The present invention relates to a compound, a pharmaceutical composition comprising or consisting of said compound, a kit comprising or consisting of said compound or pharmaceutical composition for use in the in vivo diagnosis of ischemia/cardiac remodelling.

Background of the Invention

Injury to the heart results in changes to size and function of the heart, a process generally referred to as cardiac remodeling (or ventricular remodeling). Cardiac remodelling is commonly defined as a physiological or pathological state that may occur after conditions such as myocardial infarction, pressure overload, idiopathic dilated cardiomyopathy or volume overload.

While the assessment of left ventricular systolic function and the coronary arteries is ever increasing, cardiac remodeling still lacks a reliable imaging modality in patients (Curley et al 2018; Basic Res Cardiol 2018; 113: 10.) In 2018, Lindner et al. (Protein. J Nucl Med 2018;59:1415-1422) developed a tracer for positron emission scans targeting fibroblast activation protein (FAP). The tracer consists of a quinolone-based fibroblast activation protein inhibitor (FAPI) labelled with a detection moiety such as a radio nucleoid and showed superior imaging characteristics when compared to fludeoxyglucose (Giesel et al. 2019; J Nucl Med 2019;60:386-392; Loktev et al. 2018; J Nucl Med 2018;59:1423-1429). FAP plays an important role in cancer growth and is discussed as a hot candidate for targeted oncotherapy. TGFpi induced FAP expression is also crucial for cardiac wound healing and remodeling (Aghajanian et al. 2019; Nature 2019;573:430-433; Tillmanns et al. 2015, J Mol Cell Cardiol 2015;87:194-203). Serial small animal FAPI PET-CT scans revealed a steady increase FAP activity after myocardial infarction until day 6 followed by a steady decline (Varasteh et al. 2019, J Nucl Med 2019). Moreover, a recent study used chimeric antigen receptor T cells targeting FAP to reduce cardiac fibrosis and restore systolic function after myocardial infarction (Aghajanian et al supra).

The present invention discloses the use of FAPI tracers in the diagnosis of cardiac remodeling/ischemia, in particular of early stages before myocardial infarction.

Summary of the Invention

In a first aspect, the present invention provides a compound of Formula (I) wherein

Q, R, U, V, W, Y, Z are individually present or absent under the proviso that at least three of Q, R, U, V, W, Y, Z are present;

Q, R, U, V, W, Y, Z are independently selected form the group consisting of O, CH ₂ , NR ⁴ , C=0, C=S, C=NR ⁴ , HCR ⁴ and R ⁴ CR ⁴ _. with the proviso that two Os are not directly adjacent to each other;

R ¹ and R ² are independently selected from the group consisting of -H, -OH, halo, C _1-6 -alkyl, -

O-C _{1 -6} -alkyl, S-C _1-6 -alkyl;

R ³ is selected from the group consisting of -H , -CN , -B(OH)2, -C(O) -alkyl, -C(O) -aryl-, -

C=C-C(0) -aryl, -C=C-S(0) ₂ -aryl, -C0 ₂ H , -S0 ₃ H , -S0 ₂ NH ₂ ,-P0 ₃ H ₂ , and 5-tetrazolyl;

R ⁴ is selected from the group consisting of -H, -C _1-6 -alkyl, -O-C _1-6 -alkyl, -S-C _1-6 -alkyl, aryl, and - C _1-6 -aralkyl, each of said -C _1-6 -alkyl being optionally substituted with from 1 to 3 substituents selected from -OH, oxo, halo and optionally connected to Q, R, U, V, W, Y or Z; R ⁵ is selected from the group consisting of -H, halo and C _1-6 -alkyl;

R ⁶ , and R ⁷ are independently selected from the group consisting of-H, under the proviso that R ⁶ and R ⁷ are not at the same time H, wherein L is a linker, wherein D, A, E, and B are individually present or absent, preferably wherein at least A, E, and B are present, wherein when present:

D is a linker;

A is selected from the group consisting of NR ⁴ , O, S, and CH ₂ ;

E is selected from the group consisting of

wherein i is 1, 2, or 3; wherein j is 1, 2, or 3; wherein k is 1, 2, or 3; wherein m is 1, 2, or 3;

A and E together form a group selected from a cycloalkyl, heterocycloalkyl, aryl and heteroaryl, wherein A and E can be mono-, bi- and multicyclic, preferably monocyclic. Each A and E being optionally substituted by 1 to 4 residues from the group consisting of -H, -C _1-6 -alkyl, -O-C ₁ - ₆ - alkyl, -S-C _1-6 -alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, and -C _1-6 -aralkyl, each of said -C _1-6 -alkyl being optionally substituted with from 1 to 3 substituents selected from -OH, oxo, halo; and optionally connected to A, B, D, E or

B is selected from the group consisting of S, NR ⁴ , NR ⁴ -0, NR ⁴ -C ₁ - ₆ -alkyl, NR ⁴ -C _1-6 -alkyl-NR ⁴ , and a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein NR ⁴ -C _I - ₆ -alky 1-NR ⁴ and the N-containing heterocycle is substituted with 1 to 3 substituents selected from the group consisting of C _1-6 alky 1, aryl, C _1-6 -aralkyl; and;

R ⁸ is selected from the group consisting of radioactive moiety, chelating agent, fluorescent dye, a contrast agent and combinations thereof; is a 1-naphtyl moiety or a 5 to 10- membered N-containing aromatic or non- aromatic mono- or bicyclic heterocycle, wherein there are 2 ring atoms between the N atom and X; said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S; and X is a C atom; or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate, or salt thereof; for use in a method of in vivo diagnosis of ischemia. In a second aspect, the present invention relates to a pharmaceutical composition comprising or consisting of at least one compound of the first aspect, and, optionally, a pharmaceutically acceptable carrier and/or excipient for use in a method of in vivo diagnosis of ischemia.

In a third aspect, the present invention relates to a kit comprising or consisting of the compound of the first aspect or the pharmaceutical composition of the second aspect and instructions for use in a method of in vivo diagnosis of ischemia.

List of Figures

In the following, the content of the figures comprised in this specification is described. In this context please also refer to the detailed description of the invention above and/or below.

Figure 1: Logistic regression and linear prediction model for fibroblast activation protein inhibitor signal in the myocardium

(A) Multivariate logistic regression models for signal intensity and focal enrichment. Odds ratios (OR) are depicted as a dot. Lines mark the 95% confidence interval. While metabolic variables, such as increased TSH, radiation, diabetes, overweight and different chemotherapies, are associated with an increased fibroblast activation protein inhibitor (FAPI) signal, focal FAPI enrichment patterns are mainly associated with cardiovascular risk. N=185 for both analyses. (B) Linear regression model for FAPI signal intensity prediction and outlier analysis based on significant variables of the multivariate logistic regression analysis. The model was established with the initial cohort (N=185) und tested for accuracy with a confirmatory cohort (N=44). Outliers (N=12) are marked in red in both plots. Outliers were defined as patients with residuals (actual - predicted FAPI signal) above the 95% mark of the initial cohort. Patient characteristics of the outlier cohort are reported in table 6.

Figure 2: Bullseye 17 segment analysis of different subgroups with representative FAPI images.

The median signal intensity of the corresponding segment is displayed for each group. The number of patients in a group are seen in the right lower corner of each bullseye above the grey scale. The greyscale coding was converted from a spectral color scale ranging from 0.7 (blue) to 1.3 (red) with 1.0 (yellow) being the center. Representative FAPI images are displayed below. Signal intensity was generally increased in the septal basal segments. The apex exhibited the lowest signal intensity. This effect was most pronounced in arterial hypertension. Diabetes, arterial hypertension and obesity, all risk factors associated with metabolic stress and cardiac remodeling show the highest increase in fibroblast activation protein inhibitor (FAPI) signals. Figure 3: Cardiac FAPI signal kinetics

FAPI signals were measured 10, 60 and 180 minutes after administration. The median standardized uptake values (SUVs) and their respective interquartile ranges are depicted. Cardiac signals were highest 10 minutes after administration and decreased continuously in time. The median signal intensities for each segment and the number of patients scanned are reported in the corresponding bullseye.

Figure 4: FAPI and FDG signals and signal ratios

FAPI and FDG signals in all patients who had an FDG and FAPI PET-CT examination within 12 months (n=21). FAPI and FDG signals are portrayed for each patient (A) and signal ratios were calculated for different organs (B). Myocardial standardized uptake values (SUV) were generally higher using an FDG tracer. Signal ratios were similar between FDG and FAPI for heart/blood pool as well as heart/lung. Heart/brain and heart/liver ratios were significantly higher while the heart/gluteus muscle ratio was lower using a FAPI tracer. Signal ratios were compared using a Kruskal- Wallis test. * p<0.05, ****p<0.0001

Figure 5: Myocardial signal intensities by main entitiy

Standardized uptake values (SUV) are reported for each cancer entity.

Figure 6: Median FAPI signal by subgroup

Subgroups are organized thematically. The median signal intensity for each patient subgroup and the group size is reported in each bullseye.

Figure 7:

(A) Myocardial FAPI signal intensities in special subgroups prone to myocardial fibrosis. Patients with arterial hypertension, diabetes mellitus, the elderly and overweight patients showed a significant increase in FAPI signal. The difference in patients with hypothyreosis was not significant. (B) Myocardial FAPI signal intensities by chemotherapy use. After p-level adjustment only patients treated with checkpoint inhibitor showed reduced FAPI signal intensities. Wilcoxon sum test with Holm-Bonferroni p-level adjustment ns not significant, * p<0.05, ** p<0.01, ***p<0.001, **** p<0.001 . Figure 8: FAPI standardized uptake value (SUV) and echocardiogrphic findings

Echocardiographic findings of all patients where echocardiographic data were available (n=44). Patients with reduced ejection fraction showed an increase SUV while other echocardiographic findings such as septal hypertrophy and diastolic function were not associated with increased signal intensities.

Figure 9: Sequential FAPI-PET-CT scans

26 Patients received sequential scans. The first row reports median signal intensities measured during the first and second scans for each segment. Differences are reported in the third column. Each patient is reported in a separated row. While there is a general increase in signal intensity over time, there is also one patient (patient 14), who showed a clear decrease in cardiac FAPI signals in the follow scan.

Figure 10: FAPI signals in patients following myocardial infarction.

FAPI signals are reported for each segment with a short medical history of each case and a FAPI-PET-CT image followed by coronary angiography findings. Interestingly, there is no evident increase in signal intensities years after myocardial infarction.

Figure 11: Patients with FAPI scans after coronary intervention

Left ventricular FAPI signals intensities for each segment are reported with a short medical history followed by the last coronary angiography. While the severity of the coronary artery disease does not seem to correlate directly with FAPI intensities, radiotherapy of esophageal cancer in patient 4 is likely to have increased FAPI signals in the anterior wall as a sign of cardiac inflammation and remodeling.

Detailed Descriptions of the Invention

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims, which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being optional, preferred or advantageous may be combined with any other feature or features indicated as being optional, preferred or advantageous.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being “incorporated by reference ” . In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Definitions

In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

The term “ischemia” refers in general to a restriction in or complete blockade of blood supply to a tissue, resulting in a shortage of oxygen as well as other components (nutrients etc.) required for cellular metabolism. This shortage in supply may result in damage to or death of the affected tissue. The term “coronary ischemia” refers to an ischemia that directly affects the heart, in particular myocardial tissue and may lead inter alia to myocardial infarction.

The terms “presymptomatic” and “prodromal” refer to very early stages of a disease. In the presymptomatic stage the subject is without any symptoms of the disease. Preferred examples of presymptomatic stages are subjects with an increased risk of developing a certain disease that have not yet displayed any symptoms of this disease. In the prodromal stage of a disease early symptoms of a disease are already present although those symptoms are not diagnostically specific symptoms of this disease and may occur in other diseases as well.

In the following definitions of the terms: alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkenyl and alkynyl are provided. These terms will in each instance of its use in the remainder of the specification have the respectively defined meaning and preferred meanings.

The term “alkyl” refers to a saturated straight or branched carbon chain. Preferably, the chain comprises from 1 to 10 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 e.g. methyl, ethyl methyl, ethyl, propyl, iso-propyl , but yl,tyslo-b.0u0tyl, tert- butyl, pentyl, hexyl, pentyl, or octyl. Alkyl groups are optionally substituted.

The term “heteroalkyl” refers to a saturated straight or branched carbon chain. Preferably, the chain comprises from 1 to 9 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 e.g. methyl, ethyl, propyl, iso- propyl, butyl, Ao-butyl, sec-butyl, tertbutyl, pentyl, hexyl, pentyl, octyl, which is interrupted one or more times, e.g. 1, 2, 3, 4, 5, with the same or different heteroatoms. Preferably the heteroatoms are selected from O, S, and N, e.g. -O-CH ₃ , -S-CH ₃ , -CH ₂ -O-CH ₃ , -CH ₂ -0-C ₂ H ₅ , -CH ₂ -S-CH , -CH ₂ -S-C ₂ H ₅ , -C ₂ H ₄ -0-CH3, -C ₂ H ₄ -O-C ₂ H ₅ , -C ₂ H ₄ -S-CH , - C ₂ H ₄ -S-C ₂ H ₅ etc. Heteroalkyl groups are optionally substituted.

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively, with preferably 3, 4, 5, 6, 7, 8, 9 or 10 atoms forming a ring, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl etc. The terms "cycloalkyl" and "heterocycloalkyl" are also meant to include bicyclic, tricyclic and polycyclic versions thereof. The term “heterocycloalkyl” preferably refers to a saturated ring having five of which at least one member is a N, O or S atom and which optionally contains one additional O or one additional N; a saturated ring having six members of which at least one member is a N, O or S atom and which optionally contains one additional O or one additional N or two additional N atoms; or a saturated bicyclic ring having nine or ten members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms. “Cycloalkyl” and “heterocycloalkyl” groups are optionally substituted. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl, spiro[5,4]decyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, and the like. Examples of heterocycloalkyl include l-(l,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,8 diazo-spiro-[4,5] decyl, 1,7 diazo-spiro-[4,5] decyl, 1,6 diazo-spiro-[4,5] decyl, 2,8 diazo-spiro[4,5] decyl, 2,7 diazo-spiro[4,5] decyl, 2,6 diazo-spiro[4,5] decyl, 1,8 diazo-spiro-[5,4] decyl, 1,7 diazo-spiro- [5,4] decyl, 2,8 diazo-spiro-[5,4] decyl, 2,7 diazo-spiro[5,4] decyl, 3,8 diazo-spiro[5,4] decyl, 3,7 diazo-spiro[5,4] decyl, 1-azo-7,11-dioxo-spiro[5,5] undecyl, 1,4-diazabicyclo[2.2.2]oct-2- yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, and the like.

The term “aryl” preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms or an aromatic tricyclic ring system containing 14 carbon atoms. Examples are phenyl, naphtyl or anthracenyl. The aryl group is optionally substituted.

The term “aralkyl” refers to an alkyl moiety, which is substituted by aryl, wherein alkyl and aryl have the meaning as outlined above. An example is the benzyl radical. Preferably, in this context the alkyl chain comprises from 1 to 8 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, or 8, e.g. methyl, ethyl methyl, ethyl, propyl, /.so- propyl, butyl, /.so-butyl, .svc-butenyl, tert- butyl, pentyl, hexyl, pentyl, octyl. The aralkyl group is optionally substituted at the alkyl and/or aryl part of the group.

The term “heteroaryl” preferably refers to a five or six-membered aromatic monocyclic ring wherein at least one of the carbon atoms are replaced by 1, 2, 3, or 4 (for the five membered ring) or 1, 2, 3, 4, or 5 (for the six membered ring) of the same or different heteroatoms, preferably selected from O, N and S; an aromatic bicyclic ring system wherein 1, 2, 3, 4, 5, or 6 carbon atoms of the 8, 9, 10, 11 or 12 carbon atoms have been replaced with the same or different heteroatoms, preferably selected from O, N and S; or an aromatic tricyclic ring system wherein 1, 2, 3, 4, 5, or 6 carbon atoms of the 13, 14, 15, or 16 carbon atoms have been replaced with the same or different heteroatoms, preferably selected from O, N and S. Examples are oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3,-thiadiazolyl, 1,2,5-thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-benzofuranyl, 2- benzofuranyl, indoyl, isoindoyl, benzothiophenyl, 2-benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, 2,1-benzosoxazoyl, benzothiazolyl, 1,2- benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, quinolinyl, 1,2,3-benzotriazinyl, or 1,2,4-benzotriazinyl.

The term “heteroaralkyl” refers to an alkyl moiety, which is substituted by heteroaryl, wherein alkyl and heteroaryl have the meaning as outlined above. An example is the 2- alklypyridinyl, 3-alkylpyridinyl, or 2-methylpyridinyl. Preferably, in this context the alkyl chain comprises from 1 to 8 carbon atoms, i.e. 1, 2, 3, 4, 5, 6, 7, or 8, e.g. methyl, ethyl methyl, ethyl, propyl, iso - propyl, butyl, iso -butyl, sec- butenyl, tert- butyl, pentyl, hexyl, pentyl, octyl. The heteroaralkyl group is optionally substituted at the alkyl and/or heteroaryl part of the group.

The terms “alkenyl” and “cycloalkenyl” refer to olefmic unsaturated carbon atoms containing chains or rings with one or more double bonds. Examples are propenyl and cyclohexenyl. Preferably, the alkenyl chain comprises from 2 to 8 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, or 8, e.g. ethenyl, 1 -propenyl, 2-propenyl, iso- propenyl, 1-butenyl, 2-butenyl, 3-butenyl, iso -butenyl, sec- butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, hexenyl, pentenyl, octenyl. Preferably the cycloalkenyl ring comprises from 3 to 8 carbon atoms, i.e. 3, 4, 5, 6, 7, or 8, e.g. 1-cyclopropenyl, 2-cyclopropenyl, 1-cyclobutenyl, 2-cylcobutenyl, 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, cyclohexenyl, cyclopentenyl, cyclooctenyl.

The term “alkynyl” refers to unsaturated carbon atoms containing chains or rings with one or more triple bonds. An example is the propargyl radical. Preferably, the alkynyl chain comprises from 2 to 8 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, or 8, e.g. ethynyl, 1-propynyl, 2- propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, hexynyl, pentynyl, octynyl.

In one embodiment, carbon atoms or hydrogen atoms in alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl radicals may be substituted independently from each other with one or more elements selected from the group consisting of O, S, N or with groups containing one or more elements selected from the group consisting of O, S, N. Embodiments include alkoxy, cycloalkoxy, arykoxy, aralkoxy, alkenyloxy, cycloalkenyloxy, alkynyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkenylthio, cycloalkenylthio, alkynylthio, alkylamino, cycloalkylamino, arylamino, aralkylamino, alkenylamino, cycloalkenylamino, alkynylamino radicals.

Other embodiments include hydroxyalkyl, hydroxycycloalkyl, hydroxyaryl, hydroxyaralkyl, hydroxyalkenyl, hydroxycycloalkenyl, hydroxyalinyl, mercaptoalkyl, mercaptocycloalkyk, mercaptoaryl, mercaptoaralkyl, mercaptoalkenyl, mercaptocycloalkenyl, mercaptoalkynyl, aminoalkyl, aminocycloalkyl, aminoaryl, aminoaralkyl, aminoalkenyl, aminocycloalkenyl, aminoalkynyl radicals.

In another embodiment, hydrogen atoms in alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl radicals may be substituted independently from each other with one or more halogen atoms. One radical is the trifluoromethyl radical.

If two or more radicals or two or more residues can be selected independently from each other, then the term “independently” means that the radicals or the residues may be the same or may be different.

As used herein a wording defining the limits of a range of length such as, e. g., “from 1 to 6” means any integer from 1 to 6, i. e. 1, 2, 3, 4, 5 and 6. In other words, any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.

The term “halo” as used herein refers to a halogen residue selected from the group consisting of F, Br, I and C1. Preferably, the halogen is F.

The term “linker” as used herein refers to any chemically suitable linker. Preferably, linker are not or only slowly cleaved under physiological conditions. Thus, it is preferred that the linker does not comprise recognition sequences for proteases or recognition structures for other degrading enzymes. Since it is preferred that the compounds of the invention are administered systemically to allow broad access to all compartments of the body and subsequently enrichment of the compounds of the invention wherever in the body the tumor is located, it is preferred that the linker is chosen in such that it is not or only slowly cleaved in blood. The cleavage is considered slowly, if less than 50% of the linkers are cleaved 2 h after administration of the compound to a human patient. Suitable linkers, for example, comprises or consists of optionally substituted alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, aralkyl, heteroaralyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, sulfonyl, amines, ethers, thioethers phosphines, phosphoramidates, carboxamides, esters, imidoesters, amidines, thioesters, sulfonamides, 3-thiopyrrolidine-2,5-dion, carbamates, ureas, guanidines, thioureas, disulfides, oximes, hydrazines, hydrazides, hydrazones, diaza bonds, triazoles, triazolines, tetrazines, platinum complexes and amino acids, or combinations thereof. Preferably, the linker comprises or consists of 1,4-piperazine, 1,3-propane and a phenolic ether or combinations thereof.

The expression “optionally substituted” refers to a group in which one, two, three or more hydrogen atoms may have been replaced independently of each other by the respective substituents.

As used herein, the term "amino acid" refers to any organic acid containing one or more amino substituents, e.g. a-, b- or g-amino, derivatives of aliphatic carboxylic acids. In the polypeptide notation used herein, e.g. Xaa5, i.e. Xaa1Xaa2Xaa3Xaa4Xaa5, wherein Xaal to Xaa5 are each and independently selected from amino acids as defined, the left hand direction is the amino terminal direction and the right hand direction is the carboxy terminal direction, in accordance with standard usage and convention.

The term "conventional amino acid" refers to the twenty naturally occurring amino acids, and encompasses all stereomeric isoforms, i.e. D,L-, D- and L-amino acids thereof. These conventional amino acids can herein also be referred to by their conventional three- letter or one-letter abbreviations and their abbreviations follow conventional usage (see, for example, Immunology — A Synthesis, 2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)).

The term "non-conventional amino acid" refers to unnatural amino acids or chemical amino acid analogues, e.g. a,a-disubstituted amino acids, N-alkyl amino acids, homo-amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), and ortho-, meta- or para-aminobenzoic acid. Non-conventional amino acids also include compounds which have an amine and carboxyl functional group separated in a 1,3 or larger substitution pattern, such as b-alanine, g-amino butyric acid, Freidinger lactam, the bicyclic dipeptide (BTD) , amino-methyl benzoic acid and others well known in the art. Statine- like isosteres, hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art may also be used. The use of analogues or non-conventional amino acids may improve the stability and biological half-life of the added peptide since they are more resistant to breakdown under physiological conditions. The person skilled in the art will be aware of similar types of substitution which may be made. A non-limiting list of non- conventional amino acids which may be used as suitable building blocks for a peptide and their standard abbreviations (in brackets) is as follows: a-aminobutyric acid (Abu), L-N- methylalanine (Nmala), α-amino-a-methylbutyrate (Mgabu), L-N-methylarginine (Nmarg), aminocyclopropane (Cpro), L-N-methylasparagine (Nmasn), carboxylate L-N-methylaspartic acid (Nmasp), aniinoisobutyric acid (Aib), L-N-methylcysteine (Nmcys), aminonorbomyl (Norb), L-N-methylglutamine (Nmgln), carboxylate L-N-methylglutamic acid (Nmglu), cyclohexylalanine (Chexa), L-N-methylhistidine (Nmhis), cyclopentylalanine (Cpen), L-N- methylisolleucine (Nmile), L-N-methylleucine (Nmleu), L-N-methyllysine (Nmlys), L-N- methylmethionine (Nmmet), L-N-methylnorleucine (Nmnle), L-N-methylnorvaline (Nmnva), L-N-methylomithine (Nmorn), L-N-methylphenylalanine (Nmphe), L-N-methylproline (Nmpro), L-N-methylserine (Nmser), L-N-methylthreonine (Nmthr), L-N-methyltryptophan (Nmtrp), D-ornithine (Dorn), L-N-methyltyrosine (Nmtyr), L-N-methylvaline (Nmval), L-N- methylethylglycine (Nmetg), L-N-methyl-t-butylglycine (Nmtbug), L-norleucine (NIe), L- norvaline (Nva), α-methyl-aminoisobutyrate (Maib), α-methyl-γ-aminobutyrate (Mgabu), D-α- methylalanine (Dmala), α-methylcyclohexylalanine (Mchexa), D-α-methylarginine (Dmarg), α-methylcylcopentylalanine (Mcpen), D-α-methylasparagine (Dmasn), α-methyl-α- napthylalanine (Manap), D-α-methylaspartate (Dmasp), a-methylpenicillamine (Mpen), D-α- methylcysteine (Dmcys), N-(4-aminobutyl)glycine (Nglu), D-α-methylglutamine (Dmgln), N- (2-aminoethyl)glycine (Naeg), D-α-methylhistidine (Dmhis), N-(3 -aminopropyl)glycine (Norn), D-α-methylisoleucine (Dmile), N-amino-a-methylbutyrate (Nmaabu), D-α- methylleucine (Dmleu), α-napthyl alanine (Anap), D-α-methyllysine (Dmlys), N-benzylglycine (Nphe), D-α-methylmethionine (Dmmet), N-(2-carbamylethyl)glycine (Ngln), D-a- methylornithine (Dmorn), N-(carbamylmethyl)glycine (Nasn), D-α-methylphenylalanine (Dmphe), N-(2-carboxyethyl)glycine (Nglu), D-α-methylproline (Dmpro), N- (carboxymethyl)glycine (Nasp), D-α-methyl serine (Dmser), N-cyclobutylglycine (Ncbut), D- α-methylthreonine (Dmthr), N-cycloheptylglycine (Nchep), D-α-methyltryptophan (Dmtrp), N-cyclohexylglycine (Nchex), D-α-methyltyrosine (Dmty), N-cyclodecylglycine (Ncdec), D- α-methylvaline (Dmval), N-cylcododecylglycine (Ncdod), D-N-methylalanine (Dnmala), N- cyclooctylglycine (Ncoct), D-N-methylarginine (Dnmarg), N-cyclopropylglycine (Ncpro), D- N-methylasparagine (Dnmasn), N-cycloundecylglycine (Ncund), D-N-methylaspartate (Dnmasp), N-(2,2-diphenylethyl)glycine (Nbhm), D-N-methylcysteine (Dnmcys), N-(3,3- diphenylpropyl)glycine (Nbhe), D-N-methylglutamine (Dnmgln), N-(3 guanidinopropyl)glycine (Narg), D-N-methylglutamate (Dnmglu), N-( 1 hydroxyethyl)glycine (Ntbx), D-N-methylhistidine (Dnmhis), N-(hydroxyethyl))glycine (Nser), D-N-methylisoleucine (Dnmile), N-(imidazolylethyl))glycine (Nhis), D-N- methylleucine (Dnmleu), N-(3 -indolylyethyl)glycine (Nhtrp), D-N-methyllysine (Dnnilys), N- methyl-γ-ami nobutyrate (Nmgabu), N-methylcyclohexylalanine (Nmchexa), D-N- methylmethionine (Dnmmet), D-N-methylomithine (Dnmorn), N-methylcyclopentylalanine (Nmcpen), N-methylglycine (Nala), D-N-methylphenylalanine (Dnmphe), N- methylaminoisobutyrate (Nmaib), D-N-methylproline (Dnmpro), N-( 1 -methylpropyl)glycine (Nile), D-N-methylserine (Dnmser), N-(2-methylpropyl)glycine (Nleu), D-N-methylthreonine (Dnmthr), D-N-methyltryptophan (Dnmtrp), N-(1-methylethyl)glycine (Nval), D-N- methyltyrosine (Dnmtyr), N-methyla-napthylalanine (Nmanap), D-N-methylvaline (Dnmval), N-methylpenicillamine (Nmpen), g-aminobutyric acid (Gabu), N-(p-hydroxyphenyl)glycine (Nhtyr), L-/-butylglycine (Tbug), N-(thiomethyl)glycine (Ncys), L-ethylglycine (Etg), penicillamine (Pen), L-homophenylalanine (Hphe), L-α-methylalanine (Mala), L-α- methylarginine (Marg), L-a-methylasparagine (Masn), L-α-methylaspartate (Masp), L-α- methyl-t-butylglycine (Mtbug), L-α-methylcysteine (Mcys), L-methylethylglycine (Metg), L- α-methylglutamine (Mgln), L-α-methylglutamate (Mglu), L-α-methylhistidine (Mhis), L-α- methylhomophenylalanine (Mhphe), L-a-methylisoleucine (Mile), N-(2- methylthioethyl)glycine (Nmet), L-α-methylleucine (Mleu), L-α-methyllysine (Mlys), L-α- methylmethionine (Mmet), L-α-methylnorleucine (Mnle), L-α-methylnorvaline (Mnva), L-α- methylornithine (Mom), L-α-methylphenylalanine (Mphe), L-α-methylproline (Mpro), L-α- methylserine (Mser), L-α-methylthreonine (Mthr), L-α-methyltryptophan (Mtrp), L-α- methyltyrosine (Mtyr), L-α-methylvaline (Mval), L-N-methylhomophenylalanine (Nmhphe), N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine (Nnbhm), N-(N-(3 ,3 -diphenylpropyl)- carbamylmethyl)glycine (Nnbhe), 1 -carboxy- 1 -(2,2-diphenyl-ethylamino)cyclopropane (Nmbc), L-O-methyl serine (Omser), L-O-methyl homoserine (Omhser).

The term “N-containing aromatic or non-aromatic mono or bicyclic heterocycle” as used herein refers to a cyclic saturated or unsaturated hydrocarbon compound which contains at least one nitrogen atom as constituent of the cyclic chain.

The term “radioactive moiety” as used herein refers to a molecular assembly which carries a radioactive nuclide. The nuclide is bound either by covalent or coordinate bonds which remain stable under physiological conditions. Examples are [ ¹³¹ I]-3-iodobenzoic acid or ⁶⁸ Ga- DOTA.

A “fluorescent isotope” as used herein emits electromagnetic radiation after excitation by electromagnetic radiation of a shorter wavelength.

A “radioisotope” as used herein is a radioactive isotope of an element (included by the term “radionuclide”) emitting α-, β-, and/or γ-radioation. The term “radioactive drug” is used in the context of the present invention to refer to a biologic active compound which is modified by a radioisotope. Especially intercalating substances can be used to deliver the radioactivity to direct proximity of DNA (e.g. a ¹³¹ I- carrying derivative of Hoechst-33258).

The term “chelating agent” or “chelate” are used interchangeably in the context of the present invention and refer to a molecule, often an organic one, and often a Lewis base, having two or more unshared electron pairs available for donation to a metal ion. The metal ion is usually coordinated by two or more electron pairs to the chelating agent. The terms, “bidentate chelating agent”, “tridentate chelating agent, and “tetradentate chelating agent” refer to chelating agents having, respectively, two, three, and four electron pairs readily available for simultaneous donation to a metal ion coordinated by the chelating agent. Usually, the electron pairs of a chelating agent forms coordinate bonds with a single metal ion; however, in certain examples, a chelating agent may form coordinate bonds with more than one metal ion, with a variety of binding modes being possible.

The term “fluorescent dye” is used in the context of the present invention to refer to a compound that emits visible or infrared light after excitation by electromagnetic radiation of a shorter and suitable wavelength. It is understood by the skilled person, that each fluorescent dye has a predetermined excitation wavelength.

The term “contrast agent” is used in the context of the present invention to refer to a compound which increases the contrast of structures or fluids in medical imaging. The enhancement is achieved by absorbing electromagnetic radiation or altering electromagnetic fields.

The term “paramagnetic” as used herein refers to paramagnetism induced by unpaired electrons in a medium. A paramagnetic substance induces a magnetic field if an external magnetic field is applied. Unlike diamagnetism the direction of the induced field is the same as the external field and unlike ferromagnetism the field is not maintained in absence of an external field.

The term “nanoparticle” as used herein refers to particles preferably of spheric shape, with diameters of sizes between 1 and 100 nanometers. Depending on the composition, nanoparticles can possess magnetical, optical or physico-chemical qualities that can be assessed. Additionally surface modification is achievable for many types of nanoparticles.

The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention. Suitable pharmaceutically acceptable salts of the compound of the present invention include acid addition salts which may, for example, be formed by mixing a solution of choline or derivative thereof with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compound of the invention carries an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include but are not limited to: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, Berge, S. M., 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.

The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide a compound of formula (I). A prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of this invention following administration of the prodrug to a patient. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. For a general discussion of prodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews 16.5 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985). Examples of a masked carboxylate anion include a variety of esters, such as alkyl (for example, methyl, ethyl), cycloalkyl (for example, cyclohexyl), aralkyl (for example, benzyl, p- methoxybenzyl), and alkylcarbonyloxyalkyl (for example, pivaloyloxymethyl). Amines have been masked as arylcarbonyloxymethyl substituted derivatives which are cleaved by esterases in vivo releasing the free drug and formaldehyde (Bungaard J. Med. Chem. 2503 (1989)). Also, drugs containing an acidic NH group, such as imidazole, imide, indole and the like, have been masked with N-acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)). Hydroxyl groups have been masked as esters and ethers. EP 0 039 051 (Sloan and Little, Apr. 11, 1981) discloses Mannich-base hydroxamic acid prodrugs, their preparation and use.

Compounds according to the invention can be synthesized according to one or more of the following methods. It should be noted that the general procedures are shown as it relates to preparation of compounds having unspecified stereochemistry. However, such procedures are generally applicable to those compounds of a specific stereochemistry, e.g., where the stereochemistry about a group is (S) or (R). In addition, the compounds having one stereochemistry (e.g., (R)) can often be utilized to produce those having opposite stereochemistry (i.e., (S)) using well-known methods, for example, by inversion.

Certain compounds of the present invention can exist in unsolvated forms as well as in solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention. 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 be radiolabeled with radioactive isotopes, such as for example tritium ( ³ H ), iodine-125 ( ¹²⁵ I) or carbon-14 ( ¹⁴ C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term “pharmaceutical composition” as used in the present application refers to a substance and/or a combination of substances being used for the identification, prevention or treatment of a tissue status or disease. The pharmaceutical composition is formulated to be suitable for administration to a patient in order to prevent and/or treat disease. Further a pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making the composition suitable for therapeutic use. Pharmaceutical compositions can be formulated for oral, parenteral, topical, inhalative, rectal, sublingual, transdermal, subcutaneous or vaginal application routes according to their chemical and physical properties. Pharmaceutical compositions comprise solid, semisolid, liquid, transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powder, granulate, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also comprised are liquid compositions, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion or solutions of the carrier systems of the present invention. Semisolid compositions that can be used in the context of the invention comprise emulsion, suspension, creams, lotions, gels, globules, buccal tablets and suppositories.

“Pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term “carrier”, as used herein, refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

The term “fibroblast activation protein (FAP)” as used herein is also known under the term “seprase”. Both terms can be used interchangeably herein. Fibroblast activation protein is a homodimeric integral protein with dipeptidyl peptidase IV (DPPIV)-like fold, featuring an alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain.

Embodiments

In the following different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

In a first aspect, the present invention provides a compound of Formula (I) wherein

Q, R, U, V, W, Y, Z are individually present or absent under the proviso that at least three of Q, R, U, V, W, Y, Z are present;

Q, R, U, V, W, Y, Z are independently selected form the group consisting of O, CH2, NR ⁴ , C=0, C=S, C=NR ⁴ , HCR ⁴ and R ⁴ CR ⁴ , with the proviso that two Os are not directly adjacent to each other; preferably out of the six four groups are present of which two are C=0, one is CH2 and one is NH; more preferably four groups are present of which two are C=0, one is CH2 and one is NH; most preferably, V, W, Y and Z are present of which V and Z are C=0 and W and Y are independently selected from CH2 and NH;

R ¹ and R ² are independently selected from the group consisting of -H, -OH, halo, C _1-6 -alkyl, - O-C _{1 -6} -alkyl, S-C _1-6 -alkyl;

R ³ is selected from the group consisting of -H , -CN , -B(OH)2, -C(O) -alkyl, -C(O) -aryl-, - C=C-C(0) -aryl, -C=C-S(0) ₂ -aryl, -CO2H , -SO3H , -S02NH ₂ ,-PO ₃ H ₂ , and 5-tetrazolyl;

R ⁴ is selected from the group consisting of -H, -C _1-6 -alkyl, -O-C _{1 -6} -alkyl, -S-C _1-6 -alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, and -Ci-6-aralkyl, each of said - C _1-6 -alkyl being optionally substituted with from 1 to 3 substituents selected from -OH, oxo, halo and optionally connected to Q, R, U, V, W, Y or Z;

R ⁵ is selected from the group consisting of -H, halo and C _1-6 -alkyl;

R ⁶ , and R ⁷ are independently selected from the group consisting of-H, , under the proviso that R ⁶ and R ⁷ are not at the same time H, preferably R ⁶ is attached to the 7- or 8-quinolyl position and R ⁷ is attached to the 5- or 6- quinolyl position; more preferably R ⁶ is attached to the 7-quinolyl position and R ⁷ is attached to the 6-quinolyl position, wherein L is a linker, wherein D, A, E, and B are individually present or absent, preferably wherein at least A, E, and B are present, wherein when present:

D is a linker;

A is selected from the group consisting of NR ⁴ , O, S, and CH2;

E is selected from the group consisting of C _1-6 -alkyl, wherein i is 1, 2, or 3; wherein j is 1, 2, or 3; wherein k is 1, 2, or 3; wherein m is 1, 2, or 3; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

A and E together form a group selected from: a cycloalkyl, heterocycloalkyl, aryl and heteroaryl, preferably heterocycloalkyl, wherein A and E can be mono-, bi- and multicyclic, preferably monocyclic. Each A and E being optionally substituted with 1 to 4 substituents selected from -H, -C _1-6 -alkyl, -O-C _1-6 -alkyl, -S-C _1-6 -alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, and -C _1-6 -aralkyl, each of said -C _1-6 -alkyl being optionally substituted with from 1 to 3 substituents selected from -OH, oxo, halo; and optionally connected to A, B, D, E or

B is selected from the group consisting of S, NR ⁴ , NR ⁴ -0, NR ⁴ -C _{1 -6} -alkyl, NR ⁴ -C _1-6 -alkyl-NR ⁴ , and a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein NR ⁴ -C ₁ - ₆ -alky 1-NR ⁴ and the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, C _1-6 -aralkyl; and

R ⁸ is selected from the group consisting of radioactive moiety, chelating agent, fluorescent dye, a contrast agent and combinations thereof; is a 1-naphtyl moiety or a 5 to 10- membered N-containing aromatic or non- aromatic mono- or bicyclic heterocycle, wherein there are 2 ring atoms between the N atom and X; said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S; and X is a C atom; or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate, or salt thereof for use in a method of in vivo diagnosis of ischemia. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a preferred embodiment R ⁸ is a radioactive moiety.

In a preferred embodiment R ⁸ is a chelating agent.

In a preferred embodiment, the ischemia is a coronary ischemia.

In another preferred embodiment, the ischemia, preferably coronary ischemia, is in a presymptomatic or prodromal stage. The present invention is particular useful for the in vivo diagnosis in the very early stages, i.e. presymptomatic or prodromal stage, before the occurrence of typical symptoms/disease stages of ischemia, in particular coronary ischemia. The present invention therefore allows to predict harmful consequences of ischemia, in particular myocardial infarction. A diagnosis is therefore possible in a presymptomatic or prodromal stage and allows to identify patients at risk of suffering from the harmful consequences of coronary ischemia such as myocardial infarction.

In a preferred embodiment the ischemia is in a presymptomatic or prodromal stage and allows to predict in advance acute ischemic complications, in particular myocardial infarction. Preferably the prediction can be made at least 6, 5, 4, 3, 2, 1 month(s), preferably at least 3 months, more preferably 3 months, in advance. Patients that benefit particularly from the method of diagnosis of the present invention are those fulfilling at least one of the following criteria: (a) critical ischemia, preferably critical coronary ischemia, (b) acute risk of myocardial tissue damage, (c) coronary heart disease, (d) lesions in the myocardial tissue, in particular multiple lesions, (e) overweight, in particular obesity, (f) diabetes, (g) metabolic syndrome, (h) anti-cancer treatment, in particular chemotherapy and/or radiation therapy.

The present invention is also particularly useful in preparation of any interventions, in particular surgical interventions. The present invention allows for improved risk assessment and/or selection of most suited intervention.

In a preferred embodiment, A and E together form a group selected from the group consisting of a C ₃ , C ₄ , C ₅ , C ₆ , C ₇ and C ₈ monocyclic, preferably C5 or C6 monocyclic, or C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ or C ₁₂ bicyclic, preferably C ₇ , C ₈ , C ₉ and C ₁₀ bicyclic heterocycloalkyl, comprising 1, 2, 3, or 4, preferably 1 or 2 heteroatoms independently selected from the group consisting of N, O and S, preferably N and O, most preferably 1 or 2 N.

In a preferred embodiment of the first aspect of the present invention the use of a compound of Formula (I) is provided: wherein

Q, R, U, V, W, Y, Z are individually present or absent under the proviso that at least three of Q, R, U, V, W, Y, Z are present;

Q, R, U, V, W, Y, Z are independently selected form the group consisting of O, CH ₂ , NR ⁴ , C=0, C=S, C=NR ⁴ , HCR ⁴ and R ⁴ CR ⁴ , with the proviso that two Os are not directly adjacent to each other; preferably out of the six four groups are present of which two are C=0, one is CH ₂ and one is NH; more preferably four groups are present of which two are C=0, one is CH ₂ and one is NH; most preferably, V, W, Y and Z are present of which V and Z are C=0 and W and Y are independently selected from CH ₂ and NH;

R ¹ and R ² are independently selected from the group consisting of -H, -OH, halo, C _1-6 -alkyl, -

O-C _{1 -6} -alkyl, S-C _1-6 -alkyl;

R ³ is selected from the group consisting of -H , -CN , -B(OH)2, -C(O) -alkyl, -C(O) -aryl-, -

C=C-C(0) -aryl, -C=C-S(0) ₂ -aryl, -CO2H , -SO3H , -S02NH ₂ ,-P0 ₃ H ₂ , and 5-tetrazolyl; R ⁴ is selected from the group consisting of -H, -C _1-6 -alkyl, -O-C _1-6 -alkyl, -S-C _1-6 -alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, and -C _1-6 -aralkyl, each of said - C _1-6 -alkyl being optionally substituted with from 1 to 3 substituents selected from -OH, oxo, halo and optionally connected to Q, R, U, V, W, Y or Z;

R ⁵ is selected from the group consisting of -H, halo and C _1-6 -alkyl;

R ⁶ , and R ⁷ are independently selected from the group consisting of-H, under the proviso that R and R are not at the same time H, preferably R ⁶ is attached to the 7- or 8-quinolyl position and R ⁷ is attached to the 5- or 6- quinolyl position; more preferably R ⁶ is attached to the 7-quinolyl position and R ⁷ is attached to the 6-quinolyl position, wherein L is a linker, wherein D, A, E, and B are individually present or absent, preferably wherein at least A, E, and B are present, wherein when present:

D is a linker;

A is selected from the group consisting of NR ⁴ , O, S, and CH2;

E is selected from the group consisting of C _1-6 -alkyl, wherein i is 1, 2, or 3; wherein j is 1, 2, or 3; wherein k is 1, 2, or 3; wherein m is 1, 2, or 3; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is selected from the group consisting of S, NR ⁴ , NR ⁴ -0, NR ⁴ -C _I - ₆ -alkyl, NR ⁴ -C _1-6 -alkyl-NR ⁴ , and a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein NR ⁴ -C ₁ - ₆ -alky 1-NR ⁴ and the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, Ci- ₆ -aralkyl; and

R ⁸ is selected from the group consisting of radioactive moiety, chelating agent, fluorescent dye, a contrast agent and combinations thereof; is a 1-naphtyl moiety or a 5 to 10- membered N-containing aromatic or non- aromatic mono- or bicyclic heterocycle, wherein there are 2 ring atoms between the N atom and X; said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S; and X is a C atom; or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate, or salt thereof. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In another preferred embodiment of the first aspect of the present invention A and E together form a group consisting of a C ₃ , C ₄ , C ₅ , C ₆ , C ₇ and Cx monocyclic, preferably C ₅ or C ₆ monocyclic, or C ₇ , C ₈ , C ₉ , C ₁₀ , C1 or C ₁₂ bicyclic, preferably C ₇ , C ₈ , C ₉ and C ₁₀ bicyclic heterocycloalkyl, preferably comprising 1, 2, 3, or 4, more preferably 1 or 2 heteroatoms independently selected from the group consisting of N, O and S, preferably N and O, most preferably 1 or 2 N. Preferred monocyclic heterocycloalkyls are selected from the group consisting of pyrrolidinyl, piperidinyl, imidazolidinyl, 1,2-diazacyclohexanyl, 1,3- diazacyclohexanyl, piperazinyl, 1-oxo-2-azacyclohexanyl, 1-oxo-3-azacyclohexanyl, or morpholinyl, preferably piperidinyl, piperazinyl, and pyrrolidinyl. Preferred bicyclic heterocycloalkyls are selected from the group consisting of bicyclo[2.2.1] 2,5-diazaheptanyl, 3,6-diazabicyclo[3.2.1]octanyl, 3,6-diazabicyclo[3.2.2]nonyl, octahydropyrrolo[2,3- b]pyrrolyl, octahydropyrrolo[3,2-b]pyrrolyl, octahydropyrrolo[3,4-b]pyrrolyl, octahydropyrrolo[3,4-c]pyrrolyl, 9-methyl-3,7,9-triazabicyclo[3.3.1]nonanyl.

The bond between the heterocycle formed by A and E and B on one hand and/or R ⁶ or R ⁷ on the other is preferably through the heteroatom, preferably through N.

Particularly, preferred examples of the heterocycle formed by A and E are selected from the group consisting of In a preferred embodiment of the first aspect of the present invention,

Q, R, U are CH2 and are individually present or absent; preferably, Q and R are absent;

V is CH2, C=0, C=S or C=NR ⁴ ; preferably, V is C=0;

W is NR ⁴ ; preferably, W is NH;

Y is HCR ⁴ ; preferably, Y is CH2; and

Z is C=0, C=S or C=NR ⁴ , preferably, Z is C=0.

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent;

V is CH ₂ ;

W is NH;

Y is CH2; and Z is C=0.

In a further preferred embodiment of the first aspect of the present invention,

R ¹ and R ² are independently selected from the group consisting of -H and halo; preferably, R ¹ and R ² are halo; more preferably, R ¹ and R ² are F;

R ³ is selected from the group consisting of -H, -CN, and -B(OH)2; preferably, R ³ is -CN or - B(OH)2; more preferably, R ³ is -CN;

R ⁴ is selected from the group consisting of -H and -C _1-6 -alkyl, wherein the -C _1-6 -alkyl is optionally substituted with from 1 to 3 substituents selected from -OH. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert- butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent;

V is CH ₂ ;

W is NH;

Y is CH ₂ ;

Z is C=0;

R ¹ and R ² are independently selected from the group consisting of -H and halo; preferably, R ¹ and R ² are halo; more preferably, R ¹ and R ² are F;

R ³ is selected from the group consisting of -H, -CN, and -B(OH)2; preferably, R ³ is -CN or - B(OH)2; more preferably, R ³ is -CN;

R ⁴ is selected from the group consisting of -H and -C _1-6 -alkyl, wherein the -C _1-6 -alkyl is optionally substituted with from 1 to 3 substituents selected from -OH. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert- butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

Q, R, U are absent; V is CH ₂ ;

W is CH ₂ ;

Y is NH;

Z is C=0;

R ¹ and R ² are independently selected from the group consisting of -H and halo; preferably, R ¹ and R ² are halo; more preferably, R ¹ and R ² are F;

R ³ is selected from the group consisting of -H, -CN, and -B(OH) ₂ ; preferably, R ³ is -CN or - B(OH) ₂ ; more preferably, R ³ is -CN;

R ⁴ is selected from the group consisting of -H and -C _1-6 -alkyl, wherein the -C _1-6 -alkyl is optionally substituted with from 1 to 3 substituents selected from -OH. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert- butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention, optionally further comprising 1 or 2 heteroatoms selected from O, N, and S.

In a further preferred embodiment of the first aspect of the present invention, , optionally further comprising 1 or 2 heteroatoms selected from O, N, and S.

In a further preferred embodiment of the first aspect of the present invention,

. R , and R are independently selected from the group consisting of-H, and under the proviso that R ⁶ and R ⁷ are not at the same time H and preferably R ⁶ and R ⁷ are attached on positions 5, 6 or 7. In a preferred embodiment, is selected from the group consisting of

In a further preferred embodiment of the first aspect of the present invention, R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is absent;

A is O, S, CH ₂ , NH, NC¾;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl; or

A and E together form a group selected from:

B is NR ⁴ -C ₁ - ₆ -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, Ci- ₆ -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is absent;

A is O;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -C _I - ₆ -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, Ci- ₆ -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is absent;

A is S;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -C ₁ - ₆ -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, C _1-6 -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R is preferably R is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is absent;

A is CH ₂ ;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -C _{1 -6} -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, Ci- ₆ -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is absent;

A is H;

E is C _1-6 -alkyl or wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -C _{1 -6} -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, Ci- ₆ -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R is preferably R is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is an amino acid, preferably carrying a charged side chain;

A is O;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -C _I - ₆ -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, Ci- ₆ -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is an amino acid, preferably carrying a charged side chain;

A is S;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl; B is NR ⁴ -C _{1 -6} -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, Ci- ₆ -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is an amino acid, preferably carrying a charged side chain;

A is CH ₂ ;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -C _{1 -6} -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, C _1-6 -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is an amino acid, preferably carrying a charged side chain;

A is NH; E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is NR ⁴ -C ₁ - ₆ -alkyl or a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected the group consisting of C _1-6 -alkyl, aryl, Ci- ₆ -aralkyl. Preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is absent;

A is O;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; Preferably, E is C1-6- alkyl and C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 nitrogen atoms.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H;

R ⁷ is , preferably R ⁷ is attached to the 5- or 6-quinolyl position; more preferably R ⁷ is attached to the 6-quinolyl position, wherein D is absent;

A is O;

E is C3 or C4 alkyl; more preferably, E is propyl or butyl; B is a 5- to 10-membered N-containing aromatic or non-aromatic mono- or bicyclic heterocycle, preferably further comprising 1 or 2 nitrogen atoms.

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H; and

R ⁷ is

In a further preferred embodiment of the first aspect of the present invention,

R ⁵ and R ⁶ are H; and

R ⁷ is and B is a 5- to 10-membered N-containing aromatic or non- aromatic mono-or bicyclic heterocycle, preferably further comprising 1 or 2 heteroatoms selected from O, N, and S, preferably further comprising 1 or 2 nitrogen atoms.

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is an aromatic or non-aromatic monocyclic heterocycle:

, wherein the heterocycle optionally further comprises 1 or 2 heteroatoms selected form O, N and S, optionally further comprises 1 nitrogen; is attached to position 1, 2, or 3, preferably to position 2;

1 is 1 or 2.

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is an aromatic or non-aromatic monocyclic heterocycle:

, wherein the heterocycle optionally further comprises 1 or 2 heteroatoms selected form O, N and S, optionally further comprises 1 nitrogen; is attached to position 1, 2, or 3, preferably to position 2;

1 is 1 or 2; wherein the N-containing heterocycle is substituted with a C _1-6 -alkyl.

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of: wherein the N-containing heterocycle is substituted with a C _1-6 -alkyl wherein if the N-containing heterocycle comprised in B is the heterocycle optionally further comprises 1 or 2 heteroatoms selected from O, N and S, optionally further comprises 1 nitrogen, optionally compromises one or more (e.g. amino acid derived) side chains; is attached to position 1, 2, or 3, preferably to position 2; o is 1 or 2; preferably, if the N-containing heterocycle comprised in B is , the N- containing heterocycle comprised in B is selected from the group consisting of

; more preferably, if the N-containing heterocycle comprised in B is , the N-containing heterocycle comprised in B is

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of: the heterocycle optionally further comprises 1 or 2 heteroatoms selected from O, N and S, optionally further comprises 1 nitrogen, optionally compromises one or more (e.g. amino acid derived) side chains; is attached to position 1, 2, or 3, preferably to position 2; o is 1 or 2; preferably, if the N-containing heterocycle comprised in B is , the N- containing heterocycle comprised in B is selected from the group consisting of ; more preferably, if the N-containing heterocycle comprised in B is , the N-containing heterocycle comprised in B is

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of:

In a further preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of: wherein B is substituted with a C _1-3 alkyl.

In a further preferred embodiment of the first aspect of the present invention, R ⁵ and R ⁶ are H;

R ⁷ is preferably R ⁷ is attached to the 6-quinolyl position, wherein

D is absent; A is O;

E is propyl or butyl;

B is is

In a further preferred embodiment of the first aspect of the present invention, Q, R, U are absent;

V is C=0;

W isNH;

Y is CH ₂ ;

Z is C=0; R ¹ and R ² are independently selected from the group consisting of -H and halo; preferably, R ¹ and R ² are independently selected from the group consisting of -H and F; more preferably, R ¹ and R ² are the same and are selected from the group consisting of -H and F;

R ³ is -CN;

R ⁵ and R ⁶ are H;

R is , preferably R is attached to the 6-quinolyl position, wherein

D is absent;

A is O;

E is C _1-6 -alkyl or , wherein m is 1, 2, or 3; preferably, E is C _1-6- alkyl; preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i- propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C _1-6 -alkyl, most preferably, E is C3 or C4 alkyl;

B is NH- C _1-6 -alkyl preferably, C _1-6 -alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; preferably, B is ; and

In a further preferred embodiment of the first aspect of the present invention, Q, R, U are absent;

V is C=0;

W is NH; Y is CH ₂ ;

Z is C=0;

R ¹ and R ² are the same and are selected from the group consisting of -H and F; R ³ is -CN;

R ⁵ and R ⁶ are H; R ⁷ is preferably R is attached to the 6-quinolyl position, wherein

D is absent;

A is O, S, CH ₂ , NH, NOE;

E is methyl, ethyl, propyl or butyl; A and E together form a group selected from: B is optionally B is substituted with a C1-3 alkyl; preferably, B is ; and

In a further preferred embodiment of the first aspect of the present invention, Q, R, U are absent;

V is C=0;

W is NH;

Y is CH ₂ ;

Z is C=0;

R ¹ and R ² are the same and are selected from the group consisting of -H and F; R ³ is -CN;

R ⁵ and R ⁶ are H; R ⁷ is preferably R is attached to the 6-quinolyl position, wherein

D is absent; A is O; E is methyl, ethyl, propyl or butyl;

B is preferably, B is and

In a further preferred embodiment of the first aspect of the present invention, Q, R, U are absent;

V is C=0;

W isNH;

Y is CEE;

Z is C=0;

R ¹ and R ² are the same and are selected from the group consisting of -H and F;

R ³ is -CN;

R ⁵ and R ⁶ are H;

R ⁷ is , R is attached to the 6-quinolyl position, wherein D is absent;

A is O;

E is methyl, ethyl, propyl or butyl;

; preferably, B is In a further preferred embodiment of the first aspect of the present invention, Ci-6-alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert- butyl, pentyl and hexyl.

In a further preferred embodiment of the first aspect of the present invention, C1-3-alkyl is selected from the group consisting of methyl, ethyl, propyl and i-propyl.

In a further preferred embodiment of the first aspect of the present invention, C1-6- aralkyl is selected from the group consisting of benzyl, phenyl-ethyl, phenyl-propyl, and phenyl-butyl.

In a preferred embodiment of the first aspect of the present invention, the compound is selected from the compounds of table 1. More preferably, the compound of the first aspect of the invention is selected from the compounds of table 2. More preferably, the compound of the first aspect of the invention is selected from the group consisting of FAPI-02 and FAPI-04. Most preferably, the compound of the first aspect of the invention is FAPI-04.

In a preferred embodiment of the first aspect of the present invention, the compound is selected from the compounds of table 1 and/or table 3. More preferably, the compound of the first aspect of the invention is selected from the compounds of table 2 and/or table 4. More preferably, the compound of the first aspect of the invention is selected from the group consisting of FAPI-02, FAPI-04, FAPI-46, FAPI-34, FAPI-42, FAPI-52, FAPI-69, FAPI-70, FAPI-71, F API-72 and FAPI-73.

Table 1: Preferred compounds of the first aspect of the invention.

§ fluorescent compounds; $ ^99m Tc-chelators; * Pb-chelators; R ¹ and R ² are located at the 4- pyrrolidine position; Q, R, U are absent; R ⁵ is H; R ⁶ is attached to the 7-quinolyl position; R ⁷ is attached to the 6-quinolyl position; indicates that R ⁶ or R ⁷ being H; ‘+’ indicates R ⁶ or R ⁷ being V is C=0; W is NH; Y is

CH2; Z is C=0; R is -CN; A is O (except F API-01 : A is absent, R is attached to the 5-quinolyl position).

Table 2: Compounds of special interest. Q, R, U, D are absent; R ¹ and R ² are located at the 4- pyrrolidine position; , R ⁵ , R ⁶ are H; R ⁷ is attached to the 6- quinolyl position; V is C=0; W is NH; Y is CH2; Z is C=0; R ³ is -CN; B is 1,4-piperazine; E is 1,3-propane; A is O. Table 3: Further preferred compounds of the first aspect of the invention.

§ fluorescent compounds; $ ^99m Tc-chelators; * precursors for ¹⁸ F-labeling; Q, R, U are absent; R ¹ and R ² are located at the 4-pyrrolidine position; ,R ⁵ and R ⁶ are H; R ⁷ is attached to the 6-quinolyl position and is V is C=0; W is NH; Y is CH ₂ ; Z is C=0; R ³ is -CN.

Table 4: Compounds of special interest. Q, R, U, D are absent; R ¹ and R ² are fluorine atoms located at the 4-pyrrolidine position; R ⁵ , R ⁶ are H; R ⁷ is attached to the 6-quinolyl position; V is C=0; W is NH; Y is CH2; Z is C=0; R ³ is -CN; B is 1,4-piperazine; E is 1,3-propane; A is O.

Table 5: Preferred precursors for radiolabelling with ^§ F-18; ^$ Cu-64; ^€ Ga-68; ^£ Tc-99m, Re- 188; ^* Y-90, Sm-153, Lu-177.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is a radioactive moiety, wherein the radioactive moiety is a fluorescent isotope, a radioisotope, a radioactive drug or combinations thereof. Preferably, the radioactive moiety is selected from the group consisting of alpha radiation emitting isotopes, beta radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescence emitting isotopes, such as ⁶⁸ Ga, ^U C, ¹⁸ F, ⁵¹ Cr, ⁶⁷ Ga, ^m In, ^99m Tc, ¹⁸⁶ Re, ¹⁸⁸ Re, 1 ³⁹ La, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁵³ Sm, ¹⁶⁶ Ho, ⁸⁸ Y, ⁹⁰ Y, ¹⁴⁹ Pm, ¹⁶ Dy, ¹⁶⁹ Er, ¹⁷⁷ Lu, ⁴⁷ Sc, ¹⁴² Pr, ¹⁵⁹ Gd, ²¹ Bi, 2 ¹³ Bi, ⁷² As, ⁷² Se, ⁹⁷ RU, ¹⁰⁹ Pd, ¹⁰⁵ Rh, ^101m Rh, ¹¹⁹ Sb, ¹² ¾a, ¹²³ 1, ¹²⁴ 1, ¹³¹ 1, ¹⁹⁷ Hg, ²¹¹ At, ¹⁵¹ Eu, ¹⁵³ Eu, 1 ⁶⁹ EU, ²⁰¹ T1, ²⁰³ Pb, ²¹² Pb, ⁶⁴ Cu, ⁶⁷ Cu, ¹⁸⁸ Re, ¹⁸⁶ Re, ¹⁹⁸ Au, ²²⁵ Ac, ²²⁷ Th and ¹⁹⁹ Ag. Preferably

⁶⁸ Ga, ¹⁸ F, ⁶⁴ CU, ⁹⁰ Y, ^99m Tc, ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁸⁸ Re.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is a fluorescent dye select from the group consisting of the following classes of fluorescent dyes: Xanthens, Acridines, Oxazines, Cynines, Styryl dyes, Coumarines, Porphines, Metal-Ligand- Complexes, Fluorescent proteins, Nanocrystals, Perylenes, Boron-dipyrromethenes and Phtalocyanines as well as conjugates and combinations of these classes of dyes.

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is a chelating agent which forms a complex with divalent or trivalent metal cations. Preferably, the chelating agent is selected from the group consisting of 1,4,7, 10-tetraazacyclododecane- N,N',N,A'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7- triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA), bis- (carboxymethylimidazole)glycine and 6-Hydrazinopyridine-3-carboxylic acid (HYNIC).

In a further preferred embodiment of the first aspect of the present invention, R ⁸ is a contrast agent which comprises or consists of a paramagnetic agent, preferably, wherein the paramagnetic agent comprises or consists of paramagnetic nanoparticles.

In a further preferred embodiment of the first aspect of the invention, R ⁸ is selected from any R ⁸ of tables 1 to 5.

In a second aspect, the present invention relates to a pharmaceutical composition comprising or consisting of at least one compound of the first aspect, and, optionally, a pharmaceutically acceptable carrier and/or excipient for the in vivo diagnosis of ischemia, preferably coronary ischemia. Preferably the ischemia, in particular coronary ischemia, is in a prodromal stage..

In a third aspect, the present invention relates to a kit comprising or consisting of the compound of the first aspect or the pharmaceutical composition of the second aspect and instructions for the in vivo diagnosis of ischemia, preferably coronary ischemia. Preferably the ischemia, in particular coronary ischemia, is in a prodromal stage.

Examples

Fibroblast activation protein (FAP) plays an important role in cardiac wound healing and remodeling. Although initially developed as a theranostic ligand for metastasized cancer, FAPI tracers have recently been used to study cardiac remodeling following myocardial infarction in small animal models. Disclosed herein is the evaluation of the activity of fibroblast activation protein via FAP inhibitor (FAPI) PET-CT scans in human hearts.

Methods

The inventors retrospectively analyzed FAPI-PET-CT scans from adult patients presenting in a nuclear medicine department between 2017 and 2019 suffering from various cancer entities. The study was conducted according to the principles of the Declaration of Helsinki. Approval for this research was granted by the local research ethics committee (S- 286/2017). Patient related information was pseudonymized upon data extraction. FAPI-PET-CT scans

FAPI-PET-CT (Siemens Biograph, Siemens Healthcare Diagnostics, Eschbom, Germany) scans were performed according to a standard protocol ([4], Giesel et al. 2019, J Nucl Med 2019;60:386-392). 122 to 336 mBq of Ga-68 labelled fibroblast activation protein inhibitor (F API-04; see table 1 and 5 above) were administered intravenously 60 minutes before the examination. We also scanned a subgroup of patients 10 minutes and 180 minutes after FAPI administration. A low dose whole body CT scan (130keV, 30mAs, CareDose; reconstructed with a soft-tissue kernel to a slice thickness of 5mm) was used for attenuation correction and image fusion. A 3-D emission scan (matrix 200 x 200) was performed, subsequently using FlowMotion (Siemens). The emission data was corrected for randoms, scatter and decay. Reconstruction was performed with an ordered subset expectation maximization (OSEM) algorithm with 2 iterations / 21 subsets and Gauss-filtered to atransaxial resolution of 5 mm at full-width at half-maximum (FWHM) and analyzed using regions of interest (ROI) measuring standardized uptake values (SUV). Signals were measured in the free left ventricular wall, the blood pool, the aorta, the gluteus muscle, the liver, the lung and the brain. The mean SUV of the region of interest (ROI) was reported. The ROI was selected in an area of homogenous signal intensity based on the anatomical structures depicted in the CT.

In addition to measuring the free lateral wall in an area without focal signal enrichment, all 17 segments were measured based on the anatomic structure regardless of focal signal enrichment. According to the imaging criteria of the American Heart Association, segments 1,2,7,8,13,14 and 17 were attributed to the left anterior descending artery, segments 5,6,11,12 and 16 to the left circumflex artery and segments 3,4,9,10,15 to the right anterior artery. [10,11] Septal segments were segments 2,3, 8,9 and 14. Lateral segments were 5,6,11,12 and 16. Graphs were built in R version 3.6.2 with inhouse scripting using the shape and RColorBrewer packages. [12-14]

FDG-PET-CT scans

All FDG-PET-CT scans performed within 12 months of the FAPI-PET-CT scan were also analyzed. FDGPET-CT scans were performed as previously described [15] Measurements were performed as in FAPIPET-CT scans. SUVs and SUV ratios of different organs were compared between FDG and FAPI using a Kruskal- Wallis test. The Spearman method was used to analyze the correlation between FDG and FAPI SUVs. Patient characteristics

Patient characteristics included age, sex, cancer entity, body mass index (BMI), glomerular filtration rate (GFR-CDK-EPI), thyroid stimulating hormone (TSH), cardiovascular risk factors (CvRF), diabetes mellitus (DM), arterial hypertension (aHT), known coronary artery disease (CAD), known atrial fibrillation (aFib), previous radiation to the chest, chemotherapy (anthracyclines, platin derivatives, alkylating agents, antimetabolites, taxanes, topoisomerase inhibitors), checkpoint inhibitor use, FAPI signal pattern, cardiac medication (statins, aspirin, angiotensin converting enzyme inhibitor (ACEi) or angiotensin-receptor- blocker (ARB) and betablocker use). For multivariate analyses, patients were divided into two cohorts, an initial cohort comprising 80% of patients and a confirmatory cohort comprising 20% of patients. Patients were assigned consecutively to each cohort. Whenever available, echocardiographic findings were also reported.

FAPI PET-CT scans from representative patients were selected using the next neighbor algorithm. Applicable patients with the smallest distance to the median were chosen.

Table 6:

Statistical Analysis

Statistical analyses were performed using R version 3.6.2 with the help of the MASS and ggplot2 packages. [12, 16, 17] Non-normal distributed values are reported as median ± interquartile range (IQR) and were compared using Wilcoxon rank sum tests unless stated otherwise. Normal distributed values are reported as mean ± standard deviation (SD). An analysis of variance test was performed to test for differences within the groups. Tukey’s honest significant difference method was applied for p-level adjustment.

As SUVs did not follow a standard distribution, SUVs were compared with a pairwise Wilcoxon rank sum test using Holm’s method for p-value adjustment. When available, repetitive measurements were displayed for each patient. When necessary (e.g. for linear and logistic regression models), SUVs were logarithmised to achieve normal distribution. Univariate logistic regression models were established using a signal intensity cut-off of 1.3, which was determined calculating the mean signal intensity and adding ½ of its standard deviation. Odds ratios, and 95% confidence intervals were calculated. P level adjustment was carried out using the Holm-Bonferroni method. Multivariate models were created selecting the variables according to Akaike’s information criterion in a step-down approach. [15, 18] Logistic regression results were reported with odds ratio (OR) and a 95% confidence interval (95%CI). Hosmer-Lemeshow goodness of fit test was performed for each model. A multivariate linear regression model was established for signal intensity using all variables significantly tested in the multivariate logistic approach. Standardized residuals were calculated the dataset used to create the model. Outliers were defined as patients with high signal intensities not accurately predicted by our models with calculated residuals above the 95th percentile. The predictive model based on our modeling cohort was subsequently applied to the confirmatory cohort to test for reproducibility. Outliers were further analyzed by a permutation test comparing patient characteristics of the outlier cohort to 100.000 randomly selected cohorts of the same dataset. The number of times a characteristic occurred as frequently of more frequently in the randomly selected cohorts divided by the number of permutations was used as a p-value. A p-value below 0.05 was considered significant. The model was applied to the confirmatory cohort. Standardized residuals were calculated. Outliers in the second cohort were identified using the same values as cut-off for residuals as in the first cohort.

Results

Between 2017 and April 2019 a total of 185 consecutive patients suffering from more than 20 different metastasized solid tumour entities were recruited and analyzed. Pancreatic carcinoma (n=42), bronchial carcinoma (n=20), colorectal carcinoma (n=14), oropharyngeal cancer (n=13), prostate cancer (n=11), esophageal and gastric carcinoma (n=l l) and ovarian cancer (n=10) were most frequently observed. Patient characteristics are summarized in Table 6

The highest cardiac signal intensities were measured 10 minutes after FAPI administration (median SUV 1.39 [IQR: 0.62]). Signals decreased more rapidly within the first hour until a median SUV of 1.01 [IQR: 0.39] was measured. SUVs further decreased to median levels of 0.72 [IQR: 0.22] three hours after administration (see Figure 3). After one hour, the median blood pool SUV was 0.93 [IQR: 0.32] Signal intensity ratios between the heart and other organs differed greatly: heart/blood pool 0.93 [IQR: 0.38], heart/brain 12.44 [IQR: 13.29], heart/gluteal muscle 0.99 [IQR: 0.48], heart/liver 1.17 [IQR: 0.56], heart/lung 0.84 [IQR: 0.29] 21 patients also had data from FDG PET-CT scans. Left ventricular signal intensities were higher using an FDG tracer (Figure 4). Comparing FDG and FAPI signals in each measured segment, we were able to see a relatively weak but consistent positive correlation (rs=0.36, p<0.001). Heart/blood and heart/lung signal ratios were comparable, heart/liver and heart/brain signal ratios were significantly higher in FAPI scans and heart/gluteus ratios were significantly higher in FDG scans as FDG signals are indicative of glycolytic activity.

Patients suffering from prostate cancer and ovarian cancer showed nominally increased cardiac SUVs (1.08 [IQR: 0.63] and 1.04 [IQR: 1.84], respectively) while pancreatic cancer patients exhibited a nominal decrease (0.79 [IQR: 0.57]). Comparing solely these groups cardiac signals in patients suffering from ovarian and prostatic cancer were significantly higher than in hearts from pancreatic cancer patients (p<0.05, Wilcoxon rank sum test, Figure 5 and 6)· Signal intensity correlates with diabetes, different chemotherapies and radiation to the chest

In an univariate regression analysis high left ventricular signals correlated with the presence of cardiovascular risk factors (Odds Ratio (OR): 4.3, p<0.001), a focal myocardial signal pattern (OR: 3.9, p<0.01), diabetes (OR: 4.1, p<0.05) and betablocker use (OR: 3.8, p<0.05).

Multivariate regression models were established following model optimization using Akaike’s information criterion as described in the method section. This model revealed a positive correlation of left ventricular signals with TSH levels above 4 pU/ml (OR: 8.6, p< 0.05), a BMI above 25kg/m ² (OR: 2.6, p<0.05), previous radiation to the chest (OR: 3.5, p<0.05), previous intake of platin derivatives (OR: 3.0, p<0.05) and a history of diabetes (OR: 2.9, p<0.05) while checkpoint inhibitor use was associated with a decreased signal intensity (OR: 0.17, p<0.05) (see Figure 1). A direct comparison comprising both cohorts for each subgroup is reported in Figure 7. Echocardiographic findings were available in a subgroup of patients (n=44) showing some evidence for increased FAPI signals in patients with reduced ejection fraction (see Figure 8).

Multivariate linear prediction model and outlier analysis reveals doxorubicin therapy as potentially relevant variable in cardiac FAP-activity

To test for reliability of the retrospective analysis, the inventors further created a linear multivariate regression model based on the results of the logistic regression analyses. This model was used to find outliers in the dataset by selecting 5% of patients with the highest residuals (cut-off: 1.64). A permutation test with 100.000 permutations revealed that patients with ovarian and prostate cancer patients (p<0.01), as wells as patients receiving anthracyclines (p<0.05) and/or alkylating agents (p<0.05) were significantly overrepresented in the outlier cohort of patients with unexpectedly high cardiac FAPIsignals.

The prediction model was confirmed in another cohort of 44 consecutive patients, which were scanned after the first cohort. In this confirmatory cohort, the inventors were not able to find any outliers using the same metrics as in the first cohort (see Figure 1). There was no significant difference between the mean residuals of the two cohorts (p=0.16, Student’s T-test). The standard deviation was even slightly smaller in the second cohort. Patient characteristics of the confirmatory cohort are reported in Table 6. Focal signal enrichment and increased signal intensity associated with cardiovascular risk and disease

Signal patterns differed greatly between the patients. Five distinct patterns were noted: weak, homogenous, diffuse, focal on diffuse, and focal enrichment. Significantly more patients with known cardiovascular risk factors exhibited a focal signal pattern (p<0.0001, Yate’s χ ² - test). A focal myocardial enrichment pattern was negatively correlated with female sex (OR: 0.13, p<0.01) whereas a positive correlation was seen for patients with hypertension (OR: 3.7, p<0.01), known cardiac disease (OR: 3.8, p<0.05), known coronary heart disease (OR: 4.0, p<0.05) and medication with aspirin (OR: 4.8, p<0.01) or statins (OR: 5.7, p<0.01) in univariate logistic regression. In an optimized multivariate logistic regression model, only female sex (OR: 0.12, p<0.001), known cardiovascular risk factors (OR: 5.1, p<0.001), and aspirin use (OR: 3.0, p<0.05) were significantly associated (see Figure 1).

FAPI signal increases with cardiovascular risk factors associated with metabolic disease and cardiac remodeling.

By analyzing FAPI signals in a 17-segment model of the left ventricle, a significantly higher signal in septal signals than in the lateral segments were noticed (septal vs. lateral wall: p<0.01; septal vs. anterior/posterior wall: p<0.001; lateral vs. anterior/posterior wall p=0.31). Interestingly, while cardiovascular risk per se seemed to have a mild but notable impact on signal intensity, cardiovascular risk factors associated with metabolic stress and cardiac remodeling, e.g. arterial hypertension, diabetes mellitus, overweight and obesity, showed a more pronounced increase in signal intensity (see Figure 2). Further analyzing patients grouped by cardiovascular risk (Figure 6) showed a substantial significant increase in patients taking aspirin comparing the median segment intensities (1.25 [IQR: 0.52, n=32] vs. 0.95 [IQR: 0.31, n=197], p<0.001 ,Wilcoxon rank sum test with continuity correction).

Considering all 17 segments, repetitive scans in 26 patients revealed an increase in FAP density over time in our cancer patient cohort in all segments (p<0.001, Figure 9). Of note, one patient showed a very high signal intensity under pazopanib therapy, an endothelial growth factor receptor tyrosine kinase inhibitor. Signal intensity normalized again, once pazopanib was discontinued due to hemotoxic side effects and hypertension (see patient 14, Figure 9).

Interestingly, a series of patients with a history of myocardial infarction did not show increased signal intensities years following myocardial infarction (see Figure 10). Further investigation of all patients with known coronary artery disease showed that coronary artery disease does not correlate directly with FAPI intensities. One patient scanned while on radiotherapy in close proximity to the heart showed a clear increase in myocardial FAPI intensities (see Figure 11). Taken together these findings suggest that FAPI might not be enriched in myocardial scars but rather marks the process of myocardial remodeling.

Concepts and evidence behind the invention

In this disclosure the inventors report a correlation between fibroblast activation (FAP) density measured by fibroblast activation protein inhibitor PET-CT scans and cardiovascular disease and risk factors, particularly in cancer patients. An increase in signal intensity was associated with metabolic stress such as hypothyroid metabolic state, overweight, diabetes, the use of platin derivatives, and radiation to the chest while a focal enrichment pattern was correlated with cardiovascular disease as indicated by the presence of cardiovascular risk factors or aspirin intake. Western blot analyses, microarrays, cardiac sections and most recently FAPI-PET-CT scans in small animal models showed that FAP expression in fibroblast is significantly increased in affected segments following myocardial infarction [8,9] So far, there is currently no method established to image the activation of fibroblasts in the human heart.

Immunotherapy using chimeric antigen receptor T cells against FAP significantly reduced FAP expressing activated fibroblasts after myocardial infarction. The elimination of these cells led to a significant reduction in cardiac fibrosis and an increased systolic function in mice following myocardial infarction underlying the prominent role of FAP overexpressing activated fibroblast in pathologic cardiac remodeling and dysfunction [7] In pressure overload, the development of excessive fibrosis and activation of fibroblasts are based on a direct crosstalk between fibroblasts and cardiomyocytes involving paracrine secretion of TGFP and CAMKII activation in cardiomyocytes. These processes seem to determine cardiac function and discriminate adaptive from maladaptive cardiac remodeling [19,20] In line with previous studies showing the suppressive effect of thyroid hormone on TGFP signaling, hypothyroid patients showed higher myocardial FAPI signal levels in this study [21] Although aspirin is also known to suppress TGFP signaling, patients taking aspirin exhibited much higher cardiac FAPI signal intensities and were more likely to show focal enrichment patterns than patients not taking aspirin (see Figure 1 and Figure 6). The increase in fibroblast activation seen in this patient subgroup is most likely explained by other contributing factors as aspirin use can be seen as a surrogate for an existing cardiovascular disease. Furthermore, patients with known arterial hypertension displayed higher cardiac FAP density with a marked septal signal enrichment, the most susceptible area for pathological hypertrophy followed by diastolic dysfunction in these patients (Figure 2) [22]

Arterial hypertension and pressure overload are associated with profound metabolic changes [23] FAPI signal enrichment was most noticeable in patients with arterial hypertension and metabolic diseases such as diabetes and obesity. Although metabolic disease and arterial hypertension are often present concomitantly, diabetes and obesity both promote cardiac hypertrophy and excessive fibrosis by activating inflammatory pathways and TGFp/SMAD signaling [24] These changes can be successfully documented by the cardiac FAPI-imaging of the present invention as disclosed herein.

Although FAP is mainly expressed in granulation tissue, reactive stromal fibroblasts and malignant cells, it cannot be rule out completely that other cell types might contribute to the cardiac FAPI signal [25] Inflammation induced FAP activation in cardiomyocytes, fibroblasts or even endothelial cells as seen in vascular disease might be a contributory factor in some of the patients especially since CRP levels are increased in the outlier cohort [26]

Surprisingly and in contrast to other imaging modalities for cardiac fibrosis, FAPI PET- CT scans of the present invention display active cardiac remodeling and not fibrotic scars. Analyzing patients before and after myocardial infarction, the inventors noted increased FAPI signals just before the event while signals disappeared months to years after. In addition, a patient scanned while receiving chest radiation due to esophageal cancer showed high FAPI signals in the segments affected (Figures 10 and 11).

Furthermore, a patient displayed high FAPI signals while on pazopanib treatment (patient 14, Figure 9). A follow-up scan revealed normal signal intensities once the medication was stopped due to hypertension and hematotoxic side effects. Although arterial hypertension was associated with an increase in signal intensity in general, the signal enrichment exhibited in this patient exceeded the expected effect of arterial hypertension alone. Thus, off-target inhibition of fibroblast growth factors and other kinases might have led to this FAPI up-flare indicative of cardiac remodeling [27] Taken together the present invention discloses the use of F APIs in in vivo diagnosis, which might be particularly suitable to find previously undetected cardiovascular disease or identify cardiotoxic effects of therapies, such as anti-cancer therapy. The present invention could also be used to evaluate e.g. reverse cardiac remodeling after aortic valve replacement. It might also represent a new modality for benefit stratification for asymptomatic patients with high grade mitral valve regurgitation assessed for mitral valve correction. Furthermore, as fibroblast activation is a hallmark of connective tissue diseases and sarcoidosis, FAPI-PET-CT scans might be used to identify patient with cardiac involvement.

References

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