, s), 4.40(1 H, m), 4.30-4.23 (2H, m), 3.98 - 3.86 (2H, m), 3.72 - 3.66 (3H, m), 3.57 - 3.41 (2H, m), 1.44 (3H, s), 1.22 - 1.09 (27 H, m) and 1.06 - 0.91 (6H, m), 3H obscured by the water peak.

LCMS (Method 4): 1.98 min, [M]⁺ = 846 and [M-H]’ = 845 observed

Step 11: DIEA (32 pL) was added to a solution of methyl (2S)-2-amino-3-[2(triphenylmethyl)-2/7-tetrazol-5-yl]propanoate (50 mg), 3-{[2-(1-(triphenylmethyl)-1/7imidazol-4-yl)ethyl]amino}-2,2-dimethyl-3-oxopropanoic acid (57 mg) and HATU (69 mg) in CH₂CI₂ (10 mL). The resulting mixture was stirred for 3 hours at room temperature. The mixture was washed with 10% citric acid and the aqueous washed with EtOAc (3x15 mL), the organics were combined, washed with saturated NaHCO₃ solutions, dried over MgSO₄ and the solvent removed to give a yellow residue (0.22 g). Purification by mass-directed preparative HPLC gave methyl (2S)-2-[2,2-dimethyl-2-({2-[1-(triphenylmethyl)-1/7-imidazol-4yl]ethyl}carbamoyl)acetamido]-3-[1-(triphenylmethyl)-1/7-1,2,3,4-tetrazol-5-yl]propanoate as a clear glassy solid (113 mg, 60%).

¹H NMR (400 MHz, CDCI₃): δ ppm 8.01 (1H, d, J= 4 Hz), 7.41 -7.22 (21H, m), 7.16-7.02 (10H, m), 6.58 (1H, s), 4.92 (1H, m), 3.37 (3H, s) and 3.50-3.40 (4H, m), 2.69-2.6 (2H, m), 1.30 (3H, s) and 1.22 (3H, s)

LCMS (Method 4): 1.750 min, [M]⁺ = 863 observed

Step 12: A solution of methyl (2S)-2-[2,2-dimethyl-2-({2-[1-(triphenylmethyl)-1/7-imidazol-4yl]ethyl}carbamoyl)acetamido]-3-[1-(triphenylmethyl)-1/7-1,2,3,4-tetrazol-5-yl]propanoate (65.7 mg) and LiOH (3.6 mg) in THF/water (10 mL; 1:1, v:v) was stirred for 3 hours at room temperature. The solvent was removed and the residue azeotroped with acetonitrile to give a glassy solid. The material was purified by mass-directed preparative HPLC to give ((28)-2[2,2-dimethyl-2-({2-[1-(triphenylmethyl)-1/7-imidazol-4-yl]ethyl}carbamoyl)acetamido]-3-[1(triphenylmethyl)-1/7-1,2,3,4-tetrazol-5-yl]propanoic acid as a clear glassy solid (80.2 mg, 72 %).

¹H NMR (400 MHz, cfe-DMSO): δ ppm 8.01 (1H, d), 7.51 (1H, s, br), 7.41 -6.90 (31H, m), 6.59 (1H, s), 4.63 (1H, m), 1.11 (3H, s) and 1.09 (3H, s) 6H are obscured by the solvent peaks, CO₂H not observed

LCMS (Method 4): 1.75 min, [M+H]⁺ = 849 and [M-H] = 847 observed

Step 13: Pd(PPh₃)₂CI₂ (0.35 g, 0.70 mmol) and Cul (0.19 g, 1.0 mmol) were added to a solution of 5-iodo-m-xylene (4.64 g, 20 mmol), 3-butyn-1-ol (1.68 g, 24 mmol) and triethylamine (8.4 mL, 60 mmol) in THF (30 mL). The solution was then heated to reflux for 1 hour. The mixture was cooled and stirred overnight at RT. The mixture was diluted with ether and suspension filtered through celite, the orange solution concentrated down to give a red oil. Purification by column chromatography (hexane/EtOAc, 5:1, 3:1 and 1:1) afforded 4-(3,5-dimethylphenyl)but-3-yn-1-ol as an orange oil (3.14 g, 90 %).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.05 (1H, s), 6.93 (2H, s), 3.80 (2H, q, J= 4 Hz), 2.68 (2H, t, J = 4 Hz), 2.27 (6H, s) and 1.81 (1H, t, J = 4 Hz)

LCMS (Method 4): 1.65 min, [M+H]⁺ = 175 observed

Step 14: A solution of 4-(3,5-dimethylphenyl)but-3-yn-1-ol (3.15 g, 18 mmol) in ethanol (70 mL) was treated with palladium on activated charcoal (0.192 g; 10% Pd by weight), transferred to an autoclave and stirred overnight at room temperature at 20 bar of H₂ gas. The mixture was filtered through celite, washed with ethanol and the orange solution concentrated to give a dark orange oil. The material was dissolved in ethanol (100 mL), treated with fresh palladium on activated charcoal (0.40 g; 10% Pd by weight), and the mixture stirred at 20 bar of H₂ gas, 40 °C over the weekend. The catalyst was filtered off, washed with ethanol and the solution concentrated to give 4-(3,5-dimethylphenyl)butan-1-ol as a dark green oil (3.14 g, 97 %).

¹H NMR (400 MHz, CDCI₃): δ ppm 6.82 (1H, s), 6.80 (2H, s), 3.65 (2H, dd, J= 12 Hz, J= 8 Hz), 2.57 (2H, t, J= 8 Hz), 2.28 (6H, s), 1.71 -1.52 (4H, m) and 1.25 (1H, m)

Step 15: A solution of (4-(3,5-Dimethylphenyl)butan-1-ol) (0.446 g, 2.5 mmol), phthalimide (0.386 g, 2.6 mmol) and triphenylphosphine (0.984 g, 3.8 mmol) in THF (10 mL) was cooled to 0 °C and treated with DIAD (0.74 mL, 1.5 eq.). The resulting solution was warmed and stirred at room temperature for 3 hours. The mixture was concentrated and the residue stirred in ether/pentane (1:1, 50 mL) overnight. The solid was filtered off, washed with pentane, then ether and pentane. The solution was concentrated down and purified by column chromatography, eluting with hexane/EtOAc (10:1 then 5:1) to afford 2-[4-(3,5dimethylphenyl)butyl]-2,3-dihydro-1/7-isoindole-1,3-dione as a white solid (608 mg, 79 %) ¹H NMR (400 MHz, CDCI₃): δ ppm 7.83 (2H, dd, J= 8 Hz J= 4Hz), 7.70 (2H, dd, J= 8 Hz, J = 4 Hz), 6.80 (1H, s), 6.78 (2H, s), 3.71 (2H, t, J= 8 Hz), 2.57 (2H, t J= 8 Hz), 2.27 (6H, s) and 1.80-1.51 (4H, m)

LCMS (Method 4): 2.01 min, [M+H]⁺ = 308 observed

Step 16: Hydrazine monohydrate (0.48 mL, 9.60 mmol) was added to a solution of 2-[4-(3,5dimethylphenyl)butyl]-2,3-dihydro-1/7-isoindole-1,3-dione (608 mg, 1.98 mmol) in ethanol (20 mL). The solution was stirred overnight at room temperature. The resulting solid was filtered, washed with ether and the solution concentrated to give an oil. 2 M HCI (30 mL) was added to the residue and the product extracted with ether (2 x 10 mL) and CH₂CI₂ (2 x 10 mL). The organic layers were then washed with 2 M HCI (15 mL) and the aqueous layers combined, then neutralised with K₂CO₃. The product was then extracted with CH₂CI₂ (5x10 mL), dried over MgSO₄ and the solvent removed to give 4-(3,5-dimethylphenyl)butan-1amine as a yellow oil (0.142 g, 40 %).

¹H NMR (400 MHz, CDCI₃): δ ppm 6.82 (1H, s), 6.80 (2H, s), 2.71 (2H, t, J= 8 Hz), 2.55 (2H, t, J= 8 Hz), 2.28 (6H, s), 1.70-1.55 (2H, m), 1.51 - 1.42 (2H, m) and 1.31 (2H, s, br)

Step 17: HATU (0.294 g) and DIEA (180 pL) were added to a solution of (2S)-3-(2'-ethyl-4'methoxybiphenyl-4-yl)-2-[(te/Y-butoxycarbonyl)amino]propanoic acid (205 mg) and (4-(3,5dimethylphenyl)butan-1-amine) (91.3 mg) in DMF (5 mL). The solution was then stirred for 3 hours at room temperature. The mixture was added to brine (25 mL) and the product extracted with EtOAc (5x15 mL). The organics were combined, washed with saturated NaHCO₃ solution (25 mL), 2 M HCI solution (25 mL) and brine (5 x 25 mL), dried over MgSO₄ and the solvent removed to give a brown oil. Purification by column chromatography (hexane/EtOAc, 5:1 then 3:1) gave tert-butyl Λ/-[(1 S)-1 -{[4-(3,5dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4-methoxyphenyl)phenyl]ethyl]carbamate as a clear oil (96.3 mg, 34%).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.24-7.18 (4H, m), 7.07 (1H, d, J= 8.4 Hz), 6.84 (1H, d, 4 = 2.3 Hz), 6.81 (1H, s, br), 6.79-6.73 (3H, m), 5.69 (1H, s), 5.06 (1H, s), 4.29 (1H, m), 3.84 (3H, s), 3.24-3.16 (2H, m), 3.11 -3.05 (2H, m), 2.59-2.46 (4H, m), 2.26 (6H, s), 1.54 (2H, m), 1.41 (11H, m) and 1.08 (3H, t, J= 8.4)

LCMS (Method 4): 2.13 min, [M-Boc+H]⁺ = 459 observed

Step 18: A solution of tert-butyl /V-[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2ethyl-4-methoxyphenyl)phenyl]ethyl]carbamate (86.3 mg) in MeOH (5 mL) was treated with HCI (4 M solution in dioxane, 0.8 mL). The resulting solution was stirred over the weekend. The solvent was removed, and the residue triturated in pentane. The gum was dissolved in MeOH and concentrated to give (2S)-2-amino-/V-[4-(3,5-dimethylphenyl)butyl]-3-[4-(2-ethyl4-methoxyphenyl)phenyl]propanamide hydrochloride as a clear glassy solid (74.3 mg, 97%). ¹H NMR (400 MHz, cfe-DMSO): δ ppm 8.28 (1H, m), 7.79 (3H, s, br), 7.30-7.17 (4H, m),

7.01 (1H, d, 4 = 8 Hz) 6.88 (1H, s), 6.81 -6.71 (4H, m), 3.91 (1H, m), 3.78 (3H, s), 3.21 2.97 (5H, m), 2.18 (6H, s), 1.54-1.26 (4H, m) and 1.03 (3H, t, 4 = 8 Hz). 3H partially obscured by DMSO solvent peak

LCMS (Method 4): 1.54 min, [M+H]⁺ = 459 observed

Step 19: DIEA (110 pL) was added to a solution of (2S)-2-amino-/V-[4-(3,5dimethylphenyl)butyl]-3-[4-(2-ethyl-4-methoxyphenyl)phenyl]propanamide hydrochloride (102 mg), CBz-AsptO'BujOH (66.8 mg) and HATU (118 mg) in DMF (5 mL). The resulting solution was stirred overnight at room temperature. The mixture was added to brine (50 mL) and extracted with EtOAc (5x10 mL). The organics were combined, washed with saturated NaHCO₃ (50 mL) and 10% citric acid (50 mL) solutions, then with brine (5 x 50 mL), dried over MgSO₄ and the solvent removed. Purification of the orange solid by column chromatography (Hexane/EtOAc, 5:1 then 3:1 and 1:1) gave tert-butyl (3S)-3{[(benzyloxy)carbonyl]amino}-3-{[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2ethyl-4-methoxyphenyl)phenyl]ethyl]carbamoyl}propanoate as a white solid (95.3 mg, 60.4 %).

¹H NMR (400 MHz, CDCI₃): δ ppm 7.48-7.16 (9H, m), 7.08 (1H, d), 6.85-6.69 (6H, m), 5.98 (1H, s, br), 5.72 (1H, s, br), 5.19-5.00 (2H, m), 4.62-4.40 (2H, m), 3.87 (3H, s), 3.27 -3.03 (4H, m), 2.88-2.40 (6H, m), 2.28 (6H, s), 1.62-1.31 (13H, m) and 1.08 (3H, t) LCMS (Method 4): 2.152 min, [M-'Buf = 708 observed

Step 20: A solution of tert-butyl (3S)-3-{[(benzyloxy)carbonyl]amino}-3-{[(1S)-1-{[4-(3,5dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4methoxyphenyl)phenyl]ethyl]carbamoyl}propanoate (95.3 mg) in MeOH (25 mL) was treated with palladium on active charcoal (50 mg; 10% Pd by weight) and stirred under 30 bar of hydrogen gas in the autoclave at room temperature for 2 hours. The catalyst was filtered off and the solvent removed to afford a white residue which was purified by column chromatography (hexane/EtOAc, 1:1 then 100% EtOAc) to give tert-butyl (3S)-3-amino-3{[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4methoxyphenyl)phenyl]ethyl]carbamoyl}propanoate as a clear residue (38.1 mg, 49%).

¹H NMR (400 MHz, φ-MeOH): δ ppm 7.27 (2H, d), 7.14 (2H, d), 7.01 (1H, d), 6.85 (1H, s), 6.8-6.72 (4H, m), 4.59 (1H, t), 3.80 (3H, s), 3.60 (1H, t), 3.21-2.98 (5H, m), 2.65-2.39 (7H, m), 2.21 (6H, s), 1.56- 1.40 (13H, m) and 1.05 (3H, t), NH₂ not observed LCMS (Method 4): 1.70 min, [M+H]⁺ = 630 observed

Step 21: HATU (32 mg) was added to a solution of (2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)-2-(2{[(benzyloxy)carbonyl]amino}acetamido)-3-(tert-butoxy)butanamido]-3-(2-fluorophenyl)-2methylpropanamido]-3-(tert-butoxy)butanamido]-3-(tert-butoxy)propanoic acid (47.6 mg), tert-butyl (3S)-3-amino-3-{[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4methoxyphenyl)phenyl]ethyl]carbamoyl}propanoate (32 mg) and DIEA (19.3 pL) in DMF (5 mL). The resulting solution was stirred overnight at room temperature. Brine (50 mL) and NaHCO₃ (2 mL) were added to the solution and the solid filtered off, washed with water and dried to give tert-butyl (3S)-3-[(2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)-2-(2{[(benzyloxy)carbonyl]amino}acetamido)-3-(tert-butoxy)butanamido]-2-[(2fluorophenyl)methyl]propanamido]-3-(tert-butoxy)butanamido]-3-(tert-butoxy)propanamido]3-{[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-467 methoxyphenyl)phenyl]ethyl]carbamoyl}propanoate as an off-white solid (70.1 mg, 95%).

¹H NMR (400 MHz, DMSO): δ ppm 7.89-7.79 (2H, m), 111 (1H, m), 7.69-7.48 (3H, m), 7.45-6.94 (15H, m), 6.86 (1H, s), 6.81 -6.71 (4H, m), 5.06 (2H, s), 4.54 (1H, m), 4.43 (1H, m), 4.32 - 4.22 (2H, m), 4.10 - 3.82 (3H, m), 3.80 - 3.65 (5H, m), 3.62 - 3.50 (2H, m), 3.32 (1H, m), 3.19 (1H, m), 3.12-3.00 (3H, m), 2.82 (1H, m), 2.48 (2H, m) 2.20 (6H, s), 1.531.30 (18H, m) and 1.20-0.89 (39H, m)

Step 22: A solution of tert-butyl (3S)-3-[(2S)-2-[(2S,3R)-2-[(2S)-2-[(2S,3R)-2-(2{[(benzyloxy)carbonyl]amino}acetamido)-3-(fert-butoxy)butanamido]-2-[(2fluorophenyl)methyl]propanamido]-3-(tert-butoxy)butanamido]-3-(fert-butoxy)propanamido]3-{[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4methoxyphenyl)phenyl]ethyl]carbamoyl}propanoate (82.4 mg) in MeOH (20 ml_) was treated with palladium on activated charcoal (50 mg; 10% Pd by weight) and stirred under 30 bar of hydrogen gas in an autoclave at room temperature for 1 hour. The catalyst was filtered off and the solvent removed to give a clear glassy solid. Purification by column chromatography (1%, 2%, 5% and 10% MeOH in CH₂CI₂) gave tert-butyl (3S)-3-[(2S)-2-[(2S,3R)-2-[(2S)-2[(2S,3R)-2-(2-aminoacetamido)-3-(tert-butoxy)butanamido]-2-[(2fluorophenyl)methyl]propanamido]-3-(tert-butoxy)butanamido]-3-(tert-butoxy)propanamido]3-{[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4methoxyphenyl)phenyl]ethyl]carbamoyl}propanoate as a clear residue (35.9 mg).

LCMS (Method 4): 1.90 min, [M+H]⁺ = 1323 and [M-H] = 1321 observed

Step 23: HATU (15 mg) was added to a solution of ((2S)-2-[2,2-dimethyl-2-({2-[1(triphenylmethyl)-1/7-imidazol-4-yl]ethyl}carbamoyl)acetamido]-3-[1-(triphenylmethyl)-1/71,2,3,4-tetrazol-5-yl]propanoic acid (23.3 mg), tert-butyl (3S)-3-[(2S)-2-[(2S,3R)-2-[(2S)-2[(2S,3R)-2-(2-aminoacetamido)-3-(tert-butoxy)butanamido]-2-[(2fluorophenyl)methyl]propanamido]-3-(tert-butoxy)butanamido]-3-(tert-butoxy)propanamido]3-{[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4methoxyphenyl)phenyl]ethyl]carbamoyl}propanoate (35.9 mg) and DIEA (10 pL) in DMF (3 mL). The resulting solution was stirred overnight at room temperature. The solvents were removed in vacuo, the white solid dissolved in water and the product extracted with EtOAc (5 x 10 mL), the organics combined, dried over MgSO₄ and the solvent removed.

The residue was dissolved in TFA (5 mL) and stirred overnight at room temperature. The

TFA was removed and the residue purified by mass-directed preparative HPLC to give (3S)3-{[(1S)-1-{[4-(3,5-dimethylphenyl)butyl]carbamoyl}-2-[4-(2-ethyl-4methoxyphenyl)phenyl]ethyl]carbamoyl}-3-[(2S)-2-[(2S,3R)-2-[(2S)-2-[(2fluorophenyl)methyl]-2-[(2S,3R)-3-hydroxy-2-{2-[(2S)-2-(2-{[2-(1H-imidazol-4yl)ethyl]carbamoyl}-2,2-dimethylacetamido)-3-(2/7-1,2,3,4-tetrazol-568 yl)propanamido]acetamido}butanamido]propanamido]-3-hydroxybutanamido]-3hydroxypropanamido]propanoic acid (5.09 mg) as a white solid.

LCMS (Method 5): 4.450 min, [M+H]⁺ = 1446 observed

BIOLOGICAL ASSAYS

One or more of the peptides of the invention were tested in one or more of the following assays:

a) In vitro Human GLP-1 R affinity (radioligand binding)

In order to demonstrate the affinity of the exemplified compounds radioligand binding studies were performed on HEK-293 cell membrane homogenate transiently expressing the human GLP-1 R.

HEK-293 cells were transfected with cDNA encoding the human GLP-1 R using GeneJuice (Novagen) method. Cells were harvested 48 hours after transfection and washed with icecold phosphate-buffered saline. The resulting pellet was re-suspended in ice-cold buffer (20 mM Hepes, 10 mM EDTA, pH 7.4), homogenised and the suspension was centrifuged (40,000xg for 15 min at 4 °C). The supernatant was removed and the pellet was resuspended in 20mM Hepes, 0.1 mM EDTA, pH 7.4, re-homogenised and centrifuged again (40,000 x g for 45 min at 4 °C). The resulting pellet was resuspended in 20mM Hepes, 0.1 mM EDTA, pH 7.4. Protein concentration was determined using the BCA protein assay (Merck Chemicals Ltd) and stored frozen at -80 °C before use.

Serial compound dilutions were prepared as 20 times concentrated stock solutions in assay buffer (50mM Hepes, 150mM NaCI, 0.05mg/ml bacitracin, 0.1% fresh protease inhibitor cocktail, 0.1% BSA and 0.15% CHAPS pH 7.4) at a final DMSO concentration of 5% and 25μΙ transferred into 96well assay plates. Radioligand [³H]-{H-[Aib]-E-G-T-[a-Me-Phe]-T-S-D[(2’-Et-4’-OMe)Bip]-[(3,5-dimethyl)hhPhe]-NH₂} was diluted to a final concentration of approximately 3nM in assay buffer and 50μΙ added to each well.

For radioligand [³H]-{H-[Aib]-E-G-T-[a-Me-Phe]-T-S-D-[(2’-Et-4’-OMe)Bip]-[(3,5dimethyl)hhPhe]-NH₂}:

a-Me-Phe =

Aib =

GLP-1 receptor HEK-293 membrane pellets were diluted in assay buffer and 40pg protein per well added in a volume of 425μΙ. The assay plates were incubated for 2 hours at room temperature (25°C). Non-specific binding (NSB) was defined by 1μΜ exendin-4 (Tocris bioscience, catalogue number 1933). Assays were terminated by filtration onto GF/C (Perkin Elmer) filter plates pre-soaked for 1 hour in 0.3% PEI and washed eight times with 1ml cold wash buffer (PBS + 0.15% CHAPS) and allowed to dry. Radioactivity was counted on a Trilux Microbeta counter and data were analysed in GraphPad PRISM (GraphPad Prism

Software, Inc., San Diego, CA). The IC50 was defined as the concentration required to inhibit 50% of the radioligand binding and was corrected using the Cheng-Prusoff equation to obtain the equilibrium dissociation constant (Ki). Data on homologous competition binding in WT GLP-1 R transiently transfected HEK cells using the radioligand are shown in Figure 3 and Table 4. The Kd was determined to be ~20nM.

Table 4: in vitro pharmacology data to determine the dissociation constant (Kd) for the radioligand [³H]-{H-[Aib]-E-G-T-[a-Me-Phe]-T-S-D-[(2’-Et-4’-OMe)Bip]-[(3,5dimethyl)hhPhe]-NH2}

	5.2	2.8	1.3	Gtobaf (shared)
togKD	-7 533	-7,583	-7.583	-7.583
NS	5353	585.3	535.3	585,3
HotnM	= 5.200	= 2.300	= 1.300
Bntax	53263	53263	53263	53263
Kd	2,S77e-003	2.077e-00S	2.G77e-608	2.077e-008

Figure 3 and Table 4 indicate the results obtained from homologous competition radioligand 20 binding assays using 3 concentrations of [³H]-{H-[Aib]-E-G-T-[a-Me-Phe]-T-S-D-[(2’-Et-4’OMe)Bip]-[(3,5-dimethyl)hhPhe]-NH₂}. After analysis GraphPad Prism. Data was fitted globally to define one shared value for logKD, Bmax and non-specific binding (NS) from all sets of data and to generate an equilibrium binding constant (Kd) equivalent to approximately 20nM.

b) In vitro human GLP-1 R agonistic activity (Cyclic AMP determination)

In order to demonstrate efficacy of the peptides described in the current embodiment, their ability to stimulate formation of cAMP in a cell line expressing the cloned human GLP-1 receptor was tested. Binding of GLP-1 to its receptor leads to production of cAMP (adenosine 3’-5’ monophosphate) via Gas-dependent activation of adenylate cyclase. The cAMP content of cells was determined using a kit from Cisbio Corp. (cat. no. 62AM6PEJ) based on HTRF (Homogenous Time Resolved Fluorescence) measured in HEK-293 cell lines transiently expressing human GLP-1 receptor, CHO-K1 cells stably expressing the GLP-1 R or mouse NIT-1 cells endogenously expressing the GLP-1 R.

For transient transfections HEK-293 cells (cultured in DMEM/10% FCS) were plated on day 1 incubated for 24 hours at 37°C at 5% CO₂. On day 2 cells were transfected with a transfection mixture generated by addition of 6pg of WT human GLP-1 R DNA to 18 pi GeneJuice (Calbiochem cat no. 70967-4) in 500 μΙ OptiMEM and cells incubated for a further 24 hours. On day 3 medium was removed and cells washed with PBS lacking calcium and magnesium, followed by addition of dissociation solution (Sigma-Aldrich cat. no. C5914). Detached GLP-1 R expressing cells were resuspended in HBSS assay buffer (IxHBSS; 20 mM HEPES, 0.1% BSA) supplemented with 0.5mM IBMX with a final concentration of 0.5% DMSO and diluted to 1x10⁶ cells per ml.

Test compound dilutions were prepared in HBSS buffer (Lonza) containing 3% DMSO. For measurement, 2.5 μΙ of IBMX buffer solution and 2.5pL of test compound diluted in assay buffer (final DMSO concentration of 0.3%) are added to the wells followed by 20 μΙ of the cell suspension mix and incubated at 37°C for 30 min. The assay was stopped by addition of Cisbio kit reagents and plates incubated at room temperature for 1 hour in the dark before reading on Pherastar FS using cAMP HTRF protocol (set to read at two wavelengths 620nM and 665nM). GLP-1 (7-36) peptide amide (Tocris, Cat number-2082) was used as a positive. In vitro potency of agonists was quantified by determining the concentrations that caused 50% activation of maximal response (EC₅₀). The intrinsic efficacy of compounds was expressed as a percentage effect of the maximal GLP-1 (7-36) peptide amide response.

The specificity of GLP-1 R agonists was confirmed through counter testing in HEK cells transiently transfected with empty-vector.

Table 5: In vitro pharmacology data for peptides of the present invention

Example	hGLP-1 R HEK-293 EC5o (nM)	hGLP-1 R HEK-293 Ki (nM)
1	0.03	26
2	0.06	81

3	0.014	3.5
4	0.009	48
5	0.013	7.3
6	0.3	668
8	3.1
9	0.019	26
10	0.06
11	0.24	2035
12	0.0097	32
13	0.08	1507
14	0.6	182
15	0.011	3.4
17	51
18	0.018	5.2
19	0.012	63
20	0.018	17
21	0.012	48
22	0.031	46
23	0.39	791
24	0.017	434

The results of Table 5 indicate that the peptide conjugates of the invention are potent agonists of the GLP-1 receptor.

c) In vitro mouse NIT-1 activity (Cyclic AMP determination and glucose stimulated insulin secretion)

Anti-diabetic effects of compounds were determined in an in vitro model of mouse pancreatic β-cells (NIT-1). NIT-1 cells were obtained from ATCC, and were cultured in Gibco Glutamax medium supplemented with 10 % fetal calf serum. All experiments were done with cells at less than passage 19, in accordance with the literature, which describes altered glucose sensitivity of this cell line at passage numbers above 19 (Hamaguchi et al., Diabetes. 1991 vol.40, p. 842-849).

cAMP assay

Agonist potency determinations for peptides inducing cAMP production were measured in

NIT-1 cells. NIT-1 cells were plated in standard culture medium in 96-well plates at 20 000 cells/0.1 mL/well and cultured for 24 hrs. The medium was discarded and cells were incubated for 15 min at room temperature with 100 ml stimulation buffer (Hanks buffered salt solution, 5 mM HEPES, 0.5 mM IBMX, pH 7.4). This was discarded and replaced with compound dilutions over the range 10 mM - 1 pM in stimulation buffer in the presence of 0.2 % DMSO. Cells were incubated at room temperature for 30 min. Following incubation, the assay was terminated and cAMP levels measured using the HTRF dynamic d2 cAMP assay kit from Cisbio Bioassays, according to the manufacturers’ instructions. Plates were read on PheraStaR fluorescence plate readers.

Insulin secretion assay

NIT-1 cells were plated in standard culture medium in 96-well plates at 50 000 cells/0.1 ml/well and cultured for 24 hr in complete medium. To depress high basal levels of hormone secretion, cells were re-fed in medium minus glucose and PBS for an additional 24 hrs. The medium was then discarded and cells were washed x 2 with supplemented Krebs-Ringer buffer (KRB) containing 119 mM NaCL, 4.74 mM KCI, 2.54 mM CaCI₂, 1.19 mM MgSO₄,

1.19 mM KH₂PO₄, 25 mM NaHCO₃ and 10 mM HEPES at pH 7.4 and 600 KlU/ml aprotinin. Cells were incubated with 100 mL KRB at 37 C which was then discarded. This was followed by a second incubation with KRB for 30 min, which was collected and used to measure basal insulin secretion levels for each well. Compound dilutions (10 mM - 1 pM) were then added to duplicate wells in 100 mL KRB, supplemented with 5.6 mM glucose. After 30 min incubation at 37 C, samples were removed for determination of insulin levels. Measurement of insulin was done using the insulin HTRF kit from Cisbio Corp, following the manufacturers’ instructions. For each well, insulin levels were corrected by subtraction of the basal secretion level from the pre-incubation in the absence of glucose.

d) In vitro human GLP-1R internalisation and β-arrestin recruitment

GLP-1 PathHunter cell lines (DiscoverX Corp.,) were expanded from freezer stocks according to standard procedures. Cells were seeded in a total volume of 20 pL into white walled, 384-well microplates and incubated at 37°C for the appropriate time prior to testing. For agonist determination, cells were incubated with sample to induce response.

Intermediate dilution of sample stocks was performed to generate 5X sample in assay buffer. 5 pL of 5x sample was added to cells and incubated at 37°C or room temperature for 90 or 180 minutes. Final assay vehicle concentration was 1%. Assay signal was generated through a single addition of 12.5 or 15 pL (50% v/v) of PathHunter Detection reagent cocktail, followed by a one hour incubation at room temperature. Microplates were read following signal generation with a PerkinElmer EnvisionTM instrument for chemiluminescent signal detection. Compound activity was analysed using CBIS data analysis suite (Chemlnnovation, CA).

e) In vitro rat Islet of Langerhans agonistic insulinotropic activity

The effects of peptides of the invention on glucose stimulated insulin secretion were performed on rat Islets of Langerhans. The pancreas was isolated from Sprague Dawley rats and digested with type V collagenase (Sigma) followed by purification of islets with Histopaque layering. Islets were cultured overnight in RPM I 1640 complete media and the next day starved in low glucose (2.8 mM) EBSS buffer for 30 min at 37°C with 5% CO2.

After starvation approximately 4 equal sized islets were picked and transferred into low glucose EBSS buffer (2.8 mM) followed by incubation with or without peptide in the presence of alternative glucose concentrations (up to 15.7 mM). At an appropriate time a portion of the supernatant is removed and its insulin content is measured using the Mercodia rat ELISA kit (Cat.No:10-1250-01) according to kit protocol.

f) Demonstration of in vivo efficacy of test compounds (peptides) in Blab/c mice (in vivo)

The anti-diabetic activity of controlling blood-sugar levels can be determined by measuring blood-sugar levels in mice after administration of a test compound followed by an oral glucose load. Confirmation of the pharmacodynamic activity of the compounds was demonstrated in a study using Balb/c mice. Animals were acclimatised for 1 week, after which they were weighed and randomised into groups based on body weight and fasted overnight (16 hours; during which the animals had free access to drinking water). On the study day compounds were dosed via the subcutaneous route of administration 30 minutes prior to an oral glucose challenge (2g/kg). For glucose monitoring (estimated using Glucometer) blood samples were from the tail nip capillary in heparinised glass capillary tubes at 10, 30, 60 and 120 minutes post glucose challenge. Additionally at 10 minutes post glucose challenge 50μΙ of blood was collected separately and stored for insulin estimation using Mercodia insulin kit. At the end of the experiment (120 minutes post glucose challenge) final blood samples were collected and used to estimate the levels of compound dosed using either the Phoenix EIA kit (for exendin-4) or LC/MS-MS (for agonist peptides of the invention).

Figure 1 depicts a graph showing the results of measuring, by Oral Glucose Tolerance Test (OGTT), the effect of suppressing rise in blood-sugar levels by the administration of the control GLP-1 peptide exendin-4 or the peptide of Example 3. The peptide of Example 3 was administered at doses of 30, 300 and 3000 pg/kg via subcutaneous injection while the control Exendin-4 was administered at a dose of 5 nmol/kg. Both the 300 and 3000 pg/kg doses of Example 3 fully blocked the effects of oral administration of glucose and were equivalent to the 5 nmol/kg dose of Exendin-4. Oral glucose tolerance test data are summarized in Tables 6 and 7. These results confirm the anti-diabetic effects of Examples 3,

5 and 15 peptide and demonstrate equivalence to a current clinical standard agent.

Figure 2 depicts a graph showing the results of measuring insulin levels during the Oral Glucose Tolerance Test (OGTT). Ten (10) minutes after dosing exendin-4 at 5 nmol/kg or the peptide of Example 3 at 30, 300 and 3000 pg/kg 50pl of blood was sampled for insulin levels. Data demonstrate a statistically significant increase in detectable plasma insulin by peptide example 1 at all doses tested. The control peptide exendin-4, demonstrated significant increases in plasma insulin at the 5 nmol/kg and 50 nmol/kg doses only.

Table 6: Oral glucose tolerance test data

Treatment n AUC mean SEM Comparison to (0-120min) vehicle mg/dl*min % p decrease

Vehicle	6	25077	1032
Exendin-4	5 nmol/kg sc	6	11376	336	55	<0.001***
Example 3	30pg/kg sc	6	20044	845	20	<0.001***
Example 3	300pg/kg sc	6	12359	917	51	<0.001***
Example 3	3000pg/kg sc	6	10595	611	58	<0.001***

Glucose AUC was measured over 0-120 minutes during an oral glucose tolerance test (OGTT) with glucose alone (vehicle) or glucose after pre-treatment with peptides dosed subcutaneously. The data in Table 6 demonstrates statistically significant reductions in glucose disposal over the measured time period for all peptides tested. Statistical comparisons versus vehicle were by the One way ANOVA followed by Dunnett’s test.

Table 7: Oral glucose tolerance test data

Treatment n AUC mean SEM Comparison to (0-120min) vehicle mg/dl*min % p decrease

Vehicle		6	25704	714
Example 3	30pg/kg	sc	6	14093	932	46	<0.001***
Example 3	300pg/kg	sc	6	21666	684	16	<0.001***
Example 5	300pg/kg	sc	6	15452	885	40	<0.001***
Example 15	300pg/kg	sc	6	11271	353	56	<0.001***

Glucose AUC was measured over 0-120 minutes during an oral glucose tolerance test (OGTT) with glucose alone (vehicle) or glucose after pre-treatment with peptides dosed subcutaneously. The data in Table 7 demonstrates statistically significant reductions in glucose disposal over the measured time period for all peptides tested. Statistical comparisons versus vehicle were by the One way ANOVA followed by Dunnett’s test.

q) Demonstration of systemic exposure of peptides in Blab/c mice (in vivo)

The present invention provides novel modifications to improve the pharmacokinetic properties of the peptides. For determination of plasma elimination half-life of a peptide of the invention in rats the peptide was dissolved in an isotonic buffer, pH 7.4, PBS or any other suitable buffer. The compounds, singly or in combination, were dosed via the dorsal foot vein into overnight fasted male Sprague Dawley rats using a formulation consisting of 10% DMAC,10% Solutol HS15 and 80% saline. Dosing was at 0.5mg/kg or 1 mg/kg amounts in a volume of 1ml/kg. No abnormal clinical symptoms were observed during the in-life phase. Pharmacokinetic parameters were estimated by non-compartmental model using WinNonlin. Blood samples for determination of peptide levels are taken at frequent intervals, and for a sufficient duration to cover the terminal elimination part (e.g. pre-dose, 0.25, 0.5, 1,2,4, 8, and 24 hours post dose). An aliquot of 30 pl_ plasma sample was added with 150 μΙ_ acetonitrile for protein precipitation. The sample is vigorously vortexed for about 10 min and allowed to sit on ice for 10 min. The sample is again vortexed for about 1 min, and then centrifuged for 10 min in a microcentrifuge at about 6,000 rpm. An aliquot comprising 50 μΙ_ of the supernatant is mixed with 50 μΙ_ water and 3 μΙ_ of the resultant mixture injected onto

LC-MS/MS. Derived pharmacokinetic parameters are calculated from the concentration-time data for each individual subject by use of non-compartmental methods, using the commercially available software WinNonlin Version 2.1 (Pharsight, Cary, NC, USA). The terminal elimination rate constant is estimated by log-linear regression on the terminal log5 linear part of the concentration- time curve, and used for calculating the elimination half-life.

Table 8: Pharmacokinetic properties following intravenous administration

Example	Clearance (ml/min/kg)	Terminal T1/2 (hours)
1	11.8	0.68
3	5.2	10.2
4	12.6	0.84
5	12.8	0.48
9	2.1	0.41
12	1.2	0.67
15	0.8	2.64
16	0.7	2.77

The results of Table 8 present the clearance (volume of plasma cleared of the drug per unit time) and terminal half-life (the time required for the concentration of the drug to reach half of its original value). These data highlight the effects of modifications to the peptides contained within this claim on their pharmacokinetic properties. Significantly reduced clearance and increased terminal half-life is evident for examples 15 and 16.

Claims (37)

1. According to a first aspect of the invention, there is provided a compound of formula

Q

O aai-G-T-aas-T-W-aas-aas-aasK (l) wherein Q represents phenyl or a monocyclic heteroaryl ring each of which may be optionally substituted by one or more Rq groups;

Rq represents halogen, hydroxyl, amino or Ci.6 alkyl having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S; x represents an integer selected from 1 to 3;

R1 and R2 independently represent hydrogen or a Ci_6 alkyl group, or together with the carbon to which they are attached join to form a C3.8 cycloalkyl or a heterocyclyl group; aa! represents a -NH-C(H)(R3)-CO- group;

R3 represents a-(CH2)y-COR11 or-(CH2)y-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more halogen groups or Ci.6 alkyl groups having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S; y represents an integer selected from 1 or 2;

R11 represents hydroxyl, amino or Ci.2O alkoxy having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

G represents a glycine residue;

T represents an L-threonine residue;

aa2 represents a -NH-C(R4a)(R4b)-CO- group;

R4a represents hydrogen or a Ci.6 alkyl group having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

R4b represents a Ci.w alkyl group having an alkyl chain that optionally contains one or more heteroatoms, selected from Ο, N, or S, or a benzyl group optionally substituted by one or more Ci.6 alkoxy groups, halogen atoms, or Ci.6 alkyl groups having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

W is selected from L-serine and 2,3-diaminopropionic acid residues;

aa3 represents a -NH-C(H)(R5)-CO- group;

R5 represents a-CH2-COOR12 or-(CH2)y-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more halogen groups or Ci_6 alkyl groups having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

R12 represents hydrogen or a Ci.2O alkyl group having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

aa4 represents a -NH-C(H)(R6)-CO- group;

R6 represents a -(CH2)k-Ar1 group or a Ci_i6 alkyl group having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S, k represents an integer selected from 1 to 5; such that when k represents an integer greater than 2, one CH2 group may be replaced by-O-; aa5 represents a -NH-C(H)(R7)- group;

R7 represents a -(CH2)d-Ar3 group or a Ci_i6 alkyl group having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S, and wherein the alkyl chain or heteroatom can be optionally substituted by a Ci.6 alkyl group, a -(CH2)d-Ar3 group or a C(O)-Ci-6 alkyl or alkenyl group;

Ar1 and Ar3 represents an aromatic or heteroaromatic ring system selected from phenyl, naphthyl, indolyl, pyridyl, pyrazinyl, pyrimidinyl, oxazolyl, imidazolyl, pyrazolyl, isoxazolyl, quinolinyl or isoquinolinyl which may be optionally substituted by one or more halogen, Ci.6 alkoxy, Ar2 groups, or Ci_6 alkyl having an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

Ar2 represents an aromatic or heteroaromatic ring system selected from phenyl, naphthyl, indolyl, pyridyl, pyrazinyl, pyrimidinyl, oxazolyl, imidazolyl, pyrazolyl, isoxazolyl, quinolinyl or isoquinolinyl wherein said Ar2 groups may be optionally substituted by one or more Ci.2O alkyl or Ci.2O alkoxy groups, wherein the Ci.2O alkyl or Ci.2O alkoxy groups have an alkyl chain that optionally contains one or more heteroatoms selected from Ο, N, or S;

d represents an integer selected from 1 to 5;

Z represents hydrogen, -COOR8 or -CONR9R10; and

R8, R9 and R10 independently represent hydrogen or Ci_6 alkyl;

or a tautomeric or stereochemically isomeric form or a prodrug, salt or zwitterion thereof.

2. The compound as defined in claim 1, wherein Q represents phenyl or a 5 membered monocyclic heteroaryl ring each of which may be optionally substituted by one or more Rq groups.

3.

The compound as defined in claim 2, wherein Q is selected from:

Qa

N

N

Qb

Qc

5 Qd.

wherein Rq is as defined in claim 1 and n represents an integer selected from 0 or 1.

4. The compound as defined in claim 2 or claim 3, wherein Q is selected from:

wherein Rq is as defined in claim 1 and n represents 0 or 1.

5. The compound as defined in claim 4, wherein Q is selected from:

15 Qa ; or Qb1.

6. The compound as defined in any one of claims 1 to 5, wherein x represents 2.

7. The compound as defined in any one of claims 1 to 6, wherein R1 and R2

20 independently represent hydrogen or a Ci_6 alkyl group, or together with the carbon to which they are attached join to form a C3.8 cycloalkyl (such as cyclopropyl or cyclobutyl) or a heterocyclyl group (such as oxetanyl).

8. The compound as defined in claim 7, wherein R1 and R2 independently represent hydrogen or a Ci.6 alkyl group.

9. The compound as defined in claim 8, wherein R1 and R2 both represent a Ci.6 alkyl group, such as methyl.

10. The compound as defined in any one of claims 1 to 9, wherein R3 represents a (CH2)2-COR11 or-CH2-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more Ci.6 alkyl or halogen groups.

11. The compound as defined in any one of claims 1 to 10, wherein R11 represents hydroxyl, amino or methoxy.

12. The compound as defined in any one of claims 1 to 11, wherein R3 represents a (CH2)2-CO2Me, -(CH2)2-CO2H, -(CH2)2-CONH2 or-CH2-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more Ci.6 alkyl or halogen groups.

13. The compound as defined in claim 12, wherein R3 represents a -(CH2)2-CO2Me, (CH2)2-CO2H, -(CH2)2-CONH2 or -CH2-tetrazolyl group, wherein said tetrazolyl group is unsubstituted.

14. The compound as defined in any one of claims 1 to 13, wherein R4a represents hydrogen or a methyl group.

15. The compound as defined in any one of claims 1 to 14, wherein R4b represents a C8 alkyl group or a benzyl group optionally substituted by one or two fluorine atoms.

16. The compound as defined in claim 15, wherein R4b represents a benzyl group optionally substituted by one fluorine atom (such as 2-fluorobenzyl).

17. The compound as defined in any one of claims 1 to 16, wherein R12 represents hydrogen or a Cmo alkyl group.

18. The compound as defined in any one of claims 1 to 17, wherein R5 represents a CH2-COOH, -CH2-COOMe, -CH2-COO-decan-2-yl or-CH2-tetrazolyl group, wherein said tetrazolyl group may be optionally substituted by one or more Ci_6 alkyl or halogen groups.

19. The compound as defined in claim 18, wherein R5 represents a -CH2-COOH, -CH2COOMe, -CH2-COO-decan-2-yl or -CH2-tetrazolyl group, wherein said tetrazolyl group is unsubstituted.

20. The compound as defined in any one of claims 1 to 19, wherein R6 represents a C6-15 alkyl group or a -(CH2)k-Ar1 group, such as -(CH2)k-phenyl, wherein said phenyl group may be optionally substituted by one or more methyl groups or a further phenyl group wherein said further phenyl group may also be substituted by one or more ethyl, methoxy or(OCH2CH2)5-O-CH3 groups.

21. The compound as defined in any one of claims 1 to 20, wherein k represents an integer selected from 1 to 4.

22. The compound as defined in any one of claims 1 to 21, wherein R6 represents a C7 alkyl group, a -(CH2)3-phenyl group substituted by one or more methyl groups or a -CH2phenyl group optionally substituted by a further phenyl group wherein said further phenyl group may also be substituted by one or more ethyl or methoxy groups.

23. The compound as defined in any one of claims 1 to 22, wherein R7 represents C6-15 alkyl or a -(CH2)d-Ar3 group, such as -(CH2)d-phenyl, wherein said phenyl group may be optionally substituted by one or more methyl groups or a further phenyl group wherein said further phenyl group may also be substituted by one or more ethyl or methoxy groups.

24. The compound as defined in any one of claims 1 to 23, wherein R7 is -(CH2)d-Ar3 and d represents an integer selected from 1 to 4.

25. The compound as defined in any one of claims 1 to 24, wherein R7 represents a C7 alkyl group, a Ci4 alkyl group or a -(CH2)3-phenyl group substituted by one or more methyl groups.

26. The compound as defined in any one of claims 1 to 22, wherein R7-(CH2)4-NH2 optionally substituted with a -C(O)Ci_6 alkenyl group.

27. The compound as defined in any one of claims 1 to 26, wherein Z represents hydrogen -COOR8 or -CONR9R10, such as hydrogen or-NH2.

5

28. The compound as defined in any one of claims 1 to 26, wherein each of aa!, aa2, W, aa3, aa4, and aa5in combination with Z, are in the (S)-configuration.

29. The compound as defined in any one of claims 1 to 28, wherein the compound is a PEGylated derivative thereof.

.0

30. The compound as defined in claim 1, which is selected from a compound of Examples 1-24.

31. The compound as defined in claim 1, which is or a tautomer, salt or zwitterion thereof.

32. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of claims 1 to 31 with one or more pharmaceutically acceptable carrier(s),

20 diluents(s) and/or excipient(s).

33. A compound of formula (I) as defined in any one of claims 1 to 31 for use in therapy.

34. A compound of formula (I) as defined in any one of claims 1 to 31 for use in the treatment of a disease or condition mediated by insulin, such as diabetes mellitus type 1 or 2.

35. A method of treating a disease or condition mediated by insulin, such as diabetes mellitus type 1 or 2, which comprises administering to an individual in need thereof a therapeutically effective amount of a compound of formula (I) as defined in any one of claims 1 to 31.

36. The method as defined in claim 35, wherein said administration comprises noninvasive administration of said compound.

37. The method as defined in claim 35 or claim 36, wherein said administration comprises administration via any one of the following routes: intranasal, pulmonary or oral.

Intellectual

Property

Office

GB 1522431.4

1-37

Application No: Claims searched: