WO2014122228A1

WO2014122228A1 - Labelled compounds that bind to alpha-v-beta-3 integrin

Info

Publication number: WO2014122228A1
Application number: PCT/EP2014/052364
Authority: WO
Inventors: Carsten Hoeltke; Christoph Bremer; Wael ALSIBAI
Original assignee: Westfaelische Wilhelms-Universitaet Muenster
Priority date: 2013-02-07
Filing date: 2014-02-06
Publication date: 2014-08-14

Abstract

The present invention concerns in vivo imaging and in particular a novel in vivo imaging agent of formula (I). Also provided by the present invention is a method for the preparation of the in vivo imaging agent of the invention, and precursor compounds useful in said method. The in vivo imaging agent of the invention is useful in the diagnosis of conditions where there is a deviation from normal in the expression of integrin α_vβ₃.

Description

LABELLED COMPOUNDS THAT BIND TO ALPHA-V-BETA-3 INTEGRIN

Technical Field of the Invention The present invention concerns in vivo imaging and in particular a novel in vivo imaging agent. Also provided by the present invention is a method for the preparation of the in vivo imaging agent of the invention, and precursor compounds useful in said method. The in vivo imaging agent of the invention is useful in the diagnosis of conditions where there is a deviation from normal in the expression of integrin a_vl¾3.

Description of Related Art

The integrins are a family of membrane-bound glycoproteins made up of a and β subunits. Integrins are protein molecules that are found in all animal cells, with the exception of red blood cells. They are firmly anchored to the cell membrane and through the membrane. They belong to the group of the transmembrane proteins.

Integrins associate with other cells and with the extracellular matrix. Furthermore, they are important for signaling between cells and their environment. They are also known as adhesion molecules. At least three other proteins are involved in cell-cell and cell-matrix interaction and communication play an important role - the cadherins, CAMs (cell adhesion molecules) and selectins.

The extracellular domain of these transmembrane proteins have binding sites with the RGD recognition feature such as fibronectin in fibroblasts, or "non-RGD proteins" such as intercellular adhesion molecules (ICAMs), collagen and laminin (in epithelial cells). Integrins are glycoproteins. Structurally, they are heterodimers, thus consist of two interconnected glycoprotein chains. In humans can be constructed from the previously known 18 alpha-and 8 beta- subunits can bild 24 different integrins, in other studies it has been assumed that 19 alpha-and 8 beta-subunits constitute 25 integrin heterodimers. Integrins play an important role in many processes in the body. For example they can bind to viruses to allow the migration of leukocytes to the inflammation site or mediate specific steps in blood clotting.

The change of binding between integrins and binding molecules, it has become a major objective of the development of new drugs. Possible applications include the treatment of inflammatory diseases and of cancer.

Natalizumab is a humanized monoclonal antibody against the cell adhesion molecule a4-integrin.lt is an inhibitor of the binding between the α4β1 integrin (VLA-4) present on the white cell and VCAM-1 and fibronectin. It has already been approved as a drug for the treatment of relapsing forms of multiple sclerosis. α_νβ₃ integrin is a member of the integrin family and is the receptor for vitronectin. α_νβ₃ consists of two components, integrin alpha V and integrin beta 3 (CD61), and is found on many different cell types such as platelets, monocytes, endothelial and smooth muscle cells. α_νβ₃ mediates the adhesion of cells to specific substrates of the extracellular matrix and is involved in the migration of endothelial cells and muscle cells. The vitronectin receptor is also involved in the processes of angiogenesis, apoptosis and proliferation. The vitronectin receptor is involved in various physiological and pathological processes. Examples are the migration of smooth muscle cells, angiogenesis, tumor metastasis, intimal hyperplasia, osteoporosis, and the restenosis after percutaneous transluminal coronary angioplasty.

The α_νβ₃ integrin is (over)expressed in many human tumours. It is known to be associated with angiogenesis and metastasis.

Kubota et al, Bioorg Med Chem, 2006, 14 (12): 4158-81, "Tricyclic pharmacophoric-based molecules as novel integrin α_νβ₃ antagonists. Part IV: preliminary control of α_νβ₃ selectivity by meta-oriented substitution" describes a chemical backbone that will serve as a starting point for α_νβ₃ integrin antagonists.

U.S. 6,451,800, "Phenylpiperazine derivatives as integrin α_νβ₃ antagonists" disclose a^₃integrin antagonists.

U.S. 2005/0059669, "M-substituted benzoic acid derivatives having integrin α_νβ₃ antagonistic activity" describes a chemical lead with an antagonistic activity against α_νβ₃ integrin. The compounds described in the document substances intended for the treatment or prevention of such cardiovascular, cerebrovascular, angiogenesis-related diseases, cancer metastasis.

The relevance of α_νβ₃ integrin in the angiogenic process and in the development of cancer render it a potentially a perfect target for imaging and therapy approaches. There is the need of new α_νβ₃- labelled targeted compounds for in vivo optical imaging that allows a precise diagnosis of the diseases associated with angiogenesis process such as cardiovascular diseases, angiogenesis-related diseases, cerebrovascular diseases, cancers and metastasis thereof.

Summary of the Invention

The present invention provides alternative in vivo imaging agents suitable for use in the detection of α_νβ₃ expressed in a subject. The invention also provides a method for obtaining said in vivo imaging agents, and use of the in vivo imaging agents in determining α_νβ₃ integrin expressed in a subject.

The present inventors have found new labeled compounds of formula (I) that are useful in the in vivo diagnostic imaging of a range of diseases states (for example cardiovascular diseases, angiogenesis- related diseases, cerebrovascular diseases, cancers and metastasis thereof, immunological diseases, and osteopathy) where α„β₃ integrins are known to be involved.

The compounds of the present invention may be "cold" or comprise "hot" radioactive atoms or molecules. Imaging compounds disclosed herein may have radiolabels selected from the group I, ^y c, ^/ Br, ""Gel and "P. They can also comprise dyes such as DY-495, DY675, Cy 5, Cy 5.5, Cy7, C3, Cy3.5, fluorescein (FITC), heptamethylene thiocyanine, ROX, TAMRA, CAL Red, Red 640, FAM, TET, HEX, Oregon Green, TRITC, APC, DY-751, ATTO 740, ATTO 725 and ATTO 700.

The compounds of formula (I) are compounds that selectively binds to α„β₃ integrin.

The compounds of the invention can be used in Positron Emission Tomography (PET), single photon emission computed tomography (SPECT), Magnetic resonance imaging (MRI), optical imaging (eg FRI, FMT), Photoacoustic imaging (eg MSOT), composite imaging (eg PET-CT, PET-MRI), treatment (eg, radionuclide) for the diagnosis, follow up/monitoring of a_vl¾3 integrins conditions.

Figures

Fig.l shows competition binding curves as analyzed for integrin affinity for selected compounds.

Fig.2 shows the affinity values for selected precursor compounds (WA548, WA579, WA581) in comparison to the lead (WA436), a selected building block (WA493) and the previously established peptidic precursor cyc/oCRGDCGK.

Fig. 3: it is shown the in vitro cell binding assay using Cy 5.5 labeled WA 548 on a_vls₃-positive U-87 glioblastoma and a_vl?3-negative MCF-7 breast adenocarcinoma cells.

Detailed Description of the Invention

In one aspect, the present invention provides an in vivo imaging agent of Formula I:

or a salt or solvate or tautomer thereof, wherein:

n and m are independently an integer from 1-4; preferably n is 2 and m is 1;

X is selected from OH, 0-(d-C₆) alkyl, NH₂, NH-(d-C₆)alkyl, SH, S-(d-C₆), alkyl; preferably X is OH;

Ri is one of:H, (Ci-C₆) alkyl, OH, 0-(d-C₆) alkyl, perfluoro(Ci-C₆) alkyl, 0-perfluoro(Ci-C₆)alkyl, COOH, COO-(Ci-C₆)alkyl, CH₂OH, CH₂0-(d-C₆) alkyl , Br, CI, F, I, SH, S-(d-C₆) alkyl, NH₂, NH- (Ci-C₆) alkyl; preferably, Ri is CF₃; and R₂ is one of:

wherein p is an integer from 0-12; preferably p is an integer from 3-8 and

Y describes an in vivo imaging moiety

or

wherein q is an integer from 0-12, preferably q is an integer from 0-4;

Z is 0, S or NH, preferably Z = NH;

Y describes an in vivo imaging moiety:and

R₂ is

The term in vivo imaging moiety refers to an atom or to a group of atoms that may be detected externally of a subject's body following administration to said subject.

A preferred in vivo imaging moiety "Y" of Formula (I) is selected from:

(i) a radioactive metal ion

(ii) a paramagnetic metal ion;

(iii) a gamma emitting radioactive halogen;

(iv) a positron emitting radioactive non-metal ion

(v) a reporter molecule suitable for in vivo optical imaging.

When the imaging moiety "Y" is a radioactive metal ion, i.e. a radiometal, suitable radiometals can be either positron emitters such as ⁶⁴Cu, ⁴⁸V, ⁵²Fe, ⁵⁵Co, ^94mTc or ⁶⁸Ga; γ emitters such as ^99mTc, ^mln, ^113mln, or ⁶⁷Ga. Preferred radiometals are ^99mTc, ⁶⁴Cu, ⁶⁸Ga and ^mln. When the imaging moiety "Y" is a paramagnetic metal ion, suitable such metal ions include: Gd(lll), Mn(ll), Cu(ll), Cr(lll), Fe(lll), Co(ll), Er(ll), Ni(ll), Eu(lll) or Dy(lll). Preferred paramagnetic metal ions are Gd(lll), Mn(ll) and Fe(lll), with Gd(lll) being especially preferred.

When the imaging moiety "Y" is a gamma-emitting radioactive halogen, the radiohalogen is suitably chosen from ¹²³l, ¹³¹l or ^uBr. ¹²⁵l is specifically excluded as it is not suitable for use as an imaging moiety for diagnostic imaging. A preferred gamma-emitting radioactive halogen is ¹²³l.

When the imaging moiety "Y" is a positron-emitting radioactive non-metal, suitable such positron emitters include: ⁿC, ¹³N, ¹⁵0, ¹⁷F, ¹⁸F, ⁷⁵Br, ⁷⁶Br or ¹²⁴l. Preferred positron- emitting radioactive non- metals are ⁿC, ¹³N, ¹⁸F and ¹²⁴l, especially ^UC, and ¹⁸F, most especially ¹⁸F.

When the imaging moiety "Y" is a reporter suitable for in vivo optical imaging, the reporter is any moiety capable of detection either directly or indirectly in an optical imaging procedure. The reporter might be a light scatterer (e.g. a coloured or uncoloured particle), a light absorber or a light emitter. More preferably the reporter is a dye such as a chromophore or a fluorescent compound. The dye can be any dye that interacts with light in the electromagnetic spectrum with wavelengths from the ultraviolet light to the near infrared. Most preferably the reporter has fluorescent properties. Preferred organic chromophoric and fluorophoric reporters include groups having an extensive delocalized electron system, e.g. cyanines, merocyanines, indocyanines, phthalocyanines, naphthalocyanines, triphenylmethines, porphyrins, pyrilium dyes, thiapyriliumdyes, squarylium dyes, croconium dyes, azulenium dyes, indoanilines, benzophenoxazinium dyes, benzothiaphenothiazimum dyes, anthraquinones, napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, intramolecular and intermolecular charge-transfer dyes and dye complexes, tropones, tetrazines, b/s(dithiolene) complexes, £>/s(benzene-dithioIate) complexes, iodoaniline dyes, 6/s(S,0-dithiolene) complexes. Fluorescent proteins, such as green fluorescent protein (GFP) and modifications of GFP that have different absorption/emission properties are also useful. Complexes of certain rare earth metals (e.g., europium, samarium, terbium or dysprosium) are used in certain contexts, as are fluorescent nanocrystals (quantum dots). Particular examples of chromophores which may be used include: fluorescein, sulforhodamine 101 (Texas Red), rhodamine B, rhodamine 6G, rhodamine 19, indocyanine green, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Marina Blue, Pacific Blue, Oregon Green 88, Oregon Green 514, tetramethylrhodamine, and Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Particularly preferred are dyes which have absorpti on maxima in the visible or near infrared (NIR) region, between 400 nm and 3μηι particularly between 600 and 1300 nm. Optical imaging modalities and measurement techniques include, but not limited to: luminescence imaging; endoscopy; fluorescence endoscopy; optical coherence tomography; transmittance imaging; time resolved transmittance imaging; confocal imaging; nonlinear microscopy; photoacoustic imaging; acousto- optical imaging; spectroscopy; reflectance spectroscopy; interferometry; coherence interferometry; diffuse optical tomography and fluorescence mediated diffuse optical tomography (continuous wave, time domain and frequency domain systems), and measurement of light scattering, absorption, polarisation, luminescence, fluorescence lifetime, quantum yield, and quenching. A preferred a reporter suitable for in vivo optical imaging is a Cy dye.

A most preferred in vivo imaging moiety "Y" is a radioactive metal ion, a gamma-emitting radioactive halogen, a positron-emitting radioactive non-metal, or a reporter molecule suitable for in vivo optical imaging each as suitably and preferably defined above. Preferably, for the in vivo imaging agent of Formula I, Y is ¹⁸F or ¹²³l. Alternatively preferably, for the in vivo imaging agent of Formula I, Y is an imaging moiety selected from ¹⁸F, a metal complex comprising either a radioactive metal ion or a paramagnetic metal ion or a reporter suitable for optical imaging.

By the term "metal complex" is meant a coordination complex of the metal ion with one or more ligands. It is strongly preferred that the metal complex is "resistant to transchelation", i.e. does not readily undergo ligand exchange with other potentially competing ligands for the metal coordination sites. Potentially competing ligands may be in the precursor compound itself, or in other excipients in the preparation in vitro (e.g. radioprotectants or antimicrobial preservatives used in the preparation), or endogenous compounds in vivo (e.g. glutathione, transferrin or plasma proteins).

In an embodiment, a preferred in vivo imaging agent of Formula I

or a salt or solvate or tautomer thereof, is

wherein the piperidine ring is attached to the phenyl ring in position 3 or 4; R_1; R₂ and X being as defined above.

In an embodiment, a preferred in vivo imaging agent of Formula I

or a salt or solvate or tautomer thereof, is

wherein the sterochemistry at the amino acid residue is either R or S as indicated in above formula (I); preferably the sterochemistry is S and wherein R_i; R₂ and X are as defined above.

In an embodiment, a preferred in vivo imaging agent of Formula I

or a salt or solvate or tautomer thereof, is

wherein

n is 2 and m is 1:

X is OH,

the piperidine ring is attached to the phenyl ring in position 3 or 4; preferably 3

the sterochemistry at the amino acid residue is either R or S as indicated in above formula (I); preferably the sterochemistry is S and

wherein R_1; R₂ and X are as defined above.

In I one of the preferred meaning of R₂ is

Preferably the amide substituent is in para or meta positions.

In another aspect, the present invention provides a method for the preparation of the in vivo imaging agent of Formula I as suitably and preferably defined herein, wherein said method comprises reacting a suitable source of an in vivo imaging moiety with a precursor compound of Formula II:

a salt or solvate or tautomer thereof, wherein: n and m are independently an integer from 1-4; preferably n is 2 and m is 1;

X is selected from OH, 0-(d-C₆) alkyi, NH₂, NH-id-Qalkyl, SH, S-id-Q), alkyi; preferably X is OH;

Ri is one of:H, (Ci-C₆) alkyi, OH, 0-(Ci-C₆) alkyi, perfluoro(Ci-C₆) alkyi, 0-perfluoro(Ci-C₆)alkyl, COOH, COO-id-Q alkyl, CH₂OH, CH₂0-(d-d) alkyi , Br, CI, F, I, SH, S-(d-d) alkyi, NH₂, NH- (Ci-C₆) alkyi; preferably, Ri is CF₃; and

R₂ is one of:

wherein p is an integer from 0-12; preferably p is an integer from 3-8

R is one of:

■Y

X O. . to

q

wherein q is an integer from 0-12, preferably q is an integer from 0-4;

Z is 0, S or NH, preferably Z = NH

and

R₂ is

and wherein Y in any of the above moiety of formula (II) is

Y NH₂, SH, OH, OTS (Tosylate) preferably, Y is NH₂or OTs or Y is one precursor group selected from

(i) one or more ligands capable of complexing a metallic imaging moiety; hence, Y can be for example

as exemplified in compound B below;

(ii) an organometallic derivative such as a trialkylstannane or a trialkylsilane; (iii) a derivative containing an alkyl halide, alkyl tosylate or alkyl mesylate for nucleophilic substitution;

(iv) a derivative containing an aromatic ring activated towards nucleophilic or electrophilic substitution; (v) a derivative containing a functional group susceptible to acylation;

(vi) a derivative containing a functional group that takes part in oxime formation when reacted with an aldehyde;

(vii) a derivative containing a vinylsulfone functional group;

(viii) a derivative containing a functional group which undergoes facile alkylation.

Hence the present invention is directed to compounds of formula (II) as defined above. Preferred compounds of formula (II) are compounds wherein Y is NH2 or OTs.

An embodiment of Formula II

or a salt or solvate or tautomer thereof, is

wherein the piperidine ring is attached to the phenyl ring in position 3 or 4; R_1; R₂ and X being as defined above in formuala II.

An embodiment of Formula II

II

or a salt or solvate or tautomer thereof, is

wherein the sterochemistry at the amino acid residue is either R or S as indicated in above formula (II); preferably the sterochemistry is S and wherein R_l7 R₂ and X are as defined above. An embodiment of Formula II

or a salt or solvate or tautomer thereof, is

wherein

n is 2 and m is 1:

X is OH,

wherein R_1; R₂ and X are as defined above.

In all the embodiment of formula II one of the preferred meaning of R₂ is

Preferably the amide substituent is in para or meta positions.

Preferred precursor compounds are:

in particular compound A is a precursor for F radiofluorination to be used in PET and compound B contains a cheletor for chelating metal ions like Gd, or Ga for Magnetic Resonace Imaging (MRI)

Broadly speaking, the step of "reacting" the precursor compound with the suitable source of an in vivo imaging moiety involves bringing the two reactants together under reaction conditions suitable for formation of the desired in vivo imaging agent in as high a yield as possible.

A "suitable source of an in vivo imaging moiety" means the in vivo imaging moiety in a chemical form that is reactive with a precursor group in the precursor compound such that the in vivo imaging moiety becomes covalently attached, resulting in the in vivo imaging agent of the invention.

A "precursor compound" comprises a derivative of the in vivo imaging agent, designed so that chemical reaction with a convenient chemical form of an in vivo imaging moiety occurs site- specifically; can be conducted in the minimum number of steps (ideally a single step); and without the need for significant purification (ideally no further purification), to give the desired in vivo imaging agent. Such precursor compounds are synthetic and can conveniently be obtained in good chemical purity. The precursor compound may optionally comprise one or more protecting groups for certain functional groups, or "reactive groups", in order to avoid unwanted reactions. By the term "protecting group" is meant a group which inhibits or suppresses undesirable chemical reactions, but which is designed to be sufficiently reactive that it may be cleaved from the functional group in question under mild enough conditions that do not modify the rest of the molecule. After deprotection the desired product is obtained. Protecting groups are well-known to those skilled in the art and are suitably chosen from, for amine groups: Boc (where Boc is tert-butyloxycarbonyl), Fmoc (where Fmoc is fluorenylmethoxycarbonyl), trifluoroacetyl, allyloxycarbonyl, Dde (l-(4,4- dimethyl-2,6-dioxocyclohexylidene)ethyl) or Npys (3-nitro-2-pyridine sulfenyl); and for carboxyl groups: methyl ester, tert-butyl ester or benzyl ester. For hydroxyl groups, suitable protecting groups are: methyl, ethyl or tert-butyl; alkoxymethyl or alkoxyethyl; benzyl; acetyl; benzoyl; trityl (Trt) or trialkylsilyl such as tetrabutyldimethylsilyl. For thiol groups, suitable protecting groups are: trityl and 4-methoxybenzyl. A substituent herein comprising a protecting group is "chemically protected". The use of protecting groups is described in 'Protective Groups in Organic Synthesis', Theorodora W. Greene and Peter G. M. Wuts, (Fourth Edition, John Wiley & Sons, 2006).

A "precursor group" is a chemical group that preferentially reacts with the suitable source of an in vivo imaging moiety in order to obtain the in vivo imaging agent.

Where the precursor group of the precursor compound of Formula II is either (i) or (ii), the precursor compound of Formula II forms another aspect of the present invention. It is well-known in the art of in vivo imaging agents which precursor group to select for reaction with a particular source of in vivo imaging moiety. This is described hereunder in more detail to guide the reader as to how to obtain particular in vivo imaging agents of the invention.

Where the in vivo imaging moiety is a metal ion, the precursor group comprises one or more ligands capable of complexing a metallic imaging moiety. Suitable ligands for use in the present invention which form metal complexes resistant to transchelation include: chelating agents, where 2-6, preferably 2-4, metal donor atoms are arranged such that 5- or 6- membered chelate rings result (by having a non-coordinating backbone of either carbon atoms or non-coordinating heteroatoms linking the metal donor atoms); or monodentate ligands which comprise donor atoms which bind strongly to the metal ion, such as isonitriles, phosphines or diazenides. Examples of donor atom types which bind well to metals as part of chelating agents are: amines, thiols, amides, oximes, and phosphines. Phosphines form such strong metal complexes that even monodentate or bidentate phosphines form suitable metal complexes. The linear geometry of isonitriles and diazenides is such that they do not lend themselves readily to incorporation into chelating agents, and are hence typically used as monodentate ligands. Examples of suitable isonitriles include simple alkyl isonitriles such as tert- butylisonitrile, and ether-substituted isonitriles such as MIBI (i.e. l-isocyano-2-methoxy-2- methylpropane). Examples of suitable phosphines include Tetrofosmin, and monodentate phosphines such as ir/s(3-methoxypropyl)phosphine. Examples of suitable diazenides include the HYNIC series of ligands i.e. hydrazine- substituted pyridines or nicotinamides.

Suitable chelating agents which form metal complexes resistant to transchelation include, but are not limited to:

(i) diaminedioximes;

(ii) N₃S ligands having a thioltriamide donor set such as MAG₃ (mercaptoacetyltriglycine) and related ligands; or having a diamidepyridinethiol donor set such as Pica; (iii) N₂S₂ ligands having a diaminedithiol donor set such as BAT or ECD (i.e. ethylcysteinate dimer), or an amideaminedithiol donor set such as MAMA;

(iv) N₄ ligands which are open chain or macrocyclic ligands having a tetramine, amidetriamine or diamidediamine donor set, such as cyclam, monoxocyclam dioxocyclam; and,

(vi) N₂0₂ ligands having a diaminediphenol donor set.

The above described ligands are particularly suitable for complexing technetium e.g. ^94mTc or ^99mTc, and are described more fully by Jurisson et al (1999 Chem Rev; 99: 2205-2218). The ligands are also useful for other metals, such as copper (⁶⁴Cu or ⁶⁷Cu), vanadium (e.g. ⁴⁸V), iron (e.g. ⁵²Fe), or cobalt (e.g. ⁵⁵Co). Other suitable ligands are described in WO91/01144, including ligands which are particularly suitable for indium, gallium, yttrium and gadolinium, especially macrocyclic amino carboxylate and aminophosphonic acid ligands. Ligands which form non-ionic (i.e. neutral) metal complexes of gadolinium are known and are described in US 4,885,363. Particularly preferred for gadolinium are chelates including DTPA, ethylene diamine tetraacetic acid (EDTA), triethylene tetraamine hexaacetic acid (TTHA), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), 10-(2-hydroxypropyl)- 1,4,7,10-tetraazacyclododecane- 1 ,4,7-triacetic acid (D03A) and derivatives of these.

For ^99mTc labelling of a precursor compound of Formula II comprising a suitable chelating agent, the usual technetium starting material is pertechnetate, i.e. Tc0₄ which is technetium in the Tc(VII) oxidation state. Pertechnetate itself does not readily form metal complexes, hence the preparation of technetium complexes usually requires the addition of a suitable reducing agent such as stannous ion to facilitate complexation by reducing the oxidation state of the technetium to the lower oxidation states, usually Tc(l) to Tc(V). The solvent may be organic or aqueous, or mixtures thereof. When the solvent comprises an organic solvent, the organic solvent is preferably a biocompatible solvent, such as ethanol or DMSO. Preferably the solvent is aqueous, and is most preferably isotonic saline.

A precursor compound of Formula II suitable for preparing radioiodinated in vivo imaging agents of Formula I comprises a derivative which either undergoes electrophilic or nucleophilic radioiodination or undergoes condensation with a labelled aldehyde or ketone. Examples of the first category are:

(a) organometallic derivatives such as a trialkylstannane (e.g. trimethylstannyl or tributylstannyl), or a trialkylsilane (e.g. trimethylsilyl) or an organoboron compound (e.g. boronate esters or organotrifluoroborates);

(b) a non-radioactive alkyl bromide for halogen exchange or alkyl tosylate, mesylate or triflate for nucleophilic iodination;

(c) aromatic rings activated towards nucleophilic iodination (e.g. aryliodonium salt aryl diazonium, aryl trialkylammonium salts or nitroaryl derivatives).

A preferred such precursor compound comprises: a non-radioactive halogen atom such as an aryl iodide or bromide (to permit radioiodine exchange); an organometallic precursor compound (e.g. trialkyltin, trialkylsilyl or organoboron compound); or an organic precursor such as triazenes or a good leaving group for nucleophilic substitution such as an iodonium salt. Preferably for radioiodination, the precursor compound comprises an organometallic precursor compound, most preferably trialkyltin.

Precursor compounds and methods of introducing radioiodine into organic molecules are described by Bolton (2002 J Lab Comp Radiopharm: 45: 485-528). Suitable boronate ester organoboron compounds and their preparation are described by Kabalka et al (2002 Nucl Med Biol; 29: 841-843; and 2003 Nuc Med Biol; 30: 369-373). Suitable organotrifluoroborates and their preparation are described by Kabalka et al (2004 Nucl Med Biol; 31 : 935-938).

Examples of aryl groups to which radioactive iodine can be attached are given below:

Both contain substituents which permit facile radioiodine substitution onto the aromatic ring. Hence Y in above formula Y can be SnBu3 or OH.

Alternative substituents containing radioactive iodine can be synthesised by direct radioiodination via radiohalogen exchange, e.g.

The radioiodine atom is preferably attached via a direct covalent bond to an aromatic ring such as a benzene ring, or a vinyl group since it is known that iodine atoms bound to saturated aliphatic systems are prone to in vivo metabolism and hence loss of the radioiodine.

Precursor compounds suitable for radiofluorination may be designed to be directly labelled with [¹⁸F] -Fluoride, or to be reactive with a ¹⁸F-containing prosthetic group.

Radiofluorination may be carried out via direct labelling using the reaction of [¹⁸F]-Fluoride with a suitable precursor group in the precursor compound of Formula II. The precursor group may be a good leaving group, such as an alkyl bromide, alkyl mesylate or alkyl tosylate. Direct radiofluorination with [¹⁸F] -fluoride may also be carried out by nucleophilic aromatic substitution. For aryl systems, ¹⁸F -fluoride nucleophilic displacement from an aryl diazonium salt, aryl nitro compound or an aryl quaternary ammonium salt are suitable routes to aryl-¹⁸F F derivatives. Alternatively, the precursor compound may contain a chloro nicotinamide precursor group where ¹⁸F -fluoride nucleophilic displacement at the chloro leads to the [¹⁸F]fluoronicotinamide compounds (Greguric et al 2009 J Med Chem; 52: 5299-5302). Preferably where direct labelling is carried out with [ F]-Fluoride, the precursor group is present at Y of Formula II.

To obtain in vivo imaging agents of the invention comprising a reporter suitable for optical imaging, the reader is directed to the methods described by Licha et al (2002 Topics Curr Chem; 222: 1-29; 2005 Adv Drug Deliv Rev; 57: 1087-1108). For reviews and examples of labelling using fluorescent dye labelling reagents, see "Non-Radioactive Labelling, a Practical Introduction", Garman, A.J. Academic Press, 1997; "Bioconjugation - Protein Coupling Techniques for the Biomedical Sciences", Aslam, M. and Dent, A., Macmilian Reference Ltd, (1998). Cyanine dyes (Cy ) functionalised suitable for conjugation are commercially available from GE Healthcare Limited, Atto- Tec, Dyomics, Molecular Probes and others. Most such dyes are available as NHS esters, which can react with an amine in the precursor compound of Formula II as defined herein to form the desired in vivo imaging agent. Alexa Fluor(TM) 647 functionalised with hydrazide, maleimide or succinimidyl ester groups are commercially- available from Molecular Probes. Cy^D functionalised with carboxyl or maleimide groups can be prepared according to methods described in EP1816475. These functionalised Cy^D compounds can be reacted with precursor compounds comprising hydroxy or amine precursor groups to result in the desired in vivo imaging agent of Formula I.

The method for preparation of the in vivo imaging agent of Formula I may also be carried out by solid phase synthesis. A "solid phase" is a cross-linked, insoluble polymeric material that is chemically inert to the conditions of the synthesis. The solid phase typically takes the form of spherical particles e.g. beads of diameter between 0.04- 0.15mm, but sheets, pin-shaped particles and disc-shaped particles are also used. The whole precursor compound can be synthesized on the solid phase, and cleaved off by standard methods, for example trifluoroacetic acid in dichloromethane. Alternatively, depending on the chemistries involved, an intermediate can be cleaved off at a suitable stage and subsequent steps run in solution.

In a preferred embodiment, the method for preparation of the in vivo imaging agent of the invention is automated. Automated synthesis may be conveniently carried out by means of an automated synthesis apparatus, e.g. Tracerlab(TM) and Fastlab(TM) (both available from GE Healthcare). Fastlab(TM) represents the state of the art in automated positron-emission tomography (PET) radiotracer synthesis platforms, and it is desirable in the development of a new PET radiotracer that its synthesis is compatible with Fastlab(TM). The radiochemistry is performed on the automated synthesis apparatus by fitting a "cassette" to the apparatus. Such a cassette normally includes fluid pathways, a reaction vessel, and ports for receiving reagent vials as well as any solid-phase extraction cartridges used in post-radiosynthetic clean up steps. In a further aspect of the present invention there is therefore provided a cassette for carrying out the automated method of the invention comprising:

(i) a vessel containing a precursor compound, wherein said precursor compound is as suitably and preferably defined above for the method of the invention; and,

(ii) means for eluting the vessel with a suitable source of an in vivo imaging moiety, said in vivo imaging moiety as suitably and preferably defined herein.

The cassette may also comprise an ion-exchange cartridge for removal of excess in vivo imaging moiety. The reagents, solvents and other consumables required for the automated synthesis may also be included together with a data medium, such as a compact disc carrying software, which allows the automated synthesizer to be operated in a way to meet the end user's requirements for concentration, volumes, time of delivery etc. The cassette of the invention is particularly suitable for preparation of in vivo imaging agents of the invention where the in vivo imaging moiety is ¹⁸F.

In a further aspect, the present invention provides a "pharmaceutical composition", which is defined as a composition comprising the in vivo imaging agent as defined herein together with a biocompatible carrier, in a form suitable for mammalian administration.

The "biocompatible carrier" is a fluid, especially a liquid, in which the in vivo imaging agent is suspended or dissolved, such that the composition is "suitable for mammalian administration", i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier medium is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic polyol materials (e.g. polyethyleneglycols, propylene glycols and the like). The biocompatible carrier medium may also comprise biocompatible organic solvents such as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or formulations. Preferably the biocompatible carrier medium is pyrogen- free water for injection, isotonic saline or an aqueous ethanol solution. The pH of the biocompatible carrier medium for intravenous injection is suitably in the range 4.0 to 10.5.

The pharmaceutical composition of the invention is suitably supplied in a container which is provided with a seal which is suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on septum seal closure) whilst maintaining sterile integrity. Such containers may contain single or multiple patient doses. Preferred multiple dose containers comprise a single bulk vial (e.g. of 10 to 30 cm³ volume) which contains multiple patient doses, whereby single patient doses can thus be withdrawn into clinical grade syringes at various time intervals during the viable lifetime of the preparation to suit the clinical situation. Pre-filled syringes are designed to contain a single human dose, or "unit dose" and are therefore preferably a disposable or other syringe suitable for clinical use. Where the pharmaceutical composition is a radiopharmaceutical composition, the pre- filled syringe may optionally be provided with a syringe shield to protect the operator from radioactive dose. Suitable such radiopharmaceutical syringe shields are known in the art and preferably comprise either lead or tungsten.

The pharmaceutical composition of the present invention may be prepared from a kit. Alternatively, the pharmaceutical composition may be prepared under aseptic manufacture conditions to give the desired sterile product. The pharmaceutical composition may also be prepared under non-sterile conditions, followed by terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). Preferably, the pharmaceutical composition of the present invention is prepared from a kit.

Such kits comprise a suitable precursor of the invention, preferably in sterile non- pyrogenic form, so that reaction with a sterile source of an in vivo imaging moiety gives the desired pharmaceutical composition with the minimum number of manipulations. Where the kit comprises a precursor compound of the invention, said kit itself forms a further aspect of the invention. Such considerations are particularly important for radiopharmaceuticals, especially where the radioisotope has a relatively short half-life, and for ease of handling and hence reduced radiation dose for the radiopharmacist. Hence, the reaction medium for reconstitution of such kits is preferably a biocompatible carrier as defined above, and is most preferably aqueous. The precursors for use in the kit may be employed under aseptic manufacture conditions to give the desired sterile, non-pyrogenic material. The precursors may also be employed under non-sterile conditions, followed by terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). Preferably, the precursors are employed in sterile, non-pyrogenic form.

The kits may optionally further comprise additional components such as a radioprotectant, antimicrobial preservative, pH-adjusting agent or filler.

By the term 'radioprotectant' is meant a compound which inhibits degradation reactions, such as redox processes, by trapping highly-reactive free radicals, such as oxygen-containing free radicals arising from the radiolysis of water. Suitable radioprotectants are chosen from: ascorbic acid, para- aminobenzoic acid (i.e. 4- aminobenzoic acid), gentisic acid (i.e. 2,5-dihydroxybenzoic acid) and salts thereof with a biocompatible cation.

By the term "antimicrobial preservative" is meant an agent which inhibits the growth of potentially harmful micro-organisms such as bacteria, yeasts or moulds. The antimicrobial preservative may also exhibit some bactericidal properties, depending on the dose. The main role of the antimicrobial preservative(s) of the present invention is to inhibit the growth of any such micro-organism in the pharmaceutical composition post- reconstitution, i.e. in the imaging agent product itself. The antimicrobial preservative may, however, also optionally be used to inhibit the growth of potentially harmful microorganisms in one or more components of the kit prior to reconstitution. Suitable antimicrobial preservative(s) include: the parabens, i.e. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred antimicrobial preservative(s) are the parabens.

The term "pH-adjusting agent" means a compound or mixture of compounds useful to ensure that the pH of the reconstituted kit is within acceptable limits (approximately pH 4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting agents include pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS (i.e. tns(hydroxymethyl)aminomethane), and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof.

By the term "filler" is meant a pharmaceutically acceptable bulking agent which may facilitate material handling during production and lyophilisation. Suitable fillers include inorganic salts such as sodium chloride, and water soluble sugars or sugar alcohols such as sucrose, maltose, mannitol or trehalose.

In a yet further aspect, the present invention provides an in vivo imaging method to determine the quantity and/or location of α_νβ₃ integrin expressed in a subject, wherein said method comprises:

(i) administering to said subject the in vivo imaging agent of formula (I) as suitably and preferably defined herein;

(ii) allowing said administered in vivo imaging agent of step (i) to bind to α_νβ₃ integrin expressed in said subject; (iii) detecting signals emitted by an in vivo imaging moiety comprised in said bound in vivo imaging agent of step (ii); and,

(iv) generating an image of the location and amount of said signals, wherein said signals represent the quantity and/or location of α_νβ₃ integrin expressed in said subject. In yet another embodiment, the present invention provides a method for detecting or imaging integrin a_vls₃ receptor-presenting cells in a mammalian patient, the method comprising administering to the patient a diagnostic composition comprising a compound of formula (I) in an amount and for a time sufficient to detect or image at least one integrin ct_vls₃ receptor-presenting cell in the patient to which the labeled compound or composition is bound.

In yet another embodiment, the present invention provides a method for imaging a first population of integrin α„β -expressing cells, or a first population of tumor cells, the method comprising administering to the patient an amount of a diagnostic composition comprising a compound of formula (I), and for a time effective to image the first population of integrin a_vls3-expressing cells, or the first population of tumor cells. The "subject" of the invention can be any human or animal subject. Preferably the subject of the invention is a mammal. Most preferably, said subject is an intact mammalian body in vivo. In an especially preferred embodiment, the subject of the invention is a human.

The step of 'administering' the in vivo imaging agent is preferably carried out parenterally, and most preferably intravenously. The intravenous route represents the most efficient way to deliver the in vivo imaging agent throughout the body of the subject and into contact with a_vls₃ integrin expressed in said subject. The in vivo imaging agent of the invention is preferably administered as the pharmaceutical composition of the invention, as defined herein.

Following the administering step and preceding the detecting step, the in vivo imaging agent is allowed to bind to α_νβ₃ integrin. The in vivo imaging agent moves dynamically through the subject's body, coming into contact with various tissues therein. Once the in vivo imaging agent comes into contact with α_νβ₃ integrin a specific interaction takes place such that clearance of the in vivo imaging agent from tissue with α_νβ₃ integrin takes longer than from tissue without, or expressing less α_νβ₃ integrin. A certain point in time is reached when detection of in vivo imaging agent specifically bound to α_νβ₃ integrin is enabled as a result of the ratio between in vivo imaging agent bound to tissue with α_νβ₃ integrin versus that bound in tissue expressing less (or no) α_νβ₃ integrin.

The step of "detecting signals" involves detection of signals emitted by the in vivo imaging moiety by means of a detector sensitive to said signals. Such detectors are well-known in the art. For example, where the in vivo imaging moiety is a gamma emitter, detection can be carried out using a single- photon emission computed tomography (SPECT) camera, and where the in vivo imaging moiety is a paramagnetic metal ion, detection can be carried out using a magnetic resonance imaging (MRI) camera.

The step of "generating an image" is carried out by a computer which applies a reconstruction algorithm to the acquired signal data to yield a dataset. This dataset is then manipulated to generate an image showing the location and/or amount of signals emitted by said in vivo imaging moiety. An "α_νβ₃ integrin condition" means a pathological condition characterized by abnormal expression of α_νβ₃ integrin, typically over-expression of α_νβ₃ integrin. Examples of such conditions where the method of in vivo imaging of the present invention would be of use include inflammation, cancer and fibrosis, atherosclerosis and coronary artery diseases, stroke and cerebral degenerative diseases. The in vivo imaging method of the invention is preferably carried out wherein said subject is known or is suspected to have an α_νβ₃ integrin condition, most preferably said subject is known to have an α„β₃ integrin condition. Where the subject is known to have an α_νβ₃ integrin condition, said in vivo imaging method is preferably carried out repeatedly during the course of a treatment regimen for said subject, said treatment regimen comprising administration of a drug to treat said α_νβ₃ integrin condition.

In a preferred embodiment, the in vivo imaging method as suitably and preferably defined herein further comprises the step (v) of attributing the quantity and/or location of α_νβ₃ integrin receptors to a particular clinical picture.

In a yet further aspect, the present invention provides the in vivo imaging agent as suitably and preferably defined herein for use in the in vivo imaging method as suitably and preferably defined herein.

Suitable salts according to the term "salt or solvate thereof" include (i) physiologically acceptable acid addition salts such as those derived from mineral acids, for example hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and those derived from organic acids, for example tartaric, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycollic, gluconic, succinic, methanesulphonic, and para- toluenesulphonic acids; and (ii) physiologically acceptable base salts such as ammonium salts, alkali metal salts (for example those of sodium and potassium), alkaline earth metal salts (for example those of calcium and magnesium), salts with organic bases such as triethanolamine, N-methyl-D-glucamine, piperidine, pyridine, piperazine, and morpholine, and salts with amino acids such as arginine and lysine. Suitable solvates according to the term "salt or solvate thereof include those formed with ethanol, water, saline, physiological buffer and glycol.

The term "alkyl" means straight-chain or branched-chain alkyl radical containing preferably from 1 to 4 carbon atoms. Examples of such radicals include methyl, ethyl, and propyl.

The term "aryl" refers to a cyclic aromatic radical having 5 to 6 carbon atoms, in the ring system, e.g. phenyl or naphthyl. A "heteroaryl" substituent is an aryl as defined above wherein at least one of the carbon atoms of the ring has been replaced with a "heteroatom" selected from N, S or 0.

The term "nitrogen-containing heteroaryl ring" refers herein to a heteroaryl as defined above wherein the cycle comprises one or two nitrogen heteroatoms.

The term "halogen" encompasses the substituents iodine, bromine, chlorine and fluorine, as well as isotopes thereof suitable for in vivo imaging.

The term "substituent comprising an in vivo imaging moiety" refers either to a substituent which is itself an in vivo imaging moiety, or to a chemical group in which is comprised said in vivo imaging moiety. More detail is provided below when specific in vivo imaging moieties are discussed. The term "amino acid residue" refers to meant a bivalent residue of an L- or a D-amino acid, amino acid analogue (e.g. napthylalanine) or amino acid mimetic which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers. Preferably the amino acids of the present invention are optically pure. The term "carbohydrate residue" refers to a bivalent aldehyde or a ketone derivative of a polyhydric alcohol. It may be a monomer (monosaccharide), such as fructose or glucose, or two sugars joined together to form a disaccharide. Disaccharides include sugars such as sucrose, which is made of glucose and fructose. The term "sugar" includes both substituted and non- substituted sugars, and derivatives of sugars. Preferably, the sugar is selected from glucose, glucosamine, galactose, galactosamine, mannose, lactose, fucose and derivatives thereof, such as sialic acid, a derivative of glucosamine. The sugar is preferably a or β. The sugar may especially be a manno- or galactose pyranoside. The hydroxyl groups on the sugar may be protected with, for example, one or more acetyl groups. The sugar moiety is preferably N-acetylated. Preferred examples of such sugars include N-acetyl galactosamine, sialic acid, neuraminic acid, N-acetyl galactose, and N-acetyl glucosamine.

Where a particular in vivo imaging agent of the present invention comprises a chiral centre, the scope of the invention also includes compositions comprising the racemic mixture of the two enantiomers, as well as compositions comprising each enantiomer individually substantially free of the other enantiomer. Thus, for example, contemplated herein is a composition comprising the S enantiomer substantially free of the R enantiomer, or a composition comprising the R enantiomer substantially free of the S enantiomer.

By "substantially free" it is meant that the composition comprises less than 10%, or less than 8%, or less than 5%, or less than 3%, or less than 1% of the minor enantiomer. If the particular in vivo imaging agent comprises more than one chiral center, the scope of the present disclosure also includes compositions comprising a mixture of the various diastereomers, as well as compositions comprising each diastereomer substantially free of the other diastereomers. The recitation of an in vivo imaging agent, without reference to any of its particular diastereomers, includes compositions comprising all four diastereomers, compositions comprising the racemic mixture of R,R and S,S isomers, compositions comprising the racemic mixture of R,S and S,R isomers, compositions comprising the R,R enantiomer substantially free of the other diastereomers, compositions comprising the S,S enantiomer substantially free of the other diastereomers, compositions comprising the R,S enantiomer substantially free of the other diastereomers, and compositions comprising the S,R enantiomer substantially free of the other diastereomers.

The invention will now be described in the following non-limiting examples.

Examples

Example 1: Synthesis of compound WA548 and of compound WA548 labeled with Cy 5.5.

1.1 (S)-Methyl-2,3-diaminopropanoate

Thionyl chloride (100 mL, 1.43 mol, 20 eq) was added dropwise to cold methanol (200 mL, -10°C) in presence of argon. After the addition was completed the solution was warmed to room temperature for 20 min. (S)-2,3-Diaminopropionic acid (10.0 g, 0.074 mol) was added to the solution, then the reaction mixture was heated under reflux for 48h. An excess portion of methanol (100 mL) and thionyl chloride (72 mL) was prepared as before, and added to the reaction at room temperature. The reaction was then stirred overnight at room temperature. The solvent was evaporated at 40°C by using a rotary evaporator to obtain product 2 as a white powder (13.0 g, 96%).

*H NMR (400 MHz, DMSO):

6[ppm] 8.96 (s, 6H), 4.44 (t, J = 5.9, 1H), 3.73(s, 3H) , 3.40 - 3.26 (m, 2H).

¹³C NMR (101 MHz, DMSO):

6[ppm] 166.91 , 53.40, 49.91, 38.21.

1.2 MethyI-(S)-2-amino-3(/V-t-butyloxycarbonylamino)propanoate

An amount of 10.0 g (0.053 mol) of compound 1 was suspended in 1 L of CH₂CI₂ and kept in presence of argon at -78°C. Triethylamine (30 ml, 0.20 mol) was added dropwise to the cold suspension to obtain a solution mixture. Di-r-butyldicarbonate (11.6 g, 0.054 mol) was dissolved in 100 mL CH₂CI₂ and added dropwise to the mixture. After the addition was completed the reaction was placed in an ice bath and stirred for 2h. The reaction was transferred to a separation funnel and extracted with 2 x 50 mL of 10% Na₂S0₄ solution. After the aqueous layer was washed with 3 x 10 mL CH₂CI₂, the pH was adjusted to 10 using saturated NaHC0₃ and 3N NaOH solutions, it was extracted with 10 x 100 mL of CH₂CI₂. The organic layer was dried over MgS0_4; filtered and concentrated to obtain 4.9 g of a pale yellow oil. Column chromatography on silica gel in 2.5%MeOH/ EtOAc resulted 4.32g of the product (53%).

HRMS: 219.1324 (M+H⁺), 241.11436 (M+Na⁺).

*H NMR (300 MHz, CDCI₃): 6[ppm]= 5.11 (s, 1H), 3.78 - 3.69 (s, 3H), 3.60 - 3.46 (m, 2H), 3.31- 3.15 (m, 1H), 1.64 (s, 2H),

1.42 (s, 9H).

1³C MR (75 MHz, CDCI₃):

5[ppm]= 174.46, 155.94, 79.48, 54.36, 52.26, 44.16, 28.30.

1.3 4-(Chiorosuifonyl)benzoyl chloride

The potassium salt of 4-sulfobenzoic acid (1.8 g, 0.0042 mol) was added to thionyl chloride (20 mL) along with one drop of DMF. The suspension was refluxed in presence of argon for about 4h. Excess of thionyl chloride was removed by repeated co-evaporation in vacuum with dry toluene to yield 1.7 g (95%) of the product (melting point = 55,7 °C). *H NMR (300 MHz, CDCI₃)

6[ppm] 8.40 - 8.34 (m, 2H), 8.25 - 8.18 (m, 2H).

¹³C NMR (75 MHz, CDCI₃)

6[ppm] 167.01, 148.94, 138.47, 132.24, 127.54. 1.4 4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)benzene-l-sulfonyl chloride

4-(Chlorosulfonyl)benzoyl chloride (3 g, 0.0125 mol) was added to anhydrous THF (35 mL) and stirred under argon at -78°C. A solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl amine (2.61 g, 0.0119 mol), triethylamine (1.2 g, 0.0118 mol) and 4-dimethylamino pyridine (0.1 g) in dry THF (35 mL) were added dropwise during lh. The reaction was then allowed to warm to 0°C and stirred at this temperature for 4h and then diluted with 100 mL dichloromethane and washed with 2 x IN HCI. The organic phase was dried over magnesium sulfate, filtered, and evaporated. The residue was purified by column chromatography on silica gel (cyclohexan / ethylacetate = 1/2) to yield 3.6 g (72%) of the title compound.

HRMS: 421.0949 (M+H⁺), 443.0767 (M+Na⁺). *H NMR (300 MHz, CDCI₃)

5[ppm] 8.09- 7.9 (m, 4H), 7.13 (s, 1H), 3.82 - 3.58 (m, 14H), 3.35 (m, 2H).

¹³C NMR (75 MHz, CDCI₃)

6[ppm] 165.19, 146.11, 140.82, 128.57, 127.21, 70.60, 70.46, 70.23, 69.99, 69.45, 50.60,

40.12.

1.5. (S)-methyl 2-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyI)carbamoyI)phenyI- sulfonamido)-3-((tert-butoxycarbonyl)amino)propanoate

The mixture of (S)-methyl 2-amino-3-((tert-butoxycarbonyl)amino)propanoate (0.93 g, 0.0042 mol,l eq) and triethylamine (1 mL) in dichloromethane (15 mL) was added dropwise to a solution of 4-((2- (2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)benzene-l-sulfonyl chloride (1.8 g, 0.0042 mol, 1 eq) in CH₂CI₂ (15 mL) at 0°C. The reaction mixture was stirred at that temperature for 2h, and then for 5h by room temperature. Then, the reaction mixture was washed with water and brine. The organic layers were dried over MgS0₄ and concentrated to get a residue, which was purified by column chromatography on silica gel (in-house system, cyclohexane / ethyl acetate = 1:1 ) to yield the title compound (2.3 g, 93%).

HRMS: 625.2255 (M+Na⁺).

*H NMR (400 MHz, CDCI₃)

6[ppm] 7.98 - 7.84 (m, 4H), 5.75 (s, 1H), 4.99 (s, 1H), 4.02 (s, 1H), 3.78 - 3.61 (m, 14H), 3.59

(s, , 3H), 3.54 - 3.41 (m, 2H), 3.36 (t, 7=5.0, 2H), 1.95 (s, 1H), 1.54 - 1.34 (m, 9H). ¹³C NMR (101 MHz, CDCI₃)

6[ppm] 170.06 , 165.84, 156.12, 142.11, 138.72, 127.99, 127.32, 80.24, 70.65, 70.52, 70.26,

70.03, 69.54, 56.11, 53.04, 50.64, 43.27, 40.01, 28.25.

(S)-methyl 3-amino-2-(4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyI)carbamoyI)- Isulfonamidojpropanoate

HCI in dioxane (15 mL, 4M) was added to a solution of compound 5 (2.3 g, 0.0038 mol) in dioxane (4 mL) and the mixture was stirred overnight at room temperature. The solvents were removed under vacuum and the resulting solid was used in the next step without further purification (2 g, 98%).

HRMS: 503.1927 (M+H⁺), 525.1747(M+ Na⁺)

*H NMR (300 MHz, CD₃OD)

5[ppm] 8.04 - 7.93 (m, 4H), 4.32 (dd, J = 9.0, 4.9, 1H), 3.72 - 3.56 (m, 16H), 3.44 (s, 3H), 3.34

(dd, J = 8.9, 3.9, 3H), 3.11 (m, 1H).

1³C NMR (101 MHz, CD₃OD)

5[ppm] 169.47, 168.66, 143.97, 139.92, 129.22, 128.61, 71.63, 71.50, 71.34, 71.13, 70.44,

68.17, 55.02, 53.47, 51.79, 42.14, 41.19. 1.7 (S)-methyl 2-(4-({2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyI)carbamoyI)phenyI- suIfonamido)-3-(3-(4-(pyrimidin-2-ylamino)piperidin-l-yl)-5-(trifluoromethyl)- benzamido)propanoate

A mixture of 3-(4-(pyrimidin-2-ylamino)piperidin-l-yl)-5-(trifluoromethyl)benzoic acid, prepared as described in [Kobuta 2006] (1 g, 95.8% purity, 0.0027 mol), and W-methylmorpholine (0.6 mL, 0.0059 mol) were suspended in DMF (10 mL) and kept at 0 °C; (benzotriazol- 1- yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) (0.97 g, 0.0022 mol) was slowly added to the mixture while the temperature was maintained below 4 °C. After 30 min compound 6 (1.4 g, 0.0027 mol) was slowly added to the mixture at a rate that kept the temperature below 4 °C. The mixture was stirred for an additional 23h at room temperature. A saturated aqueous sodium hydrogen carbonate solution (30 mL) was added to the mixture, and 30 mL of chloroform were added. The organic layer was separated, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography using reversed phase C-18 silica gel (35-60 μιη, in-house system) to give 1.3 g (61%) of the product.

HRMS: 851.3166 (M+H⁺), 873.2964 (M+Na⁺)

*H NMR (600 MHz,CDCI₃)

5[ppm] 8.26 (d, J = 4.8, 2H), 7.87 - 7.78 (m, 4H), 7.47 (s, 1H), 7.36 (t, J = 6.0, 1H), 7.31 (s, 1H),

7.14 (s, 1H), 7.09 (t, J = 5.4, 1H), 6.50 (t, J = 4.8, 1H), 5.81 (d, J = 7.7, 1H), 4.20 (s, 1H), 4.01 - 3.94 (m, 1H), 3.83 - 3.73 (m, 2H), 3.68 (m, 2H), 3.66 - 3.56 (m, 16H), 3.55 (s, 3H), 3.32 - 3.28 (m, 2H), 3.02 - 2.92 (m, 2H), 2.08 (t, J = 8.6, 2H), 1.60 - 1.52 (m, 2H). ¹³C NMR (151 MHz, CDCI₃)

6[ppm] 170.10, 167.40, 165.86, 161.35, 158.02, 151.40, 142.26, 135.40, 131.59(q, J=32.0),

127.89, 127.10, 123.85(if, J=272.9), 117.56, 114.75, 113.17, 110.48, 70.55, 70.54, 70.44, 70.18, 69.91, 69.47, 55.68, 53.02, 50.57, 47.66, ,47.44, 42.48, 40.00, 31.37.

¹⁹F NMR (564 MHz, CDCI₃)

6[ppm] -62.68.

1.8 (S)-2-(4-((2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyI)carbamoyI)phenyIsulfonamido)-3-(3- (4-((l,4,5,6-tetrahydropyrimidin-2-yl)amino)piperidin-l-yl)-5-(trifIuoromethyI) benzamido ) propanoic acid (WA548)

A solution was prepared by adding methanol (20 mL) and 0.7 mL of concentrated hydrochloric acid to (0.8 g, 0.9 mmol) of compound 7. Pt0₂ (0.01 g) was added to the solution. The mixture was vigorously stirred at room temperature for 4h under hydrogen pressure of 15 atm. The insoluble Pt0₂ was collected by filtration followed by concentration under reduced pressure. A mixture of 5 mL of methanol and 5 mL of THF was added to the filtrate, followed by adding 0.1 g (0.004 mol) of lithium hydroxide and 7 mL of water to the final solution. The mixture was stirred at room temperature for 6h. The system was adjusted to pH 6 by addition of IN hydrochloric acid and was concentrated under reduced pressure. The residue was purified by preparative HPLC to yield the title compound. (0.2 g, 26% yield, purity 96.5%). HRMS: 815.33709 (M+H⁺)

*H NMR (600 MHz, DMSO-d₆)

δ [ppm] 8.64 (dt, J = 45.4, 5.4, 1H), 8.37 - 8.23 (m, 1H), 7.92 - 7.81 (m, 2H), 7.54 (s, 1H), 7.50 -

7.43 (m, 1H), 7.40 (s, 1H), 7.28 (s, 1H), 3.60 - 3.32 (m, 14H), 3.24 (m, 3H), 2.93 (m, 3H), 1.91 (m,lH), 1.86 - 1.78 (m, 2H).

1³C NMR (151 MHz, DMSO-d₆)

6[ppm] 171.37, 168.88, 166.07, 159.01, 158.77(q, J=35.5), 152.41, 151.17, 143.81, 137.98,

136.34, 128.22, 126.83, 117.81, 117.32, 115.38, 70.24, 70.17, 70.14, 70.07, 70.02, 69.64, 69.19, 67.10, 55.39, 52.79, 50.50, 47.78, 47.03, 41.97, 40.97, 39.12, 38.55, 31.28, 20.18.

1⁹F NMR (564 MHz, DMSO-d₆)

5[ppm] -61.20.

1.9 Cy 5.5-IabelIed tracer from WA548

The amino functionalized derivative 8 (« 1.0 mg, 1.5 μιηοΙ) was dissolved in 100 μΙ of DMSO and 400 μΙ of a bicarbonate buffer (0.1 M NaHC0₃, pH 8.6). To this was added a solution of Cy 5.5 NHS-ester (« 1.6 mg, 1.4 μιηοΙ) in 300 μΙ DMSO. The solution was stirred for lh at rt in the dark and stored at - 20°C. Purification was carried out by semipreparative HPLC, the appropriate product fractions were collected, evaporated and reconstituted in saline.

Example 2: Competition binding curves as analyzed for integrin affinity for selected precursor compounds. Selected precursor compounds exhibiting labeling moieties like an amino group attached to a PEG- spacer were tested for their integrin binding potencies using a radiopeptide-assay based on ¹²⁵I- echistatin. Plates (96 well) are coated with integrin a„l¾₃or aIIb(S₃, l^g/mL, ΙΟΟμΙ.) in coating buffer (Tris · HCI 25mM, NaCI 150mM, CaCI₂ ImM, MgCI₂ 500μΜ, MnCI₂ ImM) at 4°C overnight. Surfaces are blocked with 1% BSA in coating buffer for 2h at RT and washed twice with binding buffer (0.1% BSA, Tris · HCI 25mM, NaCI 150mM, CaCI₂ ImM, MgCI₂ 500μΜ, MnCI₂ ImM). The antagonist (lpM - ΙΟΟμΜ) and ¹²⁵l-echistatin (60pM) were added and incubation was maintained for 3h at RT. After washing twice with binding buffer, the remaining radioactivity was removed with hot 2N NaOH (200μί), transferred to counting tubes and determined with a S-counter (Wallac Wizard 3; Perkin Elmer Life Sciences). Determination of inhibition values was possible by non-linear regression analysis of the competition curves using GraphPad 4.0 software (Fig. 1).

The affinity values for selected precursor compounds (WA548, WA579, WA581) in comparison to the lead compound (WA436), a selected building block (WA493) and the previously established peptidic precursor cyc/oCRGDCGK were determined (von Wallbrunn A., Holtke C, Ziihlsdorf M., Heindel W., Schafers M., Bremer C. In vivo imaging of integrin alpha(v)beta3 expression using fluorescence mediated tomography. Eur J Nucl Med Mol Imaging. 2007;34(5):745-54) (Fig. 2).

The precursor molecules were tested to determine whether the binds to the integrin α_νβ₃. The data clearly show that the precursor molecules selective bind to integrin α_νβ₃.

Example 3: In vitro cell binding assay using Cy 5.5 labeled WA 548. Compound WA548 was labeled with Cy 5.5 and established in initial cell binding studies. Cells were seeded on slides in growth medium and incubated overnight. Medium was removed and cells were washed with PBS at RT and fixated. Blocking solution (0.1% BSA in PBS) was added and incubated for 15 min at RT. After removing blocking buffer and washing the cells with PBS twice, binding buffer and tracer (0.2nmol) were added and incubation was maintained for 4h at 4°C. After washing with PBS and counterstaining with DAPI cells could be visualized by fluorescence microscopy. In Fig.3 it is shown the in vitro cell binding assay using Cy 5.5 labeled WA 548 on a„ls₃-positive U-87 glioblastoma and a_vls₃-negative MCF-7 breast adenocarcinoma cells.

Claims

An vivo imaging agent of Formula (I):

a salt or solvate or tautomer thereof, wherein:

n and m are independently an integer from 1 to 4; preferably n is 2 and m is 1;

X is selected from -OH, -0-{Ci-C₆)alkyl, -NH₂, -NH-(Ci-Cs)alkyl, -SH, -S-(C C₆), alkyi; preferably X is -OH;

R_! is one of: -H, -(Ci-C₆)alkyl, -OH, -0-(Ci-C₆)alkyl, perfluoro(Ci-C₆)alkyl, -0-perfluoro(Ci- C₆)alkyl, -COOH, -COO-(d-C₆)alkyl, -CH₂OH, -CH₂0-(Ci-C₆) alkyi, -Br, -CI, -F, -I, -SH, -S-(d- C₆)alkyl, -NH₂, -NH-(d-C₆)alkyl; preferably, R_x is -CF₃; and

R₂ is one of:

in

p is an integer from 0 to 12; preferably p is an integer from 3 to 8 Y describes an in vivo imaging moiety

wherein q is an integer from 0 to 12, preferably q is an integer from 0 to 4; Z is -0-, -S- or -NH-, preferably Z = -NH-;

Y describes an in vivo imaging moiety and

R₂ is

2. The compound according to claim 1 wherein Y is an atom or to a group of atoms that may be detected externally of a subject's body following administration to said subject.

3. The compound according to claims 1 or 2 wherein Y is selected from:

(vii) a radioactive metal ion;

(viii) a paramagnetic metal ion;

(ix) a gamma emitting radioactive halogen;

(x) a positiron emitting radioactive non-metal ion

(xi) a reporter suitable for in vivo optical imaging.

4. The compound according to claim 1 or 2 or 3(i) wherein Y is a radioactive metal ion selected from ⁶⁴Cu, ⁴⁸V, ⁵²Fe, ⁵⁵Co, ^94mTc or ⁶⁸Ga or a y emitters atom selected from ^99mTc, ^mln, ^113mln, or ⁶⁷Ga.

5. The compound according to claim 1 or 2 or 3(ii) wherein Y is a paramagnetic metal ion selected from Gd(lll), Mn(ll), Cu(ll), Cr(lll), Fe(lll), Co(ll), Er(ll), Ni(ll), Eu(lll) or Dy(lll)

6. The compound according to claim 1 or 2 or 3(iii) wherein Y is a gamma-emitting radioactive halogen selected from ¹²³l, ¹³¹l or ⁿBr

7. The compound according to claim 1 or 2 or 3(iv) wherein Y is a positron-emitting radioactive non-metal selected from C, ¹³N, ¹⁵0, ¹⁷F, ¹⁸F, ⁷⁵Br, ⁷⁶Br or ¹²⁴l.

8. The compound according to claim 1 or 2 or 3(v) wherein Y is a reporter for in vivo optical imaging selected from cyanines, merocyanines, indocyanines, phthalocyanines, naphthalocyanines, triphenylmethines, porphyrins, pyrilium dyes, thiapyriliumdyes, squarylium dyes, croconium dyes, azulenium dyes, indoanilines, benzophenoxazinium dyes, benzothiaphenothiazimum dyes, anthraquinones, napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, intramolecular and intermolecular charge- transfer dyes and dye complexes, tropones, tetrazines, £>/s(dithiolene) complexes, / /s(benzene-dithiolate) complexes, iodoaniline dyes, Ws(S,0-dithioIene) complexes, green fluorescent protein (GFP) and modifications thereof having a have different absorption/emission properties are also useful, complexes of europium, samarium, terbium or dysprosium, fluorescein, sulforhodamine 101 (Texas Red), rhodamine B, rhodamine 6G, rhodamine 19, indocyanine green, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Marina Blue, Pacific Blue, Oregon Green 88, Oregon Green 514, tetramethylrhodamine, and Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750.

9. The compound of any of the preceding claims wherein the piperidine ring is attached to the phenyl ring in position 3 or 4.

10. The compound of any of the preceding claims wherein the sterochemistry at the amino acid residue as indicated in above formula (I) is either R or S.

11. The compound of any of the preceding claims wherein n is 2 and m is 1 and

X is -OH.

12. A precursor compound of Formula II:

wherein R_l7 R_2, X, n and m are as defined in any of the preceding claims and wherein Y of Ri or of R₂ is selected from -NH₂, -SH, -OH, or Y is one precursor group selected from one or more ligands capable of complexing a metallic imaging moiety;

(ii) an organometallic derivative such as a trialkylstannane or a trialkylsilane;

(iii) a derivative containing an alkyi halide, alkyi tosylate or alkyi mesylate for nucleophilic substitution;

(iv) a derivative containing an aromatic ring activated towards nucleophilic or electrophilic substitution;

(v) a derivative containing a functional group susceptible to acylation;

(vi) a derivative containing a functional group that takes part in oxime formation when reacted with a benzaldehyde;

(vii) a derivative containing a vinylsulfone functional group;

(viii) a derivative containing a functional group which undergoes facile alkylation; or,

(ix) a derivative which alkylates thiol-containing compounds to give a thioether- containing product. The compounds of claim 12 selected from

Compound according to claims 1 to 11 for use in the diagnosis or follow up/monitoring of a ct_vl¾3 integrin condition.

Compound for use according to claim 14 wherein the α_νβ₃ integrin condition is selected from inflammation, cancer and fibrosis, atherosclerosis and coronary artery diseases, stroke and cerebral degenerative diseases.