WO2012156511A1 - Bombesin receptor targeting peptide incorporating a 1, 2, 3-triazole group in the backbone for preparing in vivo diagnostic and therapeutic agents - Google Patents

Abstract

The invention relates to linear stabilized peptides, wherein one, two or more carboxamide functional groups located in the backbone are replaced by a 1,4-or 1,5-substituted 1,2,3- triazole, in particular, wherein the replaced carboxamide functional groups are at or near amide bond cleavage sites. These peptides have similar properties as the peptides from which they are derived, but show increased serum stability. Examples of peptides considered are receptor targeting peptides, such as regulatory peptides. Corresponding peptides carrying non-metallic radioisotopes, non-metallic and metallic dyes, paramagnetic metals, or radioactive metals are useful as diagnostic probes and/or therapeutic agents for cancer imaging and in cancer treatment.

Description

BOMBESIN RECEPTOR TARGETING PEPTIDE INCORPORATING A 1 , 2 , 3 - TRIAZOLE GROUP IN THE BACKBONE FOR PREPARING IN VIVO DIAGNOSTIC AND THERAPEUTIC AGENTS

Field of the Invention

5 The invention relates to stabilized peptides wherein carboxamide functions are replaced by triazoles, corresponding stabilized peptides conjugated with a radioactive or nonradioactive reporter probe and/or therapeutic agent, and the use thereof in diagnosis and therapy. 0 Background Art

Regulatory peptides (both agonists and antagonists) represent a class of high affinity ligands for GPC receptors over-expressed by cancer cells. In combination with reporter probes, they display ideal characteristics for the development of molecular imaging

5 probes, e.g., targeted radiopharmaceuticals (M. Schottelius and H. J. Wester, Methods, 2009, 48, 161 -177). The major drawback of vectors based on regulatory peptides for the selective delivery of attached radionuclides to tumours and metastasis is their short biological half life as the result of rapid in vivo degradation by intra- and extracellular peptidases. Studies have shown that the enhanced stability of peptidic radiotracer

0 correlates with increased tumour uptake. Consequently, substantial research efforts have been made in the past years in order to stabilize regulatory peptides against enzymatic degradation while not impacting their high affinity to the receptors and favourable biological properties. A number of reports describe structural modifications of different peptides by well-established methods in peptide chemistry. While stabilized cyclic

5 peptides (e.g. [Tyr³]octreotide) have found application in the clinic, such efforts have so far been of limited success for linear peptides. Because rapid degradation of linear peptides by cleavage of amide bonds is still observed in vivo, new strategies for their stabilization are needed for radiotracer development. One possible approach is represented by backbone modifications of peptides, e.g. by the introduction of amide bonds surrogates0 which are not prone to enzymatic or hydrolytic degradation. It has been suggested that 1 ,4- and 1 ,5-disubstituted 1 ,2,3-triazoles, readily prepared from corresponding alkyne and azide derivatives of amino acids by employment of Cu(l)- or Ru(l)-catalysis (M. Meldal and C.W. Torn0e, Chemical Reviews, 2008, 108, 2952 ; A. Tarn, U. Arnold, M. Soellner et al. Journal of the American Chemical Society, 2007, 129, 12670) could serve as stable amide5 bond surrogates because of the similar dimensions, planarity and electronic properties (Y.

L. Angell and K. Burgess, Chemical Society Reviews, 2007, 36, 1674-1689). Reported examples include the employment of 1 ,2,3-triazoles for the formation of cyclic peptides, peptidomimetics without reported biological function, modifications of amino acid side chains, and applications to non-peptidic structures (D.S. Pedersen and A. Abell, Eur. J. Org. C em. 201 1 , 2399-241 1 ). The systematic replacement of amide bonds with 1 ,2,3- triazoles in high affinity, linear peptides has not yet been described.

Summary of the Invention

The invention relates to linear stabilized peptides, wherein one, two or more carboxamide functional groups located in the backbone are replaced by a 1 ,4- or 1 ,5-substituted 1 ,2,3- triazole, in particular, wherein the replaced carboxamide functional groups are at or near amide bond cleavage sites. These peptides have similar properties as the peptides from which they are derived, but show increased serum stability. Examples of peptides considered are receptor targeting peptides, such as regulatory peptides.

The invention also relates to variants and fragments of the mentioned peptides, for example peptides wherein further carboxamide functional group are replaced by suitable carboxamide mimics, multimers, peptides carrying suitable substituents, such as solubilizing substituents and chelators, optionally connected through spacers, and peptides carrying non-metallic radioisotopes, non-metallic and metallic dyes,

paramagnetic metals, or radioactive metals.

The invention further relates to the use of the linear stabilized peptides and variants carrying non-metallic radioisotopes, non-metallic and metallic dyes, paramagnetic metals, or radioactive metals in diagnosis and therapy, in particular diagnosis of cancer and therapy of cancer and/or reduction of side effects in cancer treatment.

Brief Description of the Figures Figure 1 : Internalization of ¹⁷⁷Lu labelled bombesin derivatives into GRP-receptor expressing PC3-cells.

% = percent of internalized radiolabeled peptide; t(min) = time (in minutes)

- - · - - ¹⁷⁷Lu-reference peptide ([¹⁷⁷Lu]-DOTA-PEG₄-[Nle¹⁴]-Bombesin(7-14) ) —■— [¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹V[Trz]His¹²,Nle¹⁴]-Bombesin(7-14)

■ ^■ A ^{■ ■} [¹⁷⁷Lu]-DOTA-PEG₄-[Ala⁹( /[Trz]Val¹⁰,Nle¹⁴]-Bombesin(7-14) Figure 2: Determination of the GRP receptor affinity of ¹⁷⁷Lu-labelled bombesin derivatives B (nM) = specific binding (nanomolar); C (nM) = concentration of radioligand (nanomolar)

- - ·- - ¹⁷⁷Lu-reference peptide ([¹⁷⁷Lu]-DOTA-PEG₄-[Nle¹⁴]-Bombesin(7-14) )

—■— [¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹V[Trz]His¹²,Nle¹⁴]-Bombesin(7-14)

■ ^■ A ^{■ ■} [¹⁷⁷Lu]-DOTA-PEG₄-[AlaV[Trz]Val¹⁰,Nle¹⁴]-Bombesin(7-14)

Figure 3: Serum stability of the investigated ¹⁷⁷Lu labelled bombesin peptides

% = percent of remaining peptide; t(h) = time (in hours)

- - ·- - ¹⁷⁷Lu-reference peptide ([¹⁷⁷Lu]-DOTA-PEG₄-[Nle¹⁴]-Bombesin(7-14) )

—■— [¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹V[Trz]His¹²,Nle¹⁴]-Bombesin(7-14)

^{■ ■} A ^{■ ■} [¹⁷⁷Lu]-DOTA-PEG₄-[AlaV[Trz]Val¹⁰,Nle¹⁴]-Bombesin(7-14)

Figure 4: Biodistribution in athymic nude mice bearing PC3 xenografts

% ID/g = Percent of injected dose per gram of tissue

Dashed bars: ¹⁷⁷Lu-reference peptide ([¹⁷⁷Lu]-DOTA-PEG₄-[Nle¹⁴]-Bombesin(7-14) )

Plain bars: [¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹ V[Trz]His¹²,Nle¹⁴]-Bombesin(7-14)

Biodistribution in blood (BL), liver (L), kidney (K), intestine (I), pancreas (P), bone (BO) and PC3 xenograft tumour (PC3). For details see Example 16.

This in vivo experiment demonstrates that the enhanced stability of triazole-stabilized [¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹V[Trz]His¹²,Nle¹⁴]-Bombesin(7-14) results in higher

accumulation of the radiotracer in receptor positive tumors and organs.

Detailed Description of the Invention The invention relates to linear stabilized peptides wherein one, two or more, e.g. three, four or five, carboxamide functional groups located in the backbone are replaced by a 1 ,4- or 1 ,5-substituted 1 ,2,3-triazole. In particular, the invention relates to such peptides wherein one, two or more carboxamide functional groups at or near amide bond cleavage sites are replaced by 1 ,4- or 1 ,5-substituted 1 ,2,3-triazole. More specifically, the invention relates to such peptides, which target receptors, for example cell membrane receptors of cancer cells, in particular, regulatory peptides.

A "naturally occurring amino acid" is one of the 22 oarmino acids that are genetically encoded and thus usually found in natural proteins. These are Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Pyl (pyrrolysine) and Sec (selenocysteine). A "non-proteinogenic amino acid" is an amino acid not usually present in natural proteins, i.e. an amino acid different from the mentioned 22 oarmino acids above.

An "amide bond cleavage site" as understood in the present invention is an amide bond between two amino acids of a peptide's amino acid sequence, or of a peptide fragment's amino acid sequence, respectively, which is prone to enzymatic or hydrolytic cleavage in vivo.

Examples of such amide bond cleavage sites are those prone to cleavage by intra- and extracellular peptidases involved in the activation or inactivation of regulatory peptides. Particular bonds are those prone to hydrolysis. General examples of particular bonds of interest and their corresponding hydrolysing enzymes are shown in Table 1.

Table 1 : Examples of particular bonds of interest and the corresponding enzymes responsible for the cleavage

Xaa means any amino acid

Preferred are the known bonds of regulatory and signalling peptides that undergo hydrolysis in vivo. Examples of preferred bonds of interest and their hydrolysing enzymes are shown in Table 2. Table 2: Examples of preferred bonds of interest and corresponding enzymes responsible for the proteolysis

P1 -[cleavage site]- PV

Cleavable bond Enzyme associated

P Gin and P1 ' Trp

P Trp and PV Ala

P His and P1 ' ^: Leu

P Leu and PV Met

P Leu and PV Nle

P Tyr and PV lie

P Pro and PV Tyr

P Gin and PV Phe

P Phe and PV ^: Phe

P Gly and PV Leu

P Tyr and PV ^: Leu 3.4.24.1 1

P Gly and PV Trp

P Asp and PV Phe

P Glu and PV Ala

P Ala and PV Tyr

P Asp and PV ^■ Val

P Ser and PV Tyr

P Tyr and PV Leu

P Glu and PV Phe

P Phe and PV ^: lie

P Trp and PV Leu

P1 = Gly and PV = Leu

P1 = His and PV = Leu 3.4.15.1

C-terminally amidated regulatory peptides

P1 = Arg and PV = Arg

3.4.24.15

P1 = Tyr and PV = Gly

P1 = Pro and PV = Tyr

3.4.24.16

P1 = Tyr and PV = Gly

P1 = Pro and PV = Val 3.4.16.2

P1 = Ala and PV = Glu

P1 = Pro and PV = Leu

P1 = Pro and PV = Ala

3.4.14.5

P1 = Pro and PV = Ser

P1 = Pro and PV = Lys

P1 = Pro and PV = Gin

Tyr and PV = Pro 3.4.1 1 .9 addition, the following carboxamide linkages shown in Table 3 are considered. Table 3: Specific bonds of interest whose proteolysis has not yet been reported

A carboxamide functional group "near" an amide bond cleavage site as understood in the present invention means a carboxamide functional group adjacent to any of the cleavage site depicted above.

"Regulatory peptides" as understood in the present invention are peptides which physiologically play a modulatory role in the human body in regions as varied as the brain; gastrointestinal tract; and endocrine, vascular, or lymphoid systems. They mediate their functions through high-affinity, specific, usually G-protein-coupled (GPC) receptors. In many incidences, the corresponding receptors have been shown to be massively overexpressed in numerous cancers. These regulatory peptides include variants and fragments as defined below. Particular "regulatory peptides" considered are bombesin, gastrins and mini gastrins, exendins (exendin-3 and exendin-4), neuropeptide-Y, neurotensin, substance P, alpha- MSH peptides (CCMSH), vasoactive intestinal peptides (VIP), CXCR4 peptides, gonadotropin releasing hormone (GnHR) peptides, glucagon like peptide-1 (GLP-1 ) and linear peptidic variants and fragments of such peptides.

Particular non-regulatory peptides considered are non-cyclic RGD peptides.

The invention relates to both, peptidic agonists and antagonists. Peptide agonists stimulate the function of the targeted receptor such as release of intracellular messenger substances (e.g. Ca²⁺ mobilization) or triggering the internalization of the receptor-ligand complex. Peptide antagonists do not stimulate the function of the targeted receptor.

Antagonists bind to the receptor with similar affinity as agonists and can therefore block the activity of the receptor. Preferred are peptides which target cell membrane receptors (e.g., GPC-receptors) with high affinity (Kd in the nanomolar range). Receptors (r) of interest are GRP-r, CCK₂-r, CCK r, GLP-1 -r, Y1 -r, NT1 -r, NK-1 -r, MC1 -r, VPAC-1 -r, GnRH-r, chemokine-4-r, and intergrins (e.g. ανββ)- Most preferred are peptides which target cell membrane receptors that are overexpressed by tumour cells involved in, for example, prostate, breast, lung, medullary thyroid and ovarian cancer, and insulinomas, glioblastomas, neuroblastomas, adenocarcinomas, and (neuro)endocrine tumours. "Fragments" as understood in the present invention are peptides, wherein one, two or more, for example up to 25 amino acids, are removed from either one or both ends of the peptide or within the amino acid sequence and which retain their regulatory peptide properties or high affinity to the corresponding receptor, respectively.

Preferred are fragments containing in full or in part the amino acid sequence responsible for binding to the corresponding receptors.

Most preferred are fragments with receptor affinities, cell binding, cell internalization and pharmacokinetic characteristics comparable or improved to those of the peptides from which they are formed. Examples include but are not limited to the binding sequence of bombesin (amino acids 7-14), exendin-4 (amino acids 9-39), neurotensin (amino acids 8- 13), and gastrin (amino acids 1 -14; minigastrin), and GLP-1 (amino acids 7-37).

"Variants" as understood in the present invention are peptides wherein single amino acids are replaced by other natural or unnatural amino acids, peptides wherein a single carboxamide functional group in the backbone is replaced by a carboxylic ester, sulfonate, or phosphonate functional group, peptides wherein a single or multiple carboxamide functional groups are /V-alkylated, /V-acylated, reduced to -CH₂-NH-, or replaced by a ketone -(C=0)CH₂- or an alkene -CH=CH- functional group. Alkyl groups considered are CrC₄-alkyl, in particular methyl, and benzyl. Acyl groups considered are CrC₄- alkylcarbonyl, in particular acetyl, formyl, tert-butoxycarbonyl and benzyloxycarbonyl. Preferred are variants which retain their receptor targeting peptide properties, in particular the receptor affinities, cell binding properties, and cell internalization characteristics of the peptide from which the variant is derived. Most preferred are variants which retain their receptor targeting peptide properties, display improved stabilities and pharmacokinetic profiles in comparison to the native peptide or fragments thereof as defined above. Improved pharmacokinetic profiles means favourable rate and route of excretion (e.g. fast renal clearance) and minimized unspecific uptake in non-targeted tissue. Examples of such peptides include the above described regulatory peptides, fragments thereof and RGD peptides.

Further variants as understood in the present invention are peptides carrying a

pharmacological modifier, e.g. solubilizing substituent, at the N- or C-terminal. A

"solubilizing substituent" as understood in the present invention is a pharmacological modifiers which increases the hydrophilicity and hence water solubility of the peptide.

Such substituents further modify the pharmacokinetics and the pharmacodynamics of the peptide, to which they are attached. Preferred solubilizing substituents are polyethylene glycols, carbohydrates, and poly-sulfonated or poly-hydroxylated linear or cyclic aliphatic or unsaturated hydrocarbons. Most preferred solubilizing substituent is polyethylene glycol, for example a polyoxyethylene group of 2 to 2000 polyoxyethylene units. Other highly preferred solubilizing agents are mono- or poly-carbohydrates, i.e. one to ten carbohydrates linked to the peptide via C-C, C-0 or C-N bonds.

Further variants as understood in the present invention are peptides carrying a chelator group. Chelator groups considered are multidentate, cyclic or acyclic structures with two to fifteen metal coordination sites, in particular coordination sites represented by heteroatoms (nitrogen, oxygen, sulfur, phosphorus) and the corresponding functional groups, e.g. primary, secondary, tertiary amines, amides, hydroxyamides, cyanides, isocyanides, thiols, sulfides, sulfonates, hydroxyls, carbonyls, carboxylates, phosphates, and phosphines, as well as saturated, unsaturated and aromatic heterocycles including one or more nitrogen, oxygen, sulfur, or phosphorus, or a combination of the these heteroatoms, respectively.

Saturated and unsaturated heterocycles considered are, e.g., pyrroline, pyrrolidine, oxazoline, oxazolidine, thiazoline, thiazolidine, piperidine, morpholine, piperazine, dioxane, 1 ,2,3-triazole, di- and tetrahydrofuran and di- and tetrahydropyran, and optionally substituted benzo fused derivatives of such monocyclic heterocyclyl, for example indoline and benzoxazolidine, all optionally substituted, for example by amine, hydroxy, oxo, thiono, carboxy, sulfuric or sulfonic acid, or phosphorous, phosphoric or phosphonic acid functions. Aromatic heterocycles considered are, e.g., pyrrol, thiophene, furane, pyrazole, imidazole, triazole, tetrazole, oxazole, isoxazole, oxadiazole, thiazole, isothiazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and benzo fused derivatives of such monocyclic heteroaryl groups, such as indole, benzimidazole, benzofuran, quinoline, or isoquinoline, all optionally substituted, for example by amine, hydroxy, carboxy, sulfonic acid or phosphonic acid functions.

Preferred are multidentate, cyclic or acyclic chelators which are known to form in vivo stable complexes with radioactive or non-radioactive metals, in particular with those metals listed below. Particularly preferred are cyclic or acyclic derivatives of polyamines which are covalently derivatized via either a C-C, C-O, or C-N bond with aliphatic or aromatic carboxylates, amines, thiols, and phosphonates. Examples of particularly preferred cyclic or acyclic chelators include those derived from polyamines such as cyclam (1 ,4,8,1 1 -tetraazacyclo- tetradecane), cyclen (1 ,4,7,10-tetraazacyclododecane) and crossbridged (CB) versions thereof (e.g. CB-cyclam; 1 ,4,8,1 1 -tetraazabicyclo[6.6.2]hexadecane), 1 ,5,9-triazacyclo- dodecane, 1 ,4,7-triazacyclononane, 1 ,7-dioxa-4,10-diazacyclododecane, 1 ,5,9-triaza- cyclododecane, 1 ,4,7,10-tetrazacyclotridecane, diamsar (1 ,8-diamino-3,6,10,13,16,19- hexaazabicyclo[6.6.6]eicosane), diethylene triamine, 1 ,4-diaminobutane, and 1 ,5- diaminopentane,.

Most preferred examples of chelators derived from the above described cyclic frameworks include, but are not limited to, DOTA (2,2',2",2"'-(1 ,4,7,10-tetraazacyclododecane- 1 ,4,7,10-tetrayl)tetraacetic acid), NOTA (2,2',2"-(1 ,4,7-triazacyclononane-1 ,4,7-triyl)- triacetic acid), TETA (1 ,4,8,1 1 -tetraazacyclo-dodecane-1 ,4,8,1 1 -tetraacetic acid), including cross-bridged versions thereof (e.g. CB-TE2A; 2,2'-(1 ,4,8,1 1 -tetraazabicyclo- [6.6.2]hexadecane-4,1 1 -diyl)diacetic acid), and phosphonate and sulfonate analogues thereof (e.g., DOTP= 1 ,4,7,10-Tetraazacyclododecane-1 ,4,7,10-tetra(methylene phosphonic acid)). Most preferred examples of chelators derived from the above described acyclic frameworks include, but are not limited to, DTPA (2,2',2",2"'-((((carboxymethyl)azanediyl)- bis(ethane-2,1 -diyl))bis(azanetriyl))tetraacetic acid) and desferrioxamine (DFO, or desferal; /V'-{5-[acetyl(hydroxy)amino]pentyl}-/V-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4- oxobutanoyl}amino)pentyl]-/V-hydroxysuccinamide). Most preferred chelators for Tc-99m include, but are not limited to known chelators for Tc-99m in its oxidation state +1 , +4 or +5, for example, MAG₃, PAMA, and 1 ,2,3-triazole- containing mono-, di- and tri-dentate chelators.

The mentioned chelator groups may be directly bound to the N- or C-terminus of the peptide, or connected through a spacer. Spacers considered are optionally substituted linear or cyclic aliphatic or aromatic hydrocarbons or saturated, unsaturated and aromatic heterocycles of 1 to 30 carbon atoms further comprising hydroxy, thio, amino or carboxy functional groups for connection with the peptide and/or the chelator, multiple neutral or charged amino acids, for example 1 to 10 amino acids selected from the natural 20 amino acids, or polyethylene glycol (PEG) comprising 2 to 20 polyethylene units, and

combinations thereof.

Linear aliphatic hydrocarbons may also be partially unsaturated, for example as in natural fatty acids. Cyclic aliphatic hydrocarbons are, e.g., cyclopentane or cylcohexane. Aromatic hydrocarbons considered are, in particular benzene, being further substituted in 1 ,2-, 1 ,3- or 1 ,4-position, naphthalene, or anthracene. Optional substituents are, for example, methyl, ethyl, benzyl, hydroxymethyl, methoxymethyl, aminomethyl, hydroxy, methoxy, ethoxy, amino, methyl- or dimethylamino, carboxy, aminocarbonyl, methoxycarbonyl, ethoxycarbonyl, and in case of aliphatic hydrocarbon also oxo. Saturated, unsaturated and aromatic heterocycles considered are those mentioned above. A particular heterocycle considered is succinimido. In combinations of hydrocarbons and heterocycles with other spacer components, the peptides or the chelators, these may be combined through a carboxamide function, a disulfide bridge, an ether, an amino or a thioether function.

A particular peptide variant according to the invention is compound of the formula

[A-(B)_m]_x-C

wherein

A is a chelator

B is a spacer

m is an integer from 0 to 6; x is an integer from 1 to 6; and

C is a linear stabilized receptor targeting peptide wherein one, two or more carboxamide functional groups located in the backbone are replaced by a 1 ,4- or 1 ,5-substituted 1 ,2,3- triazole.

Preferred are such peptide variants wherein C has with both agonistic and antagonistic properties.

Chelator A in the mentioned formula has the meaning of a chelator as defined above.

Spacer B in the mentioned formula may be a "solubilizing substituent" as defined above, being further connected to the chelator A. Preferred spacers B having the properties of a solubilizing substituent are polyethylene glycols, carbohydrates, and poly-sulfonated or poly-hydroxylated linear or cyclic aliphatic or unsaturated hydrocarbons, in particular a polyoxyethylene group of 2 to 2000, e.g. 2 to 20 polyoxyethylene units.

Spacer B, as defined above, is an optionally substituted linear or cyclic aliphatic or aromatic hydrocarbon or saturated, unsaturated and aromatic heterocycle of 1 to 30 carbon atoms further comprising hydroxy, thio, amino or carboxy functional groups for connection with the linear stabilized receptor targeting peptide C and/or the chelator A. Such a spacer B may be a short peptidic stretch of neutral or charged amino acids, for example of 1 to 10 amino acids selected from the natural 20 amino acids, or combinations of amino acids with polyethylene glycol (PEG) comprising 2 to 20 polyethylene units. Preferred are spacers B consisting of one to four charged or uncharged amino acids, e.g., a combination of Gly, Ala, pAla, Pro, Phe, Ser, Arg, Asp, Asn, Glu, Gin, Leu, Lys, Met, Trp, Tyr, and other aliphatic amino acid, e.g. linear aminoalkanoic acid (C₃-C₆), disubstituted carbocycles, e.g. cyclohexyl mono-, di- or tri-amine, disubstituted

heterocycles, e.g., morpholine or tetrahydrofuran, disubstituted aromatic carbocycles based on benzene or anthracene, disubstituted aromatic heterocycles, e.g. thiazole, triazole, imidazole or pyridine, succinimido, or polyethylene glycol.

Most preferred are the combinations of Gly, Ala, pAla, Glu, Asp, Asn, Lys, aminohexanoic acid, benzoic acid derivatives, e.g., aminobenzoic acid, pyridinecarboxylic acids, and polyethylene glycol. In formula [A-(B)_m]_x-C, m is an integer from 0 to 6, meaning that the spacer may have up to 6 repetitive units. Independently, x is an integer from 1 to 6, meaning that the linear stabilized receptor targeting peptide C may carry one, two, three, four, five or six chelators A connected through (optionally repetitive) spacer B. For multiple chelator-spacer groups A-(B)_n-, these groups may also be coupled via a side chain function of a natural or non- natural amino acid within the amino acid sequence of the peptide C. The chelators A may be bound directly to the side chain function (m = 0), or connected through a spacer B as defined above. Preferred amino acids with side chain functionalization for coupling with chelators are those bearing one of the following functional groups or, alternatively, those into which one of the following functional groups had been introduced: amine, thiol, carboxylate, halogen, alkyne, alkene, alcohol, aldehyde, azide, hydrazine, N-oxime, phosphate, thiol, and disulfide.

Preferred are side chain functionalities of natural amino acids such as lysine (amine), cysteine (thiol), glutamic acid (carboxylate), and serine and tyrosine (alcohol).

A particular peptide variant of the formula [A-(B)_m]_x-C is the compound wherein

C is a bombesin receptor targeting peptide of the formula

-Xaa⁶-Gln⁷-Trp⁸-Ala⁹-Val¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³-ZH

wherein

Xaa⁶ is the D-isomer of a naturally occurring amino acid or a non-proteinogenic a-D-amino acid with an aromatic side chain, e.g. phenyl, substituted phenyl such as p-hydroxyphenyl, biphenylyl, naphthyl, pyridyl, indolyl, imidazolyl, p-chlorophenyl, p-bromophenyl, thienyl, or thiazolyl, such as an amino acid selected from D-Phe, D-Tyr, D-Trp, D-Thi (D-thienyl- alanine), D-1 Nal (3-(1 -naphthyl)alanine), D-2Nal (3-(2-naphthyl)alanine), or is missing; 1¹ has the formula

wherein n is 1 or 2;

Xaa¹² is any amino acid, preferably Leu, Phe, or statin or a statin variant of the formula

wherein R-i is selected from a naturally occurring a-amino acid side chain, methyl, phenyl, cyclohexyl, propyl, butyl, pentyl and hexyl, e.g. as in statin, (3S,4S)-4-amino-3-hydroxy-6- methyl-heptanoyl, or in variants of statin, e.g. (4S, 5S)-4-amino-5-hydroxy-2-methyl-non-9- yl (Ri = isopropyl, carbonyl-C=0 replaced by -CH₂-CH₂-);

Xaa¹³ is a naturally occurring or a non-proteinogenic a-amino acid with an aliphatic side- chain selected from Leu, cyclopentylalanine, Cha (cyclohexylalanine), i-BuGly, i-BuAla, Met, Nle, and /^'-BuGly; and

Z is NH or O.

The superscripts 7 to 10 in the amino acids correspond to amino acids in bombesin, i.e the amino acids -Gln⁷-Trp⁸-Ala⁹-Val¹⁰- are those of bombesin, but other amino acids of bombesin are missing (1 -5, or 1 -6 if Xaa⁶ is missing) or are replaced by related amino acids.

If Z is O, the compound is a peptide with a free carboxyl function at the C terminal end. If Z is NH, the compound is a peptide with a carboxamide function at the C terminal end.

Preferably Xaa⁶ is D-Phe or D-Tyr. In an alternative preferred mode, Xaa⁶ is missing.

A particularly preferred peptide C is

wherein R₂ is isopropyl or phenyl.

Another particularly preferred peptide C is

A further particular peptide variant of the formula [A-(B)_m]_x-C is the compound wherein C is a bombesin receptor agonist of the formula

wherein Z is O or NH, preferably NH.

Also considered within this invention are multimers of the mentioned peptides. Multimers are compounds consisting of multiple numbers of the stabilized peptides connected through a central, multifunctional molecule which combines the peptides in a comb-like, tree-like or star-like shape. Examples of such central, multifunctional molecules are aliphatic or aromatic hydrocarbons with multiple functional groups such as carboxylates or amines, e.g., compounds derived from tris(hydroxymethyl)methylamine, tris(hydroxy- methyl)methanol, ethylene diamine tetraacetic acid, or diethylene amine pentaacetic acid. Further multimers considered are conjugates of multiple numbers of the stabilized peptides with stable or degradable, functionalized or non-functionalized, cross-linked or non-cross-linked polymers of linear, branched or dendrimeric shape. Polymers can be homopolymers, copolymers, and block copolymers. Examples include but are not limited to crosslinked or not crosslinked polyacrylates, polymethacrylates, polyvinyls, poly- ethyleneglycols, polypropyleneglycols, polyacrylamides, polymethacrylamides, poly- amides, polyesters, polycarbonates, polyurethanes, polyolefines, polyhalogenolefines, polyethers, polysaccharides, and polyethylenecarbonat.es.

Further multimers considered are conjugates of multiple numbers of the stabilized peptides with nanoparticles, attached by surface immobilization, e.g. nanoparticles made of gold, polymer, or single walled carbon nanotubes using technologies known to those skilled in the art.

Particular multimers considered are dimers, trimers and tetramers of the triazole-stabilized peptides, optionally conjugated with a radioactive or non-radioactive reporter probe for imaging applications as described below, and conjugates of the multimers with therapeutic radioisotopes, as described below, and combinations thereof.

If in a linear, receptor targeting peptide 1 ,2,3-triazoles are replacing one or more carboxamide functional groups at or near amide bond cleavage sites the resulting peptide is stabilized against peptide cleavage without significantly changing its physico-chemical properties (e.g. Log P), high affinity to the corresponding GPC receptors (Kd) and cell binding and internalization behaviour.

The invention further relates to linear stabilized peptides, wherein one or more

carboxamide functional groups are replaced by a 1 ,4- or 1 ,5-substituted 1 ,2,3-triazole described herein, carrying non-metallic radioisotopes, non-metallic and metallic dyes, paramagnetic metals, or radioactive metals.

Non-metallic radioisotopes considered are C-1 1 , F-18, Br-75, Br-76, Br-77, Br-80, 1-123, I- 125, 1-131 , and At-21 1 . The non-metallic radioisotopes may be conjugated covalently to either terminus of the peptide, functional groups of amino acid side chains, be part of a linear stabilized peptide as an additional substituent, e.g. in an amino acid phenylalanine or tyrosine carrying fluorine, bromine or iodine, or as an additional substituent carboxy or methyl, or as a replacement of any regular carbon atom in the peptide by C-1 1.

Preferred are the conjugation of non-metallic radioisotopes to the peptide using prosthetic groups (e.g. SFB, FBA, FPA, FPyMe for F-18, or methyl iodide for C-1 1 ) under formation of a carboxamide linkages or formation of oxime, hydrazone, disulfide or C-C bonds by employing technologies known to those skilled in the art. In addition, isotope-exchange technologies can be used to introduce non-metallic radioisotopes.

Particular non-metallic radioisotopes considered are C-1 1 , F-18, 1-125 and 1-131 .

The mentioned non-metallic radioisotopes are useful in peptides as positron emission tomography (PET) probes or as single-photon emission computed tomography (SPECT) probes, with the exception of 1-131 , useful in therapeutic applications. Further peptides considered as PET and SPECT probes are those carrying metallic radioisotopes (see below). Non-metallic and metallic dyes considered are organic molecules, e.g., commercial Alexa fluor dyes, fluorescein, rhodamine, or Cy5.5, complexes of transition metals, e.g. chelates of Eu³⁺, Tb³⁺, or nanoparticles (quantum dots) which adsorb and/or emit light in the visible range or in the near infrared. Organic dyes and chelating systems will be coupled to the peptides as described above for chelators. Conjugation of the peptides with quantum dots is done by procedures known to those skilled in the art. These peptides carrying dyes are useful as optical imaging probes. Paramagnetic metals considered are Gd, Fe, Mn, preferably Gd. The metals are attached to the peptides as will be described below. These peptides are useful as magnetic resonance imaging (MRI) probes.

Metallic radioisotopes (radioactive metals) considered are, for example, Tc-99m, ln-1 1 1 , Ga-67, Ga-68, Lu-177, Cu-64, and Zr-89, useful in imaging, and Re-186/188, Bi-213, Y- 90, Cu-67, Lu-177, Tb-161 , Tc-99m, and ln-1 1 1 for therapeutic applications. The metallic radioisotopes (and the paramagnetic metals mentioned above) are attached to the peptides of the invention through chelators as listed above, directly connected to the peptides or through a spacer. The chelators and spacers considered are those described above.

Preferred radioisotopes for diagnostic applications (SPECT and PET imaging) are Tc- 99m, ln-1 1 1 , Ga-68, Ga-67, and F-18. Preferred radioisotopes for therapeutic applications are Lu-177, Y-90, Tb-161 , and Re-188.

Tc-99m and ln-1 1 1 are not only useful for imaging, but also have therapeutic applications as Auger electron emitters.

Most preferred radioisotopes are Tc-99m, Ga-68, ln-1 1 1 , Cu-64, F-18, Y-90, Lu-177 and Re-188/186.

In particular, the invention relates to a radioconjugate

The invention further relates to the use of the linear stabilized peptides carrying non- metallic radioisotopes, non-metallic and metallic dyes, paramagnetic metals, and/or radioactive metals described herein in diagnosis and therapy, in particular in diagnosis and therapy in the field of oncology.

The described triazole-stabilized peptides are also of clinical use for the management of cancer without the combination with radioactive isotopes. In particular, regulatory peptides and derivatives thereof can be of clinical relevance for reducing not only the progression of the disease (tumor growth and formation of metastases), but also provide the means for a remedy for undesirable side effects associated with the disease. Linear stabilized peptides wherein one, two or three carboxamide functional groups are replaced by a 1 ,4- or 1 ,5-substituted 1 ,2,3-triazole, are preferably manufactured by cycloaddition reactions combining properly substituted azides and alkynes. Azide and alkyne building blocks are prepared according to literature procedures. a-Azido amino derivatives are prepared by the reaction of commercial amino acids with either azido triflate (J.T. Lundquist and J.C. Pelletier, Org. Lett. 2001 , 3, 781 -783) or imidazole- 1 -sulfonyl azide hydrochloride (E.D. Goddard-Borger and R.V. Stick, Org. Lett. 2007, 9, 3797-3800). Corresponding alkynes are prepared by either the Corey-Fuchs or the Seyferth-Gilbert homologation protocol (E. J. Corey and P. L. Fuchs, Tetrahedron Letters, 1972, 3769; J. C. Gilbert, U. Weerasooriya, Journal of Organic Chemistry, 1982, 47, 1837).

Reaction of alkyne and azide derivatives to form 1 ,2,3-triazoles is accomplished by Cu(l)- or Ru(l)-catalysis either in solution or on solid support (M. Meldal and C.W. Torn0e,

Chemical Reviews, 2008, 108, 2952 ; A. Tarn, U. Arnold, M. Soellner et al. Journal of the American Chemical Society, 2007, 129, 12670).

Peptide synthesis and conjugation with various spacers and/or chelators is performed by solid phase synthesis. Individual coupling steps (formation of amide bonds or triazole linkages) can also be carried out individually in solution.

In a particular example, starting from azido and alkynyl derivatives of amino acids amide bonds near or at known cleavage sites of the binding sequence of the regulatory peptide bombesin [Nle¹⁴]BBS(7-14) are replaced.

Peptide synthesis, formation of 1 ,2,3-triazoles and functionalization of the peptidomimetic with the universal chelator DOTA via a short PEG linker was accomplished on solid support. The product was radiolabeled with ¹⁷⁷Lu and compared in vitro with a reference peptide identical in all respect but lacking the triazole unit. The triazole-stabilized derivative exhibited an almost 4-fold increased stability in human blood serum and nearly identical affinity to GRP receptor expressing PC3 cells (K_D = <4 nM), internalization behaviour (25% internalization within 1 -2 h) and hydrophilicity (logP < -2.5). This demonstrates that replacement of amide bonds in labile, linear regulatory peptides is possible without altering the favourable characteristics of the peptide important for radiopeptide development. Examples

Abbreviations

Boc ie f-butoxycarbonyl

BOP (benzotriazol-1 -ylox)tris(dimethylamino)phosphonium hexafluorophosphate

DIBAL-H diisobutylaluminium hydride

DMF dimethylformamide

Fmoc 9-fluorenylmethoxycarbonyl

HATU tetramethyl-0-(7-azabenzotriazol-1 -yl)uronium hexafluorophosphate

HOBt 1 -hydroxybenzotriazole

RT room temperature

TBTU tetramethyl-0-(benzotriazol-1 -yl)uronium tetrafluoroborate

TFA trifluoracetic acid

TIS triisopropylsilane

Trt trityl, triphenylmethyl

General procedure 1 : Automated solid phase peptide synthesis Solid-phase peptide synthesis (SPPS) was run on an automated synthesizer Pioneer from PerSeptive Biosystems using standard Fmoc/iBu chemistry either at 0.1 or 0.03 mmol scale with TBTU/HOBt as coupling reagents and 20% piperidine in DMF as deprotection reagent. The elongation was carried out automatically using a 5-fold excess of protected amino acids and coupling reagents. The side-chain protecting groups used were Gln(Trt), His(Trt), Trp(Boc).

General procedure 2: Manual solid phase peptide synthesis

The Fmoc-protected amino acid (2 equiv., 0.2 mmol) was manually coupled onto the resin (0.03 or 0.1 mmol) in a syringe fitted with a polypropylene frit and a teflon tap in the presence of HATU (2 equiv.) and /^'-Pr₂NEt (5 equiv.) in DMF for 2 h. The completion of the reaction was checked by the Kaiser or the chloranyl test and repeated if necessary. General procedure 3: Copper(l) catalyzed cvcloaddition on solid support

In a syringe fitted with a frit and a tap, the resin was swollen with degassed DMF. The solvent was drained off thoroughly and the resin was added a solution of the Fmoc- protected amino alkyne (2 equiv., 0.06 mmol) and /^'-Pr₂NEt (1 equiv., 0.03 equiv.) in degassed DMF. To the suspension was added a solution of tetrakis(acetonitrile)copper(l) hexafluorophosphate (0.5 equiv., 0.015 mmol) and tris[(1 -benzyl-1 H-1 ,2,3-triazol-4- yl)methyl]amine (TBTA) (0.5 equiv., 0.015 mmol) and was vigorously shaken overnight. The resin was then successively and extensively washed with DMF, a solution of 0.5% of diethyldithiocarbamate in DMF (3x), CH₂CI₂ and DMF. The completion of the reaction was checked by colorimetric test of solid-supported azides (S. Punna and M.G. Finn, Synlett 2004, 1 , 99-100). The yield of the reaction was determined by UV titration of the fluorenylmethylpiperidine adduct after piperidine treatment of resin. General procedure 4: Synthesis of chiral a-azido acids lmidazole-1 -sulfonyl azide hydrochloride (E.D. Goddard-Borger and R.V. Stick, Org. Lett. 2007, 9, 3797-3800) (1 .2 equiv) was added to the amine (1 equiv.), K₂C0₃ (2.7 equiv.) and CuS0₄ pentahydrate (0.01 equiv.) in MeOH (0.2 M) and the mixture was stirred until completion of the reaction at RT (1 h - 18 h). The mixture was concentrated, diluted with H₂0, the aqueous phase was acidified to pH = 1 with a 1 M HCI solution and extracted three times with EtOAc or CH₂CI₂. The combined organic layers were dried over MgS0₄, filtered and concentrated under reduced pressure to afford the desired a-azido acids without any additional purification.

General procedure 5: Synthesis of Weinreb amides

To a solution of Fmoc- or Boc- amino acid (1 mmol), BOP (442 mg, 1 mmol, 1 equiv.) and /^'-Pr₂NEt (435 μΙ_, 2.5 mmol, 2.5 equiv.) in CH₂CI₂ (10 mL) was added Λ/,Ο-dimethyl- hydroxylamine hydrochloride (1 17 mg, 1.2 mmol, 1 .2 equiv.) and the solution was allowed to stir until completion of the reaction. The solution was then diluted with EtOAc and washed consecutively with a 1 M HCI solution (3 x 10 mL), a saturated aqueous solution of NaHC0₃ (3 x 10 mL), and water (1 x 10 mL). The organic phase was dried over MgS0₄, filtered and concentrated in vacuo. The residue was purified by flash

chromatography on silica gel to afford the desired Weinreb amide (J. Fehrentz and B. Castro, Synthesis 1983, 676-678). General procedure 6: Synthesis of chiral amino alkynes from Weinreb amides

(H.D. Dickson, S.C. Smith and K.W. Hinkle, Tetrahedron Lett. 2004, 45, 5597-5599)

The Weinreb amide (0.1 mmol) was dissolved in 1 mL of anhydrous CH₂CI₂, and the solution cooled to -78°C. DIBAL-H (0.3 mmol, 300 μΙ_, 1 M solution in CH₂CI₂) was added dropwise and the mixture was allowed to stir at -78°C until completion of the reduction (TLC monitor). The excess of DIBAL-H was quenched with 1 mL of anhydrous methanol, and the reaction mixture allowed to warm up to 0°C. Potassium carbonate (414 mg, 0.3 mmol), the Bestmann-Ohira reagent (300 μί, 0.2 mmol) and 1 mL of anhydrous methanol were added and the reaction stirred overnight at RT. A saturated aqueous solution of potassium sodium tartrate (10 mL) and CH₂CI₂ (20 mL) were added and the mixture stirred vigorously until both phases become clear. The organic layer was separated, washed with brine, dried over magnesium sulfate, and the solvent was removed under reduced pressure. The crude mixture was purified via silica gel flash chromatography to obtain the pure alkyne. a-Azide analogue of alanine, (S)-2-azidopropanoic acid

Obtained from commercial H-Ala-OH as a colourless oil according to general procedure 4 (78%). Spectrometric data were found to be identical to literature data (J.T. Lundquist and J.C. Pelletier, Org. Lett. 2001 , 3, 781 -783).

Example 2: g-Azide analogue of n-leucine, (S)-2-azidohexanoic acid

Obtained from commercial H-Nle-OH as a colourless oil according to general procedure 4 (quantitative yields). Spectrometric data were found to be identical to literature data (C.W. Torn0e, T. Sonke, I. Maes, H.E. Schoemaker, and M. Meldal, Tetrahedron: Asymmetry 2000, 1 1 , 1239-1248). xample 3: α-Azide analogue of valine, (S)-2-azido-3-methylbutanoic acid

Obtained from commercial H-Val-OH as a colourless oil according to general procedure 4 (quantitative yields). Spectrometric data were found to be identical to literature data (J.T. Lundquist and J.C. Pelletier, Org. Lett. 2001 , 3, 781 -783).

Example 4: g-Azide analogue of histidine, (S)-2-azido-3-(1 -trityl-1 H-imidazol-4-vQ- propanoic acid

a-Azido-His(Trt)-OMe (R = Me) was obtained from commercial H-His(Trt)-OMe by general procedure 4 with an additional silica gel column purification (CH₂Cl2/MeOH 0% to 0.7%) to afford the azide as a colourless oil (yield: 73%). ¹H NMR (400 MHz, CDCI₃): δ 7.39 (d, 1 H, J= 8.0 Hz), 7.35-7.31 (m, 9H), 7.15-7.1 1 (m, 6H), 6.65 (d, 1 H, J= 8.0 Hz), 4.26 (dd, 1 H, J= 8.4 Hz and 5.6 Hz,), 3.73 (s, 3H), 3.13 (dd, 1 H, J = 14.8 Hz and 8.4 Hz), 2.98 (dd, 1 H, J = 14.8 Hz and 5.6 Hz) ppm; ¹³C NMR (100 MHz, CDCI₃): δ 171 .0, 142.7, 139.2, 136.0, 130.1 , 128.5, 120.4, 75.7, 62.2, 53.0, 31 .4 ppm (one aromatic carbon was not observed due to overlapping signals,). HR-MS: calcd. for C2₆H2₃N₅O2 [M+H]+ m/z: 437.1851 found: 437.1843.

The free acid oazido-His(Trt) (R = H) was found to be unstable and was therefore prepared freshly directly before use by the following procedure: To a solution of the methyl ester in MeOH/H₂0 (2:1 ) was added LiOH hydrate (5 equiv.) and the reaction was allowed to stir at RT until completion of the hydrolysis (typically 20 min to 1 h). The reaction mixture was then diluted with EtOAc and washed with 1 M HCI. The organic phase was dried over MgS0₄, filtered and the solvent evaporated in vacuo to furnish the desired o azido-His(Trt)-OH in quantitative yields. ¹H NMR (400 MHz, DMSO-d₆): δ 7.38-7.35 (m, 9H), 7.27 (d, 1 H, J = 8.0 Hz), 7.09-7.06 (m, 6H), 6.51 (d, J = 8.0 Hz, 1 H), 4.02 (dd, 1 H, J = 4.4 Hz and 8.4 Hz), 2.95 (dd, 1 H, J= 4.4 Hz and 14.8 Hz), 2.98 (dd, 1 H, J= 8.4 Hz and 14.8 Hz) ppm; ¹³C NMR (100 MHz, DMSO-d₆): δ 171 .5, 142.1 , 137.9, 136.4, 129.2, 128.2, 128.0, 1 19.3, 74.6, 55.5, 30.2 ppm. HR-MS: calcd. for C₂₅H₂₁N₅0₂ [M+H]+ m/z: 423.1695 found: 423.170.

Example 5: Synthesis of an alkynyl analogue of amino acids

commercial Weinreb amide alkynyl-analog alkynyl amino acid of a-amino acid derivative for

SPPS

The Weinreb amide of leucine was obtained in quantitative yields as a colourless oil according to procedure 5. Spectrometric data of the compound were found to be identical to literature data (M. Rodriguez, J. P. Bali, R. Magous, B. Castro, and J. Martinez, Int. J. Pept. Protein Res. 1986, 27, 293-299). Boc-protected alkynyl analogue of leucine was obtained according to general procedure 6. After the aqueous work-up, the residue was purified by silica gel flash chromatography (n-hexane/EtOAc 95:5 to 9:1 ) to furnish the desired compound in 72% yield. Spectrometric data of the compound were found to be identical to literature data (E. Ko and K. Burgess, Org. Lett. 201 1 , 13, 980-983).

In order to obtain an alkynyl derivative suitable for standard Fmoc solid phase peptide synthesis (SPPS), the following protective group manipulations were carried out: To a solution of the Boc-protected alkyne (0.072 mmol) in CH2CI2 (3 mL) was added TFA (6 mL) and the solution was allowed to stir until the reaction was finished. The solvent was evaporated in vacuo, the residue was put in suspension in toluene (3 mL) and the solvent evaporated under reduced pressure (3x). The residue was then dissolved in CH2CI2 (2 mL), treated with /^'-Pr₂NEt (35 μί, 0.2 mmol, 2 equiv.) and /V-(9-fluorenylmethoxy- carbonyloxy)succimide (Fmoc-OSu) (48 mg, 0.2 mmol, 2 equiv.), and the solution was allowed to stir overnight at RT. The solution was then diluted with EtOAc and washed consecutively with a 1 M HCI solution (3 x 10 mL), a saturated aqueous solution of

NaHC0₃ (3 x 10 mL), and water (1 x 10 mL). The organic phase was dried over MgS0₄, filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (eluent: n-hexane/EtOAc (9:1 )) to afford the Fmoc-protected amino alkyne in 25% yield. Spectrometric data were found to be identical to literature data (S.W. Home, C.A. Olsen, J.M. Beierle, A. Montero, and R.M. Ghadiri, Angew. Chem. Int. Ed. 2009, 48, 4718-4724). Example 6: Alkynyl analogue of alanine, (S)-(9H-fluoren-9-yl)methyl but-3-yn-2- ylcarbamate

The alkynyl analogue of alanine was obtained following the procedure 6 described above for example 5. The Boc-protected intermediate was previously described and was found to have identical spectrometric data to those described in the literature (Reginato, G.; Mordini, A.; Messina, F.; Degl'lnnocenti, A.; Poli Giovanni Tetrahedron 1996, 5, 10985- 10996).

1H NMR (400 MHz, CDCI₃): δ 7.77 (d, J = 7.2 Hz, 2H), 7.60 (d, J = 7.2 Hz, 2H), 7.41 (bt, J = 7.6 Hz, 2H), 7.32 (dt, J = 7.6, 1 .2 Hz, 2H), 4.96 (s, 1 H), 4.56 (bs, 1 H), 4.43 (d, J = 6.6 Hz, 2H), 4.23 (t, J = 6.6 Hz, 1 H), 2.30 (d, J = 2.0 Hz, 1 H), 1 .44 (d, J = 6.4 Hz, 3H).

1³C NMR (100 MHz, CDCI₃): δ 171.5, 142.1 , 137.9, 136.4, 129.2, 128.2, 128.0, 1 19.3, 74.6, 55.5, 30.2 ppm

Example 7: Alkynyl analogue of glycine, (9H-fluoren-9-yl)methyl prop-2-yn-1 -ylcarbamate

The alkynyl analogue of the amino acid glycine corresponds to commercial propargyl- amine, the Fmoc derivatives of which was prepared by the following procedure: To an ice- cooled solution of propargylamine (1 10 mg, 2 mmol, 1 equiv.) and /^'-Pr₂NEt (383 μΙ_, 2.2 mmol, 2.2 equiv.) in CH₂CI₂ (20 mL) was added Fmoc-OSu (741 mg, 2.2 mmol, 1 .1 equiv.) in portions and the mixture was allowed to stir 1 h at RT. To the crude mixture was added silica, the solvent was removed in vacuo, and the residue was purified by silica gel flash chromatography (n-hexane/EtOAc (9:1 )) to afford the desired compound in quantitative yields. Spectrometric data were found to be identical to literature data (G. Tong, J.M. Lawlor, G.W. Tregear, and J. Haralambidis, J. Org. Chem. 1993, 58, 2223-2231 ). xample 8: Synthesis of reference peptide DOTA-PEG4-[Nle¹⁴l-Bombesin(7-14)

All amino acids were coupled by automated solid phase on a commercial Rink Amide MBHA LL resin (100-200 mesh) (0.1 mmol) by general procedure 1. The Fmoc-protected PEG linker and the macrocyclic chelator DOTA-tri(ieri-butyl) ester (1 ,4,7-tris(ie f-butoxy- carbonylmethyl)-1 ,4,7,10-tetraazacyclododecane-10-acetic acid) were coupled manually by general procedure 2. The peptide resin was then cleaved and deprotected by a standard 3 h-treatment with a mixture of TFA/H₂0/TIS/PhOH (87.5:5:2.5:5), and the peptide was precipitated with ice-cold diethyl ether, recovered by centrifugation and washed twice with cold diethyl ether. The precipitate was purified by preparative HPLC

(20-40% CH₃CIM in 12 min; rest 0.1 % aq. TFA; flow rate: 8 mL/min) to obtain the reference peptide in 35% yield (purity according to HPLC >95%). ESI-MS calcd. for C7iH₁₁₄N₁₈0₂i [M+H]+ m/z: 1556.7 found: 1556.2.

Example 9: Synthesis of stabilized peptide DOTA-PEG₄-iGlv¹ rrrzlHis¹²,Nle¹⁴l- Bombesin(7-14)

The amino acids from 13 to 14 were coupled by automated solid phase on a commercial Rink Amide MBHA LL resin (100-200 mesh) (0.03 mmol) using general procedure 1. The a-azido-His(Trt)-OH was coupled manually on the resin by general procedure 2. The coupling was followed by solid phase CuAAC with Fmoc-propargyl amine by general procedure 3. Residues 7 to 10, the spacer and the chelator DOTA-tri(ieri-butyl) ester (1 ,4,7-tris(ie f-butoxycarbonylmethyl)-1 ,4,7,10-tetraazacyclododecane-10-acetic acid) were subsequently coupled manually following general procedure 2. The peptide resin was then cleaved and deprotected by a standard 3 h treatment with a mixture of TFA/H₂0/TIS/PhOH (87.5:5:2.5:5), and the peptide was precipitated with ice-cold diethyl ether, recovered by centrifugation and washed twice with cold diethyl ether. The precipitate was purified by preparative HPLC (25-27% CH₃CN in 12 min; rest 0.1 % aq. TFA; flow rate: 8 mL/min) to obtain the peptide in 64% yield (purity according to HPLC >95%). ESI-MS [M+2H]²⁺= 790.80; calcd for C₇2H₁₁₄N₂o02o [M+H]⁺: 1579.85 and [M+2H]²⁺ m/z : 790.80.

Example 10: Synthesis of stabilized peptide DOTA-PEG₄-rAla⁹ urTrzlVal¹⁰,Nle¹⁴l- Bombesin(7-14)

The amino acids from 1 1 to 14 were coupled by automated solid phase on a commercial Rink Amide MBHA LL resin (100-200 mesh) (0.03 mmol) using general procedure 1. The oazido-Val-OH was coupled manually on the resin by general procedure 2. The coupling was followed by solid phase CuAAC with Fmoc-protected alkynyl analogue of alanine by general procedure 3. Residues 7 to 8, the spacer and the chelator DOTA-tri(ieri-butyl) ester (1 ,4,7-tris(ie f-butoxycarbonylmethyl)-1 ,4,7,10-tetraazacyclododecane-10-acetic acid) were subsequently coupled manually following general procedure 2. The peptide resin was then cleaved and deprotected by a standard 3 h treatment with a mixture of TFA/H₂0/TIS/PhOH (87.5:5:2.5:5), and the peptide was precipitated with ice-cold diethyl ether, recovered by centrifugation and washed twice with cold diethyl ether. The precipitate was purified by preparative HPLC (25-27% CH₃CN in 12 min; rest 0.1 % aq. TFA; flow rate: 8 mL/min) to obtain the peptide in 20% yield (purity according to HPLC >95%). ESI-MS [M+H]⁺: 1579.9 and [M+2H]²⁺ m/z : 790.60 ; calcd for C₇2H₁₁₄N₂o0₂o

[M+H]⁺: 1579.85 and [M+2H]²⁺ m/z : 790.80.

Example 1 1 : Radiolabelling with ¹⁷⁷Lu

Radiolabelling with ¹⁷⁷Lu was accomplished by reacting 20 μg of peptides (approx. 12.8 nmol) with -37 MBq ¹⁷⁷LuCI₃ in 300 μί 0.4 M NH₄OAc (pH 5.0) in a pre-lubricated

Eppendorf tube for 30 min at 95°C. After adding 9 nmol of a 0.1 mM ^natLuCI₃ solution the mixture was heated for additional 30 min at 95°C. Quality control was performed by reversed phase HPLC. The radiochemical yield was >99.0% at a specific activity of 2.9 GBq/μΓΤΐοΙ.

Example 12: Internalization studies

Internalization experiments were performed in six-well plates. 2.5 pmol of the

corresponding [¹⁷⁷Lu]-labelled peptide (2.9 GBq/ μηιοΙ) was added to PC-3 cells in incubation medium (DMEM supplemented with vitamins, essential and non-essential amino acids, L-glutamine, antibiotics (penicillin/streptomycin), fungicide [amphotericin B (Fungizone)] and 1 % FCS); 0.5-1 x10⁶ cells per well) and incubated at 37°C in a 5% C0₂ environment for different time points (30, 60, 120 and 240 min). A large excess of bombesin 1 -14 (Bachem) was used to determine non-specific internalization (blocking experiments). At each time point, the internalization was stopped by removal of the medium followed by washing the cells (2x) with ice-cold solution composed of 0.01 M PBS buffer pH 7.4. Cells were then treated with glycine buffer (0.05 M, pH 2.8) twice for 5 min at 4°C to determine the cell surface bound fraction. Finally, cells were detached and lysed from the plates by incubation with 1 M NaOH aqueous solution for 10 min at 37°C.

Radioactivity of all solutions was measured in a gamma counter. The percentage of added activity per million cells (% of total) was calculated and decay-corrected for each time point. Both peptides exhibited identical in vitro behavior resulting in approx. 25% internalized radiopeptide within 1 -2 h (Figure 1 ).

Example 13: Saturation binding experiments for determination of receptor affinities PC-3 cells at confluence were placed in 6-well plates (~10⁶ cells/well). To the cells was added at different concentrations the ¹⁷⁷Lu-radiolabeled peptides (1 , 5, 10, 50, 100, 500, 1000 nM) corresponding to a final concentration of peptide per well of 0.1 to 100 nM. The different plates were then incubated for 2 h at 4°C (final volume, 1 mL/well). After two washing steps with cold 0.01 M PBS pH 7.4, the cells were detached from the plates by incubation in 1 M NaOH aqueous solution for 10 min at 37°C. The radioactivity of solutions was measured in a gamma counter. A large excess of bombesin 1 -14 was used to determine non-specific binding (blocking experiments). These experiments (Figure 2 and Table 4) demonstrate that the introduction of a 1 ,2,3-triazole as an amide bond surrogate into the amino acid sequence of a peptide is possible without altering the favorable affinity of the peptide to its receptor. Table 4: K_n and Bmax of the investigated ¹⁷⁷Lu- labelled bombesin derivatives

Example 14: Log P determination

To a solution containing 500 μΙ_ of n-octanol and 500 μΙ_ of PBS (pH 7.4) (obtained from pre-saturated octanol-PBS solutions), 10 μΙ_ of 1 μΜ radiolabeled peptide (2.9 GBq/ μηιοΙ) was added. The resulting solutions were shaken vigorously at room temperature for 20 min and centrifuged at 3000 rpm for 10 min. Aliquots of 100 μΙ_ were removed from the octanol and from the saline phase, and the activity measured in a γ-counter. The lipophilicity was calculated as the average log of the ratio between the radioactivity in the organic fraction and the PBS fraction from the three samples. Log P values for both compounds were in the same favorable range indicating that the introduction of a triazole unit as amide bond surrogate does not influence significantly the lipophilicity of the peptide conjugate.

Table 5: Log P of the investigated ¹⁷⁷Lu- labelled bombesin derivatives

Compound Log P

[¹⁷⁷Lu]-DOTA-PEG₄-[Nle¹⁴]-Bombesin(7-14)

-2.71

reference compound

[¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹V[Trz]His¹² Nle¹⁴]-

-2.61

Bombesin(7-14), triazole-stabilized compound

[¹⁷⁷Lu]-DOTA-PEG₄-[Ala⁹( /[Trz]Val¹⁰,Nle¹⁴]-

-2.69

Bombesin(7-14) Example 15: Serum stability

For the determination of serum stability, 90 pmol of radioactive labeled peptide (specific activity: 6.4 nmol / 3 mCi ¹⁷⁷LuCI₃) were added to 1 mL fresh, human blood serum and incubated at 37°C in an atmosphere of 5% C0₂. At different time points (1 , 2, 4, 6, 24 h) samples of 100 μΙ_ were taken and the serum proteins precipitated by addition of 200 μΙ_ ethanol. After centrifugation at 5200 rpm for 5 min the supernatant was transferred into a tube and again precipitated with 200 μΙ_ ethanol. The supernatant was removed by centrifugation and analyzed with HPLC (Figure 3).

Table 6: Half-life in human blood serum of the investigated ¹⁷⁷Lu- labelled bombesin derivatives

It was observed that the 1 ,2,3-triazole stabilized radiopeptides exhibited a 3.5-fold increased serum stability (17.25 h versus 5 h for the non stabilized bombesin derivative). These experiments demonstrate the stabilizing effect of introduced 1 ,2,3-triazole amide bond surrogates in peptides while not altering their physiological properties and high affinity to the corresponding receptors (see above).

Example 16: Biodistribution in athymic nude mice bearing PC3 xenografts

Reference compound [¹⁷⁷Lu]-DOTA-PEG₄-[Nle¹⁴]-Bombesin(7-14) and triazole-stabilized analogue [¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹ V[Trz]His¹²,Nle¹⁴]-Bombesin(7-14) (each approx. 10 pmol / 0.2 MBq / 100 μΙ_ per mouse) were injected in PC3 tumour-bearing athymic nude mice (n= 5-6). After 4 h post injection (p.i.), the mice were sacrificed and biodistributions were performed. Accumulation of radioactivity in organs and tissues is expressed in % injected dose/gram. Accumulation of radioactivity in non-targeted tissues was negligible for both compounds (e.g., liver and bones; other organs not shown). Blood clearance and kidney uptake as the result of renal excretion was virtually identical for both radiopeptides (Figure 4). Uptake in receptor positive organs (intestines and pancreas) was significantly higher for the stabilized, triazole-containing compound [¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹ V[Trz]His¹²,Nle¹⁴]- Bombesin(7-14) as was tumor uptake (2 -fold increase). This in vivo experiment demonstrates that the enhanced stability of triazole-stabilized [¹⁷⁷Lu]-DOTA-PEG₄- [Gly¹ V[Trz]His¹²,Nle¹⁴]-Bombesin(7-14) results in higher accumulation of the radiotracer in receptor positive tumors and organs. i

Claims

Claims

1 . A linear stabilized peptide, wherein one, two or more carboxamide functional groups located in the backbone are replaced by a 1 ,4- or 1 ,5-substituted 1 ,2,3-triazole, and variants thereof.

2. The peptide according to claim 1 , wherein the replaced carboxamide functional groups are at or near amide bond cleavage sites, and variants thereof.

3. The peptide according to claim 1 or 2, which is a receptor targeting peptide, and variants thereof.

4. The peptide according to claim 1 or 2, which is a regulatory peptide, and variants thereof.

5. The peptide variant according to anyone of claims 1 to 4 wherein a further

carboxamide functional group is replaced by a carboxylic ester, sulfonate, or phosphonate functional group, or wherein a single or multiple carboxamide functional groups are N- alkylated, /V-acylated, reduced to -CH₂-NH-, or replaced by a ketone -(C=0)CH₂- or an alkene -CH=CH- functional group.

6. A peptide variant, which is a multimer of a peptide or peptide variant according to anyone of claims 1 to 5.

7. The peptide variant according to anyone of claims 1 to 6 carrying a chelator group.

8. The peptide variant according to claim 7, wherein the chelator group is connected to the N- or C-terminus of the peptide through a spacer.

9. The peptide variant according to claim 8 of the formula

[A-(B)_m]_x-C

wherein

A is a chelator

B is a spacer

m is an integer from 0 to 6;

x is an integer from 1 to 6; and C is a linear stabilized receptor targeting peptide wherein one, two or more carboxamide functional groups located in the backbone are replaced by a 1 ,4- or 1 ,5-substituted 1 ,2,3- triazole.

10. The peptide variant according to claim 9 wherein C is a bombesin receptor targeting peptide of the formula

-Xaa⁶-Gln⁷-Trp⁸-Ala⁹-Val¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³-ZH

wherein

Xaa⁶ is an a-D-amino acid with an aromatic side chain or is missing;

Xaa¹¹ has the formula

wherein n is 1 or 2;

Xaa¹² statin or a statin variant of the formula

wherein R-i is selected from a naturally occurring a-amino acid side chain, methyl, phenyl, cyclohexyl, propyl, butyl, pentyl and hexyl,

or wherein is isopropyl and carbonyl-C=0 is replaced by -CH₂-CH₂-;

Xaa¹³ is selected from Leu, cyclopentylalanine, cyclohexylalanine, i-BuGly, i-BuAla, Met, Nle, and /^'-BuGly; and

Z is NH or O.

1 1 . The peptide variant according to claim 10 wherein Xaa¹² is (3S,4S)-4-amino-3- hydroxy-6-methyl-heptanoyl.

12. The peptide variant according to claim 10 wherein C is

wherein R₂ is isopropyl or phenyl.

13. The peptide variant according to claim 10 wherein C is

14. The peptide variant according to claim 9 wherein C is a bombesin receptor targeting

wherein Z is O or NH.

15. The peptide variant according to any of the preceding claims carrying a non-metallic radioisotope, non-metallic or metallic dye, paramagnetic metal, or radioactive metal.

16. The peptide variant according to claim 15 carrying a non-metallic radioisotope selected from C-1 1 , F-18, Br-75, Br-76, Br-77, Br-80, 1-123, 1-125, 1-131 , and At-21 1 .

17. The peptide variant according to claim 15 carrying a non-metallic or metallic dye selected from Alexa fluor dyes, fluorescein, rhodamine, Cy5.5, chelates of Eu³⁺, Tb³⁺, and quantum dots.

18. The peptide variant according to according to claim 15 carrying a paramagnetic metal selected from Gd, Fe, and Mn.

19. The peptide variant according to claim 15 carrying a radioactive metal selected from Tc-99m, Ga-67, Ga-68, Lu-177, Cu-64, Zr-89, Re-186/188, Bi-213, Y-90, Cu-67, Tb-161 , and ln-1 1 1 .

20. [¹⁷⁷Lu]-DOTA-PEG₄-[Gly¹^[Trz]His¹²,Nle¹⁴]-Bombesin(7-14) according to claim 19.

21 . [¹⁷⁷Lu]-DOTA-PEG₄-[Ala¾[Trz]Val¹⁰,Nle¹⁴]-Bombesin(7-14) according to claim 19.

22. The peptide variant according to anyone of claims 15 to 21 for use in diagnosis of cancer.

23. The peptide variant according to anyone of claims 1 to 21 for use in the treatment of cancer or in the reduction of side effects in cancer treatment.