EP3490614A1

EP3490614A1 - Radiopaque compound containing diiodotyrosine

Info

Publication number: EP3490614A1
Application number: EP17748889.7A
Authority: EP
Inventors: Ngee Han LIM; Milan Mrazek FOWKES
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2016-07-28
Filing date: 2017-07-27
Publication date: 2019-06-05
Also published as: GB201613035D0; WO2018020262A1

Abstract

The present invention relates to a radiopaque compound or salt thereof comprising diiodotyrosine as defined herein. Further aspects of the invention include a conjugate comprising said radiopaque compounds and a chelating agent, a contrast agent comprising said radiopaque compounds or salts thereof and methods of X-ray imaging.

Description

RADIOPAQUE COMPOUND CONTAINING DIIODOTYROSINE

INTRODUCTION

The present invention relates to radiopaque compounds and contrast agents comprising said compounds which have particular application to X-ray imaging. BACKGROUND OF THE INVENTION

Contrast agents for medical imaging are important tools in the diagnosis and staging of many diseases, including cancer¹ , Alzheimer's² and Parkinson's³, amongst others. Major imaging modalities such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI) and the X-ray based computed tomography (CT) all utilize contrast agents and are based on a variety of designs ranging from small molecules^{4 6} to peptides^{7 9} and engineered proteins^{10 12}.

PET and SPECT radiotracers are typically targeted, as they are often developed to bind to a specific molecule of interest such as an enzyme¹³ or receptor¹⁴. Conversely, MRI contrast media tend to be non-specific and based on metal complexes of Fe¹⁵ or Gd¹⁶. This lack of specificity is also found with X-ray contrast agents^{17, 18}.

Due to the poor sensitivity of X-ray imaging, a high local concentration of final contrast agent is required to image soft tissues. Accordingly, targeted imaging approaches using antibodies or enzymes, such as those used in PET and SPECT, are not feasible as in order to deliver the minimally detectable levels of iodine (2mg l/ml) by CT imaging a 150 kDa antibody, 30 kDa single chain fragment variable or 6 kDa affibody labeled with a single iodine would have to be delivered at unviable doses of 2.4 g /ml, 472 mg /ml or 94.5 mg /ml respectively.

At present, commercially available small-molecule X-ray imaging agents tend to suffer from rapid renal clearance¹⁹, a trait that narrows the available time window for effective imaging. Furthermore, these agents are non-specific binders. Development of a radiopaque compound/contrast agent specific to biological targets (e.g. akin to that achievable by antibodies) would allow disease states to be imaged by X-ray.

There is a need in the art for a contrast agent and/or radiopaque compounds which benefit from one more of the following properties (i) suitable for X-ray imaging, (ii) biocompatible, (iii) can easily be adapted to target specific tissues, (iv) can be administered at practical doses and/or (v) have suitable solubility and/or half-life/stability to enable X-ray imaging. At least one or more of the above problems is solved by embodiments of the present invention.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a radiopaque compound or salt thereof as defined herein. In a second aspect, the present invention provides a conjugate comprising a radiopaque compound as defined herein and a chelating agent.

In a third aspect, the present invention provides a contrast agent comprising a radiopaque compound or salt thereof as defined herein.

In a fourth aspect, the present invention relates to the use of a radiopaque compound or salt thereof as defined herein in the preparation of a contrast agent.

In a fifth aspect, the present invention relates to the use a radiopaque compound or salt thereof as defined herein, or a conjugate as defined herein, or a contrast agent as defined herein in X-ray imaging.

In a sixth aspect, the present invention provides a method of X-ray imaging tissue in a human or non-human animal subject comprising administering to said subject a radiopaque compound or salt thereof as defined herein, a conjugate as defined herein or contrast agent as defined herein.

BRIEF DESCRIPTION OF THE DRA WINGS

Figure 1 depicts the conversion of μΟΤ scans to colour-coded maps of X-ray absorption. Figure 2A shows representative μΟΤ coronal views of mouse tibiae after 1 h and 24 h in saline and corresponding colour-coded maps of absorption of the lateral aspect of the plateau for pure peptides binding to freshly dissected tibiae.

Figure 2B shows a plot of the percentage decrease in contrast (mean ± SD, n=3) over time upon washing in saline used to determine the half-life (ti/₂) of pure peptides. Figure 3 shows representative μΟΤ coronal views of mouse knee joints and correspondent colour-coded maps of absorption of the lateral aspect of the joint upon intra-articular injection of Ύ^'-Aeea-WYKGKL peptide into the synovial space in vivo. DETAILED DESCRIPTION OF THE INVENTION

Amino acids and their residues are denoted herein using single letter amino acid notation. Accordingly, in the definition of compounds of the invention where single letters are used to denote structural features of the compounds these refer to the commonly accepted amino acid/residue unless specifically defined otherwise. All amino acid sequences are represented herein by formulae with left to right orientation in the conventional direction of N- terminal to C-terminal unless otherwise specified.

All optical isomers of the compounds of the invention are within the scope of the invention. In one embodiment, where the compounds comprise amino acids, each amino acid has its natural stereochemistry. In one embodiment, where the compounds comprises amino acids they have L stereochemistry.

In a first aspect the present invention provides a radiopaque compound or salt thereof comprising diiodotyrosine.

As used herein, diiodotyrosine (also abbreviated to DIT) refers to the amino acid tyrosine or its residue which is substituted on the phenyl ring with two ¹²⁷iodine atoms.

As used herein, ¹²⁷iodine (I) refers to the most naturally abundant isotope of iodine.

In one embodiment, the diiodotyrosine is 3,5-diiodotyrosine (A) or its residue (B) which are depicted below (wherein ~nsw indicates the points of attachment to adjacent residues/structures):

Ον ΑΛ

B 3,5-diiodotyrosine or its residue are denoted herein in single letter amino acid notation as Ύ' as distinct from tyrosine which is commonly denoted as Y in single letter amino acid notation.

In one embodiment, the radiopaque compounds of the invention comprise at least one diiodotyrosine. For example, 1 , 2, 3, 4, or 5 diiodotyrosine. Suitably, 1 , 2, or 3 diiodotyrosine. In another embodiment, the radiopaque compounds of the invention comprise two or more diiodotyrosine. For instance, 2, 3, 4 or 5 diiodotyrosine. Suitably, 2 or 3 diiodotyrosine.

As used herein the term radiopaque refers to the ability of said compounds to block the passage of X-rays. In the context of the present invention, a compound can be said to be radiopaque if on X-ray imaging the compound is visualized as a light or white area on exposed film (similar to bone). In contrast, if exposed film remains dark on X-ray imaging then the compound is not considered radiopaque (similar to soft tissue when visualize in the absence of contrast agent).

The degree to which a compound is radiopaque can be quantified according to Hounsfield scale. The Hounsfield unit (HU) scale is a linear transformation of the original linear attenuation coefficient measurement into one in which the radiodensity of distilled water at standard pressure and temperature (STP) is defined as zero Hounsfield units (HU), while the radiodensity of air at STP is defined as -1000 HU. In a voxel with average linear attenuation coefficient μ, the corresponding HU value is therefore given by: HU = 1000 X (μ - Mwater/Mwater - Mair) where μ_{νΐ3 βί} and are respectively the linear coefficients of water and air.

In one embodiment, a compound is radiopaque if it has a HU value of greater than or equal to about 400, suitably a HU value of greater than or equal to about 500, suitably greater than or equal to about 700, suitably a HU value of greater than or equal to about 1000, suitably a HU value of greater than or equal to about 1300, suitably a HU value of greater than or equal to about 1 500, suitably a HU value of greater than or equal to about 1 800, suitably a HU value of greater than or equal to about 2000.

In another embodiment, a compound is radiopaque if it has a HU value of from about 500 to about 4000, suitably a HU value of from about 500 to about 3000, suitably a HU value of greater than or equal to about 500 to about 2000, suitably a HU value of greater than or equal to about 500 to about 1500, suitably a HU value of greater than or equal to about 500 to about 1250, suitably a HU value of greater than or equal to about 500 to about 1000.

In another embodiment, a compound is radiopaque if it has a HU value of from about 600 to about 4000, suitably a HU value of from about 600 to about 3000, suitably a HU value of greater than or equal to about 600 to about 2000, suitably a HU value of greater than or equal to about 600 to about 1500, suitably a HU value of greater than or equal to about 600 to about 1250, suitably a HU value of greater than or equal to about 600 to about 1000.

In another embodiment, a compound is radiopaque if it has a HU value of from about 700 to about 4000, suitably a HU value of from about 700 to about 3000, suitably a HU value of greater than or equal to about 700 to about 2000, suitably a HU value of greater than or equal to about 700 to about 1500, suitably a HU value of greater than or equal to about 700 to about 1250, suitably a HU value of greater than or equal to about 700 to about 1000.

In one embodiment, the HU value of a compound can be measured using a CT scanner (e.g. Quantum FX, Perkin Elmer, USA) at a spatial resolution of 10 Mm/pixel (200 μΑ, 90 kV, 3 minutes of acquisition time) calibrated with reference to water at room temperature and pressure (e.g. 21 °C and 101 KPa).

In one embodiment, the radiopaque compound comprises/essentially consists of/consists of a peptide comprising at least one diiodotyrosine.

In one embodiment, the compound has a molecular weight of 5 kDa or less, suitably 4 kDa or less, suitably 3 kDa or less, 2 kDa or less, suitably 1 .5 kDa or less, more suitably 1 kDa or less.

In one embodiment, the compound has a molecular weight of about 433 to about 5000 Da. Suitably, the compound has a molecular weight of about 433 to about 4000 Da, suitably about 433 to about 3000 Da, suitably about 433 to about 2000 Da, suitably about 433 to about 1500 Da, suitably about 433 to about 1200 Da.

In one embodiment, the compound has a molecular weight of about 800 to about 5000 Da. Suitably, the compound has a molecular weight of about 800 to about 4000 Da, suitably about 800 to about 3000 Da, suitably about 800 to about 2000 Da, suitably about 800 to about 1500 Da, suitably about 800 to about 1200 Da. In one embodiment, the compound has a molecular weight of about 1500 to about 5000 Da. Suitably, the compound has a molecular weight of about 1500 to about 4000 Da, suitably about 1500 to about 3000 Da, suitably about 1500 to about 2000 Da. In one embodiment, the compound has a molecular weight of about 1000 to about 5000 Da. Suitably, the compound has a molecular weight of about 1000 to about 4000 Da, suitably about 1000 to about 3000 Da, suitably about 1000 to about 2000 Da, suitably about 1000 to about 1500 Da, suitably about 1000 to about 1200 Da. In one embodiment, the compound comprises/essentially consists of/consists of a chain of five or more amino acids including at least one diiodotyrosine, suitably 6 or more, 7 or more, 8 or more, 9 or more, 10 or more amino acid s including at least one diiodotyrosine. In another embodiment, the compound is polymeric in nature.

In one embodiment, the compound comprises/essentially consists of/consists of a chain of 6 to 20 amino acids, suitably 6 to 15, suitably 6 to 12, suitably 6 to 10 amino acids.

In one embodiment, at least a portion of the compound binds to a biological target, thus allowing said target to be imaged by X-ray based techniques.

Examples of biological targets include cartilage, diseased tissue (such as cancerous tissue, fibrotic tissue) liver, kidney, heart, brain. In one embodiment, the biological target is diseased tissue, such as cancerous or fibrotic tissue. In another embodiment, the biological target is cartilage.

In one embodiment, at least one diiodotyrosine is part of the portion of the compound/peptide which binds to the biological target.

In another embodiment, at least one diiodotyrosine is not part of the portion of the compound/peptide which binds to the biological target.

In one embodiment the compound/peptide comprises a group X¹ , wherein X¹ is a diiodotyrosine ('Y')-containing peptide of molecular formula (K)_r(E)_s('Y')t; wherein r and s are numbers independently selected from 0 to 5, and t is a number selected from 1 to 5. It will be understood by the skilled person that in this embodiment the molecular formula (K)_r(E)_s('Y')_t merely indicates the ratio of constituent amino acid residues and does not specify any particular sequence.

In one embodiment, s is 0.

In another embodiment, r is 0.

In another embodiment, t is 1 , 2 or 3. In another embodiment, r and t are independently selected from 1 , 2 or 3, and s is 0. In another embodiment, s and t are independently selected from 1 , 2 or 3, and r is 0. In one embodiment, the sum of r and s is greater than or equal to t.

In another embodiment the compound/peptide comprises a group X¹ , wherein X¹ is a diiodotyrosine ('Y')-containing peptide selected from (K'Y')_m and (E'Y')_m; wherein m is a number selected from 1 to 5. Suitably, m is a number selected from 1 , 2 or 3.

In one embodiment, X¹ is (K'Y')_m and m is a number selected from 1 , 2 and 3.

In one embodiment, the compound/peptide comprises a peptide of formula (lla):

WYRGRL (lla) wherein optionally at least one R residue is conservatively substituted. In another embodiment, the compound/peptide comprises/essentially consists of/consists of a peptide of formula (lib):

W'Y'RGRL (lib) wherein optionally at least one R residue is conservatively substituted. In one embodiment, the compound is of general formula (I): X²-(Nnker)n-J (|) wherein

X² is a diiodotyrosine (T) containing peptide selected from (K)_r(E)_s('Y')_t, ('Y')_q, (K'Y')_m and (E'Y')_m; wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5 m and q are independently numbers selected from 1 to 5; n is 0 or 1 ; and

J is a peptide capable of binding to a biological target. In one embodiment, X² is a peptide of molecular formula (K)_r(E)_s('Y')_t. In one embodiment, s is 0. In another embodiment, r is 0. In another embodiment, t is 1 , 2 or 3.

In another embodiment, r and t are independently selected from 1 , 2 or 3, and s is 0.

In another embodiment, s and t are independently selected from 1 , 2 or 3, and r is 0. In one embodiment, the sum of r and s is greater than or equal to t.

In another embodiment, X² is selected from ('Y')_q, (K'Y')_m and (E'Y')_m; suitably ('Y')_q or (K'Y')_m.

In one embodiment, m and q are independently a number selected from 1 , 2 and 3.

In another embodiment, n is 0. In another embodiment, n is 1 .

In one embodiment, the linker may be a peptide. In another embodiment, the linker may be non-peptidic

In another embodiment, the linker is selected from peptides, aminoethylethanolamine (Aeea), PEG-based linkers, and 4-aminobutyric acid.

In another embodiment, the linker is selected from aminoethylethanolamine, PEG-based linkers, and 4-aminobutyric acid. In another embodiment, the linker is aminoethylethanolamine.

In another embodiment, X² is (K'Y')_m, m is a number selected from 1 , 2 and 3, n is 1 and the linker is aminoethylethanolamine.

In another embodiment, X² is ('Y')_q, q is a number selected from 1 , 2 and 3, n is 1 and the linker is aminoethylethanolamine. In another embodiment, X² is (K'Y')_m, m is a number selected from 1 , 2 and 3, and n is 0.

In another embodiment, X² is (E'Y')_m, m is a number selected from 1 , 2 and 3, and n is 0.

In another embodiment, J is a peptide comprising/essentially consisting of/consisting of formula (lla):

WYRGRL (lla) wherein optionally at least one R residue is conservatively substituted. As used herein, conservatively substituted refers to substituting an amino acid belonging to a grouping of amino acids having a particular characteristics for another amino acid belonging to the same group. For example, amino acids may be grouped based on the following characteristics: Nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.

The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.

The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Such alterations are not expected to substantially affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. Exemplary conservative substitutions include, but are not limited to, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free - OH is maintained; and Gin for Asn to maintain a free NH₂.

In one embodiment at least one R in formula ( I la) is conservatively substituted with K or H, suitably K.

In another embodiment, each R in formula ( I la) is conservatively substituted with K or H, suitably K. In another embodiment, J consists of the formula (I la) wherein each R is conservatively substituted with K.

In another embodiment, J is a peptide comprising/essentially consisting of/consisting of formula (lib):

W'Y'RGRL (lib) wherein optionally at least one R residue is conservatively substituted.

In one embodiment at least one R in formula (lib) is conservatively substituted with K or H, suitably K. In another embodiment, each R in formula (lib) is conservatively substituted with K or H, suitably K.

In another embodiment, J consists of the formula (lib) wherein each R is conservatively substituted with K. In one embodiment, the compound is of general formula (III):

J-(linker)n-X³ (III) wherein

X³ is a diiodotyrosine (Ύ') containing peptide selected from (K)_r(E)_s('Y')_t, ('Y')_q, ('Y'G)_m, wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5 m and q are independently a number selected from 1 to 5; n is 0 or 1 ; and J is a peptide capable of binding to a biological target.

In one embodiment, X³ is a peptide of molecular formula (K)_r(E)_s('Y')_t.

In one embodiment, s is 0. In another embodiment, r is 0. In another embodiment, t is 1 , 2 or 3.

In another embodiment, r and t are independently selected from 1 , 2 or 3, and s is 0. In another embodiment, s and t are independently selected from 1 , 2 or 3, and r is 0. In one embodiment, the sum of r and s is greater than or equal to t.

In another embodiment, X³ is selected from ('Y')_q, ('Y'G)_m (K'Y')_m and (E'Y')_m; suitably (Y')_q, ('Y'G)_m and (K'Y')_m.

In one embodiment, m and q are independently a number selected from 1 , 2 and 3. In another embodiment, n is 0. In another embodiment, n is 1 . In one embodiment, the linker may be a peptide.

In another embodiment, the linker may be non-peptidic

In another embodiment, the linker is selected from peptides, aminoethylethanolamine, PEG- based linkers, and 4-aminobutyric acid. In another embodiment, the linker is selected from aminoethylethanolamine, PEG-based linkers, and 4-aminobutyric acid.

In another embodiment, the linker is aminoethylethanolamine.

In another embodiment, the linker is a peptide of structure aminoethylethanolamine.

In another embodiment, X³ is ('Y'G)_m, m and n are 1 , and the linker is aminoethylethanolamine. In another embodiment, X³ is ('Y'K)_m, m is a number selected from 1 , 2 and 3, n is 1 and the linker is aminoethylethanolamine.

In another embodiment, X³ is ('Y')_q, q is a number selected from 1 , 2 and 3, n is 1 and the linker is aminoethylethanolamine.

In another embodiment, X³ is ('Y'G)_m, m is 1 , and n is 0. In another embodiment, X³ is ('Y'K)_m, m is a number selected from 1 , 2 and 3, and n is 0.

In another embodiment, X³ is ('Y')_m, m is a number selected from 1 , 2 and 3, and n is 0.

In another embodiment, J is a peptide comprising/essentially consisting of/consisting of formula (II):

WYRGRL (Ma) wherein optionally at least one R residue is conservatively substituted.

In another embodiment, each R in formula ( I la) is conservatively substituted with K or H, suitably K. In another embodiment, J consists of the formula (I la) wherein each R is conservatively substituted with K. In another embodiment, J is a peptide comprising/essentially consisting of/consisting of formula (lib):

W'Y'RGRL (lib) wherein optionally at least one R residue is conservatively substituted. In one embodiment at least one R in formula (lib) is conservatively substituted with K or H, suitably K.

In another embodiment, each R in formula (lib) is conservatively substituted with K or H, suitably K.

In another embodiment, J consists of the formula (lib) wherein each R is conservatively substituted with K.

In one embodiment, the compound is of general formula (IV):

X^4a-linker-J-linker-X^4b (IV) wherein

X^4a is a diiodotyrosine ( ) containing peptide selected from (K)_r(E)_s('Y')_t, ('Y')_q, (K'Y')_m and (E'Y')_m;

X^4b is a diiodotyrosine (Ύ') containing peptide selected from (K)_r(E)_s('Y')_t, ('Y')q, ('Y'G)_m, ('Y'K)m and ('Y'E)_m; wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5 m and q are independently a number selected from 1 to 5; and

J is a peptide capable of binding to a biological target.

In one embodiment, X^4a is a peptide of molecular formula (K)_r(E)_s('Y')_t.

In one embodiment, X^4b is a peptide of molecular formula (K)_r(E)_s('Y')_t. In one embodiment, X^4a and X^4b are independently selected from a peptide of molecular formula (K)_r(E)_s('Y'),. In one embodiment, s is 0. In another embodiment, r is 0. In another embodiment, t is 1 , 2 or 3.

In another embodiment, X^4a is selected from ('Y')_q, (K'Y')_m and (E'Y')_m; suitably ('Y')_q or (K'Y')_m.

In another embodiment, X^4b is selected from ('Y')_q, ('Y'G)_m (K'Y')_m and (E'Y')_m; suitably ('Y')_q, ('Y'G)_m and (K'Y')_m.

In another embodiment, X^4a and X^4b are independently selected from ('Y')_q, (K'Y')_m and (E'Y')_m; suitably ('Y')_q or (K'Y')_m.

In one embodiment, the linker may be a peptide.

In another embodiment, the linker may be non-peptidic

In another embodiment, the linker is selected from peptides, aminoethylethanolamine, PEG- based linkers, and 4-aminobutyric acid.

In another embodiment, the linker is selected from aminoethylethanolamine, PEG-based linkers, and 4-aminobutyric acid.

In another embodiment, the linker is aminoethylethanolamine.

In another embodiment, the linker is a peptide of structure aminoethylethanolamine. In another embodiment, X^4a is (K'Y')_m, X^4b is ('Y'G)_m, m is a number selected from 1 , 2 and 3, n is 1 and the linker is aminoethylethanolamine.

In another embodiment, X^4a is ('Y')_m, X^4b is ('Y'G)_m, m and n are 1 , and the linker is aminoethylethanolamine.

In another embodiment, X^4a is ('Y')_m, X^4b is ('Y'K)_m, m is a number selected from 1 , 2 and 3, n is 1 and the linker is aminoethylethanolamine. In another embodiment, X^4a is ('Y')_m, X^4b is ('Y')_q, q is a number selected from 1 , 2 and 3, n is 1 and the linker is aminoethylethanolamine.

In another embodiment, J is a peptide comprising/essentially consisting of/consisting of formula (II): WYRGRL (Ma) wherein optionally at least one R residue is conservatively substituted.

In another embodiment, each R in formula ( I la) is conservatively substituted with K or H, suitably K.

In another embodiment, J consists of the formula (I la) wherein each R is conservatively substituted with K.

In another embodiment, J is a peptide comprising/essentially consisting of/consisting of formula (lib): W'Y'RGRL (lib) wherein optionally at least one R residue is conservatively substituted.

In one embodiment at least one R in formula (lib) is conservatively substituted with K or H, suitably K.

In one embodiment, the radiopaque compound is selected from:

W^'Y^'RGRL

^'Y^'W^'Y^'RGRL

W^'Y^'KGKL ^'Y^'W^'Y^'KGKL

'Y^' Y^'W^'Y^'KGKL

'Y^"Y^"Y^'W^'Y^'KGKL

'Y^"Y^"Y^"Y^'W^'Y^'KGKL

Ύ^'-Aeea-WYKGKL

Ύ^'-Aeea-W^'Y^'KGKL

Ύ^'Ύ^'-Aeea-W^'Y^'KGKL

Ύ^'Ύ^'-Aeea-WYKGKL

Ύ Ύ'Ύ^'-Aeea-WYKGKL

WYKGKL-Aeea-^'Y^'G

W^'Y^' KG KL-Aeea- TG

Ύ^'-Aeea-W YKG KL-Aeea- TG

K^'Y^'KW^'Y^'KGKL

K^'Y^'K^'Y^'W^'Y^'KGKL

ΚΎ^'ΚΎ^'-Aeea-WYKGKL

K^'Y^'-Aeea-W^'Y^'KGKL

in linear or cyclic form.

In one embodiment, the radiopaque compound is selected from:

W^'Y^'RGRL

^'Y^'W^'Y^'RGRL

W^'Y^'KGKL

^'Y^'W^'Y^'KGKL

^'Y^'T^'W^'Y^'KGKL

^'Y^"Y^"Y^'W^'Y^'KGKL

^'Y^"Y^"Y^"Y^'W^'Y^'KGKL

Ύ^'-Aeea-WYKGKL

Ύ^'-Aeea-W^'Y^'KGKL Ύ^'Ύ^'-Aeea-W^'Y^'KGKL

Ύ^'Ύ^'-Aeea-WYKGKL

Ύ^'Ύ^'Ύ^'-Aeea-WYKGKL

WYKGKL-Aeea-^'Y^'G

W^'Y^'KGKL-Aeea-^'Y^'G

Y^'-Aeea-WYKGKL-Aeea-^'Y^'G

K^'Y^'KW^'Y^'KGKL

K^'Y^'K^'Y^'W^'Y^'KGKL

K^'Y^'K^'Y^'-Aeea-WYKGKL

in linear or cyclic form.

In another embodiment, the radiopaque compound is selected from:

W^'Y^'KGKL

'Y^'W^'Y^'KGKL

'Y^"Y^'W^'Y^'KGKL

'Y^"Y^"Y^'W^'Y^'KGKL

Y^'-Aeea-WYKGKL

Ύ^'-Aeea-W^'Y^'KGKL

Y^"Y^'-Aeea-W^'Y^'KGKL

Ύ ^' Ύ^'-Aeea-W YKG KL

Y^"Y^"Y^'-Aeea-WYKGKL

WYKGKL-Aeea-^'Y^'G

W^'Y^'KGKL-Aeea-^'Y^'G

K^'Y^'KW^'Y^'KGKL

K^'Y^'K^'Y^'W^'Y^'KGKL

in linear or cyclic form.

In another embodiment, the radiopaque compound is selected from W^'Y^'KGKL Ύ^'-Aeea-WYKGKL

WYKGKL-Aeea-Ύ

K^'Y^'KW^'Y^'KGKL

K^'Y^'K^'Y^'W^'Y^'KGKL

ΚΎ^'ΚΎ^'-Aeea-WYKGKL

in linear or cyclic form.

In another embodiment, the radiopaque compound is selected from:

W^'Y^'KGKL

Ύ^'-Aeea-WYKGKL

K^'Y^'KW^'Y^'KGKL

K^'Y^'K^'Y^'W^'Y^'KGKL

in linear or cyclic form.

In one embodiment, the radiopaque compounds as defined above are in linear form. In another embodiment, the radiopaque compounds as defined above are in cyclic form, e.g. cyclised N-terminal to C-terminal (head-to-tail).

The radiopaque compounds of the invention may be in salt form. Suitable salt forms include acetate and hydrochloride salts.

In another aspect, the present invention relates to a conjugate comprising a radiopaque compound as defined herein and a chelating agent.

Suitable chelating agents may be selected from 1 ,4,7-triazacyclononane-triacetic acid (NOTA), 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA), suitably DOTA.

In one embodiment, the chelating agent is DOTA and the conjugate comprises up to four (e.g. 1 , 2, 3 or 4) compounds of the invention in conjugation with the DOTA.

Thus, in one embodiment, the conjugate is of formula (V) wherein U is a radiopaque compound of the invention:

(V)

In one embodiment U is selected from any of compounds defined herein.

In one embodiment, U is bonded to the chelating agent via the N-terminal residue. In another embodiment, U is selected from:

W^'Y^'RGRL

'Y^'W^'Y^'RGRL

W^'Y^'KGKL

'Y^'W^'Y^'KGKL

Ύ ^"Y^'W^'Y^'KGKL

Ύ^" Y^{' '} Y^'W Ύ^' KG KL

Y^"Y^"Y^"Y^'W^'Y^'KGKL

Ύ^'-Aeea-WYKGKL

Ύ^'-Aeea-W^'Y^'KGKL

Ύ^'Ύ^'-Aeea-W^'Y^'KGKL

Ύ^'Ύ^'-Aeea-WYKGKL

Ύ^'Ύ^'Ύ^'-Aeea-WYKGKL

WYKGKL-Aeea-^'Y^'G

W^'Y^'KGKL-Aeea-^'Y^'G

Ύ^'-Aeea-W YKG KL-Aeea- Ύ K^'Y^'KW^'Y^'KGKL

K^'Y^'K^'Y^'W^'Y^'KGKL

ΚΎ^'ΚΎ^'-Aeea-WYKGKL

ΚΎ^'- Aeea-W ^' Y^' KG KL

in linear or cyclic form, suitably linear.

In another embodiment, U is ^'Y^'W^'Y^'KGKL in linear or cyclic form, suitably linear.

In another aspect, the present invention provides a contrast agent comprising a radiopaque compound as defined any of the above embodiments. Suitably, the contrast agent will further comprise a physiologically acceptable carrier or excipient. Conventional carriers and excipients would be known to the skilled person.

The contrast agents of the invention can be formulated to be compatible with a particular route of administration or use. Contrast agents for parenteral administration can include a sterile diluent, such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. The preparation may contain one or more preservatives to prevent microorganism growth (e.g., antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity (such as sodium chloride or dextrose). Contrast agents for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, or by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Including an agent that delays absorption, for example, aluminum monostearate and gelatin can prolonged absorption of injectable compositions.

In one embodiment, the concentration of iodine in the contrast agent is greater than 5 mg/ml. Suitably greater than 10 mg/ml, greater than 20 mg/ml greater than 50 mg/ml, greater than 100 mg/ml, greater than 150 mg/ml, greater than 200 mg/ml, greater than 250 mg/ml, or greater than 300 mg/ml.

In another embodiment, the concentration of iodine in the contrast agent is between 5 mg/ml and 500 mg/ml, or between 5 mg/ml and 350 mg/ml, or between 5 mg/ml and 250 mg/ml, or between 5 mg/ml and 150 mg/ml, or between 5 mg/ml and 100 mg/ml.

In another embodiment, the concentration of iodine in the contrast agent is between 10 mg/ml and 500 mg/ml, or between 10 mg/ml and 350 mg/ml, or between 10 mg/ml and 250 mg/ml, or between 10 mg/ml and 150 mg/ml, or between 10 mg/ml and 100 mg/ml.

In another aspect, the present invention provides a method of X-ray imaging tissue in a human or non-human animal subject comprising administering to said subject a radiopaque compound as defined herein above, a conjugate as defined herein, or contrast agent as defined herein above.

The method of the invention may further comprise imaging said subject with an X-ray device, for instance, in a CT scanner. In one embodiment, the radiopaque compound, conjugate or contrast agent is administered prior to imaging with the X-ray device. In another embodiment, the radiopaque compound, conjugate or contrast agent is administered concurrently with imaging with the X-ray device.

In one embodiment, the radiopaque compound, conjugate or contrast agent is administered by injection or infusion, e.g. intraarticularly or intravascularly. In another embodiment, the radiopaque compound, conjugate or contrast agent is administered orally. For oral administration, the compound, conjugate or contrast agent may take the form of a capsule, tablet or as a liquid.

In one embodiment, the subject is a human subject.

In one embodiment, the subject is a non-human animal, suitably a non-human mammal, suitably livestock (such as sheep, cattle, goats or horses) or a companion mammal (such as a cat or dog).

In one embodiment, the tissue imaged is soft tissue. For instance, diseased tissue (such as cancerous or fibrotic tissue), cartilage, liver, kidney, heart, brain. In one embodiment, the tissue imaged is cartilage. In another embodiment, the tissue is diseased tissue (such as cancerous or fibrotic tissue). The subject matter of the invention will now be further described by means of the following numbered paragraphs:

1 . A radiopaque compound or salt thereof comprising diiodotyrosine.

2. A radiopaque compound or salt thereof according to paragraph 1 wherein the diiodotyrosine is 3,5-diiodotyrosine.

3. A radiopaque compound or salt thereof according to any preceding paragraph wherein the diiodotyrosine is of formula (B), wherein indicates the points of attachment:

Ο ΛΛΛ

(B)

A radiopaque compound or salt thereof according to any one of paragraphs 1 to 3 wherein said compound comprises a peptide comprising at least one diiodotyrosine.

A radiopaque compound or salt thereof according to any preceding paragraph wherein at least a portion of the compound binds to a biological target.

A radiopaque compound or salt thereof according to any preceding paragraph wherein the compound comprises a group X¹ , wherein X¹ is a diiodotyrosine (Ύ') containing peptide selected from a peptide of molecular formula (K)_r(E)_s('Y')_t, wherein r and s are numbers independently selected from 0 to 5, and t is a number selected from 1 to 5; or (K'Y')m and (E'Y')_m, wherein m is a number selected from 1 to 5.

A radiopaque compound or salt thereof according to any preceding paragraph wherein the compound comprises a peptide of formula (I la) :

WYRGRL (Ma) wherein at least one R residue is conservatively substituted.

A radiopaque compound or salt thereof according to any preceding paragraph wherein the compound comprises a peptide of formula (lib)

A radiopaque compound or salt thereof according to any one of paragraphs 1 to 5 of general formula (I):

X²-(linker)_n-J (I) wherein

X² is a diiodotyrosine (T) containing peptide selected from (K)_r(E)_s('Y')_t, (Ύ , (K'Y')_m and (E'Y')_m; wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5 m and q are independently numbers selected from 1 to 5; n is 0 or 1 ; and

J is a peptide capable of binding to a biological target. A radiopaque compound or salt thereof according to any one of paragraphs 1 to 5 of general formula (III):

J-(linker)n-X³ (III) wherein

X³ is a diiodotyrosine (Ύ') containing peptide selected from (K)_r(E)_s('Y')_t, ('Y')_q, ('Y'G)_m, ('Y'K)m and ('Y'E)_m; wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5; m and q are independently a number selected from 1 to 5; n is 0 or 1 ; and J is a peptide capable of binding to a biological target. A radiopaque compound or salt thereof according to any one of paragraphs 1 to 5 wherein the peptide is of general formula (IV):

X^4a-linker-J-linker-X^4b (IV) wherein X^4a is a diiodotyrosine ( ) containing peptide selected from (K)_r(E)_s('Y')_t, ( )_q, (K'Y')_m and (E'Y')_m;

X^4b is a diiodotyrosine (Ύ') containing peptide selected from (K)_r(E)_s('Y')_t, ('Y')q, ('Y'G)_m, ('Y'K)m and ('Y'E)_m; wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5 m and q are independently a number selected from 1 to 5; and J is a peptide capable of binding to a biological target. A radiopaque compound or salt thereof according to any one of paragraphs 9 to 11 wherein J comprises an amino acid sequence of formula (Ma)

WYRGRL (Ma) wherein at least one R residue is conservatively substituted. A radiopaque compound or salt thereof according to any one of paragraphs 9 to 1 1 wherein J comprises the amino acid sequence of formula (lib)

WY RGRL (lib) wherein optionally at least one R residue is conservatively substituted.

14. A radiopaque compound or salt thereof according to any one of paragraphs 7, 8, 12 and 13 wherein both R residues are conservatively substituted.

15. A radiopaque compound or salt thereof according to any one of paragraphs 7, 8, 12 to 14 wherein when R is substituted it is substituted with K.

16. A radiopaque compound or salt thereof according to any one of paragraphs 9 to 15 wherein m and q are independently a number selected from 1 to 3.

17. A radiopaque compound according to any one of paragraphs 9 to 16 wherein the linker is selected from peptides, aminoethylethanolamine (Aeea), PEG-based linkers, and 4- aminobutyric acid, suitably aminoethylethanolamine.

18. A radiopaque compound or salt thereof according to any one of paragraphs 1 to 17 comprising two or more diiodotyrosine residues.

19. A radiopaque compound or salt thereof according to paragraph 18 comprising 2 or 3 diiodotyrosine residues. 20. A radiopaque compound according to any preceding paragraph wherein the at least one diiodotyrosine is present adjacent to a K or E residue, suitably K.

21 . A radiopaque compound according to any one of paragraphs 1 to 20 wherein the at least one diiodotyrosine is present as (K'Y')_m, wherein m is a number selected from 1 to 5. 22. A radiopaque compound according to any preceding paragraph wherein the compound has a molecular weight of 5 kDa or less, suitably 2 kDa or less.

23. A radiopaque compound selected from

W^'Y^'RGRL

^'Y^'W^'Y^'RGRL

W^'Y^'KGKL

^'Y^'W^'Y^'KGKL

^'Y^"Y^'W^'Y^'KGKL

^'Y^"Y^"Y^'W^'Y^'KGKL Ύ^{' '}γ^' Ύ^'Ύ'νν Ύ^' KG KL

Ύ^'-Aeea-WYKGKL

Ύ^'-Aeea-W^'Y^'KGKL

Ύ^'Ύ^'-Aeea-W^'Y^'KGKL

Ύ^'Ύ^'-Aeea-WYKGKL

Ύ^'Ύ^'Ύ^'-Aeea-WYKGKL

WYKGKL-Aeea-^'Y^'G

W^'Y^'KGKL-Aeea-Ύ

Ύ^'-Aeea-W YKG KL-Aeea- Ύ

K^'Y^'KW^'Y^'KGKL

K^'Y^'K^'Y^'W^'Y^'KGKL

ΚΎ^'ΚΎ^'-Aeea-WYKGKL

ΚΎ^'-Aeea-W^'Y^'KGKL

in linear or cyclic form.

24. A radiopaque compound selected from

W^'Y^'KGKL

Ύ^'-Aeea-WYKGKL

K^'Y^'KW^'Y^'KGKL

K^'Y^'K^'Y^'W^'Y^'KGKL

K^'Y^'K^'Y^'-Aeea-WYKGKL

in linear or cyclic form.

25. A radiopaque compound according to any one of paragraphs 5 to 22 wherein the biological target is cartilage.

26. A conjugate comprising a radiopaque compound according to any one of paragraph 1 to 25 and a chelating agent, suitably DOTA.

27. A contrast agent comprising a radiopaque compound or salt thereof according to any one of paragraphs 1 to 25 and at least one physiologically acceptable carrier or excipient. 28. Use of a radiopaque compound or salt thereof according to any one of paragraphs 1 to 25 in the preparation of a contrast agent.

29. Use of a radiopaque composition or salt thereof according to any one of paragraphs 1 to 25 or a conjugate according to paragraph 26, or a contrast agent according to paragraph 27 in X-ray imaging.

30. A method of X-ray imaging tissue in a human or non-human animal subject comprising administering to said subject a radiopaque composition or salt thereof according to any one of paragraphs 1 to 25 or a conjugate according to paragraph 26, or a contrast agent according to paragraph 27. 31 . A method according to paragraph 30 wherein the tissue is cartilage. EXAMPLES

Herein we describe the substitution/insertion/addition of DIT residues into the peptide sequence WYRGRL and modifications thereof in order to develop a targeted contrast agent for the imaging of articular cartilage. General Information

All reagents were obtained from commercial suppliers and used without further purification. Piperidine/DMF was acquired as a 20% premix from AGTC Bioproducts. Peptides were synthesized using the CEM Liberty Blue automated microwave peptide synthesizer and purified using a Waters HPLC system. In this system, solvent A (pH = 6) consisted of 94 % H₂0, 6 % acetonitrile with 0.01 M ammonium acetate and solvent B (pH = 6) consisted of 19 % H₂0, 81 % acetonitrile and 0.01 M ammonium acetate. Purification was performed on two gradient systems: 5-50-5% or 10-90-10% solvent B over 15 minutes (flow rate = 20 ml/min). The preparative column used was a Kinetex 5u EVO C18 100A (150 x 21 .2 mm). The radio- opaque peptides were acquired as acetate salts with≥ 95% purity by HPLC. Analytical QC runs were performed on a Kinetex 5u EVO C18 100A column (100 x 3.0 mm) in water and/or DMSO solvent. Lyophilisation was carried out on a Genevac EZ-2 Elite personal evaporator. LRMS were determined using a Waters SQ Detector 2 (ESI-MS). HRMS data was obtained on an Agilent 6530 accurate-mass Q-TOF LC/MS. Vortexing was performed on a Stuart SA8 Vortex Mixer with centrifugation on an Eppendorf 581 OR centrifuge. 1 H-NMR spectra were acquired using a Bruker Ascend spectrometer at 400 MHz. Chemical shifts (δ) are referenced to residual solvent, reported in ppm with coupling constants (J) quoted in Hz. Peptide Synthesis

Fmoc-amino acids and resins were purchased from a combination of AGTC Bioproducts, Fluorochem, Chem-lmpex and Novabiochem. Peptides were synthesized using the CEM Liberty Blue automated microwave peptide synthesizer, as per manufacturer's instructions. Fmoc-deprotection of each amino acid was monitored using the internal UV detection software, with double-conventional couplings performed for residues that showed incomplete coupling.

A typical synthetic procedure is described below. 'Y'-Aeea-WYKGKL The peptide sequence was inputted into the method editor of the Liberty Blue Application Software (v. 1 .31 .5207.25525) using standard single letter amino acid abbreviations with the exception of Ύ' and Aeea, where the numbers 2 and 6 were assigned respectively. The synthesis was performed on a 0.1 mmol scale with resin swelling occurring in 50% DCM/DMF (10 ml). In a typical procedure, deprotection of the N-terminal Fmoc group (20% piperidine/DMF, 5 ml, 75 ^°C) was followed by washing (DMF, 3 x 4 ml), further deprotection (20% piperidine/DMF, 5 ml, 75 ^°C), washing (DMF, 1 x 7ml, 2 x 4 ml), coupling (2.5 ml amino acid/linker, 0.2 M, 75 ^*C, 300 s) with HCTU/DMF (1 ml, 0.45 M) and DIPEA/DMF (1 ml, 1 M) and then final washing (DMF, 1 x 5 ml, 1 x 4 ml) to complete the cycle. Removal of the N- terminal Fmoc group involved deprotection (20% piperidine/DMF, 5 ml, 75 ^°C), washing (DMF, 3 x 4 ml), deprotection (20% piperidine/DMF, 5 ml, 75 ^°C) and final washing (DMF, 1 x 5 ml, 2 x 4 ml). In the case of the Ύ residue, four washes were carried out after the second deprotection (DMF, 1 x 7 ml, 3 x 4 ml) and double-conventional coupling of this amino acid (2.5 ml, 0.2 M, 25 ^°C, 3600 s each time) used HATU/DMF (1 ml, 0.45 M). All other steps were the same as described previously. Successful couplings were monitored using the inbuilt UV detection software.

Peptide Cleavage and Isolation

After completion of peptide synthesis, cleavage cocktail (95% TFA: 2.5% TIPS: 2.5% H20) was added to Wang resin and the subsequent yellow-brown mixture vortexed for 6 hours at room temperature. The cleavage cocktail was then cooled in a freezer for 10 minutes before the addition of ice-cold diethyl ether (50 ml) and the peptide left to precipitate in a freezer overnight (-26°C). The white precipitate that formed was centrifuged (3800 rpm, 4°C, 10 mins) and washed with a further aliquot of ether (25 ml) after decanting of the supernatant. After another cycle of centrifugation and decanting to remove residual scavengers, the white precipitate was dried under low vacuum (5 hours) to deliver the peptide as a crude off-white powder. Peptide purity was assessed by LC-MS and the crude peptide was either purified and then dissolved in a mixture of DMSO/H20 at a concentration of 20 mgl/ml for use in the binding study or carried forward for use in the binding study without additional purification. Pure lodotyrosinated Peptides

W'Y'RGRL

Purification by HPLC delivered an off-white solid (5.3 mg, 4%). 1 H-NMR (400 MHz, D20): δ 7.56 (s, 2H, DIT ArH), 7.55-7.49 (m, 2H, Trp ArH), 7.30 (s, 1 H, Trp HN-CH), 7.28-7.23 (m, 1 H, Trp ArH), 7.19-7.13 (m, 1 H, Trp ArH), 4.43 (dd, J = 8.6, 5.5 Hz, 1 H, DIT Ha), 4.34 (t, J = 6.5 Hz, 1 H, Arg Ha), 4.28 (t, J = 7.0 Hz, 1 H, Trp Ha), 4.22-4.14 (m, 1 H, Leu Ha), 4.06-3.97 (m, 2H, Arg Ha, Gly Ha), 3.84 (d, J = 17.1 Hz, 1 H, Gly Ha), 3.36 (d, J = 7.1 Hz, 2H, Trp Ηβ), 3.22- 3.04 (m, 4H, Arg Ηδ), 2.93-2.85 (m, 1 H, DIT Ηβ), 2.79 (dd, J = 13.9, 8.6 Hz, 1 H, DIT Ηβ), 1 .87- 1 .69 (m, 2H, Arg 2Ηβ), 1 .69-1 .54 (m, 7H, Arg 2Ηβ, 2Ηγ, Leu 2Ηβ, Ηγ), 1 .53-1 .43 (m, 2H, Arg Ηγ), 0.90 (d, J = 6.1 Hz, 3H, Leu CH3), 0.85 (d, J = 6.1 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 551.8 [M+2H]2+; HRMS (ESI-MS): calc. for C40H58I2N13O8 [M+H]+ 1102.2615 found 1102.2642.

'Y'W'Y'RGRL

HPLC purification gave a yellow solid (1 .8 mg, 1 %). 1 H-NMR (400 MHz, (CD3)2SO): δ 10.79 (s, 1 H, Trp NH), 9.89 (s, 1 H, Tyr OH), 8.30-8.07 (m, 3H, NH), 8.01 -7.91 (m, 1 H, NH), 7.48 (d, J = 7.9 Hz, 1 H, ArH), 7.46-7.40 (m, 3H, ArH), 7.31 (d, J = 8.1 Hz, 1 H, ArH), 7.24 (d, J = 7.3 Hz, 1 H, ArH), 7.08-7.02 (m, 2H, ArH), 6.95 (d, J = 7.0 Hz, 1 H, ArH), 6.55 (s, 1 H, NH), 4.56- 4.48 (m, 1 H, Ha), 4.47-4.39 (m, 1 H, Ha), 4.22-4.08 (m, 2H, Ha), 3.94-3.79 (m, 2H, Ha), 3.72- 3.59 (m, 2H, Ha), 3.15-2.98 (m, 7H, CH2), 2.80-2.72 (m, 1 H, CH2), 2.66-2.60 (m, 1 H, CH2), 2.32-2.21 (m, 1 H, CH2), 1 .91 -1 .76 (m, 1 H, CH2), 1 .78-1 .33 (m, 10H, CH2 Leu Ηγ, 2Ηβ), 0.87 (d, J = 2.6 Hz, 3H, CH3), 0.85 (d, J = 2.7 Hz, 3H, CH3) ppm; ESI-MS (m/z): 759.1 [M+2H]2+; HRMS (ESI-MS): calc. for C49H65I4N14O10 [M+H]+ 1517.1181 found 1517.1204.

W'Y'KGKL

Purification by HPLC delivered a white crystalline solid (23.5 mg, 19%). 1 H-NMR (400 MHz, D20): δ 7.55-7.50 (m, 2H, Trp ArH), 7.50 (s, 2H, DIT ArH), 7.30 (s, 1 H, Trp HN-CH), 7.28-7.23 (m, 1 H, Trp ArH), 7.18-7.13 (m, 1 H, Trp ArH), 4.38 (dd, J = 7.8, 5.1 Hz, 1 H, DIT Ha), 4.25-4.17 (m, 3H, Lys 2Ha, Trp Ha), 4.08 (d, J = 17.2 Hz, 1 H, Gly Ha), 3.98 (t, J = 7.2 Hz, 1 H, Leu Ha), 3.80 (d, J = 17.1 Hz, 1 H, Gly Ha), 3.41 -3.29 (m, 2H, Trp Ηβ), 2.99-2.91 (m, 4H, Lys Ηε), 2.90- 2.76 (m, 2H, DIT Ηβ), 1 .78-1 .49 (m, 10H, Lys 4Ηδ, 3Ηβ, Leu Ηγ, 2Ηβ), 1 .42-1 .24 (m, 5H, Lys 4Ηγ, Ηβ), 0.90 (d, J = 6.1 Hz, 3H, Leu CH3), 0.86 (d, J = 6.1 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 524.1 [M+2H]2+; HRMS (ESI-MS): calc. for C40H58I2N9O8 [M+H]+ 1046.2498 found 1046.2489. 'Y'WGKKL

HPLC purification gave a yellow solid (22.8 mg, 19%). 1 H-NMR (400 MHz, D20): δ 7.60 (d, J = 7.9 Hz, 1 H, Trp ArH), 7.55 (s, 2H, DIT ArH), 7.49 (d, J = 8.1 Hz, 1 H, Trp ArH), 7.27-7.21 (m, 1 H, Trp ArH), 7.21 (s, 1 H, Trp HN-CH), 7.18-7.13 (m, 1 H, Trp ArH), 4.62 (t, J = 7.3 Hz, 1 H, Trp Ha), 4.27-4.21 (m, 2H, Lys Ha), 4.21 -4.16 (m, 1 H, Leu Ha), 4.13 (dd, J = 7.8, 5.3 Hz, 1 H, DIT Ha), 3.94 (d, J = 17.0 Hz, 1 H, Gly Ha), 3.68 (d, J = 17.0 Hz, 1 H, Gly Ha), 3.29-3.15 (m, 2H, Trp 2Ηβ), 3.09 (dd, J = 14.3, 5.2 Hz, 1 H, DIT Ηβ), 2.99-2.87 (m, 5H, DIT Ηβ, Lys 4Ηε), 1 .86- 1 .50 (m, 11 H, Lys 4Ηδ, 4Ηβ, Leu 2Ηβ, Ηγ), 1 .46-1 .27 (m, 4H, Lys 4Ηγ), 0.90 (d, J = 6.1 Hz, 3H, Leu CH3), 0.85 (d, J = 6.1 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 524.1 [M+2H]2+; HRMS (ESI-MS): calc. for C40H58I2N9O8 [M+H]+ 1046.2498 found 1046.2503. 'Y'W'Y'KGKL

Purification by HPLC gave an off-white solid (32.6 mg, 20%). 1 H-NMR (400 MHz, (CD3)2SO): δ 10.83 (s, 1 H, Trp NH), 8.28-8.00 (m, 4H, NH), 7.93 (s, 1 H, NH), 7.60 (s, 1 H, NH), 7.48-7.36 (m, 5H, 4DIT ArH, Trp ArH), 7.28 (d, J = 8.0 Hz, 1 H, Trp ArH), 7.05 (s, 1 H, Trp HN-CH), 6.99 (t, J = 7.6 Hz, 1 H, Trp ArH), 6.90 (t, J = 7.4 Hz, 1 H, Trp ArH), 4.47 (s, 1 H, Trp Ha), 4.42-4.32 (m, 1 H, DIT Ha), 4.21 -4.01 (m, 2H, Lys Ha), 3.98-3.89 (m, 2H, DIT Ha, Leu Ha), 3.79-3.59 (m, 3H, Gly 2Ha, Trp Ηβ), 3.33-3.25 (m, 1 H, DIT Ηβ), 3.00 (s, 1 H, Trp Ηβ), 2.82-2.55 (m, 6H, Lys 2CH2, DIT 2Ηβ), 2.31 -2.19 (m, 1 H, DIT Ηβ), 1 .71 -1 .35 (m, 11 H, Lys 4CH2, Leu Ηγ, 2Ηβ), 1 .34-1.10 (m, 4H, Lys CH2), 0.80 (d, J = 7.1 Hz, 6H, Leu CH3) ppm; ESI-MS (m/z): 731 .5 [M+2H]2+; HRMS (ESI-MS): calc. for C49H65I4N10O10 [M+H]+ 1461 .1064 found 1461 .1040. 'V'Y'W'Y'KGKL

Purification by HPLC gave a yellow solid (9.8 mg, 5%). 1 H-NMR (400 MHz, (CD3)2SO): δ 10.81 (s, 1 H, Trp NH), 8.69-8.44 (m, 1 H, NH), 8.25-7.90 (m, 4H, NH), 7.66-7.56 (m, 2H, NH, Trp ArH), 7.56-7.46 (m, 4H, DIT ArH), 7.41 (s, 2H, DIT ArH), 7.30 (d, J = 8.0 Hz, 1 H, Trp ArH), 7.13 (s, 1 H, Trp HN-CH), 7.04 (t, J = 7.5 Hz, 1 H, Trp ArH), 6.96 (t, J = 7.4 Hz, 1 H, Trp ArH), 4.55-4.45 (m, 2H, Ha), 4.44-4.34 (m, 1 H, Ha), 4.21 -4.12 (m, 1 H, Ha), 4.01 -3.93 (m, 1 H, Ha), 3.83-3.74 (m, 2H, Ha), 3.71 -3.63 (m, 2H, Ha), 3.31 -3.23 (m, 2H, CH2), 3.15-3.07 (m, 2H, CH2), 2.79-2.64 (m, 6H, CH2), 2.35-2.23 (m, 2H, CH2), 1 .73-1 .13 (m, 15H, 6CH2 Leu 2Ηβ, Ηγ), 0.85 (2d, J = 6.5 Hz, 6H, Leu CH3) ppm; ESI-MS (m/z): 939.2 [M+2H]2+; HRMS (ESI- MS): calc. for C58H72I6N11012 [M+H]+ 1875.9625 found 1875.9599.

'Y"Y"Y'W'Y'KGKL

HPLC purification furnished a yellow solid (5.7 mg, 2%). 1 H-NMR (400 MHz, (CD3)2SO): δ 10.81 (s, 1 H, Trp NH), 8.42 (s, 1 H, NH), 8.22-7.83 (m, 4H, NH), 7.71 -7.43 (m, 9H, DIT 8ArH, Trp ArH), 7.31 (d, J = 8.0 Hz, 1 H, Trp ArH), 7.16 (s, 1 H, Trp HN-CH), 7.05 (t, J = 7.5 Hz, 1 H, Trp ArH), 6.97 (t, J = 7.5 Hz, 1 H, Trp ArH), 4.60-4.51 (m, 1 H, Ha), 4.50-4.33 (m, 3H, Ha), 4.24- 4.12 (m, 1 H, Ha), 4.03-3.93 (m, 1 H, Ha), 3.82-3.60 (m, 4H, Ha), 3.35-3.25 (m, 6H, CH2), 2.87- 2.66 (m, 6H, CH2), 2.38-2.28 (m, 2H, CH2), 1 .77-1 .07 (m, 15H, 6CH2 Leu Ηγ 2Ηβ), 0.88 (d, J = 6.5 Hz, 3H, Leu CH3), 0.85 (d, J = 6.5 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 2291 .4 [M+H]+; HRMS (ESI-MS): calc. for C67H79I8N12014 [M+H]+ 2290.8191 found 2290.8213.

WYKGKL-Aeea-'Y'G

HPLC purification furnished an off-white solid (29.1 mg, 18%). 1 H-NMR (400 MHz, D20): δ 7.51 (d, J = 7.8 Hz, 1 H, Trp ArH), 7.45 (s, 1 H, Trp ArH), 7.43 (s, 2H, DIT ArH), 7.22 (s, 1 H, Trp HN-CH), 7.17 (t, J = 7.6 Hz, 1 H, Trp ArH), 7.07 (t, J = 7.5 Hz, 1 H, Trp ArH), 6.93 (d, J = 8.1 Hz, 2H, Tyr ArH), 6.73 (d, J = 8.0 Hz, 2H, Tyr ArH), 4.63→4.57 (m, 1 H, DIT Ha), 4.47 (t, J = 7.2 Hz, 1 H, Tyr Ha), 4.34^1.26 (m, 2H, Lys Ha), 4.20 (t, J = 6.7 Hz, 1 H, Trp Ha), 4.15-4.09 (m, 1 H, Leu Ha), 4.02-3.69 (m, 6H, Aeea CH2, Gly 4Ha), 3.55-3.20 (m, 10H, Aeea 4CH2, Trp 2Ηβ), 2.99-2.90 (m, 3H, Lys CH2, DIT Ηβ), 2.91 -2.80 (m, 4H, Lys CH2, Tyr 2Ηβ), 2.69-2.58 (m, 1 H, DIT Ηβ), 1 .83-1 .67 (m, 3H, Leu Ηβ, Lys CH2), 1 .66-1 .46 (m, 8H, Lys 3CH2, Leu Ηβ, Leu Ηγ), 1 .42-1 .29 (m, 2H, Lys CH2), 1 .30-1 .13 (m, 2H, Lys CH2), 0.80 (d, J = 5.4 Hz, 3H, Leu CH3), 0.76 (d, J = 5.3 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 706.9 [M+2H]2+; HRMS (ESI-MS): calc. for C57H81 12N12014 [M+H]+ 1411 .4085 found 1411 .4063.

W'Y'KGKL-Aeea-'Y'G HPLC purification delivered an off-white solid (5.9 mg, 4%). 1 H-NMR (400 MHz, (CD3)2SO): 5 10.90 (s, 1 H, Trp NH), 8.43-8.37 (m, 1 H, NH), 8.30 (s, 1 H, NH), 8.27-8.20 (m, 1 H, NH), 8.10 (s, 1 H, NH), 7.99-7.93 (m, 1 H, NH), 7.89-7.81 (m, 3H, NH), 7.56 (d, J = 8.1 Hz, 1 H, Trp ArH), 7.47 (s, 2H, DIT ArH), 7.37 (s, 2H, DIT ArH), 7.34 (d, J = 8.1 Hz, 1 H, Trp ArH), 7.20-7.16 (m, 1 H, Trp HN-CH), 7.06 (t, J = 7.3 Hz, 1 H, Trp ArH), 6.97 (t, J = 7.2 Hz, 1 H, Trp ArH), 4.57-4.44 (m, 2H, DIT Ha), 4.28-4.18 (m, 3H, Trp Ha, Lys Ha, Leu Ha), 4.17-4.07 (m, 1 H, Lys Ha), 3.88 (d, J = 15.5 Hz, 2H, Gly Ha), 3.81 (d, J = 15.5 Hz, 2H, Gly Ha), 3.57-3.46 (m, 13H, Aeea 5CH2, DIT Ηβ, Trp 2Ηβ), 3.10-3.06 (m, 1 H, DIT Ηβ), 2.86-2.78 (m, 1 H, DIT Ηβ), 2.78-2.59 (m, 5H, Lys 2CH2, DIT Ηβ), 1 .68-1 .40 (m, 11 H, Lys 4CH2, Leu 2Ηβ, Ηγ), 1 .30-1 .15 (m, 4H, Lys CH2), 0.83 (d, J = 6.5 Hz, 3H, Leu CH3), 0.78 (d, J = 6.4 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 832.2 [M+2H]2+; HRMS (ESI-MS): calc. for C57H79I4N12014 [M+H]+ 1663.2018 found 1663.2043. 'Y'-Aeea-WYKGKL

HPLC purification yielded an off-white solid (40.4 mg, 26%). 1 H-NMR (400 MHz, D20): δ 7.51 - 7.49 (m, 1 H, Trp ArH), 7.48 (s, 2H, DIT ArH), 7.44 (d, J = 8.1 Hz, 1 H, Trp ArH), 7.20-7.14 (m,

I H, Trp ArH), 7.14 (s, 1 H, Trp HN-CH), 7.08 (t, J = 7.4 Hz, 1 H, Trp ArH), 6.90 (d, J = 8.4 Hz, 2H, Tyr ArH), 6.72 (d, J = 8.4 Hz, 2H, Tyr ArH), 4.64 (t, J = 6.7 Hz, 1 H, Trp Ha), 4.46 (t, J = 7.1 Hz, 1 H, Tyr Ha), 4.30 (t, J = 7.2 Hz, 1 H, Lys Ha), 4.20 (t, J = 7.1 Hz, 1 H, Leu Ha), 4.14 (dd, J = 8.3, 6.1 Hz, 1 H, Lys Ha), 3.98 (dd, J = 9.0, 6.1 Hz, 1 H, DIT Ha), 3.95-3.83 (m, 4H, Aeea CH2, Gly 2Ha), 3.51 -3.28 (m, 6H, Aeea CH2), 3.23-3.03 (m, 4H, Aeea CH2, Trp 2Ηβ), 3.01 - 2.88 (m, 5H, DIT Ηβ, Lys 2CH2), 2.87-2.72 (m, 3H, Tyr 2Ηβ, DIT Ηβ), 1 .87-1 .69 (m, 2H, Lys CH2), 1 .68-1 .52 (m, 9H, Lys 3CH2, Leu Ηγ, 2Ηβ), 1 .45-1 .32 (m, 2H, Lys CH2), 1 .33-1 .21 (m, 2H, Lys CH2), 0.86 (d, J = 5.9 Hz, 3H, Leu CH3), 0.82 (d, J = 5.7 Hz, 3H, Leu CH3) ppm; ESI- MS (m/z): 678.2 [M+2H]2+; HRMS (ESI-MS): calc. for C55H78I2N11013 [M+H]+ 1354.3870 found 1354.3870.

'Y'-Aeea-W'Y'KGKL

HPLC purification delivered a yellow solid (9.4 mg, 5%). 1 H-NMR (400 MHz, (CD3)2SO): δ 11 .04 (s, 1 H, Trp NH), 8.29 (s, 1 H, NH), 8.18-8.06 (m, 2H, NH), 8.04-7.93 (m, 2H, NH), 7.65 (d, J = 7.1 Hz, 2H, NH), 7.56-7.49 (m, 3H, DIT 2ArH, Trp ArH), 7.41 (s, 2H, DIT ArH), 7.34 (d, J = 8.0 Hz, 1 H, Trp ArH), 7.12 (s, 1 H, Trp HN-CH), 7.03 (t, J = 7.4 Hz, 1 H, Trp ArH), 6.95 (t, J = 7.4 Hz, 1 H, Trp ArH), 4.54 (dd, J = 7.9 Hz, 1 H, Trp Ha), 4.44 (dd, J = 6.8 Hz, 1 H, DIT Ha), 4.26-4.09 (m, 2H, Lys Ha), 3.97 (dd, J = 7.8 Hz, 1 H, Leu Ha), 3.83 (d, J = 15.4 Hz, 1 H, Gly Ha), 3.77-3.67 (m, 2H, Gly Ha, DIT Ha), 3.51 -3.01 (m, 14H, Aeea 5CH2, DIT 2Ηβ, Trp 2Ηβ), 2.85-2.57 (m, 6H, Lys 2CH2, DIT 2Ηβ), 1 .74-1 .19 (m, 15H, Lys 6CH2, Leu Ηγ, 2Ηβ), 0.85 (d, J = 7.1 Hz, 6H, Leu CH3) ppm; ESI-MS (m/z): 803.7 [M+2H]2+; HRMS (ESI-MS): calc. for C55H76I4N11013 [M+H]+ 1606.1797 found 1606.1819.

Ύ'Ύ'-Aeea-WYKGKL HPLC purification furnished a white solid (9.2 mg, 5%). 1 H-NMR (400 MHz, (CD3)2SO): δ

I I .04 (s, 1 H Trp NH), 8.33 (d, J = 7.9 Hz, 1 H, NH), 8.14 (d, J = 7.2 Hz, 1 H, NH), 8.08-7.87 (m, 4H, NH), 7.67 (d, J = 7.2 Hz, 1 H, NH), 7.57 (d, J = 7.8 Hz, 1 H, NH), 7.53 (d, J = 7.9 Hz, 1 H, Trp ArH), 7.49 (s, 2H, DIT ArH), 7.36-7.32 (m, 3H, Trp ArH, DIT 2ArH), 7.08 (s, 1 H, Trp HN- CH), 7.06-7.01 (m, 1 H, Trp ArH), 7.00 (d, J = 8.5 Hz, 2H, Tyr ArH), 6.95 (t, J = 7.4 Hz, 1 H, Trp ArH), 6.63 (d, J = 8.4 Hz, 2H, Tyr ArH), 4.56 (dd, J = 7.5 Hz, 1 H, Trp Ha), 4.47 (dd, J = 7.4 Hz,

I H, Tyr Ha), 4.40-4.31 (m, 1 H, DIT Ha), 4.30-4.17 (m, 2H, Lys Ha), 4.00-3.91 (m, 1 H, Leu Ha), 3.80 (d, J = 15.5 Hz, 1 H, Gly Ha), 3.75-3.65 (m, 2H, Gly Ha, DIT Ha), 3.39-3.33 (m, 4H, Aeea CH2, DIT 2Ηβ), 3.32-3.16 (m, 6H, Aeea CH2), 3.13-3.01 (m, 4H, Aeea CH2, Trp 2Ηβ), 2.98- 2.87 (m, 1 H, Tyr Ηβ), 2.80-2.61 (m, 7H, Lys 2CH2, DIT 2Ηβ, Tyr Ηβ), 1 .77-1 .39 (m, 11 H, Lys 4CH2, Leu Ηγ, 2Ηβ), 1 .39-1 .21 (m, 4H, Lys CH2), 0.86 (d, J = 6.6 Hz, 3H, Leu CH3), 0.83 (d, J = 6.6 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 885.9 [M+2H]2+; HRMS (ESI-MS): calc. for C64H85I4N12015 [M+H]+ 1769.2431 found 1769.2459. Ύ'Ύ'-Aeea-W'Y'KGKL

HPLC purification gave an off-white solid (4.9 mg, 2%). 1 H-NMR (400 MHz, (CD3)2SO): δ

I I .05 (s, 1 H, Trp NH), 8.36-8.26 (m, 1 H, NH), 8.15-7.98 (m, 4H, NH), 7.62 (d, J = 7.1 Hz, 2H, NH), 7.57-7.48 (m, 5H, DIT 4ArH, Trp ArH), 7.38 (s, 2H, DIT ArH), 7.34 (d, J = 8.1 Hz, 1 H, Trp ArH), 7.15-7.09 (m, 1 H, Trp HN-CH), 7.03 (t, J = 7.6 Hz, 1 H, Trp ArH), 6.95 (t, J = 7.3 Hz, 1 H, Trp ArH), 4.58-4.51 (m, 1 H, Trp Ha), 4.51 -4.43 (m, 1 H, DIT Ha), 4.43-4.34 (m, 1 H, DIT Ha), 4.22-4.11 (m, 2H, Lys Ha), 4.00-3.92 (m, 1 H, Leu Ha), 3.83 (d, J = 15.4 Hz, 1 H, Gly Ha), 3.75- 3.67 (m, 2H, Gly Ha, DIT Ha), 3.39-3.32 (m, 6H, Aeea CH2, DIT 4Ηβ), 3.33-3.17 (m, 6H, Aeea CH2), 3.15-3.01 (m, 4H, Aeea CH2, Trp 2Ηβ), 2.78-2.61 (m, 6H, Lys 2CH2, DIT 2Ηβ), 1 .74- 1 .20 (m, 15H, Lys 6CH2, Leu Ηγ, 2Ηβ), 0.85 (2d, J = 6.6 Hz, 6H, Leu CH3) ppm; ESI-MS (m/z): 1011.1 [M+2H]2+; HRMS (ESI-MS): calc. for C64H83I6N12015 [M+H]+ 2021 .0363 found 2021 .0372.

'Y"Y"Y'-Aeea-WYKGKL

HPLC purification furnished an off-white solid (10.2 mg, 4%). 1 H-NMR (400 MHz, (CD3)2SO): 5 11 .01 (s, 1 H, Trp NH), 8.38-8.26 (m, 1 H, NH), 8.24 (d, J = 8.0 Hz, 1 H, NH), 8.12 (d, J = 7.6 Hz, 1 H, NH), 8.07-7.94 (m, 2H, NH), 7.91 (d, J = 7.2 Hz, 1 H, NH), 7.68 (d, J = 7.0 Hz, 1 H, NH), 7.58-7.50 (m, 3H, DIT 2ArH, Trp ArH), 7.47 (s, 2H, DIT ArH), 7.40 (s, 2H, DIT ArH), 7.33 (d, J = 8.1 Hz, 1 H, Trp ArH), 7.09 (s, 1 H, Trp HN-CH), 7.04 (t, J = 7.7 Hz, 1 H, Trp ArH), 7.00 (d, J = 8.4 Hz, 2H, Tyr ArH), 6.95 (t, J = 7.5 Hz, 1 H, Trp ArH), 6.63 (d, J = 8.4 Hz, 2H, Tyr ArH), 4.61 -4.51 (m, 1 H, Trp Ha), 4.51 -4.42 (m, 2H, DIT Ha, Tyr Ha), 4.35 (q, J = 7.1 Hz, 1 H, DIT Ha), 4.30-4.16 (m, 2H, Lys Ha), 3.99-3.91 (m, 1 H, Leu Ha), 3.80 (d, J = 15.1 Hz, 1 H, Gly Ha), 3.73-3.66 (m, 2H, Gly Ha, DIT Ha), 3.40-3.25 (m, 10H, Aeea CH2), 3.12-3.04 (m, 3H, DIT 2Ηβ, Trp Ηβ), 3.03-2.97 (m, 1 H, Trp Ηβ), 2.96-2.88 (m, 2H, DIT Ηβ, Tyr Ηβ), 2.80-2.60 (m, 8H, Lys 2CH2, DIT 3Ηβ, Tyr Ηβ), 1 .77-1 .41 (m, 11 H, Lys 4CH2, Leu 2Ηβ, Ηγ), 1 .40-1 .24 (m, 4H, Lys CH2), 0.86 (d, J = 6.6 Hz, 3H, Leu CH3), 0.84 (d, J = 6.5 Hz, 3H, Leu CH3) ppm; ESI- MS (m/z): 1092.5 [M+2H]2+; HRMS (ESI-MS): calc. for C73H91 16N13017 [M+H]+ 2184.0997 found 2184.1012.

K'Y'K'Y'-Aeea-WYKGKL

HPLC purification delivered a white solid (6.2 mg, 3%). 1 H-NMR (400 MHz, D20): δ 7.46 (d, J = 7.9 Hz, 1 H, Trp NH), 7.44-7.39 (m, 3H, DIT 2ArH, Trp ArH), 7.21 (s, 1 H, Trp HN-CH), 7.17 (t, J = 7.6 Hz, 1 H, Trp ArH), 7.12 (s, 2H, DIT ArH), 7.04 (t, J = 7.3 Hz, 1 H, Trp ArH), 6.89 (d, J = 8.3 Hz, 2H, Tyr ArH), 6.65 (d, J = 8.5 Hz, 2H, Tyr ArH), 4.93 (t, J = 6.3 Hz, 1 H, Trp Ha), 4.64 (dd, J = 8.1 , 5.9 Hz, 1 H, DIT Ha), 4.44 (t, J = 7.7 Hz, 1 H, DIT Ha), 4.39-4.30 (m, 2H, Lys Ha), 4.28-4.22 (m, 1 H, Lys Ha), 4.20-4.15 (m, 1 H, Leu Ha), 4.03-3.94 (m, 3H, Tyr Ha, Gly 2Ha), 3.93-3.88 (m, 1 H, Lys Ha), 3.52-3.35 (m, 4H, Aeea CH2), 3.34-3.11 (m, 6H, Aeea 2CH2, Trp 2Ηβ), 3.01 -2.82 (m, 12H, Tyr 2Ηβ, Aeea CH2, Lys 4CH2), 2.81 -2.74 (m, 2H, DIT Ηβ), 2.72- 2.64 (m, 1 H, DIT Ηβ), 2.39-2.30 (m, 1 H, DIT Ηβ), 1 .86-1 .74 (m, 4H, Lys CH2), 1 .73-1 .50 (m, 15H, Lys 6CH2, Leu 2Ηβ, Ηγ), 1 .45-1 .35 (m, 4H, Lys CH2), 1 .36-1 .17 (m, 4H, Lys CH2), 0.87 (d, J = 5.8 Hz, 3H, Leu CH3), 0.84 (d, J = 5.8 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 1013.2 [M+2H]2+; HRMS (ESI-MS): calc. for C76H109I4N16O17 [M+H]+ 2025.4330 found 2025.4327.

K'Y'W'Y'KGKL

Purification by HPLC gave an off-white solid (8.4 mg, 5%). 1 H-NMR (400 MHz, D20): δ 7.59- 7.38 (m, 4H, ArH), 7.38-7.25 (m, 2H, ArH), 7.25-7.13 (m, 2H, ArH), 7.13-7.00 (m, 1 H, ArH), 4.69-4.53 (m, 2H, Ha), 4.52-4.32 (m, 1 H, Ha), 4.32-4.16 (m, 2H, Ha), 4.16-3.97 (m, 2H, Ha), 3.97-3.75 (m, 2H, Ha), 3.26-3.05 (m, 2H, CH2), 3.05-2.84 (m, 5H, CH2), 2.85-2.60 (m, 5H, CH2), 1 .80-1 .45 (m, 14H, 6CH2, Leu Ηγ, Ηβ), 1 .45-1 .11 (m, 7H, 3CH2, Leu Ηβ), 0.94-0.77 (m, 6H, Leu CH3) ppm; ESI-MS (m/z): 795.4 [M+2H]2+; HRMS (ESI-MS): calc. for C55H77I4N12011 [M+H]+ 1589.2008 found 1589.1987. K'Y'KW'Y'KGKL

HPLC purification afforded a white solid (8.5 mg, 4%): 1 H-NMR (400 MHz, D20): δ 7.57-7.50 (m, 3H, Trp ArH, DIT 2ArH), 7.46 (d, J = 8.1 Hz, 1 H, Trp ArH), 7.32-7.25 (m, 3H, DIT 2ArH, Trp HN-CH), 7.22 (t, J = 7.6 Hz, 1 H, Trp ArH), 7.12 (t, J = 7.3 Hz, 1 H, Trp ArH), 4.64-4.52 (m, 2H, DIT Ha), 4.43-4.33 (m, 1 H, Trp Ha), 4.32-4.17 (m, 4H, Lys 3Ha, Leu Ha), 4.10 (d, J = 17.1 Hz, 1 H, Gly Ha), 3.92 (t, J = 6.7 Hz, 1 H, Lys Ha, br, 1 H, Gly Ha), 3.36-3.26 (m, 1 H, DIT Ηβ), 3.26- 3.18 (m, 1 H, ΟΙΤ Ηβ), 3.00-2.82 (m, 9H, Lys 4CH2, DIT Ηβ), 2.83-2.73 (m, 1 H, DIT Ηβ), 2.68- 2.50 (m, 2H, Trp Ηβ), 1 .84-1 .74 (m, 2H, Lys CH2), 1 .73-1 .50 (m, 15H, Lys 6CH2, Leu Ηγ, 2Ηβ), 1 .43-1 .05 (m, 10H, Lys 5CH2), 0.90 (d, J = 6.0 Hz, 3H, Leu CH3), 0.86 (d, J = 5.9 Hz, 3H, Leu CH3) ppm; ESI-MS (m/z): 859.2 [M+2H]2+; HRMS (ESI-MS): calc. for C61 H89I4N14012 [M+H]+ 1717.2958 found 1717.2936.

K'Y'K'Y'W'Y'KGKL

HPLC purification furnished a white solid (23.4 mg, 10%). 1 H-NMR (400 MHz, D20, 333K): δ 7.89-7.74 (m, 6H, ArH), 7.73-7.64 (m, 2H, ArH), 7.55-7.45 (m, 2H, ArH), 7.44-7.36 (m, 1 H, ArH), 5.10-4.86 (m, 2H, Ha), 4.63-4.50 (m, 3H, Ha), 4.42-4.30 (m, 2H, Ha), 4.08-4.00 (m, 1 H, Ha), 3.62-3.48 (m, 3H, Ha), 3.39-3.20 (m, 10H, CH2), 3.09-3.02 (m, 2H, CH2), 2.21 -1 .87 (m, 21 H, 10CH2, Leu Ηγ), 1 .80-1 .50 (m, 10H, 4CH2, Leu 2Ηβ), 1 .28-1 .16 (m, 6H, Leu CH3). ESI- MS (m/z): 1066.6 [M+2H]2+; HRMS (ESI-MS): calc. for C70H96I6N15O14 [M+H]+ 2132.1524 found 2132.1545.

Animal work and μΟΤ scanning

All animal work was conducted according to the Animals (Scientific Procedures) Act 1986. C57BI/6 mice (male, 10-weeks old) purchased from Charles River (UK Ltd, Margate, UK) were housed in individually ventilated cages, maintained under regular light/dark conditions and had ad libitum access to water and food.

All scans were reconstructed using the built-in software from the scanner manufacturer (version 2.3, Perkin Elmer, USA) and subsequently resliced for analysis using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Generation of color-coded maps of absorption For calibration of the μΰΤ scans in Hounsfield units (HU) of X-ray absorption, air and water were scanned and reconstructed using the same settings of the specimens. The average grey level intensity within a cylindrical volume of interest (VOI) was subsequently obtained from the image histograms in ImageJ (-832.85 and 152.54 for air and water, respectively). Based on the knowledge of the average grey level intensity and the correspondent HU units (-1000 and 0 for air and water, respectively), the following linear transformation was derived:

HU = 1.01 x Grey level intensity - 154.80

Using this calibration, the grey level intensity of each pixel in the μΰΤ images was linearly transformed into the correspondent absorption by means of Matlab (R2014b, The MathWorks Inc., Natick, MA, USA). To enhance the difference between the X-ray absorption of each tissue, colour-coded maps were generated in ImageJ by assigning a look up table (LUT) of 256 colour entries to the absorption images. Briefly, black was assigned to absorption levels below 525 HU, red (R) to absorptions above 525 HU, green (G) to absorptions ranging between 2100 and 2950 HU and blue (B) to absorptions above 2950 HU. The LUT editor then produced colour gradations between these entries by interpolation methods according to the RGB colour model (Figure 1 ). Final maps were expressed in HU and had the articular cartilage with colours ranging from red to yellow, while bone varied from yellow/green to blue.

Half-lives of the pure peptide sequences To determine the half-lives of the peptide sequences, a VOI extending 500 μηι across the anterior-posterior axis was manually placed in the tibial articular cartilage and the average grey level intensity within it measured. The amount of remaining peptide in the articular cartilage at a certain time point, t, upon saline wash was defined as the ratio between the average grey level intensities at t and at equilibrium, expressed as a percentage, and calculated by the following equation: grey level intensity_t

Remaining peptide_t(%) = - - x 100, t

grey level intensity^■equilibrium

= number of hours in saline

These quantifications provided the percentage decrease in contrast over time and were used to determine the half-lives of the sequences by fitting one-phase decay models to the data (Graph Pad Prism 5 software, San Diego, CA, USA).

Ex vivo Imaging

For ex vivo testing of contrast agent binding to articular cartilage, mice were sacrificed by inhalation of C0₂, knee joints harvested and split under a dissection microscope. Tibiae (n=3 per contrast agent) were finely dissected to remove the soft tissue and incubated to equilibrium in 20 μΙ of contrast agent. Proximal tibiae were imaged in a μΟΤ scanner (Quantum FX, Perkin Elmer, USA) at a spatial resolution of 10 μηι/ρίχβΙ (200 μΑ, 90 kV, 3 minutes of acquisition time) to determine the contrast achieved at equilibrium. Binding was evaluated by immersion of the articular cartilage from freshly explanted murine tibias in a solution containing the peptidic contrast agents at a concentration of 20 mg I /ml (in H₂0 containing up to 20% DMSO for crude peptides and up to 10% DMSO for pure peptides) until equilibration. Upon imaging, tibiae were washed in saline and subsequently imaged within saline until the articular cartilage was no longer detectable. Results & Discussion

A series of peptides comprising DIT were prepared and their binding to murine cartilage assessed (Table 1 ) ex vivo as described above. The binding was initially assessed with crude peptide (50-94% purity by HPLC) before characterization of positive "hits" with the pure compound (≥ 95% by HPLC).

Table 1. Peptide sequences, purities and half-lives

Entry Peptide Sequence Purity Half-life

t1/2 (h)

(HPLC)a

18 W^'Y^'KGKL-Aeea-^'Y^'G ≥ 99% M

19 Ύ^'-Aeea-WYKGKL- ≥ 50% P

Aeea-^'Y^'G

20 K^'Y^'W^'Y^'KGKL ≥ 99% 33.86

21 K^'Y^'KW^'Y^'KGKL ≥ 96% 95.70

22 K^'Y^'K^'Y^'W^'Y^'KGKL ≥ 97% 298.00

23 ΚΎ^'ΚΎ^'-Aeea-WYKGKL ≥ 99% 93.80

aPeptide purity was established by integration of the area under the curve for each chromatogram at all wavelengths. P: Peptide precipitated during testing at 50-94% purity. M: peptide showed binding at 50- 94% purity but was insoluble at > 95% purity at [2 mg I /ml] in 10% DMSO 90% H20, the lowest detectable concentration. Introduction of one or two Ύ' residues into the N-terminus of WYRGRL (Table 1 , entries 1 and 2 respectively) disrupted the binding to cartilage, where there was no visible binding to ex vivo murine articular cartilage by μΟΤ (ti/₂ reported as < 0.05 h, the time it takes to obtain an image).

Integration of DIT into the sequence by replacing the 2^nd Y amino acid with furnished crude peptides that displayed binding to articular cartilage (entries 3 and 4). This signified that the replacement of aryl hydrogens for iodine atoms in tyrosine did not affect binding to articular cartilage. However, both pure peptides were insoluble in 10% DMSO at 2 mg I /ml so this result could not be confirmed at the higher purity.

Conservative substitution of the Arg residues with Lys residues (entry 5) yielded a peptide that retained articular cartilage binding at crude level and was soluble in 10% DMSO as a pure peptide. W^'Y^'KGKL was cleared from the tissue with a ti/₂ of 2.6h. This represented an approximately 2-fold higher ex vivo half-life than the clinically approved anionic contrast agent ioxaglate (ti/₂ = 1 .12 h) and a 1 .5-fold higher ex vivo half-life than the preclinical contrast agent CA4+ (ti/2 = 1 .74 h). The scrambled derivative ^'Y^'WGKKL (entry 6) showed poor binding, indicating the importance of the specific amino acid sequence in collagen-ll binding. Representative μCT coronal views of mouse tibiae 1 h and 24 h after washing in saline are shown in Figure 2A (upper rows). The correspondent color-coded maps (expressed in Hounsfield units and focused on the lateral aspect of the tibial plateau) highlight the difference between the X-ray absorption of each tissue and enhance the clearance of the peptides from the articular cartilage over time. After 1 h of saline wash, W^'Y^'KGKL peptides visualizes the articular cartilage at an absorption range between 1800-2200 HU. After 24 h in saline, W^'Y^'KGKL is cleared and the articular cartilage is no longer detectable. The curves used to calculate the half-life is shown in Figure 2B.

Additional DITs were added to the N-terminus of W^'Y^'KGKL to decrease the dose of peptide required for visualization (entries 7-10). A maximum of three N-terminal DITs were tolerated before the peptide precipitated during testing at the crude level. Binding to articular cartilage was detected with peptides 7-9 at crude level. These peptides were however not soluble in 10% DMSO at 2 mg l/ml. C-terminal insertion of DIT to WYKGKL disrupted binding to articular cartilage (entry 11 ).

The Fmoc-Aeea-OH linker was used to separate the signaling (Ύ^') and targeting entities (WYKGKL) at the N-terminus (entries 12-16), the C-terminus (entries 17-18) and both terminii (19). Ύ^'-Aeea-W YKG KL had the highest half-life of the single Ύ^' derivatives of 13.52 h. The other sequences within the series displayed binding at crude level but had solubility problems at pure level when two or more DITs were introduced either with (entries 13-14) or without (entries 15-16) an integrated design. Separation of the signaling Ύ^' with the Aeea linker at C- terminus allowed the peptide to retain articular cartilage binding, with a half-life of 4.95 hours (entry 17). The version of peptide 17 with an integrated DIT (entry 18), retained binding at crude level, but was not soluble in 10% DMSO at the pure level. The N- and C-terminal modified peptide 19 precipitated during testing.

To address the solubility problems associated with the introduction of multiple DIT residues, a series of lysine rich derivatives were constructed (entries 20-23). The first compound in this series was found to have a half-life of 33.86 h (entry 20).This value was found to almost triple with the insertion of an additional lysine residue (entry 21 , ti/₂ = 95.70 h), probably due to the increase in aqueous solubility afforded by this amino acid. The addition of a lysine residue for every DIT introduced into the peptide sequence enabled the synthesis of peptide 22 with three Ύ^' amino acids, with excellent solubility and half-life (ti/₂ = 298.00 h) as well as retention of articular cartilage binding. Finally, the use of the flexible Aeea linker furnished peptide 23 with a half-life comparable to that of 21 (cf. ti/₂ = 93.80 h vs ti/₂ = 95.70 h respectively) indicating both a linker and lysine may be used successfully in tandem to increase solubility. These lysine-rich derivatives allow the synthesis of WYKGKL peptides containing multiple DIT residues without having to sacrifice binding strength and solubility. In vivo Imaging

The ability of the peptide sequence Ύ^'-Aeea-WYKGKL (ex vivo ti/₂ 13.52h) to image cartilage in vivo was tested.

For in vivo imaging, mice were anaesthetised by inhalation of isoflurane (3% in 100% oxygen for induction and 2% in 100% oxygen for maintenance). 20 μΙ of contrast agent in the right knee joint and 20 μΙ of saline solution in the left knee joint. Upon injections, animals were placed in the μΟΤ scanner and knee joints imaged also at a spatial resolution of 10 μηι/ρίχβΙ (Figure 3).

Colour-coded maps of absorption highlight the binding of the peptidic contrast agent to articular cartilage when compared to an injection of the same volume of saline. This result suggests that peptide sequences, such as Ύ^'-Aeea-WYKGKL, show promise for visualizing articular cartilage in vivo in murine preclinical disease models via binding to collagen type II.

REFERENCES

1 . Hussain, T.; Nguyen, Q. T. Molecular imaging for cancer diagnosis and surgery. Adv. Drug Delivery Rev. 2014, 66, 90-100.

2. Villemagne, V. L. Ageing Research Reviews, in press. 3. Brooks, D. J. Ageing Research Reviews, in press.

4. Pillarsetty, N.; Punzalan, B.; Larson, S. M. J. Nucl. Med. 2009, 50, 1709-1714.

5. Postema, E.; McEwan, A. B.; Riauka, T.; Kumar, P.; Richmond, D.; Abrams, D.; Wiebe, L. Eur. J. Nucl. Med. Mol. Imaging 2009, 36, 1565-1573.

6. Rivlin, M.; Tsarfaty, I.; Navon, G. Magn. Reson. Med. 2014, 72, 1375-1380. 7. Ocampo-Garcia, B. E.; Santos-Cuevas, C. L; De Leon-Rodriguez, L. M.; Garcia-Becerra, R.; Ordaz-Rosado, D.; Luna-Guitierrez, M. A.; Jimenez-Mancilla, N. P.; Romero-Pina, M. E.; Ferro-Flores, G. Nucl. Med. Biol. 2013, 40, 481 -487.

8. Dong, C; Zhao, H.; Yang, S.; Shi, J.; Huang, J.; Cui, L; Zhong, L; Jin, X.; Li, E; Liu, Z.; Jia, B. ; Wang, F. Mol. Pharmaceutics 2013, 10, 2925-2933. 9. Ogawa, K.; Yu, J.; Ishizaki, A.; Yokokawa, M.; Kitamura, M.; Kitamura, Y; Shiba, K.; Odani, A. Bioconjugate Chem. 2015, 26, 1561 -1570.

10. Wang, H.; Li, D.; Liu, S.; Liu, R.; Yuan, H.; Krasnoperov, V.; Shan, H.; Conti, P. S.; Gill, P. S.; Li, Z. J. Nucl. Med. 2015, 56, 908-913.

11. Zhang, Y; Hong, H.; Orbay, H.; Valdovinos, H.; Nayak, T.; Theuer, C; Barnhart, T.; Cai, W. Eur. J. Nucl. Med. Mol. Imaging 2013, 40, 759-767.

12. BIykers, A.; Schoonooghe, S.; Xavier, C; D'hoe, K.; Laoui, D.; D'Huyvetter, M.; Vaneycken, I.; Cleeren, F; Bormans, G.; Heemskerk, J.; Raes, G.; De Baetselier, P.; Lahoutte, T.; Devoogdt, N.; Van Ginderachter, J. A.; Caveliers, V. J. Nucl. Med. 2015, 56, 1265-1271 .

13. Yang, X.; Mease, R. C; Pullambhatla, M.; Lisok, A.; Chen, Y; Foss, C. A.; Wang, Y; Shallal, H.; Edelman, H.; Hoye, A. T.; Attardo, G.; Nimmagadda, S.; Pomper, M. G. J. Med.

Chem. 2016, 59, 206-218.

14. Yan, Y; Xiao, Z.; Song, Y; Kang, Z.; Wang, P. ; Sun, X. ; Shen, B. Bioorg. Med. Chem. Lett. 2015, 25, 1647-1652. 15. Bates, D.; Abraham, S.; Campbell, M.; Zehbe, I.; Curiel, L. PLoS ONE 2014, 9, e97220.

16. Freedman, J. D.; Lusic, H.; Wiewiorski, M.; Farley, M.; Snyder, B. D.; Grinstaff, M. W. Chem. Commun. 2015, 51 , 11166-11169.

17. Stewart, R. C; Bansal, P. N.; Entezari, V.; Lusic, H.; Nazarian, R. M.; Snyder, B. D.; Grinstaff, M. W. Radiology 2013, 266, 141 -150.

18. Freedman, J. D.; Lusic, H.; Snyder, B. D.; Grinstaff, M. W. Angew. Chem. Int. Ed. 2014, 53, 8406-8410.

19. Lusic, H.; Grinstaff, M. W. X-ray-Computed Tomography Contrast Agents. Chem. Rev. 2013, 113, 1641 -1666.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law). All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise paragraphed. No language in the specification should be construed as indicating any non-paragraphed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. This invention includes all modifications and equivalents of the subject matter recited in the paragraphs appended hereto as permitted by applicable law.

Claims

1 . A radiopaque compound or salt thereof comprising diiodotyrosine,

wherein the compound comprises a group X¹ , wherein X¹ is a diiodotyrosine (Ύ') containing peptide selected from a peptide of molecular formula (K)_r(E)_s('Y')_t, (K'Y')_m and (E'Y')_m; wherein r and s are numbers independently selected from 0 to 5, and t is a number selected from 1 to 5; and wherein m is a number selected from 1 to 5.

2. A radiopaque compound or salt thereof according to claim 1 wherein the diiodotyrosine is 3,5-diiodotyrosine.

A radiopaque compound or salt thereof according to any preceding claim wherein the diiodotyrosine is of formula (B), wherein indicates the points of attachment:

Ο ΛΛΛ

(B)

4. A radiopaque compound or salt thereof according to any preceding claim wherein at least a portion of the compound binds to a biological target.

5. A radiopaque compound or salt thereof according to any preceding claim wherein the sum of r and s is greater than or equal to t.

6. A radiopaque compound or salt thereof according to any preceding claim wherein the compound comprises a peptide of formula (I la):

WYRGRL (Ma) wherein at least one R residue is conservatively substituted. A radiopaque compound or salt thereof according to any preceding claim wherein the compound comprises a peptide of formula (lib)

A radiopaque compound or salt thereof according to any one of claims 1 to 4 of general formula (I):

X²-(linker)_n-J (I) wherein

X² is a diiodotyrosine (T) containing peptide selected from (K)_r(E)_s('Y')_t, (Ύ , (K'Y')_m and (E'Y')_m; wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5 m and q are independently numbers selected from 1 to 5; n is 0 or 1 ; and J is a peptide capable of binding to a biological target.

A radiopaque compound or salt thereof according to any one of claims 1 to 4 of general formula (III):

J-(linker)n-X³ (III) wherein

X³ is a diiodotyrosine (Ύ') containing peptide selected from (K)_r(E)_s('Y')_t, ('Y')_q, ('Y'G)_m, ('Y'K)m and ('Y'E)_m; wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5; m and q are independently a number selected from 1 to 5; n is 0 or 1 ; and J is a peptide capable of binding to a biological target.

10. A radiopaque compound or salt thereof according to any one of claims 1 to 4 wherein the peptide is of general formula (IV):

X^4a-linker-J-linker-X^4b (IV) wherein

X^4a is a diiodotyrosine ( ) containing peptide selected from (K)_r(E)_s('Y')_t, ( )_q, (K'Y')_m and (E'Y')_m;

X^4b is a diiodotyrosine (T) containing peptide selected from (K)_r(E)_s('Y')_t, (T)_q, ('Y'G)_m, wherein r and s are independently numbers selected from 0 to 5, and t is a number selected from 1 to 5 m and q are independently a number selected from 1 to 5; and J is a peptide capable of binding to a biological target.

11 . A radiopaque compound or salt thereof according to any one of claims 8 to 10 wherein J comprises an amino acid sequence of formula (Ma)

WYRGRL (Ma) wherein at least one R residue is conservatively substituted.

12. A radiopaque compound or salt thereof according to any one of claims 8 to 10 wherein J comprises the amino acid sequence of formula (lib)

13. A radiopaque compound or salt thereof according to any one of claims 6, 7, 1 1 and 12 wherein both R residues are conservatively substituted.

14. A radiopaque compound or salt thereof according to any one of claims 6, 7, 1 1 to 13 wherein when R is substituted it is substituted with K.

15. A radiopaque compound or salt thereof according to any one of claims 8 to 14 wherein m and q are independently a number selected from 1 to 3.

16. A radiopaque compound according to any one of claims 8 to 15 wherein the linker is selected from peptides, aminoethylethanolamine (Aeea), PEG-based linkers, and 4- aminobutyric acid, suitably aminoethylethanolamine.

17. A radiopaque compound or salt thereof according to any one of claims 1 to 16 comprising two or more diiodotyrosine residues.

18. A radiopaque compound or salt thereof according to claim 17 comprising 2 or 3 diiodotyrosine residues.

19. A radiopaque compound according to any preceding claim wherein the at least one diiodotyrosine is present adjacent to a K or E residue, suitably K.

20. A radiopaque compound according to any one of claims 1 to 19 wherein the at least one diiodotyrosine is present as (K'Y')_m, wherein m is a number selected from 1 to 5.

21 . A radiopaque compound according to any preceding claim wherein the compound has a molecular weight of 5 kDa or less, suitably 2 kDa or less.

22. A radiopaque compound selected from

W^'Y^'RGRL

Y^'W^'Y^'RGRL

W^'Y^'KGKL

Y^'W^'Y^'KGKL

'Y^"Y ^'W^'Y^'KGKL

Y ^'Y ^'Y^'W^'Y^'KGKL

Ύ Ύ Ύ ^"Y^'W^'Y^'KGKL

Y^'-Aeea-WYKGKL Ύ^'-Aeea-W Ύ KGKL

Ύ^'Ύ^'-Aeea-W^'Y^'KGKL

Ύ ^" Y ^'- Aeea- WYKG KL

Ύ ^"Y ^"Y -Aeea-WYKGKL

WYKGKL-Aeea-^'Y^'G

W Ύ ^'KG KL- Aeea- Ύ ^'G

Ύ -Aeea- WYKG KL- Aeea- Ύ G

ΚΎ KW Y KGKL ΚΎ K Ύ W Ύ KGKL ΚΎ ΚΎ -Aeea-WYKGKL

K^'Y^'-Aeea-W^'Y^'KGKL

in linear or cyclic form.

23. A radiopaque compound selected from

W^'Y^'KGKL

Y^'-Aeea-WYKGKL

ΚΎ KW Y KGKL K Y K Ύ W^'Y KGKL

ΚΎ ΚΎ -Aeea-WYKGKL

in linear or cyclic form.

24. A radiopaque compound according to any one of claims 1 to 21 wherein the biological target is cartilage.

25. A conjugate comprising a radiopaque compound according to any one of claim 1 to 24 and a chelating agent, suitably DOTA.

26. A contrast agent comprising a radiopaque compound or salt thereof according to any one of claims 1 to 24 and at least one physiologically acceptable carrier or excipient.

27. Use of a radiopaque compound or salt thereof according to any one of claims 1 to 24 in the preparation of a contrast agent.

28. Use of a radiopaque composition or salt thereof according to any one of claims 1 to 24 or a conjugate according to claim 25, or a contrast agent according to claim 26 in X-ray imaging.

29. A method of X-ray imaging tissue in a human or non-human animal subject comprising administering to said subject a radiopaque composition or salt thereof according to any one of claims 1 to 24 or a conjugate according to claim 25, or a contrast agent according to claim 26.

30. A method according to claim 29 wherein the tissue is cartilage.