NL2033714B1 - Multiplexing targeting and imaging agents - Google Patents

Abstract

Provided herein are compounds of formula | and formula ll that are useful in the targeting and/or imaging of biological specimens, in particular as agents that may be used for targeting and 5 imaging the hepatobiliary system and related cells. The compounds represent multiplexing tracers comprising a fluorescent dye-based targeting moiety and a secondary moiety that may include a second imaging label and/or a therapeutic agent. R1 R2 R4 R3 R7 R10 R5 R8 Re R6 / N [ÎICB L I R11 R12 (I) R1 R2 R4 Ra R7 R8 RQ R10 R5 Re / N @ |/ | N X n R14 R14 (ll) 10

Description

MULTIPLEXING TARGETING AND IMAGING AGENTS

[2061] This invention relates to compounds that are useful in the targeting and/or imaging of biological specimens, in particular as agents that may be used for targeting and imaging the hepatobiliary system and related cells. The compounds represent multiplexing tracers comprising a fluorescent dye-based targeting moiety and a secondary moiety that may include a second imaging label and/or a therapeutic agent.

BACKGROUND

[3002] Biological imaging, including medical imaging, permits the rapid, non-invasive and longitudinal visualisation of the structure, physiology and cellular processes of biological specimens. The three main classes of imaging agents include small molecules, proteins, and nanoparticles. [G03] Within the class of small molecules, fluorescent chromophores (or “fluorophores”) play an important role in molecular and cell imaging both in vitro and in vivo. As an example of an in vivo dye, the water-soluble anionic tricarbocyanine dye indocyanine green (ICG), which was originally developed as a photography dye, has been used for several decades in a variety of medical diagnostic applications, including the assessment of cardiac output, hepatic function, liver and gastric blood flow, and for use in fluorescent angiography, such as ophthalmic angiography. ICG absorbs light in the 600-900 nm wavelength range with a maximum, depending on medium, of around 800 nm (e.g. about 820 nm in serum), and emits (by fluorescence) in the 750 nm to 950 wavelength range with a maximum around 820 nm. After intravenous injection, ICG non-covalently binds to native proteins and is rapidly cleared from the bloodstream by the liver, where the dye-protein complex is metabolised by hepatocytes and excreted into bile. In healthy individuals, ICG is eliminated from the body with a biological half- life of about 3 to 5 minutes. In areas with liver dysfunction or disease (meaning damage to the liver anatomy), a prolongation of IGC half-life is usually observed. As such, ICG clearance is not only used during bile duct surgery, but also facilitates dynamic liver function assessments and the identification of intrahepatic tumour lesions. However, a shortcoming of fluorescence imaging is that it typically only allows for accurate identification of features that are present less than about 1 centimetre from the surface on which the imaging occurs. [G04] Also within the category of small molecules, radio-labelled (or “radioisotopically labelled”) compounds have been extensively used in in vitro and in vivo medical and biological imaging and tracking techniques. Such agents generally comprise a molecule wherein one or more atoms have been replaced by a radionuclide, or wherein the molecule contains one or more moieties that are capable of chelating a radionuclide. Radio-labelled tracer molecules are very valuable agents for non-invasive studying of drug targeting and drug metabolism. Additionally, radiotracers targeting a specific cell component (such as nuclear, cytoplasmic or membrane proteins) have been found particularly useful for the localisation and characterisation of a variety of diseases and disorders. For example, positron emission tomography (PET) tracers targeting the mitochondria via the translocator protein (TSPO) as a molecular biomarker of inflammation can be employed to study peripheral sterile inflammatory diseases such as Crohn’s disease, viral, cholestatic and autoimmune hepatitis, inflammatory vasculopathies, and rheumatoid arthritis [Largeau et al., Contrast Media & Molecular Imaging, Volume 2017, Article ID 6592139].

As a marker of specifically neuroinflammation, binding to TSPO has also been used to investigate various inflammatory, neurodegenerative and neoplastic brain disorders [Chauveau et al., Eur. J. Nucl. Med. Mol. Imaging (2008) 35:2304-23]. However, the most widely used tracer for all these purposes, [11 C]JPK11195, suffers from a short half-life of the 11C radioiosotope, non-specific binding, and a poor signal-to-noise ratio which complicates its quantification. The in-depth non-invasive imaging characteristics of diagnostic nuclear agents means they are often employed as ‘scout’ scan for dosimetry and subsequent therapeutic delivery that is based on the same targeting vector. This concept of identifying sites of high uptake of a diagnostic version of a therapeutic agent and subsequent targeting of the therapeutic agent is commonly referred to as “theranostics”.

[3005] The above fluorescent and/or radioactive labelling approaches have furthermore enabled the visualisation, in particular the (real-time) tracking and quantification of (micro)organisms and cells while present in organs and/or tissue.

[0008] For example, sporozoitic malaria parasites labelled with a fluorescent cyanine-based dye targeting mitochondria via the translocator protein (TSPO) have been individually monitored while migrating through human skin [Winkel et al., Theranostics 2019; 9(10): 2768-2778]; using the same dye, skin invasion of fluorescently-labelled Schistosoma Cercariae has been monitored in human skin explants [Winkel et al., Front Immunol., 2018 Oct 31;9:2510]. [A007] Using 9°"Tc or "In chelating molecules for labelling white blood cells, the presence of such radiolabelled cells in different organs has been utilised to localise and quantify infection in living subjects [Roca et al., Eur. J. Nucl. Med. Mol. Imaging (2010) 37(4): 835-841; De Vries et al. Eur. J. Nucl. Med. Mol. Imaging (2010) 37:842-848].

[3608] Hybrid concepts have been pursued to combine the advantages of fluorescent and radioactive agents via multiplexing. Such agents contain complementary diagnostics labels and can even combine diagnostic with therapeutic labels. A combination of both fluorescent and radioactive labelling approaches is employed in WO2020/074705A1, wherein a prostate-specific membrane antigen (PSMA) targeting compound containing a fluorophore moiety and a moiety that may be radiolabelled is disclosed.

[2003] Such bimodal tracking and imaging agents are particular useful in the emergent, interdisciplinary field of so-called interventional/intraoperative molecular imaging. Interventional molecular imaging technologies are employed, among others, for the management of primary tumours and their metastases. Cancers such as liver cancer and lymphatic and/or liver metastases of various cancers have a relatively high prevalence and treatment is technically difficult. Image-guided resection of the primary tumour as well as, e.g. lymphatic and/or hepatic, metastases is thought to enhance the surgical outcome. One approach to providing image- guided resection involves the provision of molecules that can act as a labelled tracer enabling both pre-operative detection of cancerous material and other lesions, and intra-operative imaging thereof. [33107 In view of the foregoing, it is apparent that there is a general need in the art to develop further and improved agents for the targeting, tracking and/or imaging of biological structures and species, such as hybrid agents that can perform the same functions as ICG but do not suffer from the shortcomings described above and/or have other enhanced features.

[3611] Accordingly, an object of the invention is to provide compounds that can be used for the in vitro an in vivo tracking of cellular processes.

[3312] A further object of the invention is to provide compounds for the targeting and imaging of specific cell components, such as nuclear, cytoplasmic or membrane proteins. In particular, it is an object to provide compounds specifically capable of binding to mitochondria via translocator protein (TSPO), and/or of imaging of mitochondrial activity.

[0014] Another object of the invention is to provide compounds enabling the labelling of (micro)organisms for cell tracking studies, particularly the visualisation of the spatial and temporal dynamics, and/or the quantification of pathogens in living specimens.

[0814] Another object of the invention is to provide compounds that are useful for the assessment of liver function, particularly hepatic metabolism and/or clearance.

[3615] Another object of the invention is to provide compounds capable of enabling intraoperative surgical guidance, for example in the detection and image-guided resection of primary and secondary tumours, in particular for the illumination of liver lesions.

BRIEF SUMMARY OF THE DISCLOSURE

[3318] In one aspect, the invention provides compounds that are useful as tracers. In particular, compounds of the invention combine a targeting moiety (“probe”) with a fluorophore, and with one or more moieties that may be radiolabelled. These compounds, when they comprise a radiolabelled nucleotide, may be considered a hybrid, “bimodal” or “multimodal” tracer, as the probe portion is conjugated to two or more labels that can serve multiple purposes: a fluorophore, as well as a radiolabel.

[3817] Accordingly, in a first aspect, the present invention relates to a compound of formula | or formula Il, or a pharmaceutically acceptable salt, or solvate thereof:

R, Rs R4 Rs

R7 Rio

Rs Rg Rg Re

NZ N

© Cb

Ris Riz 0),

R, R2 Ra R3

R7 Re Rg Rio

Rs Rs 2

NG rr | N

X Jh

Ris Ria (In wherein bridging group L is selected from the group consisting of

NE NENG = NGF

Riz Riz Ris Ris

Ris Ria SE od , , , and

Ris

NF NF wherein

Ri is selected from hydrogen, halogen, C1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine and azide, and R; is selected from hydrogen, halogen, C4-Cs-alkyl, sulfonate, carboxyl, phosphonate, amine and azide; or

Rs and R2 together form an aryl that is optionally substituted with one or two groups each independently selected from C4-Cs-alkyl, halogen, sulfonate, carboxyl, phosphonate, amine and azide;

R3 is selected from hydrogen, halogen, C:1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine 5 and azide, and Ry is selected from hydrogen, halogen, C1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine and azide; or

R3 and Ra together form an aryl that is optionally substituted with one or two groups each independently selected from C4-Cy-alkyl, halogen, sulfonate, carboxyl, phosphonate, amine and azide; each of Rs and Re is independently selected from hydrogen, halogen, C1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine and azide; each of R7, Rs, Re and Rao is independently selected from hydrogen, methyl, ethyl, halogen, sulfonate, carboxyl, phosphonate, amine and azide;

R11 is selected from C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, (CH2).-Y, (CH2)a-NH-Y, (CHs)a-C{=O)-Y, (CH2)a-Z, (CH2)a-NH-Z, (CH2)a-C{(=0)-Z;

R12 is selected from C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, (CHa).-Y, (CH2)a-NH-Y, (CHs)a-C{=O)-Y, (CH2)a-Z, (CH2)a-NH-Z, (CH2)a-C{=0)-Z;

Ris is selected from H, C4-Cs-alkyl, C1-C4-haloalkyl, C1-Cas-alkoxy, C1-C4-haloalkoxy, (CHz)a-Y, (CH2)a-NH-Y, (CH2)a-C(=0)-Y, (CH2)a-Z, (CH2)a-NH-Z, (CH2)a-C(=0)-Z; wherein at least one of R11, R12 and Rus is selected from (CHz)a-Y, (CH2)a-NH-Y, (CH2)-C(=0)-

Y; and wherein no more than two of R11, R12 and R43 are selected from (CHz)}a-Y, (CHz)a-

NH-Y, (CH2)a-C(=0)-Y, (CH2)a-Z, (CH2)s-NH-Z and (CH:).-C(=0)-Z; each Ry, is independently selected from H, C+-Cy-alkyl, C1-Ca4-haloalkyl, C4-Cs-alkoxy, C1-C4- haloalkoxy, (CHz)a-Y, (CH2)a-NH-Y, (CH2)s-C(=0)-Y, (CH2)s-Z, (CH2)a-NH-Z, (CH2).-C(=0)-

Z; wherein at least one R14 is selected from (CHa)a-Y, (CHz)4-NH-Y; and wherein no more than two

R14 are selected from (CH2)a-Y, (CH2)a-NH-Y, (CH2)s-C(=0)-Y, (CH2)s-Z, (CH2)a-NH-Z and (CH2)s-C(=0)-Z; one X is O and each other X is CHR 14;

Y is a chelating moiety;

Z is a therapeutic moiety or H; ais 3to 6; nis 2,3 ord.

[3318] In a second aspect, the present invention relates to a formulation comprising a compound of the invention and optionally a pharmaceutically acceptable carrier. In an embodiment, the compound of the invention is a compound of the first aspect. [G3149] In another aspect, the present invention relates to a use in imaging of a compound of the first aspect, or a formulation of the second aspect. Specifically, the invention relates to a method for imaging a specimen, including a cell, a tissue, a sample, an organ or a subject, comprising - contacting the specimen with a compound of the first aspect or formulation of the second aspect; and - measuring a fluorescent and/or a radioactivity signal emerging from the compound, thereby imaging the specimen. [D020] It was furthermore surprisingly found by the present inventors that the compounds as disclosed herein are rapidly metabolized by hepatocytes and are pharmacologically cleared from the body via the bile /hepatic route. This allows the compound to be used to determine liver function and to visualise hepatic abnormalities (such as tumours or lesions) that affect bile clearance.

[0321] Accordingly, in a further aspect, the present invention relates to a method for determining abnormalities in liver function or bile clearance comprising administering to a subject a compound of the first aspect or formulation of the second aspect, and after a predetermined time imaging the liver. The imaging may be non invasive (e.g. SPECT imaging) and/or performed during surgery (e.g. using a probe configured for fluorescence or radiolabel detection).

[022] As will be demonstrated herein, compared to imaging agents known in the art, such as indocyanine green (ICG), which only allow for the detection of lesions less than 1 cm deep from the surface of the liver, the radioactive signature of the compound of the invention would allow for the identification of significantly deeper lesions. Advantageously, the multimodal tracer compound of the present invention allows far the preoperative localisation and visualisation of such lesions in 3D nuclear medicine scans, which in turn allows surgical planning and intraoperative surgical guidance, for example in the detection and image-guided resection of primary and secondary tumours. [D023] Another aspect of the invention provides a method for imaging a tumour, comprising administering to a subject a compound of the first aspect or formulation of the second aspect, and after a predetermined time imaging the tumour.

[3024] A further aspect provides a compound of the first aspect, or a formulation of the second aspect, for use as a medicament. In an embodiment, the compound comprises a chelated radiolabel. In an embodiment, the compound comprises a therapeutic moiety.. [G25] A further aspect provides a method for the treatment of cancer, comprising administering a compound of the first aspect or a formulation of the second aspect. In an embodiment, the compound comprises a chelated radiolabel and/or a therapeutic moiety.

[0628] A further aspect provides a method for the treatment of a primary or a secondary tumour, comprising administering a compound of the first aspect or a formulation of the second aspect. In an embodiment, the compound comprises a chelated radiolabel and/or a therapeutic moiety. [D027] A further aspect provides a method for the treatment of a primary or a secondary tumour, comprising administering a compound of the first aspect or a formulation of the second aspect. In an embodiment, the compound comprises a chelated radiolabel and/or a therapeutic moiety. [0U28] The invention will now be described further by reference to the following examples and figures. These are not intended to be limiting but merely exemplary of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which;

Figure 1 illustrates a general reaction scheme 1 for the synthesis of exemplary compounds of the invention. Figure 1A displays a synthesis scheme for the dye moieties. Figure 1B displays a synthesis scheme for the chelate moieties. Figure 1C displays a synthesis scheme for the hybrid compounds.

Figure 2 illustrates the chemical structure of the hybrid tracer *"Tc¢-Cy5-Methyl-Amine-

CMAS: (97"Tc-Me-Cy5-MAS:3) which contains a Cy5 dye and a MAS; chelate containing *™Tc.

Figure 3 illustrates the results of an assessment of the in vivo biodistribution of radio- actively labelled *"Tc-Me-Cy5-MAS; between 1 and 24 hours after intravenous tracer administration in mice. Fig 3A shows a quantitative assessment of the uptake per organ (in percentage of the injected dose per gram of tissue; %ID/g). Fig 3B shows in vivo Single Photon

Emission Tomography (SPECT) imaging results obtained at 1, 2, 4 and 24 hours after tracer administration. The inserts at 2 and 24 hours show ex vivo fluorescence imaging of the liver and gall bladder (top) and intestines (bottom; glow scale, photons/sec/cm?).

Figure 4 illustrates the results of cellular uptake assays of Me-Cy5-Me (top), Me-Cy5-

COOH (middle) and Me-Cy5-MAS3 (bottom). The left panels show flow cytometry results, the right panels show fluorescent confocal microscopy images.

Figure 5 shows the results of Me-Cy5-Me (left) and **"Tc-Me-Cy5-MAS; (right) cellular uptake essays in hepatocytes (top panels) and RT4 control cells (bottom panels).

Figure 6 illustrates the results of an assessment of the hepatobiliary clearance of non- radioactively labelled Me-Cy5-MAS: in a porcine model with liver lesions. Fig. 6A shows a representation of the in vivo porcine liver lesion model, with G = gall bladder, L = liver lesion and = intestines, white arrow pointing to liver lesion. Fig. 6B shows in vivo laparoscopic fluorescence imaging at 4 hr after tracer administration, demonstrating the hepatobiliary excretion of Me-Cy5-MAS:. Fig. 6C provides a white-light image of the lesion (left), Cy5 fluorescence image (centre) and the same fluorescence image after image processing along with an intensity-based signal-to-background ratio (scalebar).

Figure 7 provides immunohistochemistry and fluorescence confocal imaging of a heat- induced liver lesion. Fig. 7A shows Haemataxylin and Eosin stain (H&E) staining of an excised liver sample containing healthy liver tissue (shown as **) and a heat-induced liver lesion (encircled). Fig. 7B shows a zoom in of the selected area within the rectangle showing a transitional rim (between dashed lines) between necrotic liver tissue (#) and the healthy liver tissue. Fig. 7C shows fluorescence confocal imaging of this same area with fluorescence uptake in this transitional rim.

Figure 8 illustrates results obtained for fluorescence confocal microscopy of hepatocytes with example compounds. HC04 hepatocytes incubated with Me-Cy5-Mass at A) 10 minutes and

B) 30 minutes incubation, C) after inhibition with the TSPO inhibitor PK11195 or D) in medium without serum. Incubation of hepatocytes with Me-Cy5-Me at E) 10 minutes and F) 30 minutes incubation, G) after inhibition with the TSPO inhibitor PK11195 or H) in medium without serum served as control measurements. | = fluorescence confocal image with tracer uptake (Cy5) in red and the nucleus (Hoechst) in blue. II = 3D representation of the distribution of the fluorescence signal throughout the cell (yellow line = orientation 3D analysis).

Figure 9 illustrates results obtained for fluorescence confocal microscopy of epithelial cells with example compounds. Geb3 epithelial cells incubated with Me-Cy5-Mas3 at A) 10 minutes and B) 30 minutes incubation, C) after inhibition with the TSPO inhibitor PK11195 or D) in medium without serum. Incubation of hepatocytes with Me-Cy5-Me at E) 10 minutes and F) 30 minutes incubation, G) after inhibition with the TSPO inhibitor PK11195 or H) in medium without serum served as control measurements. | = fluorescence confocal image with tracer uptake (Cy5) in red and the nucleus (Hoechst) in blue. Il = 3D representation of the distribution of the fluorescence signal throughout the cell (yellow line = orientation 3D analysis).

Figure 10 provides a quantitative assessment of tracer uptake. Flow cytometry results of uptake of A) Me-Cy5-Mass or B) Me-Cy5-Me after 10 minutes (red) or 30 minutes (orange) of incubation in hepatocytes, C) Me-Cy5-Mas: with (brown) and without (purple) serum and D)

Me-Cy5-Mas3 with (dark green) or without (light green) pre-incubation with the TSPO inhibitor

PK-11195 in hepatocytes. Flow cytometry results of uptake of E) Me-Cy5-Mas3 or F) Me-Cy5-

Me after 10 minutes (red) or 30 minutes (orange) of incubation in epithelial cells, G) Me-Cy5-

Mass with (brown) and without (purple) serum and H) Me-Cy5-Mas: with (dark green) or without (light green) pre-incubation with the TSPO inhibitor PK-11195 in epithelial cells.

DETAILED DESCRIPTION

[3030] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[2031] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[3032] The reader's attention is directed to all papers and documents which are filed concurrently with or before this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

DEFINITIONS

[2633] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

[0034] The invention concerns amongst other things imaging. The term “imaging” includes providing a visual representation of a sample by detecting radiation. The radiation may be a product of radioactive decay of a radiolabel. The radiation may be a product of fluorescence.

The visual representation may be provided by electronic processing of the detected radiation, for example by performing positron emission spectroscopy (PET), single photon emission computed tomography (SPECT), scintigraphy, (optionally intraoperative) gamma- tracing/imaging, (optionally intraoperative) beta-tracing. The visual representation may be provided by visual detection, for example by observing samples that fluoresce at visible wavelengths (e.g. from about 390 to 700 nm) on exposure to higher frequency electromagnetic radiation. The visual representation may be provided by fluorescence spectroscopy. [D035] The invention concerns amongst other things the treatment of a disease. The term “treatment”, and the therapies encompassed by this invention, include the following and combinations thereof: (1) hindering, e.g. delaying initiation and/or progression of, an event, state, disorder or condition, for example arresting, reducing or delaying the development of the event, state, disorder or condition, or a relapse thereof in case of maintenance treatment or secondary prophylaxis, or of at least one clinical or subclinical symptom thereof; (2) preventing or delaying the appearance of clinical symptoms of an event, state, disorder or condition developing in an animal (e.g. human) that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; and/or (3) relieving and/or curing an event, state, disorder or condition (e.g., causing regression of the event, state, disorder or condition or at least one of its clinical or subclinical symptoms, curing a patient or putting a patient into remission). The benefit to a patient to be treated may be either statistically significant or at least perceptible to the patient or to the physician. It will be understood that a medicament will not necessarily produce a clinical effect in each patient to whom it is administered; thus, in any individual patient or even in a particular patient population, a treatment may fail or be successful only in part, and the meanings of the terms “treatment”, “prophylaxis” and “inhibitor” and of cognate terms are to be understood accordingly. The compositions and methods described herein are of use for therapy and/or prophylaxis of the mentioned conditions.

[2036] The term “prophylaxis” or “prevention” includes reference to treatment therapies for the purpose of preserving health or inhibiting or delaying the initiation and/or progression of an event, state, disorder or condition, for example for the purpose of reducing the chance of an event, state, disorder or condition occurring. The outcome of the prophylaxis may be, for example, preservation of health or delaying the initiation and/or progression of an event, state, disorder or condition. It will be recalled that, in any individual patient or even in a particular patient population, a treatment may fail, and this paragraph is to be understood accordingly.

[037] The term “probe” or “targeting moiety” includes reference to a moiety or vector that targets specific cell components, such as nuclear, cytoplasmic or membrane proteins via an affinity type interaction. In an embodiment, the compounds of the invention comprise a moisty specifically capable of imaging translocator protein (TSPO) expression.

[2038] The term “tracer” includes reference to a molecule that comprises a targeting moiety and an imaging label. A tracer may comprise two imaging labels, e.g. as a hybrid tracer with two different types of imaging label. For example, in a hybrid tracer, a probe portion may be conjugated to two labels that can serve a complementary purpose: a fluorophore label and a radiolabel.

[0439] The term “alkyl” as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The term includes reference to, for example, methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl or tert-butyl), pentyl, hexyl and the like. In particular, alkyl may be a “C4-Cs alkyl”, i.e. an alkyl having 1, 2, 3 or 4 carbon atoms; or a “C+-Ce alkyl”, i.e. an alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms; or a “C+-

Csalkyl”, i.e. an alkyl having 1, 2 or 3 carbon atoms. The term “lower alkyl” includes reference to alkyl groups having 1, 2, 3 or 4 carbon atoms. [D040] The term “cycloalkyl” as used herein includes reference to an alicyclic moiety having 3, 4,5 or 6 carbon atoms. The group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

[0041] The term "heterocycloalkyl" as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus and sulphur. For example, a heterocycolalkyl may comprise 3, 4, or 5 ring carbon atoms and 1 or 2 ring heteroatoms selected from nitrogen and oxygen. The group may be a polycyclic ring system but more often is monocyclic. This term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolizidinyl and the like.

[2042] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings {preferably from 1 to 3 rings) which are fused together or linked covalently. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1- naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2- pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. “Arylene” and “heteroarylene” refers to a divalent radical derived from an aryl and heteroaryl, respectively. [G33] The term “alkoxy” as used herein include reference to -O-alkyl, wherein alkyl is straight or branched chain and comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1, 2, 3 or 4 carbon atoms, e.g. 1, 2 or 3 carbon atoms. This term includes reference to, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like. The term “lower alkoxy” includes reference to alkoxy groups having 1, 2, 3 or 4 carbon atoms. An alkoxy group, in particular a lower alkoxy (e.g. an alkoxy having 2 carbon atoms), may be provided as a polyalkoxy, i.e. as a linear or branched (e.g. as a linear) chain of repeating alkoxy units.

[0044] The term “substituted” as used herein in reference to a moiety means that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described substituents. Unless otherwise specified, exemplary substituents include —OH, -CN, -NH,, =O, -halo, -C4-Cs alkyl, -

C2-Cs alkenyl, -C+-Cs haloalkyl, -C4-Cs haloalkoxy and-C2-Cs haloalkenyl, -C1-Cs alkylcarboxylic acid (e.g. -CH3COOH or -COOH). Where the substituent is a -C4-Csg alkyl or -C+-Cs haloalkyl, the C1-Cs chain is optionally interrupted by an ether linkage (-O-) or an ester linkage (-C(O)O-).

Exemplary substituents for a substituted alkyl may include —OH, -CN, -NHz, =O, -halo, -CO:H, -

C1-Cs haloalkyl, -C1-Cs haloalkoxy and-C2-Cshaloalkenyl, -C1-Cs alkylcarboxylic acid (e.g. —

CH3COOH or -COOH). For example, exemplary substituents for an alkyl may include —OH, -CN, -NH2, =O, -halo.

[0045] It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible. For example, amino or hydroxy groups with free hydrogen may be unstable if bound to carbon atoms with unsaturated (e.g. olefinic) bonds. Additionally, it will of course be understood that the substituents described herein may themselves be substituted by any substituent, subject to the aforementioned restriction to appropriate substitutions as recognised by the skilled person.

[3348] Where steric issues determine placement of substituents on a group, the isomer having the lowest conformational energy may be preferred. For example, the preferred state for carbocyanines (e.g. Cy-dyes) may be the all-trans conformation, as this represents the ground state (see, e.g., A.M. Kolesnikov and E.A. Mikhailenko, Russian Chemical Reviews, (1987), 56, 275-287; W. West et al., Journal of Physical Chemistry, (1967), 71, 1316-1326; and P.J.

Wheatley, Journal of the Chemical Society, (1959), 4096-4100).

[3047] Where a compound, moiety, process or product is described as “optionally” having a feature, the disclosure includes such a compound, moiety, process or product having that feature and also such a compound, moiety, process or product not having that feature. Thus, when a moiety is described as “optionally substituted”, the disclosure comprises the unsubstituted moiety and the substituted moiety.

[3348] Where two or more moieties are described as being “independently” or “each independently” selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties.

[2043] The term “pharmaceutically acceptable” as used herein includes reference to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This term includes acceptability for both human and veterinary purposes.

[0650] The term “pharmaceutically acceptable salts” is meant to include salts of the compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dinydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[3051] Thus, the compounds of the present invention may exist as salts with pharmaceutically acceptable acids. The present invention includes such salts. Examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates, (-)-tartrates or mixtures thereof including racemic mixtures, succinates, benzoates and salts with amino acids such as glutamic acid. These salts may be prepared by methods known to those skilled in the art.

[052] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those which are known in the art to be too unstable to synthesize and/or isolate.

[0053] The term “chelating moiety” includes reference to a residue of a chelating agent such as mercaptoacetyltriglycine (MAG3), S-acetylmercaptoacetyltriserine (MAS3), bis(carboxymethyl)- 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl}-methylbenzoic acid (CPTA), N'-[5- [acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy )amino]-4-oxobutanoyllamino] pentyl]-N-hydroxybutandiamide (DFO), 4,11-bis{carboxymethyl}-1,4,8,11-tetraazabicyclo[6.6.2]- hexadecan (DO2A), 1,4,7,10-tetraazacyclododecan-N,N',N",N"-tetraacetic acid (DOTA), 2- [1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-pentanedioic acid (DOTAGA), N,N'- dipyridoxylethylendiamine-N,N'-diacetate-5,5'-bis(phosphat) (DPDP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethyleneglykol-O,0-bis(2-amincethyl)-N,N,N’,N'-tetraacetic acid (EGTA), N,N- bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate (HP-DOA3), 6-

hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), 1,4,7-triazacyclononan-1-succinic acid- 4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7- triazacyclononane (NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA), 4,11- bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (TE2A), 1,4,8,11- tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), terpyridin- bis(methyleneamintetraacetic acid (TMT), 1,4,7,10-tetraazacyclo-tridecan-N,N',N",N"'- tetraacetic acid (TRITA), triethylenetetraaminehexaacetic acid (TTHA), N,N'-bis[(6-carboxy-2- pyridyl)methyl]-4,13-diaza-18-crown-6 (H2macropa), and 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl- 4-ox0-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl} heptanedioic acid bis-[(3-hydroxy-1,6- dimethyl-4-oxo-1,4-dihydro-pyridin- 2-ylmethyl)-amide] (THP). Such a residue is obtainable by covalently binding a carboxyl group contained in the chelating agent to the remainder of the compound via an ester or an amide bond, preferably an amide bond.

[0054] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may have enriched levels (e.g. at least 50% or at least 75%) of stable isotopes, such as deuterium (2H) or carbon-13 (°C), at one or more atoms of the compound. For example, the compounds may comprise radioactive isotopes, such as for example tritium (3H), iodine-125 (1251) or carbon-14 (™C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention. [D058] The term “radiolabel” as used herein refers to a radioactive isotope that readily forms a cation. Exemplary radiolabels include *Sc, Sc, *'Cr, "Mn, %8Co, $2Fe, ®Ni, 5Ni, 82Cu, Cu, 57Cu, 86Ga, 88Ga, 67Ga, 897r, soy, 89y, MTC, mT Ru, 105Rh, pg, Ag, 110m, "Mn, 113m]n amp, 17mSn, 1218p, 17Te, 12pr, 143pr 149pm, 51Pm, 149Th, 538m, 157Gd, 181Tb, Ho, 185Dy, 169Er, 169Yb, 75yp, 12Tm, 17u, Re, Re, = 17Hg, 8A, 198A, 22pb, 23pp, 21At, 212g; 213Bi, 223R3, 225Ac, and 22"Th, or a cationic molecule comprising 13F, such as 18F-[AIF]2*.

Exemplary radiolabels also include “Sc, Sc, Cu, “Cu, %Ga, 9°Y, "In, 181Tb, "Ho, "Lu, 188Re, 212pp, 212Bi, 213Bj, 225Ac, and ?2’Th, or a cationic molecule comprising '8F. Preferred radiolabels may include $3Ga, 9°Y, 177Lu, 212Bi, and 213Bi A radiolabel may be a y-emitter, i.e. a radiolabel that when it decays emits y radiation; a B-emitter, i.e. a radiolabel that when it decays and emits a B particle; or and a-emitter, i.e. a radiolabel that when it decays emits an a particle.

Exemplary y-emitters include **™Tc and "In. Exemplary B-emitters include *°Y, '®Ho and Ga, 177Lu. Exemplary a-emitters include 225Ac, 22Ra and #'*Bi. A “chelated radiolabel” represents a radiolabel (e.g. radioactive cation) that is complexed with a multidentate ligand. The chelating moieties described herein represent multidentate ligands of use in accordance with the present disclosure.

[0056] Exemplary multidentate ligands of use in accordance with the present disclosure include -MAS3, -MAGs3;, -DOTA-GA, -DOTA, and -DTPA. MAS: is a preferred multidentate ligand.

[2057] The term “pharmaceutical formulation” as used herein includes reference to a formulation comprising at least one active compound and optionally one or more additional pharmaceutically acceptable ingredients, for example a pharmaceutically acceptable carrier.

Where a pharmaceutical formulation comprises two or more active compounds, or comprises at least one active compound and one or more additional pharmaceutically acceptable ingredients, the pharmaceutical formulation is also a pharmaceutical composition. Unless the context indicates otherwise, all references to a “formulation” herein are references to a pharmaceutical formulation.

[3058] The term “product” or “product of the invention” as used herein includes reference to any product containing a compound of the present invention. In particular, the term product relates to compositions and formulations containing a compound of the present invention, such as a pharmaceutical composition, for example.

[0059] The term “therapeutically effective amount” as used herein refers to an amount of a drug, or pharmaceutical agent that, within the scope of sound pharmacological judgment, is calculated to (or will) provide a desired therapeutic response in a mammal (animal or human).

The therapeutic response may for example serve to cure, delay the progression of or prevent a disease, disorder or condition.

[0080] The term “imaging effective amount” as used herein refers to an amount of a compound, or formulation, that within sound clinical or experimental experience, is calculated to (or will) provide a sufficient response for imaging, e.g. based on detection of radioactive decay or fluorescence. Compounds or formulations used for imaging may be provided in an imaging effective amount.

COMPOUNDS

[2061] In one aspect, the invention provides compounds of formula | or formula Il as previously described or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof. In embodiments, one or more of L, Rs, Ro, Rs, Ra, Rs, Re, Rz, Rs, Rs, Rig, R11, R12, R13, Rus, Ris, X,

Y, Z, a, and n are as described in the following paragraphs: iti IN NNN

[0062] L may be selected from the group consisting of ANF A , = NINE Ris Riz Ris

R13 Riz os FA ee = = = z=

Ris + 3 3

SE R13 geet A and . L may be selected from

PN = A and =~ : for example L may be

ZN AA a eN

ASD of AS . L may be selected from Ris Ris 6 = ZZ

Ria and Ris ; for example L may be Ria ;

R13

Ris , or Ria . L may be selected from ,

R43 Ris R13

AP Cr = = = ' and ‚ for

Ris rr example, L may be .

[0063] L may be NN = or = A . For example, L

ZZ maybe NTN [D064] R:1 may be selected from hydrogen, halogen, C1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine and azide. R; may be H, methyl or ethyl. Ry may be H. [D065] Re may be selected from hydrogen, halogen, C1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine and azide. R2 may be H, methyl or ethyl. R1 may be H.

[0088] Rs and R2 together may form an aryl that is optionally substituted with one or two groups each independently selected from C4-Cs-alkyl, halogen, sulfonate, carboxyl, phosphonate, amine and azide. Rs and R2 together may form an aryl that is substituted with one group selected from C:1-C4-alkyl, halogen, sulfonate, carboxyl, phosphonate, amine and azide. Rt and

R2 together may form an unsubstituted aryl. The aryl may be or comprise phenyl. The aryl may be or comprise napthyl.

[3067] Rs may be selected from hydrogen, halogen, C1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine and azide. Rs may be H, methyl or ethyl. Rs may be H.

[2068] R4 may be selected from hydrogen, halogen, C1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine and azide. R4 may be H, methyl or ethyl. R4 may be H.

[2065] Rs and Rs together may form an aryl that is optionally substituted with one or two groups each independently selected from C4-Cs-alkyl, halogen, sulfonate, carboxyl, phosphonate, amine and azide. Rs and R4 together may form an aryl that is substituted with one group selected from C1-C4-alkyl, halogen, sulfonate, carboxyl, phosphonate, amine and azide. R3 and

Ra together may form an unsubstituted aryl. The aryl may be or comprise phenyl. The aryl may be or comprise napthyl.

[0070] At least 1 of Ry and R2 may be other than H. At least 1 of Rs and Ra4 may be other than

H. At least one of R+, Rs, Rs and Rs may be other than H.

[2071] Each of Rs and Rs may be independently selected from hydrogen, halogen, C+-Cs-alkyl, sulfonate, carboxyl, phosphonate, amine and azide. Each of Rs and Rs may be independently selected from hydrogen, halogen, methyl, ethyl, sulfonate, carboxyl, phosphonate, amine and azide. Each of Rs and Rs may be independently selected from CH3, CHsCHs and H. Each of Rs and Rs may be independently selected from CHs and CH2CHs. Each of Rs and Rs may be CHa.

Each of Rs and Rs may be CH2CHs. Each of Rs and Rs may be H.

[0072] Each of Ry, Rs, Re and Rio may be independently selected from hydrogen, methyl, ethyl, halogen, sulfonate, carboxyl, phosphonate, amine and azide. Each of R7, Rs, Rs and Rio may be independently selected from CH3, CH2CH3 and H. Each of R7, Rs, Rs and Rig may be independently selected from CH3 and CH2CHs. Each of R7, Rs, Rs and Ric may be CHa. Each of

R7, Rs, Rs and Ri may be CHsCHs. Each of Ry, Rs, Rs and Rie may be H.

[0073] R1 may be selected from C+-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, (CHz)a-Y, (CH2)a-NH-Y, (CHz)a-C{3O)-Y, (CH2)s-Z, (CH2)s-NH-Z, (CH.).-C(=0)-Z.

[2074] R12 may be selected from C+-C+-alkyl, C1-Cs-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, (CH2)a-Y, (CH2)a-NH-Y, (CH2}a-C{=0O)-Y, (CH2)s-Z, (CH2)s-NH-Z, (CH2)a-C(=0)-Z. [D075] R13 may be selected from C+-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-Cs-haloalkoxy, (CHa)a-Y, (CH2)a-NH-Y, (CHz)a-C(=0)-Y, (CH2)s-Z, (CH2)a-NH-Z, (CH2)a-C(=0)-Z. Riz may be H.

[0678] At least one of R11, R12 and Rs is selected from (CHz)a-Y, (CH2)s-NH-Y and (CH2).-

C(=0)-Y, and no more than two of R11, R12 and Rs are selected from (CHz)a-Y, (CH2)a-NH-Y, (CH2)a-C(=0})-Y, (CHz)a-Z, (CH2)s-NH-Z and (CH.).-C(=0)-Z.

[3077] In an embodiment, one of R11, Riz and Ris is selected from (CH2)a-Y, (CHz)a-NH-Y, (CH2).-C(=0)-Y, and the others of R11, R12 and Ris are independently selected from C-Cs-alkyl,

C1-Cs-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy. For example, R11 may be selected from (CH2)a-Y, (CH2)a-NH-Y, (CH2).-C(=0)-Y, while R12 (and, if present R13) may be independently selected from C4-Cs-alkyl, C1-Cs-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy. In another example,

R12 may be selected from (CHz}a-Y, (CH2)a~-NH-Y, (CH2)s-C(=0)-Y, while R+: (and, if present R13) may be independently selected from C+-Cs-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4- haloalkoxy. [GGTH] One of R11, Riz and R13 may be (CH2)a-NH-Y or (CHz)a-C(=0)-Y, and the others of Ruy,

R+ and Riz may be independently selected from C+-Cs-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, Ci-

Cas-haloalkoxy. For example, R11 may be (CHz).-C(=0O)-Y or (CHz)}a-NH-Y, while R42 (and, if present R13) may be independently selected from C+-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, Ci-

C4-haloalkoxy. In another example, R12 may be selected from (CH:)a-NH-Y, while R41 (and, if present R13) may be independently selected from C4-Cs-alkyl, C1-C4-haloalkyl, C4-Cs-alkoxy, Ci-

C4-haloalkoxy.

[3379] One of R1 or R12 may be methyl and Rs (if present) may be H. R11 may be methyl. R12 may be methyl,

[0289] Each R44 may be independently selected from H, C1-Ca4-alkyl, C1-C4-haloalkyl, C1-Ca- alkoxy, Ci-Cs-haloalkoxy, (CHz)a-Y, (CH2)a-NH-Y, (CH2)-C(=0)-Y, (CH2)s-Z, (CH2)a-NH-Z, (CH2)s-C(=0)-Z. For example, one R14 may be selected from (CHz)a-Y, (CHz2)a-NH-Y, (CHs)s-

C(=0)-Y; another R14 may be selected from H, C+-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4 haloalkoxy, (CHz)s-Y, (CHz2)a-NH-Y, (CH.)s-C(=0}-Y, (CHs)a-Z, (CH2)a-NH-Z, (CH2).-C(=0)-Z; and any further R14 may be independently selected from H, C+-C4-alkyl, C1-Cs-haloalkyl, C1-C4- alkoxy, and C4-Cs-haloalkoxy. One Rs may be selected from (CHaz)a-Y, (CHz)a-NH-Y, (CHz)a-

C(=0)-Y; and each of the other R14 may be independently selected from H, C4-Cs-alkyl, C1-Cs- haloalkyl, C1-Cs-alkoxy, and C1-C4-haloalkoxy.

[3081] One R14 may be selected from (CHs)a-Y, (CH2)s-NH-Y, (CH2)s-C(=0)-Y; another R14 may be selected from H, C4-Cs-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, (CH2)2-Z, (CH2)a-NH-Z, (CH2)a-C(=0)-Z; and any further R14 may be independently selected from H, C4-

Csalkyl, C1-C4-haloalkyl, C1-C4-alkoxy, and C1-C4-haloalkoxy. One R14 may be selected from (CH2)a-Y, (CH2)a-NH-Y, (CHz)a-C{=0)-Y; and each of the other R14 may be independently selected from H, C-Cys-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, and C1-C4-haloalkoxy.

[3082] One Ris may be (CH:z)s-C{=O)-Y or (CH:)a-NH-Y; another R14 may be selected from H,

C+-C--alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, (CHz)a-Y, (CH2)a-NH-Y, (CHz)a-

C(=0)-Y, (CH2)a-Z, (CH2)a-NH-Z, (CHz)a-C{=0)-Z; and any further R14 may be independently selected from H, C4-Cs-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, and C4-Cs-haloalkoxy. One R14 may be (CH.).-C(=0)-Y or (CH2).-NH-Y; another R14 may be selected from H, C1-C4-alkyl, C1-C4- haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy, (CHz)s-Z, (CH2)a-NH-Z, (CH2).-C(=0)-Z; and any further R14 may be independently selected from H, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy, and C4-Cs-haloalkoxy. One R14 may be (CH2).-NH-Y; and each of the other R14 may be independently selected from H, C4-C4-alkyl, C1-C4-haloalkyl, C4-C4-alkoxy, and C1-C4- haloalkoxy.

[083] a may be 3, 4, 5 or 6. a may be 3. a may be 4.

[0084] n may be 2, 3 or 4. n may be 2 or 3. n may be 2. n may be 3. [C&85] One X may be O and each other X may be CHR14, such that the compounds of formula

Il comprise one oxygen containing heterocyclic ring. The additional ring structures in the compounds of formula Il compared to the compounds of formula | provide conformation restraint. Without wishing to be bound by any theory, this conformational restrain may provide improved fluorescence quantum yield, as demonstrated for conformationally restrained cyanine fluorophores in M.S Michie, et al., J. Am. Chem. Soc. (2017) 139, 12406-12409.

[0088] Y may be a chelating moiety that is a residue of a chelating agent as defined herein. For example, Y may be or comprise a residue of mercaptoacetyltriglycine (MAG3), S- acetylmercaptoacetyltriserine (MAS3), bis(carboxymethyl)-1,4,8,11- tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4- (1,4,8,11-tetraazacyclotetradec-1-yl}-methylbenzoic acid (CPTA), N'-[5- [acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]lamino] pentyl]-N-hydroxybutandiamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]- hexadecan (DO2A), 1,4,7,10-tetraazacyclododecan-N,N',N",N"-tetraacetic acid (DOTA), 2- [1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-pentanedioic acid (DOTAGA), N,N'- dipyridoxylethylendiamine-N,N'-diacetate-5,5'-bis(phosphat) (DPDP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethyleneglykol-O,0-bis(2-amincethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N- bis(hydroxybenzyl}-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate (HP-DOAS3), 6- hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), 1,4,7-triazacyclononan-1-succinic acid- 4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7- triazacyclononane (NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA), 4,11- bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (TE2A), 1,4,8,11- tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), terpyridin- bis(methyleneamintetraacetic acid (TMT), 1,4,7,10-tetraazacyclo-tridecan-N,N',N",N"'- tetraacetic acid (TRITA), triethylenetetraaminehexaacetic acid (TTHA), N,N'-bis[(6-carboxy-2-

pyridyl)methyl]-4,13-diaza-18-crown-6 (Hzmacropa), and 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl- 4-oxo-1,4-dihydro-pyridin-2-ylmethyl)-carbamoyl]-ethyl} heptanedioic acid bis-[(3-hydroxy-1,6- dimethyl-4-oxo-1,4-dihydro-pyridin- 2-ylmethyl)-amide] (THP).

[2687] Y may be -MAS;3, -MAG3, -DOTA-GA, -DOTA, -DTPA, wherein MAS3, MAG3, DOTA-GA,

DOTA, DTPA are as defined in Table 1. Y may be MAS:.

[3088] Z may be a therapeutic moiety. Examples of therapeutic moieties are anticancer molecules including chemotherapeutics (e.g. doxorubicin), checkpoint inhibitors (ipilimumab, nivolumab); immune suppressive moieties, e.g. for use after liver transplant or in auto-immune hepatitis; TNFa inhibitors; antiviral compounds; immune modulators; and toxicant antidotes such as N-acetyl-cysteine to combat paracetamol intoxication.

[0483] In an embodiment, R+, R2, Rs, Ra, Rs and Re are each hydrogen, Ry, Rs, Rs and Ryo are each methyl, R11 is methyl, Riz is (CHz)4-NH-MAS: and

Lis ZF

[2080] In an embodiment, R1, Rz, Rs, R4, Rs and Rs are each hydrogen, Rz, Rs, Rs and Rio are each methyl, R11 is methyl, Riz is (CH2)4-NH-MAS:3 and

Lis

[9991] In an embodiment, R+, Ra, Rs, R4, Rs and Rs are each hydrogen, R7, Rs, Rs and Ryo are each methyl, R+: is methyl, Riz is (CH2)4-NH-MAG: and =

Lis NFN.

[0082] In an embodiment, R, R2, Rs, R4, Rs and Rs are each hydrogen, Ry, Rs, Re and Ryo are each methyl, R11 is methyl, Riz is (CH2)}4-NH-MAG:3 and

Lis — ANF :

[0683] In the above, “(CH2)4-NH-MAS:" refers to a amide bond between the methylene bridge and the terminal carboxy groups of the chelating moiety residue.

[0084] Table 1: Exemplary substituents (chelating moieties) Y; “R-NH" or “"R-(C=0)" depicts covalent bonding with an amide or ester bond to the remainder of the compound through methylene bridge (CH). as defined herein

MAS; o HO o

H H

R. GAN

Aal L Tol oh,

H z H =

HOT 0 Sore

MAG; 0 0

H H rR RL oo ie re 0 0

DOTA-GA HO a OH \ 0 0 Ö

N N

OH

RO

DOTA 0

Aon

N

N

L N

NT Jon

Ho. 0

DTPA R

No

HO

TANT” 0 0 oy 0

OH OH

5 [0085] The compound may be a compound selected from the compounds exemplified in Table 2.

[086] Table 2: Exemplary compounds =

NY NEN N

/ LL ny (R) (BR) © ne (R) + "NH bd 0 s—( 0 mm

NY IONE N

/ ny ne (R)

HO Pl (R) © ne (B) « “NH oH? 0

Oo

BA

NY NEN N

/ LL { y © in {

NH

AT

SC

Q

Me-Cy7-MAG3

NY LN NEN N

/ L in”

NH

1 oo ad

NH en

O

[3097] The compound may comprise a chelated radiolabel. The chelated radiolabel may be selected from a y-emitter (e.g. 9°7Tc, In), a B-emitter (e.g. *Y, **Ho, %8Ga, 177Lu, ®Re), and an a-emitter (e.g. 225Ac, Ra, 213Bi), or a combination thereof. The chelated radiolabel may be 5 a y-emitter; e.g. *™Tc, "In, or a combination thereof. The chelated radiolabel may be a p- emitter; e.g. °9Y, "%®Ho, Ga, "Lu, or a combination thereof. The chelated radiolabel may be an a-emitter; e.g. 225Ac, #**Ra, 213Bi, or a combination thereof.

[0028] Exemplary compounds are provided in Table 3.

Table 3: Exemplary compounds with chelated radiolabel mT c-Me-Cy5-MAS:

NN NEN N

/ au

HN-° OOH

HO N g--%mp--N a

N

0 OH 99mTc-Me-Cy7-MAS:

NAE SENS SN

/ a

HN—° © OH

HO” N s--%my--N i (R/=O

LA

O OH mT c-Me-Cy5-MAG:

NES TN

/ nu we” Q oo 99m. _-N

S-- Me

Ln

Oo

¥mTc-Me-Cy7-MAG; Ot xD

N” = NGF TN / au ne” 9 99m LN

S-- Te

Li ©

FORMULATIONS AND ADMINISTRATION

[3099] Compounds of the invention may be administered parenterally, for example the compounds may be administered intravenously. Compounds of the invention may be administered enterally, for example by feeding small organisms a composition comprising a compound of the invention. The compounds may be administered in the form of pharmaceutical preparations comprising the compound either as a free compound or, for example, a pharmaceutically acceptable non-toxic organic or inorganic acid or base addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and imaging requirements, the compositions may be administered at varying doses.

[26108] According to a further aspect of the invention there is thus provided a pharmaceutical formulation or composition including a compound of the invention, optionally in admixture with a pharmaceutically acceptable adjuvant, diluents or carrier. The formulation or composition may also comprise a compound that influences in vivo kinetics the compound of the invention. The formulation may also comprise a compound that reduces kidney retention of the compound of the invention.

[33101] Pharmaceutical formulations or compositions of this invention for injection may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols {such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters, such as ethyl oleate.

[00102] These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Inhibition of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben,

chlorobutanol or phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars or sodium chloride, for example.

[20103] The formulations according to the present subject matter may also contain inactive components. Suitable inactive components are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of

Therapeutics, 8"Ed., Gilman et al, Eds. Pergamon Press (1990), and Remington's

Pharmaceutical Sciences, 17" Ed., Mack Publishing Co., Easton, Pa. (1990), both of which are incorporated by reference herein in their entirety.

[09104] The formulations may be used in combination with an additional pharmaceutical dosage form to enhance their effectiveness in treating any of the disorders described herein. In this regard, the present formulations may be administered as part of a regimen additionally including any other pharmaceutical and/or pharmaceutical dosage form known in the art as effective for the treatment of any of these disorders.

IMAGING AND OTHER USES

[20105] Compounds of the invention are tracers that are useful for imaging. In particular, compounds of the invention, e.g. of formula | or formula Il, are useful for imaging.

[03106] An aspect provides a method for imaging a specimen, comprising - contacting the specimen with a compound of the first aspect; and - measuring a fluorescent and/or a radioactivity signal emerging from the compound, thereby imaging the specimen.

[09107] Typically, the method comprises contacting the specimen with a detectably sufficient amount of a compound of the first aspect, and after a predetermined time imaging the specimen.

[03108] The specimen may comprise a sample, a cell, a tissue, an organ, or an organism, or a combination thereof.

[09103] The compound of the invention may be a compound of formula | and/or a compound of formula ll. The predetermined time may be a predetermined time prior to intraoperative imaging.

In some instances, the imaging could be dynamic, in which case the predetermined time may be as low as 0 hours. The predetermined time, e.g. prior to intraoperative imaging may be at least 0 hours, at least 0.25 hours, at least 0.5 hours, or at least 1 hour. The predetermined time, e.g. prior to intraoperative imaging is at least about 0.5 and not more than about 48 hours. For example, the predetermined time may be at least about 1 (or about 2) and not more than about

36 hours; e.g. the predetermined time may be at least about 3 and not more than about 24 hours.

[00118] The compound (e.g. compound of formula | or compound of formula II) may comprise a chelated radiolabel and the imaging may comprise imaging of radioactive decay. The imaging of radioactive decay may comprise at least one of positron emission tomography (PET), single photon emission computed tomography (SPECT), scintigraphy, gamma-tracing/imaging, beta- tracing, intraoperative gamma-tracing/imaging, or intraoperative beta-tracing. The imaging may comprise positron emission spectroscopy (PET), single photon emission computed tomography (SPECT), scintigraphy, (intraoperative) gamma-tracing/imaging, or (intraoperative) beta-tracing.

For example, the imaging may comprise PET or SPECT. The imaging may comprise PET. The imaging may comprise SPECT.

[33111] The imaging may comprise fluorescence imaging. The imaging may comprise fluorescence spectroscopy.

[20112] The imaging may comprise imaging of radioactive decay and fluorescence imaging.

The imaging may comprise imaging of radioactive decay prior to fluorescence imaging. For example, the imaging of radioactive decay may be performed before surgery and the fluorescence imaging may be performed during surgery.

[03113] Compounds of the invention and formulations of the invention may further be used for determining liver function in a subject. [UD114] Furthermore, compounds of the invention and formulations of the invention may be used for imaging of liver lesions. For example, the compounds of the invention and formulations of the invention may be used in hepatobiliary surgery.

[00175] Figure 3 illustrates the results of an assessment of the in vivo biodistribution of radio- actively labelled ®"Tc-Me-Cy5-MAS; between 1 and 24 hours after intravenous tracer administration in mice. Fig 3A shows a quantitative assessment of the uptake per organ (in percentage of the injected dose per gram of tissue; %ID/g). These data reveal mainly hepatobiliary clearance with high levels of uptake in the liver and gallbladder, and subsequently in the intestines at 2 hours post tracer administration. At 24 hours uptake levels are significantly reduced, underlining overall excretion. Fig 3B shows in vivo Single Photon Emission

Tomography (SPECT) imaging results obtained at 1, 2, 4 and 24 hours after tracer administration. The inserts at 2 and 24 hours show ex vivo fluorescence imaging of the liver and gall bladder (top) and intestines (bottom; glow scale, photons/sec/cm?), demonstrating the hepatobiliary clearance over time, with the highest fluorescence intensity present in the gallbladder and intestines at 2 hours post tracer administration. [D6118] Figure 4 illustrates the results of cellular uptake assays of Me-Cy5-Me (top), Me-Cy5-

COOH (middle) and Me-Cy5-MAS:3 (bottom). The left panels show flow cytometry results, the right panels show fluorescent confocal microscopy images. The fluorescence confocal microscopy reveals clear mitochondrial uptake, showing a comparable uptake pattern in different cell lines. Furthermore, while the location of uptake is identical for all compounds, a clear concentration dependent uptake relation is observed; the non-functionalized Me-Cy5-Me dye can already be effectively used at 10nm, while addition of additional additives (COOH- group or the larger MAS: chelate) required higher tracer concentrations in order to achieve similar imaging results. Flow cytometry reveals a similar concentration- and size dependent relationship.

[00117] Fig. 5 shows the results of Me-Cy5-Me (left) and 99"Tc-Me-Cy5-MAS:s (right) cellular uptake essays in hepatocytes (top panels) and RT4 control cells (bottom panels). Whereas the

RT4 controls cells yield characteristic mitochondrial staining that could be clearly differentiated from the lysosomes, within the cytosol of the hepatocytes the patchy cytosolic staining can be seen to overlay with the lysosomes. In addition, the cellular interfaces between the hepatocytes shows fluorescent hotspots (see arrows in image), suggesting that 59"Tc-Methyl-Cy5-mas3 is being excreted by the cells.

[40118] Fig. 6B shows in vivo laparoscopic fluorescence imaging at 4 hr after tracer administration, demonstrating the hepatobiliary excretion of Me-Cy5-MAS:.

[03119] Compounds of the invention and formulations of the invention may also be used for the detection and/or treatment of a disease, in particular a disease relating to hepatocytes or hepatobiliary excretion, such as hepatitis or liver cancer.

[00120] Such compounds (or formulations comprising such compounds) may comprise a radiolabel as disclosed herein. For example, the radiolabel may be selected from a B-emitter (e.g. %0Y, "®Ho, %Ga, 177Lu), and an a-emitter (e.g. 223Ac, ***Ra, 213Bi). The compounds provide hybrid tracers that may target TSPO and when said compounds comprise a radiolabel that is a

B-emitter and / or an a-emitter, the compound may be useful in radiotherapy. In such therapeutic applications, the main role of the dye portion may be to provide favourable pharmacokinetics (e.g. by extending the circulation half-life of the compound, prolonging the time window for binding to TSPO).

[00133] An aspect provides a compound of the invention or formulation of the invention for use as a medicament. A related aspect a compound of the invention or formulation of the invention for use in the treatment of cancer. The cancer may be a cancer comprising hepatic cells.

[0122] An aspect provides a method for the treatment of cancer, comprising administering a compound of the invention or formulation of the invention to a patient in need of treatment. The cancer may be a cancer comprising hepatic cells.

EXAMPLES

Example 1: Synthesis of exemplary compounds

General

[03123] All chemicals and solvents were obtained from commercial sources and used without further purification. Dimethylformamide (DMF), dichloromethane (DCM) and dimethylsulfoxide (DMSO) were dried using 4 A molecular sieves, unless stated otherwise. Column chromatography was performed using 40-63 um silica from Screening Devices (Amersfoort,

The Netherlands). Dry column vacuum chromatography (DCVC) was performed as published by

Pedersen et al. (http://dx.doi.org/10.1055/s-2001-18722) using 15-40 um silica and Hyflo

Supercell Celite (Fisher Scientific, Waltham, USA). High-performance liquid chromatography (HPLC) was performed using a Waters HPLC system using either a 1525EF or 2545 pump and a 2489 UV/VIS detector. For preparative HPLC either a Dr. Maisch GmbH Reprosil-Pur 120

C18-AQ 10 um (250 x 20 mm) column or an XBridge Prep C8 10 um OBD (250 x 30 mm) column was used. For semi-preparative HPLC a Dr. Maisch GmbH Reprosil-Pur C18-AQ 10 um (250 x 10 mm) column was used. For analytical HPLC a Dr. Maisch GmbH Reprosil-Pur C18-

AQ 5 um (250 x 4.6 mm) column was used. High-resolution mass spectrometry (HRMS) was performed on a Waters Acquity H-class UPLC (Waters, Waltham, USA) using a Acquity UPLC

BEH C18 1.7 um (2.1 x 50 mm) column coupled to a high-resolution XEVO G2S-XTOF Mass

Spectrometer (Waters, Waltham, USA). Low-resolution mass spectrometry (LRMS) was performed using a Bruker Microflex matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer. 'H and *C NMR were performed on a Bruker Ascend 850 (850 MHz) equipped with a CryoProbe (all from Bruker, Waltham, USA) in deuterated solvents.

Absorbance spectra were recorded using an Ultrospec 2100 pro (Amersham Biosciences, Little

Chalfont, UK) or a Shimadzu UV-1280 (Shimadzu, Kyoto, Japan). Fluorescence spectra were recorded using a PerkinElmer LS-55 (Perkin Elmer, Waltham, USA).

Mercaptoacetyltriserine (MAS) 0 H dios hoo

It

HO OH

[20124] Mass was synthesized using Fmoc-D-Ser-WANG resin (Iris Biotech, Marktredwitz,

Germany) and standard Fmoc-based SPPS procedures using two cycles of Fmoc-D-(Ser(tBu)-

OH (4 eq), PyBOP (4 eq), and N-methylmorpholine (NMM) (5 eq) at room temperature (RT) for 2 h and one cycle of s-acetylthioacetic acid (4 eq), PyBOP (4 eq) and N-methylmorpholine (5 eq) at RT for 2 h. The resin was cleaved by agitating with TFA/H20 (95:5; 20 mL) for 90 minutes followed by precipitating in ice cold 1:1 MTBE/Et:O (300 mL). The precipitate was centrifuged and resuspended in ice cold MTBE/Et0 twice before drying in vacuo. The crude compound was purified using preparative HPLC (595% acetonitrile in 40 minutes). The fractions of interest were combined and lyophilized yielding the title compound as a white fluffy solid (145 mg, 51% isolated yield). '"H NMR (850 MHz, D20} 5 4.52 (t, J = 5.1 Hz, 1H), 4.50 (t, J = 4.4 Hz, 1H), 4.47 (t, J = 5.4 Hz, 1H), 3.74 (dd, J = 5.2, 11.6 Hz, 1 H), 3.76 (dd, J =4.3, 12.1 Hz, 1 H), 3.78 (dd, J=6.6, 11.8 Hz, 1 H), 3.79 (dd, J = 6.0, 11.9 Hz, 1 H), 3.80 (dd, J = 5.8, 11.8 Hz, 1 H), (dd, J = 4.9, 11.8 Hz, 1 H), 3.72 (q, J = 15.6 Hz, 2H), 2.37 (s, 3H). 13C NMR (214 MHz, D20) & 200.72, 174.08, 172.77, 172.47, 172.32, 62.03, 62.00, 61.93, 61.85, 56.84, 56.38, 55.93, 33.72, 30.41. Mass calculated (C13H21N30¢S): 396.1077; measured (HRMS): m/z 396.1092 (3.79 ppm).

N-Boc-Aminophenol-Merrifield resin o © | =

SALT

[03125] As performed by Lopalco et al., (https://doi.org/10.1039/B8207 19B} chloromethyl polystyrene resin {8.3 g, 15.0 mmol), N-Boc-aminophenol (9.4 g, 45.0 mmol), TBAI (1.7 g, 4.5 mmol) and CsCO3 (14.7 g, 45.0 mmol) were dissolved in acetone and refluxed at 70 °C overnight under N2 atmosphere. The resin was then washed extensively with 100 mL of DMF,

H20, DMF, DCM and diethyl ether and dried in vacuo yielding 10.3 g of the title compound (95% isolated yield).

Methyl-Cy5-NH:

Gt 3D aN NY / L

NH,

[00126] 1,2,3,3-tetramethyl-3H-indol-1-ium (Indole-Methyl) was synthesized as described by

Hensbergen et al. (hitps://doi.org/10.2967/jnumed.119.233064) and 1-(4-(1,3-dioxoisoindolin-2- ylbutyl}-2,3,3-trimethyl-3H-indol-1-ium (Indole-Phth) was synthesized as described by

Bunschoten et al. (https://doi.org/10.1021/acs.bioconjchem.6b00093). Indole-Phth (1.5 g, 3.4 mmol) and malonaldehyde dianilide. HCI (1.0 g, 3.7 mmol) were dissolved in 1:1 acetic anhydride/acetic acid (40 mL) and stirred overnight at 60 °C. The mixture was subsequently heated to 120 °C for 60 min and then cooled down to RT. The mixture was precipitated in diethyl ether (800 mL) followed by centrifugation of the suspension. The pellet was resuspended and centrifuged three times yielding the crude hemicyanine. During this time the N-Boc-

Aminophenol-Merrifield (1.1 g, 1.8 mmol) resin was allowed to preswell in DCM for 5 minutes and the Boc protecting group was cleaved by agitating in 20% TFA/DCM for 60 minutes followed by neutralization for 15 minutes using 20% DIPEA/DCM followed by washing with DCM (2x). The dried hemicyanine was then dissolved in 1:1 DCM/DMF (40 mL), added to the resin and agitated for 60 minutes followed by washing with DMF (2x), DCM (1x) and again DMF (2x).

Subsequently, Indole-Methyl (213.0 mg, 0.8 mmol) was dissolved in 3:1 pyridine/acetic anhydride (40 mL), added to the resin followed by agitation overnight at RT. The liquid was drained and the resin was washed with DMF/DCM mixtures, where after the solvents were removed in vacuo. The dark blue solid was purified using dry column vacuum chromatography (DCVC. 0-50% methanol in ethyl acetate) (http://dx.doi.org/10.1055/s-2001-18722) followed purification using preparative HPLC (352>95% acetonitrile in 40 min). The fractions containing product were combined and lyophilized resulting in a blue solid of which 60.0 mg (105.1 umol) was dissolved in methylamine (33 wt®% in EtOH; 25 mL) and stirred at RT for 3 hours. The solvents were then removed in vacuo and crude product was purified by preparative HPLC (352>95% acetonitrile in 40 min). The fractions of interest were lyophilized and yielded the title compound as a blue solid (40.9 mg, 88% isolated yield). '"H NMR (850 MHz, MeOD) & 8.27 (q, J = 13.6 Hz, 2H), 7.51 (d, J = 7.3 Hz, 1H), 7.49 (d, J = 7.0 Hz, 1H), 7.43 (td, J = 7.9, 1.1 Hz, 1H), 7.40 (td, J=7.9,1.1 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.30 (d, J=8.0 Hz, 1 H), 7.29 (td, J=7 4, 0.7 Hz, 1 H), 7.25 (td, J = 7.4, 0.5 Hz, 1H), 6.64 (t, J = 12.4 Hz, 1H), 6.30 (t, J = 12.5, 2H), 4.16 (t, J=7.4 Hz, 2H), 3.65 (s, 3H), 2.99 (t, J = 7.6 Hz, 2H), 1.94 — 1.85 (m, 2H), 1.84 — 1.77 (m, 2H), 1.74 (s, 6H), 1.73 (s, 6H). 13C NMR (214 MHz, MeOD) ò 176.01, 174.16, 174.13, 155.99, 155.93, 155.35, 155.30, 144.16, 143.57, 142.65, 142.50, 129.78, 129.75, 126.75, 126.71, 126.55, 126.06, 123.47, 123.32, 112.08, 111.74, 104.87, 103.91, 50.70, 50.67, 50.41, 44.14, 40.38, 31.61, 28.01, 27.70, 26.02, 25.53. Mass calculated (C3o9H48N3*): 440.3066; found (HRMS): m/z 440.3087 (4.77 ppm).

Methyl-Cy5-MAS: / AGN OH

OL, ee

Lit of

Oo

[03137] A mixture of mercaptoacetyltriserine (26.9 mg, 68.1 pmol), HATU (25.9 mg, 68.1 umol) and N-methylmorpholine (37.5 uL, 340.4 pmol) in DMSO (2 mL) was stirred for 5 minutes at RT where after Methyl-Cy5-NH2 (30.0 mg, 68.1 umol) was added. After stirring at RT for 25 min, a mixture of 15% acetonitrile in H2O (0.1% TFA) was added and the crude product was purified by preparative HPLC (25-95% acetonitrile in 40 min). The fractions of interest were combined and lyophilized yielding a blue solid (30.0 mg, 54% yield) . 'H NMR (850 MHz,

DMSO) 6 8.37 — 8.29 (m, 3H), 8.15 (dd, J = 13.0, 7.2 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.87 (d,

J=79Hz 1H), 7.62 (d, J = 7.4 Hz, 1H), 7.61 (d, J = 7.4 Hz, 1H), 743 — 7.36 (m, 4H), 7.25 (t, J = 7.6 Hz, 1H), 7.24 (t, J = 7.4 Hz, 1H), 6.56 (t, J = 12.0 Hz, 1H), 6.26 (d, J = 13.8 Hz, 1H), 5.19 (t, J=4.9Hz 1H), 5.14 (t, J =4.9 Hz, 1H), 4.88 (t, J = 4.7 Hz, 1H), 4.36 (dt, J = 7.6, 6.0 Hz, 1H), 4.30 (q, J = 6.36 Hz, 1H), 4.20 (dt, J = 8.0, 5.5 Hz, 1H), 4.07 (t, J = 7.1 Hz, 2H), 3.68 (s, 2H), 3.60 (br. s, 5H), 3.57 — 3.49 (m, 2H), 3.16 (sextet, J = 7.0 Hz, 1H), 3.08 (sextet, J = 6.4 Hz, 1H), 2.33 (d, J = 4.8 Hz, 3H), 1.73 — 1.62 (m, 14H), 1.53 (p, J = 6.5 Hz, 2H). 13C NMR (214

MHz, DMSO) ò 194.57, 194.56, 173.25, 172.49, 172.47, 170.38, 170.20, 170.02, 169.99, 169.69, 169.67, 167.23, 167.20, 154.12, 154.04, 142.81, 142.01, 141.09, 141.05, 128.48, 128.39, 125.45, 124.75, 124.64, 122.44, 122.35, 111.15, 111.07, 103.35, 103.33, 103.11, 61.70, 61.46, 61.42, 61.36, 61.34, 55.76, 55.66, 55.54, 55.39, 55.33, 55.24, 48.87, 43.16, 39.98, 39.88, 39.78, 38.04, 37.99, 32.52, 32.50, 31.34, 31.08, 30.13, 27.21, 27.02, 26.25, 26.18, 24.14. Mass calculated (C43Hs7NsOsS*): 817.3959; found (HRMS): m/z 817.3946 (1.59 ppm).

Example 2: Radiolabeling

[33128] Radiolabeling of Methyl-Cy5-MAS: with technetium-99m was based on a protocol described by Robu et al. (https://doi.org/10.2967/jnumed.116.178939) with some amendments.

A solution in a 2 mL Eppendorf tube containing 10.5 uL of phosphate buffer (0.2 M, pH 8), 15.6 pL of phosphate buffer (0.25 M, pH 8), 1.44 uL of Methyl-Cy5-MAS: (992 uM, 0.81 mg/mL in

H-0), 12.5 uL disodium tartrate (50 mg/mL phosphate buffer, 0.2 M, pH 8) and 3.1 pL of SnCl2 (4 mg/mL L-ascorbic acid, 3 mg/mL in 0.1 M HCI, degassed using N2 for 20 min) was mixed with 0.1-2 mL of technetium-99m freshly eluted from a generator (10-1500 MBq, Ultra-Technekow™,

Mallinckrodt Medical B.V., The Netherlands) and heated to 100°C for 20 min. A Sep-Pak Plus

Light cartridge (WAT023501) (Waters, Waltham, USA) was activated using 5 mL of ethanol absolute followed by another 10 mL of Milli-Q. After cooling down the reaction mixture to RT it was diluted with 10 mL of Milli-Q and the product was immobilized on the Sep-Pak cartridge.

The reactants were removed by eluting 10 mL of Milli-Q followed by purging with 10 mL air. The labelled peptide was eluted from the cartridge using 1.5 mL of ethanol absolute and the blue eluate was subsequently evaporated to +/- 10 uL at 100°C and then further analyzed using iTLC-SG with acetonitrile as mobile phase. The elution of radioactivity corresponded with that of

Methyl-Cy5-MAS; was calculated using the following formula: . . radioactivitypmethyl-cys-MAS3

Radiochemical yield (%) = „100 radioactivitymethyl-cys-Mass + radioactivitYynpound

Example 3: Photophysical properties

Methods

[383128] Molar extinction coefficient, relative quantum yield, brightness, lipophilicity and serum protein binding were performed as described by Hensbergen et al. (https://doi.org/10.1016/j.dyepig.2020.108712).

Chemical stability

[30136] 125 pL of a 2 mM solution of Methyl-Cy5-MAS; in Nz sparged 0.1M HEPES pH 7.4 was added to 125 pL of a 4 mM solution of reduced L-glutathione in N2 sparged 0.1M HEPES pH 7.4 in an LCMS vial. After mixing this was placed in a temperature-controlled sample manager set to 37 °C of a Waters 2690/2695 Alliance HPLC equipped with a Symmetry C18 (3.5 um, 150 x 2.1) column and a 996 PDA (Waters, Waltham, USA). Every 35 minutes a sample was injected. The area of the product peak (Tr=14.3 min) at each timepoint was divided by that of t=0. This experiment was repeated with 5-aminovaleric acid instead of L-glutathione.

Optical stability

[33131] Two 4 mL disposable cuvettes (Kartell labware, Noviglio, Italy) were filled with 3 mL of a < 1 uM solution in PBS. Two < 1 uM solutions of control compound (Sulfonate- (SO3)Cy5(S03)-COOH) were also prepared. A cap was placed on the cuvettes and parafilm was used to prevent leaking. The cuvettes were placed on their sides at a distance of 5 cm from the tip of the STORZ laparoscope (Karl Storz Endoskope, Amersfoort, the Netherlands). At a light source strength of 20%, the cuvettes were irradiated using the Cy5 setting and measured at intervals of 5 minutes using a Perkin Elmer L355 fluorescence spectrometer.

Serum protein binding and lipophilicity of %*"Tc-bound species

[05132] These experiments performed as described by Hensbergen et al. (https://doi.org/10.1016/j.dyepig.2020.108712) with the following deviation: Instead of unlabeled compound, radiolabeled compound was used and instead of a fluorescence readout, a Wizard 2 gamma counter was used (Perkin Elmer, Waltham, USA).

Summary of Methyl-Cy5-MAS: properties

Property (measured in PBS)

Molar extinction coefficient (L:mol1-cmr1) 26 500

Relative quantum yield (OF)

Brightness (L-1-mol1-cm"1) 3445

Absorption/Emission (nm) 640/665

Stability towards glutathione after 6 hours

Stability towards aminovaleric acid after 6 hours

Optical stability after 30 minutes

Serum protein binding

Serum protein binding *™Tc-bound species

LogP 9"Tc-bound species

Example 4: Culturing and imaging

In vitro cell culture

[09133] HCO4 hepatocyte and MDAMB231 cell lines were cultured in Gibco’s minimum essential medium (MEM) enriched with 10% fetal bovine serum and penicillin/streptomycin (all

Life Technologies Inc.). Cells were kept under standard culturing conditions.

Fluorescence confocal imaging

[09134] One day prior to fluorescence confocal imaging cells were seeded on glass bottom culture dishes (for confocal imaging; MatTek corporation) and were placed in the incubator overnight. Samples were stained with Me-Cy5-Me (10 nM) or 10 uM Me-Cy5-Mas3 for 1 hour at 37°C. Hoechst and lysotracker green were added as interval control for staining of the nucleus and lysosomes. Samples were washed three times with PBS before being placed on the microscope. Fluorescence confocal microscopy on live cell cultures and excised tissue samples was performed using a Leica SP8 WLL 1 Laser Scanning Confocal Microscope (Leica

Microsystems) using a 100x magnification/1.4 Oil DIC III immersion objective. For imaging of cells sequential scanning settings were used to visualize the different fluorescent features:

Hoechst (Aex 405 nm, Aem 430-480 nm), Cy5 (Aex= 633 nm, Aem= 650-700 nm), SYBR® Green (Aex= 488 nm, Aem= 500-550 nm). For tissue samples, Cy5 imaging settings were applied. Image acquisition and processing was performed using LASX software (Leica Application Software

Suite 4.8).

Flow cytometry

[03135] Cells were trypsinized and aliquoted in portions of 300000 cells. For details on the culturing conditions, see the SI. Samples were incubated with identical concentrations as used for fluorescence microscopy. Fluorescence was measured using a LSRII flow cytometer (BD

Biosciences) with APC-A settings (635 nm laser and 750 nm long pass filter). Live cells were gated on Forward Scatter, Side Scatter and Pulse Width, and 20000 viable cells were analyzed.

All experiments were performed in triplicate (total n=6).

In vivo fluorescence imaging

Porcine model

[33138] 4 non-tumor-bearing pigs were used. Animals were bred and kept in accordance with

Belgium law in and by a licensed establishment for use of experimental animals. Pigs were housed at the animal facility at ORSI Academy (Melle, Belgium) until used for surgical training and imaging experiments (35-40 kg per animal). All animals remained under anesthesia for the entire duration of the experiment and were euthanized when the examination was completed.

Experiments were approved by the local ethics committee of Gent University (EC2019/79) and were performed in accordance with the Experiments on Animals Act (Wod, 2014), the applicable legislation in Belgium and in accordance with the European guidelines (EU directive no. 2010/63/EU) regarding the protection of animals used for scientific purposes.

[20157] Liver lesions were created using the coagulation setting of a bi-polar robotic forceps.

After placement of the forceps on the liver tissue, lesions were formed via localized heat production with subsequent tissue disruption. 4 mg Me-Cy5-MAS3 was dissolved in 1.5 mL 0.9% saline solution and administered intravenously. Imaging was performed at 4 hours after tracer administration. For imaging of white light and Cy5 a clinical modified grade IMAGE 1 S camera system equipped that contained an integrated Cy5 filter, with a 0° laparoscope was used (KARL STORZ).

[08138] In-house developed image-processing software was used to create color-coded heat-

map and real-time representation of the signal-to-background ratio (SBR) based on the intensity of the fluorescence signal. Differences in fluorescence signal intensity were represented in a intensity-based scale-bar in real-time. A pseudo-colored fluorescence overlay allowed real-time visualization of the distribution of the fluorescence signal within the tissue sample. Image- processing software was written in C++-programming language using open-source computer vision libraries (OpenCV).

Example 6: Hepatic uptake

[33138] Incubation of hepatocytes with Me-Cy5-Mass resulted in a focalized uptake positioned in between cells (Figure 8A). The location of uptake could be attributed to circular structures positioned between the cells, that correspond to the location of bile cannulas (Murray JW, et a/.,

Heterogeneous accumulation of fluorescent bile acids in primary rat hepatocytes does not correlate with their homogenous expression of ntcp. Am J Physiol Gastrointest Liver Physiol. 2011;301(1):G60-8). Over time, clear differences in localization of the fluorescence signal was seen (Figure 8B). Addition of the Mass chelate seemed to be an important factor herein, as uptake of the Me-Cy5-Me dye alone resulted in mitochondrial staining throughout the cytoplasm of the cell (Figure 8C-D). Moreover, the focalized uptake of Me-Cy5-Mas3 was shown to be specific for hepatocytes as uptake of Me-Cy5-Mass (Figure 9A-B) and Me-Cy5-Me (Figure 9C-

D) in epithelial cells revealed a comparable distribution pattern that was in line with the mitochondrial uptake of Me-Cy5-Me in hepatocytes. No difference in distribution of the Cy5 signal was seen over time in epithelial cells.

[00140] Quantitative assessment of the tracer uptake and resulting fluorescence signal using flow cytometry further underlined the differences in uptake between the two tracers seen with fluorescence confocal microscopy, but also between the two cell types (Figure 10). Overall signal intensities were lower in hepatocytes (Figure 10A-D) compared to epithelial cells (Figure 10E-H). Uptake of Me-Cy5-Me was significantly higher in hepatocytes compared to the uptake of Me-Cy5-Mass. This is in line with the cytoplasmatic uptake seen for Me-Cy5-Me (Figure 8B and Figure 9B) and the focalized uptake of Me-Cy5-Mass in hepatocytes (Figure 8A) that was localized outside of the main cell body. In both cell lines fluorescence intensities obtained after incubation with Me-Cy5-Mass were lower than after incubation with Me-Cy5-Me. Over time, an increase in signal intensity (and thus level of uptake) was seen for Me-Cy5-Me in hepatocytes (Figure 10B) and for both tracers in epithelial cells (Figure 10E-F). Interestingly, no significant increase in signal was seen for Me-Cy5-Mas: in hepatocytes (Figure 10A). Together with the differences in signal localization between, this suggests that uptake of Me-Cy5-Me is a passive process, while Me-Cy5-Massis actively processed and not taken up by hepatocytes.

[00143] In literature, uptake of compounds that show structural similarities to the dye Me-Cy5-

Me dye is contributed to TSPO, a translocator protein protein that is mainly found on the outer mitochondrial membrane (Denora N, et a/., New Fluorescent Probes Targeting the

Mitochondrial-Located Translocator Protein 18 kDa (TSPO) as Activated Microglia Imaging

Agents. Pharmaceutical Research. 2011;28(11):2820). However, no significant difference in tracer localization or signal intensity were seen after inhibition of hepatocytes (Figure 8E, Figure 10D) and epithelial cells (Figure 9E and Figure 10H) with the well-known TSPO inhibitor PK- 11195 (Wyatt SK, et al., Molecular imaging of the translocator protein (TSPO) in a pre-clinical model of breast cancer. Mol Imaging Biol. 2010;12(3):349-58). Also, no change in uptake pattern was seen for Me-Cy5-Mass in hepatocytes, where after TSPO inhibition tracer uptake remained focalized and localized between individual cells (Figure 8E). From this could be concluded that uptake of for Me-Cy5-Mass is not TSPO mediated.

[08142] As previously described as an important factor in uptake and biodistribution of tracers, the presence of serum and subsequent serum binding was shown to influence the uptake of

Me-Cy5-Mass. As a comparable shift in the uptake pattern (Figure 8F and Figure 9F) and quantitative signal intensity (Figure 10C and Figure 10G) was seen in both cell types, the contribution of serum can be designated as a more general effect on tracer uptake and does not contribute to the manner of uptake of Me-Cy5-Mas: in hepatocytes.

Claims (44)

Conclusions 1. Compound of formula | or formula II: Ry R2 Ra Rs R 7 R 4 0 xR) Rs — Ry “JT \ / Rg \ Pr N Yo vl Ry Riz (1, Ri Rs Rg Rs Ny Ry Ren 3 \ x 12 Ra Rs ACTA | | Re N zn | : | N X dy Rig Rig (in which the bridging group L is selected from the group consisting of Rea Ra Ria , Ria ‚and Rip Riz Raz 3 ' ‚and Ria and where R: is selected from hydrogen, halogen, C+-Cy-alkyl, sulfonate, carboxyl, phosphonate, amine, and azide, and R: is selected from hydrogen, halogen, C+-C5-alkyl, sulfonate, carboxyl, phosphonate, amine, and azide; or Ri and Rz together form an aryl optionally substituted with 1 or 2 groups independently selected from C1-C4 alkyl, halogen, sulfonate, carboxyl, phosphonate, amine, and azide; R3 is selected from hydrogen, halogen, C1-C8-alkyl, sulfonate, carboxyl, phosphonate, amine, and azide, and R4 is selected from hydrogen, halogen, C1-C4-alkyl, sulfonate, carboxyl, phosphonate, amine, and azide; or Rs and R4 together form an aryl optionally substituted with 1 or 2 groups independently selected from C:+-C4-alkyl, halogen, sulfonate, carboxyl, phosphonate, amine, and azide; wherein each of Rs and Rs are independently selected from hydrogen, halogen, C:-C--alkyl, sulfonate, carboxyl, phosphonate, amine, and azide; wherein each of R7, R8, R8, and R1g are independently selected from hydrogen, methyl, ethyl, halogen, sulfonate, carboxyl, phosphonate, amine, and azide; R1 is selected from C1-C4-alkyl, C+-C8-haloalkyl, C1-C4-alkoxy, C1-C8-haloalkoxy, (CH2)a-Y, (CH2)a-NH-Y, (CH2)a-C(=0) -Y, (CH2)a-Z, (CH32)a-NH-Z, (CH2)}4-C{=0)-Z; Rio is selected from Cs-Cs-alkyl, Ct-Cs-haloalkyl, C+-Cs-alkoxy, C1-Cs-haloalkoxy, (CH2)a-Y, (CH2)a-NH-Y, (CH2)a-C(=0) -Y, (CH2)a-Z, (CH2)a-NH-Z, (CH2)a-C(=0)-Z; R 8 is selected from H, C 5 -C 8 alkyl, C: -C 4 -haloalkyl, C + -C 5 -alkoxy, C: -C 4 -haloalkoxy, (CH 2 )a-Y, (CH 2 )a-NH-Y, (CH 2 )a-C (=0)-Y, (CH2)a-Z, (CH2)a-NH-Z, (CH2)a-C(=0)-Z; wherein at least one of Ris, Riz, and Ris is selected from (CH2)a-Y, (CHz)a-NH-Y, (CHz)a-C(=OO)-Y; and wherein not more than two of R41, R12, and Ra: are selected from (CHz)a-Y, (CHz)a-NH-Y, (CHz)a-C{=O)-Y, (CH2)a-Z, (CH2)a-NH-Z, (CHz) s-C{=0)-Z; each R14 is independently selected from H, C+-C4-alkyl, C1-C8-haloalkyl, C:-C4-alkoxy, C+-C8-haloalkoxy, (CH2)a-Y, (CH2)a-NH-Y, (CH2) a-C(=O)-Y, (CHs)a-Z, (CH2)a-NH-Z, (CHz)a- C(=O)-Z; wherein at least one R+4 is selected from (CHz)a-Y , (CH2)a-NH-Y; and wherein not more than two R44 are selected from (CH2)e-Y, (CHs)a-NH-Y, (CH2)o-C(=OO)-Y, (CHa)a-Z, (CH2)a-NH-Z, and (CH.)a-C(=O)-Z; one X is O, and every other X is CHR 14; Y is a chelating group; Z is a therapeutic group or H; a is 3 to 6 inclusive; n is 2, 3, or 4; or a pharmaceutically acceptable salt or solvate thereof. 2. A compound according to claim 1, wherein R y and R 2 are H. 3. A compound according to claim 1 or 2, wherein Rs and Ra are H. A compound according to any preceding claim, wherein R+, Rs, R3, Ra, Rs, and Rg are each hydrogen. 5. A compound according to claim 1 wherein R y and R 2 together form an optionally substituted phenyl ring. 6. A compound according to claim 1, wherein R 5 and R 4 together form an optionally substituted phenyl ring. A compound according to any preceding claim, wherein R7 and R8 are H or methyl. 8. A compound according to any preceding claim, wherein Rs and Rip are H or methyl. 9. A compound according to any one of the preceding claims, wherein R7, R8, Rc, Rs are each methyl. 10. A compound according to any preceding claim, wherein L is or is equal to eN 11. A compound according to any one of claims 1 to 9, wherein n is 2 or 3. 12. A compound according to any one of claims 1 to 9, wherein n is 2. 13. A compound according to any one of claims 1 to 9, wherein n is 3. A compound according to any one of claims 1 to 9 or claims 11 to 13, wherein one R 18 is selected from (CH 2 ) a -Y, (CH 2 ) a -NH -Y; optionally wherein each other R 44 is independently selected from H, C + -C 4 alkyl, C 1 -C 4 haloalkyl, C + -C 4 alkoxy, C + -C 4 haloalkoxy. A compound according to any preceding claim, wherein R+: is methyl. A compound according to any preceding claim, wherein R12 is methyl. 17. A compound according to any preceding claim, wherein a is 4. 18. A compound according to any preceding claim, wherein a is 5. A compound according to any preceding claim, wherein Y is selected from -MAS:3, -MAG:3, -DOTA-GA, -DOTA, and -DTPA, optionally from -MAS; and -MAG:. 20. A compound according to claim 19, wherein Y is -MAS:. 21. A compound according to any preceding claim, wherein the compound is: ) | i SNHeY where n is 2 or 3, and Y is MAS; or MAG:. 22. A compound according to any one of claims 1 to 21, wherein the compound is selected from: OMAS esd TT ed i i SNN i i i / Lo. i VR HD i i HON TNH | i Jr ; LS By OT, BY oo PRT i WAS, ì Ne | | onee) : i et BN TC a § i i WE nO i i FQ NET i i © ng ì x ss * a 2 i i BN” 2 i i iep ì dan i i Ne Fr TN A X ì mg) i ong i 9 £ i i PRE ; oT . 23. A compound according to any one of claims 1 to 23, further comprising a chelated radiolabel. 24. The compound of claim 24, wherein the radiolabel is selected from "Sc, "Sc, 51Cr 52m Co S2Fa SSN STN 82Cu Cy STCu Ga 853g 87Ga 897, soy 89y TTG "MTC RU 155Rh 109pg Ag Tom "Mn 1137] 1147] 117Sn 121Sn Te 142pr 143pp 49pm 151pm 149Th 538m 157Gd 181Th 16Ho "Dy 169 169yp 175yh 172Tm 77 y 188Ra 188Ra 1981 pt 197Hg 188A 188A 1 212pp 203ph 211At 212B;j 213g; 223R4 225A and 221TH: or a cationic molecule comprising '*F, such as "®F-[AIF]?*. 25. The compound of claim 23 or 24, wherein the chelated radiolabel is selected from a γ-emitter, a β-emitter, and an α-emitter, or a combination of the foregoing. 26. A compound according to any one of claims 24 to 25, wherein the chelated radiolabel is selected from $3Ga, "Lu, "In, and "Tc, or a combination of the foregoing. 27. A compound according to any one of claims 23 to 28, wherein the chelated radiolabel is *™Tc. A compound according to claim 27, wherein the compound is selected from: PE es whee i i Ee a TN Ed ì Hn og i tS ì § A ì RE I ì Nen i vn eN T ~ es ì i BT aE N i i 3 NO, N i RT FL dh ì psies i Lennannnnaaaaaannnaarasaan anna AA han AA AAA AAA AAA AAA A AAA A AAA AAA RAR A AAR A AAA AAA R RRR RRA R RRR R A RAR R RRR R A 29. A formulation comprising a compound according to any preceding claim and a pharmaceutically acceptable carrier. 30. A method of performing an imaging of a specimen, comprising e contacting the specimen with a compound according to any one of claims 1 to 28, or with a formulation comprising a compound according to any one of claims 1 to 28; and e measuring a fluorescent and/or radioactive signal emitted by the compound, so that the specimen can be imaged. 31. The method of claim 30, wherein the specimen is a sample, a cell, a tissue, an organ, a tumor, or an organism, or a combination of the foregoing; optionally wherein the sample is a liver sample. 32. The method of any one of claims 30 or 31, wherein the compound comprises a chelated radiolabel, and the imaging comprises nuclear imaging. 33. The method of claim 32, wherein the imaging comprises positron emission spectroscopy (PET), single photon emission-computed tomography (SPECT), scintigraphy, gamma-tracing/imaging, or beta-tracing. 34. The method of any of claims 30 or 31, wherein the imaging comprises fluorescence imaging, optionally wherein the imaging comprises near-IR fluorescence spectroscopy. 35. A method according to any one of claims 30 to 34, wherein the imaging comprises a combination of nuclear imaging and fluorescence imaging. 36. A method according to any one of claims 30 to 35, wherein the imaging comprises imaging liver function. 37. The method of any one of claims 30 to 35, wherein the imaging comprises imaging lesions of the liver. 38. The method of any one of claims 30 to 35, wherein the imaging comprises imaging hepatic metastases. 39. The method of any one of claims 30 to 35, wherein the method comprises intraoperative imaging during the performance of a hepatobiliary or pancreatic surgical procedure. 40. Use in imaging of a compound according to any one of claims 1 to 28 or of a formulation according to claim 29. 41. Use according to claim 40, wherein the imaging comprises intraoperative imaging, optionally imaging during the performance of a hepatobiliary surgical procedure. 42. A compound according to any one of claims 1 to 28, or a formulation according to claim 30, for use as a medicament. 43. A compound according to any one of claims 1 to 28, or a formulation according to claim 29, for use in the treatment of cancer, optionally wherein the cancer comprises primary or secondary tumors. 44. A compound according to any one of claims 1 to 28, or a formulation according to claim 29, wherein the cancer is liver cancer.