EP2268319A2 - Precursors of lipid metabolism for the diagnosis and treatment of cancer - Google Patents

Abstract

The present invention pertains to methods for the treatment and diagnosis of cancer employing prodrugs for targeting of lipid metabolism comprising either a detectable label or a therapeutic residue. The invention is based on the finding that the present prodrugs are specifically enriched in tumor tissue, in which they may be detected by suitable imaging techniques. Alternatively, a prodrug comprising a therapeutic residue, such as a suitable radioactive residue, may be used for the treatment of cancer.

Description

Precursors of lipid metabolism for the diagnosis and treatment of cancer

The present invention pertains to methods for the treatment and diagnosis of cancer employing prodrugs for targeting of lipid metabolism comprising either a detectable label or a therapeutic residue. The invention is based on the finding that the present prodrugs are specifically enriched in tumor tissue, in which they may be detected by suitable imaging techniques. Alternatively, a prodrug comprising a therapeutic residue, such as a suitable radioactive residue, may be used for the treatment of cancer.

Cancer represents one of the leading causes of death, and if current trends continue, cancer is expected to be the leading cause within some years. Lung and prostate cancer represent one of the most frequent cancer killers for men, while lung and breast cancer are the most frequent cancer killers for women. Currently available cancer therapies include surgery, chemotherapy, hormonal therapy and/or radiation treatment to eradicate neoplastic cells in a patient. Recently, advances in cancer therapy have led to employment of biological therapy and immunotherapy to treat cancer.

AU known cancer treatments pose significant drawbacks for the patient. Surgery, for example, may be contraindicated due to the health of the patient or may be unacceptable to the patient. Additionally, surgery may not completely remove the neoplastic tissue. Hormonal therapy is rarely given as a single agent and although it can be effective, is often used only to prevent or delay recurrence of cancer after other treatments have removed the majority of the cancer cells. Biological and immuno therapies are limited in number and may produce side effects such as rashes or swellings, flu-like symptoms, including fever, chills and fatigue, digestive tract problems or allergic reactions. Despite the availability of a variety of chemotherapeutic agents, chemotherapy has many drawbacks. Almost all chemotherapeutic agents are toxic, and chemotherapy causes significant, and often dangerous, side effects, such as severe nausea, bone marrow depression, and immunosuppression. Additionally, even with administration of combinations of chemotherapeutic agents, many tumor cells are resistant to or develop resistance to the chemotherapeutic agents. In fact, cells resistant to a particular chemotherapeutic agent often prove to be resistant to other drugs, even drugs that act by wholly unrelated mechanisms; the so called pleiotropic drug or multidrug resistance. Thus, many cancers prove refractory to standard chemotherapeutic treatment protocols because of drug resistance.

Accordingly, there remains a significant and unmet need for additional cancer therapies as all current treatments have significant disadvantages. Further, it is uncommon for a particular treatment to be effective to treat every instance of a given cancer.

Currently available methods for the diagnosis of cancer exhibit similar drawbacks. Known assays base for example on the detection of nucleic acids, proteins or other biomarkers released from tumor cells in body fluids. WO2006128192 discloses for example the use of free circulating DNA as a marker for diagnosis, prognosis, and treatment of cancer.

The diagnostic and prognostic tumor biomarkers in use today are not adequate in early identification of cancer due to their low sensitivity. Accordingly, there is still a need to develop more robust assays and genotypic markers that can be related to functional tumor biology. There is also a need in developing improved methods in the treatment of cancer.

The present invention seeks to overcome the problems associated with prior art methods.

The present invention provides particular prodrugs for targeting of lipid metabolism as well as their use for diagnosis and/or the treatment of cancer. The present prodrugs for targeting of lipid metabolism are selected from

wherein R represents an aliphatic, aromatic, heterocyclic, aralkylic, heteroaralkylic, or alicyclic substituent comprising a detectable label or a therapeutic residue. Rl and R2 are independently selected from lower alkyl, alkenyl or alkynyl, n = 1 - 8.

The present invention is based on the finding that the present prodrugs for targeting of lipid metabolism are enriched in tumor cells. Labeling said prodrugs for targeting of lipid metabolism with a detectable label renders in vivo or in vitro diagnosis possible, whereas marking with a therapeutic residue permits the treatment of cancer.

Without wishing to be bound by any theory, it is presently assumed that the present prodrugs for targeting of lipid metabolism are esterified to the respective Acyl-CoA derivatives with coenzyme A (CoA) and enriched in tumor cells by specific interaction with enzymes, such as racemases, of lipid metabolism of the tumor. It has been surprisingly found that the activity said enzymes is highly upregulated in tumor cells giving the possibility to quantitatively, selectively and sensitively detect tumors. Another advantage resides in the possibility to treat tumors with non-invasive techniques, i.e. by administering the present prodrugs for targeting of lipid metabolism exhibiting therapeutic residues, which are almost exclusively incorporated in tumor tissue. Another advantage of the use of the present prodrugs for targeting of lipid metabolism resides is that they may be easily adapted to the changing requirements in the clinical field since the prodrug may be easily provided with another detectable label or therapeutic residue.

In the figures

Fig. 1 shows the synthesis of 11 -aminoundecylmalonic acid and of /?-[*I]-iodobenzoyl-l l- aminoundecylmalonic acid according to an embodiment of the present invention;

Fig. 2 shows the synthesis of ⁿC-dodecylmalonic acid according to an embodiment of the present invention;

Fig. 3 shows the synthesis of 11-fluorododecylmalonic acid as well as 11-[¹⁸F]- fluorododecylmalonic acid (FSU 01) according to an embodiment of the present invention;

Fig. 4 shows the synthesis of 13-iodotridecen-12-ylmalonic acid as well as 13-[*Ij- iodotridecen-12-ylmalonic acid according to an embodiment of the present invention;

Fig. 5 shows an uptake of FSU 01 in prostate carcinoma cells according to an embodiment of the present invention, wherein only a neglectable cellular retention of FSU 01 may be observed after 24 h incubation, suggesting that said compound is metabolically degraded and eliminated from the cell in a quantitative manner; and

Fig. 6 shows the biodistribution of FSU 01 in the transgene TRAMP - prostate carcinoma - model of the mouse according to an embodiment of the present invention, wherein FSU 01 is fast and intensively enriched in the prostate carcinoma tissue of the primary tumor and in lymph nodes, wherein the remaining organs show only a minor and uniform uptake of FSU

01, with exception of the skeleton and liver and kidney as excretion organs, wherein the high activity of FSU 01 in the bladder is probably due to the degradation to low molecular excretion products and subsequent renal excretion, wherein further analysis has shown that partial enrichment of FSU 01 in lymph nodes and skeleton was due to the formation of metastasis.

Definitions:

A "prodrug" and a "prodrug for targeting of lipid metabolism", i.e. a prodrug of lipid metabolism, as used herein pertains to a pharmacological substance, i.e. a free fatty acid (FFA), which is metabolized in vivo into an active metabolite. Metabolisation is hereby performed inter alia by the action of racemases, i.e. enzymes which may catalyze the inversion around the asymmetric carbon atom in a substrate having one center of asymmetry. It will be, however, appreciated that the substrates of e.g. racemases do not necessarily exhibit a center of asymmetry. Fatty acid utilization in normal prostate tissue and in prostate carcinoma is in comparison to FFA utilization in liver (Iozzo, P. et al., Eur J Nucl Med MoI Imaging, 30(8) (2003), pp. 1160-4) and heart muscles (Reske, S. N. et al., Am J Physiol Imaging 1(4) (1986), pp. 214-229) is comparatively low (Toghrol, F. et al., Toghrol, 25(6) (1980), pp. 371-6;del Hoyo, N. et al., Biosci Rep, 10(1) (1990), pp. 105-12). The present inventors surprisingly found in cell culture experiments and in mouse model experiments a strong uptake of a radio labelled prodrug of lipid metabolism, such as 2-(l l- [¹⁸F]fluoroundecyl)malonic acid (also termed [F18]-FSU 01), in tumor tissue giving raise not only to the detection and/or localisation of the tumor, but also permits treatment of the tumor by employing a suitable therapeutic residue instead of a therapeutic label.

The expression "detectable label" refers to any atom or molecule that may be used to provide a detectable (preferably quantifiable) effect and that can be attached to the present prodrugs. A non-limiting list of these labels comprise e.g. enzymes which produce a detectable signal, for example by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, [beta]-galactosidase or glucose-6-phosphate dehydrogenase, chromophores, such as fluorescent, luminescent or dye compounds, groups with an electron density detectable by electron microscopy or by virtue of their electrical property, such as conductivity, amperometry, voltametry or impedance, detectable group, for example the molecules of which are sufficiently large to induce detectable modifications of their physical and/or chemical characteristics; this detection can be carried out by optical methods such as diffraction, surface plasmon resonance, surface variation or contact angle variation, or physical methods such as atomic force spectroscopy or the tunnel effect, radioactive molecules such as ¹¹C, ¹³N, ¹⁸F₅ ³²P₅ ³⁵S₁ ⁶⁴Cu, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,^80mBr, ⁸⁶Y, ⁸⁹SrZ⁸Y, ⁹⁰Y, ^99mTc, ¹¹¹In, ¹²¹1, ¹²³I, ¹²⁴I₅ ¹²⁷I, ¹²⁵I, ¹³¹I, ¹⁵³Sm, ¹⁶⁵Dy; ¹⁶⁹Er, ¹⁷⁷Lu, ¹⁷⁸Ta, ¹⁸⁶Re, ¹⁸⁸Re ^195mPt, ²¹¹At, ²¹³Bi, ²²⁵Ac. Indirect systems may be used as well, such as, for example, ligands capable of reacting with an anti-ligand. The ligand/anti-ligand pairs are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin/streptavidin, hapten/antibody, antigen/antibody, peptide/antibody, sugar/lectin, polynucleotide/sequence complementary to the polynucleotide. The anti-ligand may be detected directly by the labels described above or can itself be detectable by another ligand/anti-ligand pair.

An imaging technique as used herein refers to any kind of apparatus or device, capable of producing a visual signal or an image upon detecting a detectable label. Preferably the apparatus permits localization of the detectable label within a mammal. These kinds of biological imaging include for example radiology. Non-limiting examples include positron- emission-tomography (PET), which produces a three-dimensional image or map of functional processes in the body. The system detects pairs of gamma rays emitted indirectly by a positron-emitting radioisotope, which is introduced into the body on a metabolically active molecule, i.e. a prodrug. Images of metabolic activity in space are then reconstructed by computer analysis, which may be supported by a CT X-ray scan performed on the patient at the same time and in the same device in order to obtain a three dimensional image enabling localization of tissue in which the prodrug is enriched. In positron emission a proton is converted via the weak force to a neutron, a positron and a neutrino. Isotopes, which undergo this so called beta plus decay, and thereby emit positrons. Suitable positron-emitting radionuclides for this purpose include ¹¹C, ¹³N, ¹⁸F, ⁶²Cu, ⁶⁴Cu, ⁶⁸Ga, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,^80mBr, ⁸⁶Y, ¹²¹I, ¹²⁴I of which ¹¹C and ¹⁸F may be preferred. The present invention should not be assumed to be intended thereto; other radionuclides may be used. The present prodrugs may be also labeled with technetium and rhenium isotopes using known chelating complexes. Methods for the generation of radionuclides as well as radio labeling of compounds are well known to the skilled person. US2007273308 and WO2007122488 pertain e.g. to the production of radionuclides, the contents of which are incorporated herein by way of reference. Radio labeling is for example outlined in WO 2007148089 and WO 2007148083, the contents of which are incorporated herein by way of reference. PET is preferably coupled with a computer tomography (CT). Such PET/CT devices enable quantitative detection and allocation of the signals detected to particular tissues, i.e. a localization of the radionuclides employed and hence of the prodrugs attached thereto. Function and operation of PET/CT as well as devices are well known to the skilled person (cf. Reske, S.N., Der Onkologe, 13(8) (2007) pp. 677-690; Reske, S.N et al., Urologe A, 45(10) (2006), pp. 1240-1250; Reske, S.N. et al., Urologe A, 45(6) (2006) pp. 707-714; Schδder, H. and S.M. Larson, Semin Nucl Med, 34(4) (2004) pp. 274-292; Morris, MJ. and H.I., Scher Eur. J. Nucl. Med. MoI. Imaging, 34(2) (2007) pp. 181-4). Other suitable techniques comprise single photon emission computer tomography (SPECT) and techniques based on magnetic resonance tomography (MRT) wherein the quantum mechanical magnetic properties of an atom's nucleus is detected. Useable contrast agents for MRT may involve attachment of Gadolinium, C- 13, F- 19 or ferrous species to the prodrugs for targeting of lipid metabolism for enhancing MR - contrast in the targeted tissue.

"Spacers" or "linkers" are molecules that are characterized in that they have a first end attached to the prodrug and a second end attached to the detectable label or therapeutic residue. Thus, the spacer molecule separates the prodrug and the detectable label or therapeutic residue, but is attached to both. The spacers may be synthesized directly on or may be attached as a whole to the prodrug. Bindings within the spacer may include carbon- carbon single bonds, carbon-carbon double bonds, carbon-nitrogen single bonds, or carbon- oxygen single bonds. In addition, the spacer may have side chains or other substitutions. The detectable label or therapeutic residue may be reacted by suitable means to form for example preferably a covalent bound between the spacer and prodrug. Suitable linkers include alkyl, alkenyl, alkynyl chains, aromatic, polyaromatic, and heteroaromatic rings any of which may be optionally substituted for example with one or more ether, thiooether, ester, amine, sulphonamide, or amide functionality, monomers and polymers comprising ethylene glycol, amino acid, or carbohydrate subunits. The linkers may be chosen to provide good in vivo pharmacokinetics, such as favorable excretion characteristics of the prodrug upon conversion in the mammal. The use of linkers with different lipophilicities and or charge can significantly change the in vivo pharmacokinetics of the prodrug to suit the diagnostic and /or therapeutic needs. Linkers including a polyethylene glycol moiety have been found to slow blood clearance which may be desirable in some circumstances. Spacers may be also in form of chelators capable of forming a complex with the detectable label or therapeutic residue.

The term "aliphatic" or "aromatic" substituent refers to residues composed of carbon and hydrogen. Aromatic substituents include ring structures, such as benzene, whereas aliphatic compounds do not. The present aliphatic or aromatic substituents may exhibit in addition to the detectable label or therapeutic residue one or more other residues, such as a hydroxy or amino group.

The term "lower alkyl" as used herein refers either to linear alley 1 residues of 1 - 8 C atoms or to branched alkyl residues of 3 - 8 C atoms. "Lower alkenyl" designates residues exhibiting different numbers of double bounds; the length of said residues is in case linear alkenyl residues 3 - 8 C and branched alkenyl residues from 4 - 8 C atoms, respectively. "Lower alkynyl" is directed to residues exhibiting different numbers of tripple bounds; the length of said residues is 3 - 8 C. Said residues may also have one or more substituents, such as heteroatoms. The letter "n" is used to designate the number of C-atoms of lower alkyl, alkenyl and alkynyl residues. It will be appreciated that lower alkenyl residues may also contain single bonds, whereas lower alkynyl residues may contain single and/or double bonds.

The term "therapeutic residue" as used herein refers to an atom, such as ³²P, "⁷Cu₅ ⁸⁹Sr,⁸⁸Y, ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁵³Sm, ¹⁶⁵Dy; ¹⁶⁹Er, ¹⁷⁷Lu, ¹⁷⁸Ta₅ ¹⁸⁶Re, ¹⁸⁸Re, ^195mPt, ²¹¹At, ²¹³Bi, ²²⁵Ac or a complex containing such an atom in a coordinated form. It will be appreciated that respective radioactive therapeutic residues may be generated and attached to the present prodrugs for targeting of lipid metabolism in the same manner as detectable labels. The use of the present prodrugs with therapeutic residues in cancer treatment combines the advantage of target selectivity with that of being systemic, as with chemotherapy, and it may be used as part of a therapeutic strategy with curative intent or for disease control and palliation. Preferably, therapeutic residue and detectable label are identical permitting diagnosis and treatment of cancer simultaneously.

The expression "sample" is meant to include any specimen or culture of biological material from a mammal. Biological samples may be human from human origin, fluid, such as blood or urine, solid or tissue. The sample may be used as such in an assay or may be subjected to a preliminary isolation step prior to performing the assay. The samples may be for example taken in order to follow elimination of radionuclides from the body and in order to assure that complete elimination has occurred.

The term "room temperature" refers to a temperature between 21 and 23⁰C. The expression "reduced pressure" designates any pressure below atmospheric pressure. A reduced pressure is preferably below 0.1 bar. The wording "saturated saline" refers to a saturated aqueous NaCl solution. According to an embodiment of the present invention a method for the diagnosis of cancer in vivo is provided. Said method comprises the step of providing a prodrug for targeting of lipid metabolism to a mammal, wherein the prodrug comprising a detectable label, followed by detecting and/or localizing the label by an imaging technique. The prodrug is selected from

wherein R represents an aliphatic, aromatic, heterocyclic, aralkylic, heteroaralkylic, or alicyclic substituent comprising a detectable label or a therapeutic residue. Rl and R2 are independently selected from lower alkyl, alkenyl or alkynyl, n = 1 - 8. This highly selective enrichment in tumor cells may be used in diagnosis or treatment of said tumors by employing detectable labels or therapeutic residues attached to the present prodrugs.

Free fatty acids represent the substrates of lipid metabolism, lipid conversion and lipid storage particularly in the liver and adipose tissue and energy production in heart and skeletal muscles. FFA are therefore incorporated by heart and liver in great amounts after intravenous injection and dependent from work load and hormonal regulation of the skeletal muscles. FFA further represent important substrates for biosynthesis of cellular membranes, modification of proteins, transcriptional regulation and intracellular signal transduction (Berk, P. D. (1996). "How do long-chain free fatty acids cross cell membranes" Proc Soc Exp Biol Med 212(1): 1 - 4). Energy demand of the tumor and energy metabolism, of, for example, the prostate carcinoma, is very low due to a slow growth with a tumor cell growth fraction of around 1% and a very low division rate of tumor cells in comparison to energy demand of the heart muscle or skeletal muscles under workload. The contribution to lipid metabolism is insignificant in comparison to e.g. the liver and membrane biosynthesis of the slow growing tumor cells is notably reduced in comparison to fast proliferating tissue, such as hematopoietic bone marrow or mucosa of the small intestine.

The fast and intensive uptake of substrates of fatty acid metabolism in tumor cells, of e.g. the prostate cancer, is therefore unexpected and surprising. Examples of such tumors comprise inter alia prostate carcinoma, colorectal carcinoma, breast cancer, lung tumors, tumors of the male or female genitourinary system, malignant melanoma, head and neck cancer, malignant lymphoma, neoplasia of the hematopoietic system and musculoskeletal tumors. Characteristic for some of the faster growing tumors is also that they supply their energy demand by highly increased glucose consumption. (Reske, S. N. and J. Kotzerke, Eur J Nucl Med 28 (11) (2001), pp. 1707-23; Czernin, J. and M.-E. Phelps, Annu Rev Med, 53 (2001), pp. 89-112; Gatenby, R. A. and R. J. Gillies, Nature Cancer 4, November (2004), pp. 891-899).

According to another embodiment of the present invention the cancer is characterized by increased consumption of substrates of tumoral lipid metabolism. Not limiting examples of such cancers/tumors include prostate carcinoma, colorectal carcinoma, breast cancer, lung tumors, tumors of the male or female genitourinary system, malignant melanoma, head and neck cancers, malignant lymphomas, neoplasias of the hematopoietic system and musculoskeletal tumors.

Preferably, a method for the diagnosis of prostate carcinoma (PCa) is provided. It has been found that by employing the present prodrugs for targeting of lipid metabolism the differentiation from benign hyperplasia and focal chronic prostatitis is significantly facilitated.

According to an embodiment the prodrug further comprises a linker arranged between said prodrug and said detectable label, in that one end of the linker or spacer is attached to the prodrug, whereas the other end is attached to the detectable label. The linker is preferably selected in a manner to provide good in vivo pharmacokinetics, inter alia by enabling rapid uptake of the prodrug in tumor tissue.

According to yet another embodiment said detectable label is a radioactive label. By using a suitable radioactive label detection with PET, preferably PET/CT, is enabled thus facilitating the localization of tumors in healthy tissue. Another advantage resides in that even new formed metastasis of few cells may be localized due to the high sensitivity of PET. The radioactive label may be for example of ¹¹C, ¹³N, ¹⁸F, ⁶²Cu, ⁶⁴Cu, ⁶⁸Ga, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ^8OmBr, ⁸⁶Y or ¹²⁴I. Preferably, radioactive halogens are employed. More preferably, ¹¹C and ¹⁸F are used due to their minor influence on the interaction between enzyme and prodrug.

According to an embodiment said imaging technique is selected from positron-emission- tomography (PET), and positron-emission-tomography/computer tomography (PET/CT). PET/CT is preferred due to the improved possibilities of visualizing/indicating the position and dimensions of a tumor in healthy tissue. Another advantage resides in that even metastasis comprising few tumor cells may be detected and localized. The metastasis comprise preferably less than 10⁵ tumor cells, more preferably less than 10⁴ tumor cells and most preferably less than 10² tumor cells.

According to a preferred embodiment of the present invention a prodrug of lipid metabolism, wherein said prodrug is selected from

wherein R represents an aliphatic, aromatic, heterocyclic, aralkylic, heteroaralkylic, or alicyclic substituent comprising a detectable label or a therapeutic residue. Rl and R2 are independently selected from lower alkyl, alkenyl or alkynyl, n = 1 - 8. Preferred prodrugs comprise malonic, alpha - methyl - substituted carboxylic acid derivatives with a single substituent, malonic acid monomethylester and malonic acid monoamide. It will be appreciated that the synthesis of appropriate prodrugs for targeting of lipid metabolism is well within the knowledge of the skilled person and that the skilled artisan is not restricted to a particular route of synthesis.

P-Iodbenzoyl-l l-aminodecylmalonic acid (standard) and 11-Aminoundecylmalonic acid (precursor) may be produced starting from DMUSM in three steps as outlined in figure 1. The corresponding radio labelled malonic acid derivate may be obtained by acetylating e.g. 11 -aminodecylmalonic acid with p- I-benzene-N-hydroxysuccinate. It will be appreciated that other radio labels or therapeutic residues may be attached in a similar manner.

Starting from tetradecanic acid ^uC-dodecylmalonic acid may be produced (cf. fig. 2). By addition of lithium diisopropylamide (LDA) in presence of hexamethylphosphortriamide (HMPT) the precursor is generated in situ. By converting with ¹¹CO₂, followed by hydrolysis, the ¹¹C marked product is obtained. The corresponding standard is obtained by hydro lysing of the dimethylester, which is obtained by alcylation of sodium dimethylmalonate with dodecylbromide.

2-Methyl-13-(methylsulfonyloxy)methyltridecanoate, serving as precursor for the preparation of 13-[ⁱ⁸F]fluoro-2-methyItridecanic acid, is synthesised starting from 2-methyldimethyl- malonate. In the same manner 13-fluoro-2-methyltridecanic acid may be obtained. Synthesis of 3-Hydroxy-(2S)-methylmethylpropionate is known in the state of the art.

According to an embodiment, a synthesis of 13-iodotridecen-12-ylmalonic acid as well as 13- [*I]-iodotridecen-12-ylmalonic acid are synthesized are exemplary outlined in fig.4.

Quality control of the compounds may be easily checked by high performance liquid chiOmatographie (HPLC) or in case of e.g. ¹⁸F labelling or another labelling with a halogen by comparison of the retention times of the radio labelled products with those of the respective "cold" standard compounds.

The prodrug preferably comprises a linker arranged between said prodrug and said detectable label. The linker may be selected in a manner bestowing the present prodrug advantageous properties, such as good in vivo pharmacokinetics.

The detectable label preferably comprises a chemoluminescent, fluorescent, bioluminescent, radioactive label or a label detectable by PET, CT or MRT. It will be appreciated that also in case of chemoluminescent, fluorescent, bioluminescent or radioactive labels also in vitro approaches may be used. In vitro assay for the diagnosis of cancer may for example based on interaction with enzymes, e.g. racemases, characteristic for the presence of a tumor from the body. Accordingly, a method is envisaged comprising the steps of providing a sample from a mammal, optionally purifying the sample, adding a present prodrug exhibiting one of the above mentioned markers and detecting the degradation products caused by the activity of enzymes characteristic for the presence of a tumor. It will be appreciated that such methods are well known to the skilled person.

According to an embodiment of the present invention, prodrug may be alpha - methyl - carboxylic or malonic acids and their derivatives. According to another embodiment of the present invention, the prodrug is 2-[l l-[¹⁸F]fluoroundecyl]malonic acid (designated [F18]- FSU 01), which has been found to be incorporated in tumor tissue, such as prostate tumor tissue, fast and in high amounts.

According to an embodiment of the present invention, the aliphatic substituent may be selected from C8 - C20 linear alkyl or C8 - C20 branched alkyl. Instead of an aliphatic substituent also an aromatic substituent may be employed. According to another embodiment of the present invention, the aliphatic substituent may be C9, CI l, C13, C15, C17 or C19 linear alkyl. According to a further embodiment of the present invention, the aliphatic substituent may be Cl 1 linear alkyl.

According to an embodiment of the present invention, the detectable marker may be selected from the group consisting of ¹¹C, ¹³N, ¹⁸F, ³²P₅ ³⁵S₅ ⁶⁴Cu, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,^S0mBr, ^S6Y, ⁸⁹Sr, ⁸⁸Y₅ ⁹⁰Y, ^99mTc, ¹¹¹In, ¹²¹I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁷I, ¹³¹I, ¹⁵³Sm, ¹⁶⁵Dy; ¹⁶⁹Er, 1⁷⁷Lu, ¹⁷⁸Ta, ¹⁸⁶Re, ^ISSRe ^295mPt, ²¹¹At, ²¹³Bi, ²²⁵Ac. According to another embodiment of the present invention, the diagnostic residue may be ¹¹C, ¹⁸F or ¹²³I.

According to an embodiment of the present invention, the therapeutic residue may be preferably selected from the group consisting Of ³²P, ⁶⁷Cu, ⁸⁹Sr, ⁸⁸Y, ⁹⁰Y, ¹²³1, ¹²⁵1, ¹³¹I, ¹⁵³Sm, 1⁶⁵Dy; ¹⁶⁹Er, ¹⁷⁷Lu, ¹⁷⁸Ta, ¹⁸⁶Re, ¹⁸⁸Re, ^195mPt, ²¹¹At, ²¹³Bi, ²²⁵Ac. According to another embodiment of the present invention, the therapeutic residue may be ⁹⁰Y.

According to an embodiment of the present invention, the detectable marker may be ¹⁸F, ¹¹C or ¹²³I. According to an embodiment of the present invention, the detectable marker may be 1⁸F and ¹²³I. Rl and R2 may be independently selected from H, Cl - C8 linear alkyl, C2 - C8 branched alkyl, C3 - C8 linear alkenyl, C4 - C8 branched alkenyl, C3 - C8 linear alkynyl, or C4 - C8 branched alkynyl. According to an embodiment of the present invention, Rl and/or R2 may be H or Cl - C2 linear alkyl, since these residues may easily react with enzymes in tumor tissue due to a reduced steric hindrance. According to another embodiment of the present invention, Rl and/or R2 may be H.

According to still another embodiment of the present invention a method for the treatment of cancer is provided. The method comprises the steps of providing a prodrug for targeting of lipid metabolism to a mammal, wherein said prodrug comprises a therapeutic residue, administering said prodrug to a mammal. The prodrug may be selected from

wherein R represents an aliphatic, aromatic, heterocyclic, aralkylic, heteroaralkylic, or alicyclic substituent comprising a detectable label or a therapeutic residue. Rl and R2 are independently selected from lower alky L alkenyl or alkynyl, n = 1 - 8.

Since the neoplastic tissue, i.e. the tumor tissue, exhibits a considerable higher uptake of the present prodrugs, said tissue is subjected to the radiation originating from the therapeutic residue. It will be appreciated that the therapeutic residue may be selected in order to correspond to a suitable detectable label, in that upon administering of the present prodrug not only enrichment in the target tissue and elimination from the body, but also the activity of the therapeutic residue on the tumor tissue may be observed.

In addition to the good in vivo pharmacokinetics of the present prodrugs, i.e. fast and selective enrichment in tumor tissue and fast degradation and elimination from the body, the pharmacokinetics may be further influenced by choice of a suitable linker.

According to an embodiment, said therapeutic residue may be selected from the group consisting Of ³²P, ⁶⁷Cu, ⁸⁹Sr, ⁸⁸Y, ⁹⁰Y₅ ¹²³I, ¹²⁵I₅ ¹³¹I, ¹⁵³Sm, ¹⁶⁵Dy, ¹⁶⁹Er₅ ¹⁷⁷Lu, ¹⁷⁸Ta₅ ¹⁸⁶Re₅ ¹⁸⁸Re, ^195mPt, ²¹¹At₅ ²¹³Bi, and ²²⁵Ac, According to an embodiment of the present invention, the therapeutic residue may be identical with a detectable label.

According to an embodiment of the present invention, prodrug may be alpha - methyl - carboxylic or malonic acids and their derivatives.

According to an embodiment of the present invention, the aliphatic substituent may be selected from C4 - C25 linear alkyl or C4 - C25 branched alkyl. Instead of an aliphatic substituent also an aromatic substituent may be employed. According to another embodiment of the present invention the aliphatic substituent may be CI l₅ C13 or Cl 5 linear alkyl. According to a further embodiment of the present invention the aliphatic substituent may be CI l linear alkyl.

Rl and R2 may be independently selected from Cl - C8 linear alkyl, C3 - C8 branched alkyl, C3 - C8 linear alkenyl, C4 - C8 branched alkenyl, C3 - C8 linear alkynyl, and C4 - C8 branched alkynyl. According to another embodiment of the present invention Rl and/or R2 may be H or Cl - C2 linear alkyl, since these residues may easily react with enzymes in tumor tissue due to a reduced steric hindrance. According to a further embodiment of the present invention Rl and/or R2 may be H.

According to yet another embodiment, a cancer to be treated is selected from prostate carcinoma, colorectal carcinoma, breast cancer, lung tumors, tumors of the male or female genitourinary system, malignant melanoma, head and neck cancers, malignant lymphomas, neoplasias of the hematopoietic system and musculoskeletal tumors.

According to still another embodiment, the prodrug is administered by intravenous, subcutaneous, intramuscular or intracavitary injection.

According to yet another preferred embodiment the prodrug is contained in a pharmaceutical composition.

Said composition may be in the form of a parenteral formulation since due to the lipophilic nature of said compound administration via injection is advisable. However, other kind of formulations, such as oral formulations, may be envisaged as well. The methods of preparation may include the step of bringing the active compound into association with a carrier, which constitutes of one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into desired formulations.

Suitable compositions for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non- aqueous liquid; or as an oil-in- water or water-in-oil emulsion. Such compositions may be prepared by any suitable method of pharmacy, which includes the step of bringing into association the active compound and a suitable carrier. In general, the compositions are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a power or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispensing agent(s). Molded tablets may be prepared by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder. A syrup may be made by adding the active compound to a concentrated aqueous solution of a sugar, for example sucrose to which may also be added any accessoiy ingredient(s). Such accessory ingredient(s) may include flavourings, suitable preservatives, an agent to retard crystallization of the sugar, and an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol, for example glycerol or sorbitol. Compositions for oral administration may optionally include enteric coatings known in the art to prevent degradation of the compositions in the stomach and provide release of the drug in the small intestine. Compositions suitable for buccal or sub-lingual administration include lozenges comprising the active compound in a flavoured base, usually sucrose and acacia or tragacanth and pastilles comprising the compound in an inert base such as gelation and glycerin or sucrose and acacia. Compositions suitable for parenteral administration comprise sterile aqueous and nonaqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient.

These preparations may contain anti-oxidants, buffers, bacteriostats and solutes, which render the compositions isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried or a lyophilized condition requiring only the addition of the sterile liquid carrier, for example, saline or water- for- injection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

According to still another embodiment the use of the present prodrugs for targeting of lipid metabolism for the treatment and/or prevention of cancer is envisaged.

It is to be understood that the above description is intended to be illustrative only and not restrictive. Many embodiments will be apparent to those of skill in the art upon reviewing the above description. By way of example, the invention has been described preliminary with reference to diagnosis of [FlS]-FSU 01. It should be clear that all kinds of suitable therapeutic residues and detectable labels may be synthesized and attached to the present prodrugs for targeting of lipid metabolism. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

Examples

The following PCa-cell lines were used: LNCaP (human prostate carcinoma, DSMZ Cat.-No. ACC 256) and TRAMP (murine prostate adenocarcinoma, obtainable from LGC Cat. No. CRL-2730; Greenberg, N.M. et al., Proc Natl Acad Sci U S A, 92(8) (1995) pp. 3439-43). PC3 (DSMZ Cat.-No. ACC 465) was used as reference. Human colon adenocarcinoma CX-I were obtained from DSMZ Cat.-No. ACC 129.

PC3 was cultivated/maintained in 50% Ham's F12 with 50% RPMI, 10% FKS, 1% penicillin + streptomycin, 1% L-glutamate. LNCaP was cultivated/maintained in RPMI with 10% FKS, 1% penicillin + streptomycin, 1% L-glutamate, 1% sodium pyruvate, 1% not essential amino acids. TRAMP cells were cultivated/maintained in DMEM (high glucose), 5% Nu-serum IV, 5 % FKS; 1% penicillin + streptomycin, 10^"s M dihydrotestosterone, 5 μg/ml insuline. CX-I cells were cultivated/maintained in DMEM (high glucose), 10% FKS.

For analysis of standard and radiolabeled compounds a Dionex HPLC System with UV- detector (210 nm) and radio detector has been employed. Stationary phase: LiChrospher Select B 5μ, 250x4.6mm, Chromatographic Service; mobile phase: CH₃CN/H₂O 50/50 v/v (0.1% TFA); flow: 2 ml/min.

[¹⁸F]-Synthesis has been performed according to manufactures instructions with a remote controlled synthesis unit by Nuclear Interface (GE) with semi preparative Radio-HPLC- System (stationary phase: LiChrospher Select B 5μ, 250x10 mm, Chromatographic Service; mobile phase: CH₃CN/H₂O 60/40 v/v (0.1% TFA); flow: 5 ml/min.

Radioactivity was measured according to manufactures instructions with a γ-counter (Cobra 2, GR Healthcare / Packard Instruments, Palo Alto, USA).

Example 1

Synthesis of 1 -bromo- 11 -fluoroundecane:

11-Bromoundecanol-l (4.1 g, 16.32 mmol) was added (teflon bottle) to DAST (5 ml, 6.1 g, 37.84 mmol) under Ar-atmosphere. The mixture was stirred for 10 min. at room temperature and afterwards heated to 50-55 C and stirred for other 6 hours. After cooling to room temperature, the mixture is diluted with ice water (200 ml) and neutralised (pH=7 ) with sodium bicarbonate. The obtained emulsion was extracted with pentane (2 x 100 ml). The pentane solution was washed with water (3 "< 50 ml) and saturated saline (50 ml) and afterwards dried over magnesium sulfate. Following filtration, the solvent was distilled under reduced pressure. The residue was purified by column chromatography on silica gel (mobile phase: pentane). The target compound (1.95 g, 47%) was obtained as colourless liquid. R_f = 0.38 (pentane). ¹H-NMR(CDCl₃, 500 MHz): δ = 1.20-1.35 (m, 10 H), 1.35-1.46 (m, 4 H), 1.67-1.74 (m, 2 H), 1.84 (dt, J = 14.9, 6.7 Hz), 3.39 (t, J = 6.9 Hz), 4.42 (dt, J = 47.8, 6.7 Hz); ¹³C-NMR(CDCl₃, 125.77 MHz): δ = 25.1 (d, J = 5.5 Hz), 28.15, 28.7, 29.2, 29.38, 29.39, 29.44, 30.4 (d, J = 19.3 Hz), 32.8, 34.0, 84.2 (d, J = 164.0 Hz). MS (EI): m/z = 254, 252 (0.5) [M⁺], 173.2 (6) [C_nH₂₂F⁺], 151, 149 (10) [C₅H₁₀Br⁺], 135, 137 (100) [C₄H₈Br⁺], 117 (7) [C₇H₁₄F⁺], 97 (20) [C₇H₁₃ ⁺], 83 (10), 71 (15), 69 (34) [C₅H₉ ⁺], 55 (38) [C₄H₇ ⁺].

Example 2 Synthesis of dimethyl- 11-fluoroundecylmalonate:

To a solution of compound 1 (1 g, 3.95 mrnol) in butanone-2 (20 ml) sodium iodide (0.62 g, 4.15 mmol) was added. The reaction mixture was heated for 16 h under reflux. The reaction mixture was allowed to cool to room temperature and diluted with water (100 ml) and 10% Na₂S₂O₃-solution (40 ml). The resulting emulsion was extracted with pentane (2 x 100 ml). The pentane solution was washed with water (5 x 50 ml) and saturated saline (50 ml) and afterwards dried over magnesium sulfate. Following filtration, the solvent was distilled under reduced pressure. 1-Iodo-l l-fluoroundecane (1.1 g, 93%) was obtained as colourless liquid. NaH (0.119 g, 2.97 mmol; 60%ige suspension in paraffin oil) was suspended in tetrahydrofurane (THF) (10 ml) under argon atmosphere and stirred at room temperature. Dimethylmalonate (0.342 ml, 0.394 g, 2.97 mmol) was added drop wise within 3 min. (exotherm reaction, gas evolution) and stirred afterwards for 5 min. Following this, first 1- iodo-11-fluoroundecane (0.51 g, 1.70 mmol) and afterwards DMSO (10 ml) were added. The reaction mixture was stirred for 15 hours. THF was distilled under reduced pressure. The residue was diluted with 1 M HCl (10 ml) and water (200 ml). The resulting emulsion was extracted with pentane (100 ml). The pentane solution was washed with 1 M HCl (50 ml), water (10 * 50 ml) and a saturated saline solution (50 ml) and afterwards dried over magnesium sulfate. The solvent was distilled after filtration and the residue was purified by column chromatography on silica gel (mobile phase: EtOAc.hexane = 1 :10). The target compound (0.43 g, 81%) was obtained as colourless liquid. R_f = 0.29 (EtOAc :hexane = 1 :10). ¹H-NMR(CDCl₃, 500 MHz): δ = 1.15-1.35 (m, 16 H), 1.67-1.74 (m, 2 H), 1.87 (dd, J = 7.3, 7.3 Hz, 2 H), 3.34 (t, J = 7.3 Hz, 1 H), 3.72 (s, 6 H), 4.42 (dt, J = 47.6, 6.3 Hz; 2 H); ¹³C-NMR(CDCl₃, 125.77 MHz): δ = 25.1 (d, J = 5.5 Hz), 27.3, 28.83 (x 2), 29.15, 29.20, 29.3, 29.4 (x 2), 30.4 (d, J = 19.2 Hz), 51.7, 52.4, 84.2 (d, J = 164.0 Hz), 170.0. MS (ESI): positive mode m/z = 631.4 ([2M + Na]⁺), 327.2 ([M + Na]⁺), 305.2 ([M + Na]⁺); ESI HRMS: calculated for C₁₆H₂₉FO₄Na⁺: 327.19421; found: 327.19410; calculated for C₁₆H₃₀FO₄ ⁺: 305.21226; found: 305.21222.

Example 3:

Synthesis of 11-fluoroundecylmalonic acid:

To a solution of compound 2 (0.35 g, 1.21 mmol) in methanol (5 ml) 10 M NaOH (1.3 ml) and water (1.3 ml) were added. The reaction mixture was stirred for 4 hours at 50°C. After cooling to room temperature the mixture was diluted with water (25 ml). The resulting aqueous solution was underlayed with hexane (3 x 20 ml). The hexane phase was discarded after phase separation. The aqueous phase was acidified to pH 1 with 1 M HCl. The resulting suspension was extracted with ether (100 ml). The ether solution was washed with 1 M HCl (2 x 10 ml), water (3 x 20 ml) and saturated saline (2 x 20 ml) and afterwards dried over magnesium sulfate. The solvent was distilled under reduced pressure after filtration. The target compound (0.43 g, 81%) was obtained as colourless solid after recrystallization of the residue from ether/pentane. R_f = 0.20 [EtOAc±exane = 1 :3 (3% AcOH)]. ¹H- NMR[(CD₃)₂SO, 500 MHz]: δ = 1.25-1.44 (m, 16 H), 1.55-1.75 (m, 2 H)₅ 1.68 (dd, J = 7.0, 7.0 Hz, 2 H), 3.16 (t, J = 7.2 Hz, 2 H)₅ 4.40 (dt, J = 47.6, 6.2 Hz), 12.50-12.90 (br, 1 H); ¹³C- NMR[(CD₃)₂SO, 125.77 MHz]: δ = 24.6 (d, J = 5.5 Hz), 26.8, 28.4, 28.6, 28.7, 28.8, 28.86, 28.88 (x 2), 28.89, 29.8 (d, J = 19.2 Hz)₅ 51.6, 84.2 (d, J = 162.3 Hz), 171.0. MS (ESI): positive Mode m/z = 299.2 ([M + Na]⁺), 277.2 ([M + Na]⁺); ESI HRMS: calculated for C₁₄H₂₅FO₄Na⁺: 299.16291; found: 299.16293; calculated for C₁₄H₂₆FO₄ ⁺: 277.18096; found: 277.18104.

Example 4:

Synthesis of dimethyl- 11-mesyloxyundecylmalonate:

Sodium methylate (0.848 g, 15.55 mmol) was dissolved in methanol (20 ml) and heated for 15 min. under reflux. Within 10 min. dimethylmalonate (1.78 ml, 2.054 g, 15.55 mmol) was added drop wise to the receiver. After 15 min. I l-bromoundecanol-1 (3.516 g, 13.99 mmol) dissolved in methanol (10 ml) was included drop wise within 15 min. The reaction mixture was heated for 20 hours under reflux. After cooling to room temperature and concentration under reduced pressure, the obtained residue was dissolved in ether (100 ml) and 1 M HCl (40 ml). The ether solution was washed with 1 M HCl (40 ml), water (3 * 50 ml) and saturated saline (2 x 20 ml) and afterwards dried over magnesium sulfate. The solvent was distilled under reduced pressure after filtration. The residue was purified by column chromatography on silica gel (mobile phase: EtOAc:hexane = 1:3, R_f = 0.15). Dimethyl-11- hydroxyundecylmalonate (1.81 g, 43%) was obtained as colourless oil. Dimethyl-11- hydroxyundecylmalonate (1.55 g, 5.13 mmol was dissolved in dichloromethane (20 ml) and triethylamine (0.67 ml, 0.928 g, 6.67 mmol) under ice cooling, stirring and argon atmosphere. Within 5 min. mesylchloride (0.52 ml, 0.763 g) was added drop wise and the mixture was stirred for 3 hours under ice cooling. The temperature of the reaction mixture was increased to room temperature by adding of water (50 ml) and ether (100 ml). The obtained organic layer was washed with water (2 x 30 ml), 1 M KHSO₄ (2 x 30 ml), water (2 x 30 ml) and saturated saline (50 ml) and afterwards dried over magnesium sulfate. After filtration of the dried organic phase through a pad of silica gel (2 cm), distillation of the solvent was performed under reduced pressure. Toluene (50 ml) was three times distilled from the residue. The target compound (1.92 g, 98%) was obtained as yellowish oil, which crystallizes slowly as solid. R_f = 0.17 (EtOAc:hexane = 1 :3). ¹H-NMR(CDCl₃, 500 MHz): δ = 1.20-1.34 (m, 14 H)₅ 1.34-1.45 (m, 2 H), 1.72 (dt, J = 15.0, 7.2 Hz, 2 H), 1.87 (dd, J = 7.2 Hz, 2 H), 2.98 (s, 1 H), 3.33 (t, J = 7.5 Hz₅ 1 H)₅ 3.71 (s, 6 H)₅ 4.20 (t, J = 6.5 Hz; 2 H); ¹³C- NMR(CDCl₃, 125.77 MHz): δ = 25.4, 27.3, 28.8, 29.0, 29.09, 29.12, 29.2, 29.3, 29.4 (x 2), 37.3, 51.7, 52.4, 70.1, 169.9. MS (ESI): positive mode m/z = 783.4 ([2M + Na]⁺), 403.2 ([M + Na]⁺), 381.2 ([M + Na]⁺); ESI HRMS: calculated for C₁₇H₃₂O₇SK⁺: 419.15003; found: 419.15000; calculated for C₁₇H₃₂O₇SNa⁺: 403.17610; found: 403.17618; calculated for C₁₇H₃₆O₇SN⁺: 398.22070; found: 398.22074; calculated for C₁₇H₃₃O₇S⁺: 381.19415; found: 381.19419.

Example 5

Synthesis of 2-(l l-[¹⁸F]fluoiOundecyl)malonic acid ([F18]-FSU 01):

[¹⁸F]fluoride was produced by ¹⁸O(p, n)¹⁸F nuclear reaction by proton irradiation of enriched [18O] water (2 ml, RWE₅ degree of enrichment >95%) using a PETtrace cyclotron (E_p = 16,5 MeV, GE). For radiosynthesis a remote controlled synthesis unit of Nuclear Interface (GE) was employed. [¹⁸F]fluoride produced by the cyclotron was fixed on a conditioned (10 ml 1 M NaHCO₃, 10 ml water) anion exchange cartridge (Sep-Pak light, Accell Plus QMA₅ Waters) and thereby separated from the [¹⁸O]water-matrix. [^{] S}F]fluoride was eluted with 360 μL 0,066 M KOH and subsequently 20 mg Kryptofix 2.2.2. in 1 ml acetonitrile was added to a [¹⁸F] fluoride solution. The radionuclide was activated for the radiolabeling by means of subsequent azeotropic distillation for 6 min. under vacuum (approx. 6 mbar) until dryness at first 95⁰C and later 100°C. The radiolabeling was conducted by addition of compound (9 mg, 23.65 μmol) obtained in example 4 in acetonitrile (1 ml) and heating at 85⁰C for 10 min. Product was obtained afterwards by hydrolysis of the intermediate dimethyl ester with 400 μl 1 M NaOH for 10 min. at 7O⁰C. The reaction matrix was cooled to 30⁰C and 2 ml HPLC- eluent (acetonitrile/water 60/40 v/v) and 300 μL acidic acid were added. Not converted [¹⁸F] fluoride was removed from the neutralized and diluted reaction mixture by means of a Al₂θ₃-cartiϊdge (Sep-Pak light, Alumina N₅ Waters) and purified by semipreparative HPLC (stationaiy phase: LiChrospher Select B 5μ, 250x10 mm, Chromatographic Service; mobile phase: CH₃CN/H₂O 60/40 v/v, 0,1 % TFA; flow: 5 ml/min; radioactivity detector and UV- detector (210 nm)) by means of an automated injection unit (flow-detector). The product fraction was isolated after approx. 7 min. by valve switching and was diluted with 50 ml water. Afterwards solid phase extraction was carried out by means of a C- 18 cartridge (Sep- Pak light, C- 18, Waters). Elution was performed with 1 ml EtOH, followed by 10 ml isotonic NaCl-solution. Starting from approx. 45 GBq [¹⁸F]fluoride within 60 min. approx. 5 - 8 GBq product were obtained with a radiochemical purity > 95% Quality control

Quality control of the produced fluorine- 18 labelled fatty acids was performed by an analytical Radio-HPLC-system (stationary phase: LiChrospher Select B 5μ, 250x4,6 mm, Chromatographic Service; mobile CH₃CN/H₂O 50/50 v/v, 0,1 % TFA; flow: 2 ml/min; radio activity detector und UV-detector (210 nm)). Product identification was performed by comparison of the retention times of the products with those of the respective "cold" standard compounds (the compounds obtained in example 3).

Example 6

Synthesis of dimethyl-2-tridec- 12-ynylmalonate:

Tridecynyl- 12-01-1 (4.95 g, 29.71 mmol), Ph₃P (11.69 g, 44.57 mmol) and imidazole (3.034 g, 44.57 mmol) were dissolved in THF (50 ml) under argon atmosphere and ice cooling. Iodine (10.406 g₅ 41.0 mmol) in THF (20 ml) was added drop wise within 20 min. The reaction mixture was stirred for 2 hours at room temperature. Afterwards saturated Na₂S₂O₃ solution (3 ml) was added drop wise within 2 min. The reaction mixture was dried over magnesium sulfate. The obtained residue was extracted with pentane (20 x 50 ml). The solvent was distilled under reduced pressure and the residue was purified by column chromatography on silica gel (mobile phase: petrolether 40/60). 13-Iodotridecyn-l (6.4 g, 93%) was obtained as colourless liquid. R_f = 0.24 (petrolether 40/60). ¹H-NMR(CDCl₃, 500 MHz): δ = 1.23-1.33 (m, 11 H), 1.34-1.41 (m, 3 H), 1.49-1.55 (m, 2H), 1.81 (dt, J = 14.5, 7.2 Hz, 2 H) 1.92 (t, J = 2.7 Hz, 1 H), 2.17 (dt, J = 1.8, 7.2 Hz, 2 H), 3.18 (t, J = 7.0 Hz, 2 H). NaH (0.92 g, 22.86 mmol; 60% suspension in paraffin oil) was suspended in tetrahydrofurane (50 ml) under argon atmosphere and stirred at room temperature. Within 3 min. dimethylmalonate (2.62 ml, 3.02 g, 22.86 mmol) was added drop wise (exotherm reaction, gas evolution). The reaction mixture was stirred for 5 min. Afterwards, 13- iodotridecyn- 1 (4.0 g, 13.06 mmol) and DMSO (50 ml) were added successively and the reaction mixture was stirred for another 20 hours. THF was distilled under reduced pressure. The residue was diluted with 1 M KHSO₄ (25 ml) and water (500 ml). The obtained emulsion was extracted with pentane (400 ml). The pentane solution was washed successively with 1 M KHSO₄ (50 ml), water (5 x 100 ml) and saturated saline (50 ml). The organic phase was dried over magnesium sulfate. After filtration of the dried organic phase, the solvent was distilled under reduced pressure. The residue was purified by column chromatography on silica gel (mobile phase: petrolether 40/60:aceton = 10:1). The target compound (3.25 g, 80%) was obtained as colourless oil, which crystallizes slowly as colourless solid. R_f = 0.34 (petrolether 40/60:aceton = 10:1). ¹H-NMR(CDCl₃, 500 MHz): δ = 1.12-1.33 (m, 14 H), 1.33-1.42 (m, 2 H), 1.45-1.55 (m, 2 H), 1.87 (dd, J = 7.3, 7.3 Hz, 2 H)₅ 1.91 (t, J = 2.7 Hz, 1 H), 2.16 (dt, J = 2.7, 7.1 Hz₅ 2 H), 3.34 (t, J = 7.8 Hz, 1 H), 3.72 (s, 6 H); ¹³C-NMR(CDCl₃, 125.77 MHz): δ = 18.4, 27.3, 28.5, 28.7, 28.8, 29.1, 29.2, 29.3, 29.4 (x 2), 29.5, 51.7, 52.4, 68.0, 84.8, 169.9.

Example 7

Synthesis of dimethyl- 13-tributylstannyltridec- 12-enylmalonate: Compound 6 (1.0 g, 3.22 mmol), tributylstannan (1.06 ml, 1.17g, 4.02 mmol) and AIBN (20 mg)were dissolved in toluene (10 ml) under argon atmosphere. The solution was carefully degassed and then stirred for 2 hours at 8O⁰C and for 1 hour at 105⁰C. After cooling the reaction mixture to room temperature, the solvent was distilled under reduced pressure. The residue was purified by column chromatography on silica gel (0.1% CaO) (mobile phase: petrolether 40/60:aceton = 12:1). The target compound (0.95 g, 49%) was obtained as a mixture of E/Z isomers (70:30) as colourless oil. R_f = 0.14 (petrolether 40/60 : aceton = 12:1). ¹H-NMR(CDCl₃, 500 MHz): δ = 0.75-0.90 (m, 14 H), 1.20-1.40 (m, 25 H), 1.45-1.55 (m, 6 H), 1.87 (dd, J = 7.3, 7.3 Hz, 2 H), 2.01 (dt, J = 6.8, 6.8 Hz, 0.6 H), 2.11 (dt, J - 6.0, 6.0 Hz, 1.4 H), 3.34 (t J = 7.8 Hz, 1 H), 3.72 (s, 6 H), 5.35-6.05 (m, 1.4 H), 6.44-6.65 (m, 0.6 H); ¹³C-NMR(CDCl₃, 125.77 MHz): δ = 9.4, 10.25; 13.7, 27.26, 27.33, 27.35, 28.87, 28.94, 29.1, 29.2, 29.3, 29.5, 29.60, 29.62, 29.9; 37.2, 37.9; 51.7, 52.4; 126.9, 127.6; 149.3, 149.9; 170.0.

Example 8:

Synthesis of dimethyl- 13-iodtridec- 12-enylmalonate:

To a solution of compound 7 (0.3 g, 0.50 mmol) in dichloromethane (5 ml) iodine (0.216g, 0.5 mmol) in a solution of dichloromethane (3 ml) was added drop wise. The reaction mixture was stirred for 10 minutes at room temperature. Thereafter, a saturated solution of Na₂S₂O₃ (0.5 ml) was added and the reaction mixture was stirred for further 5 min. at room temperature. After dilution of the reaction mixture with dichloromethane, (30 ml) 10% KF solution (30 ml) was added and was intensively stirred for 1 hour at room temperature. The aqueous phase was discarded and the organic phase was dried over magnesium sulfate. After filtration through a pad of silica gel (1 cm) the solvent was distilled under reduced pressure and the residue was purified by column chromatography on silica gel (mobile phase: petrolether 40/60:acetone = 10:1). The target compound (0.212 g, 100%) was obtained as E/Z- isomer mixture (70:30). It was obtained as colourless oil. R_f = 0.20 (petrolether 40/60:aceton = 10:1). ¹H-NMR(CDCl₃, 500 MHz): δ = 1.15-1.33 (m, 16 H), 1.33-1.40 (m, 2 H), 1.87 (dd, J = 6.9, 6.9 Hz₅ 2 H)₅ 2.03 (dt, J = 7.2, 7.0 Hz, 1.4 H), 2.11 (dt, J = 2.3, 1.7 Hz, 0.6 H), 3.34 (t, J = 7.6 Hz, 1 H), 3.72 (s, 6 H), 5.96 (d, J = 14.3 Hz, 0.7 H), 6.13-6.19 (m, 0.6 H), 6.49 (dt, J = 14.3, 7.2 Hz, 0.7 H); ¹³C-NMR(CDCl₃, 125.77 MHz): δ = 27.3, 27.9, 28.3,

28.8, 28.9, 29.2, 29.26, 29.31, 29.47, 29.51; 34.7, 36.0; 51.7, 52.4; 74.2, 82.1; 141.5, 146.8; 170.0.

Example 9:

Synthesis of 13-iodotridec-12-enylmalonic acid: Compound 8 (0.2 g, 0.50 mmol) was dissolved in ethanol (10 ml) and 1 M NaOH (2 ml) was added and refluxed for 15 minutes. After cooling to room temperature, the reaction mixture was diluted with 1 M KHSO₄ (5 ml) and extracted with ether (70 ml). The organic phase was separated and consecutively washed with 1 M KHSO₄ (20 ml), water (3 x 20 ml) and saturated saline (2 >- 20 ml). After drying the organic phase over magnesium sulfate, the solvent was distilled by reduced pressure. The remaining residue was recrystallized from ether/pentane. Thereby, the target compound was obtained as a mixture Z/E isomers (30:70), containing less than 5 mol% of the elimination product (tridecyn-12-ylmalonic acid) (0.175 g, > 89%) as colourless solid. R_f = 0.38 [EtOAc:hexane = 3: 1 (3% AcOH)]. ¹H-NMR(CDCl₃, 500 MHz): δ = 1.0-1.45 (m, 18 H), 1.92 (dd, J = 7.2, 7.2 Hz, 2 H), 2.03 (dd, J = 7.2, 7.2 Hz, 1.4 H), 2.10- 2.25 (m, 0.6 H), 3.42 (t, J = 7.4 Hz, 1 H), 5.96 (d, J = 14.4 Hz, 0.7 H), 6.13- 6.21 (m, 0.6 H), 6.49 (dt, J = 14.4, 7.2 Hz, 0.7 H); ¹³C-NMR(CDCl₃, 125.77 MHz): δ - 27.2,

27.9, 28.3, 28.7, 28.9, 29.1, 29.2, 29.3, 29.47, 29.51 ; 34.7, 36.0: 51.6; 74.2, 82.1; 141.5, 146.8; 175.1.

Example 10:

Synthesis of radioiodinated 13-iodotridec-12-enylmalonic acid:

Na*I (5 μL; solution in 0.05 M NaOH) was added to a solution of compound 7 (20 μl, 9,31 mg/ml in methanol) and acetic acid (1 μl) in ethanol (20 μl) . Afterwards chloroamine Tsolution (4 mg/ml in 66 % methanol) was added and the mixture was stirred for 3 min. The reaction was quenched by adding saturated solution of methionine (10 μl). The protected intermediate was obtained in > 94% yield. The reaction mixture was diluted with ethanol to total volume of 200 μl. 1 M NaOH (50 μl) was added and the reaction mixture was heated for 15 min at 80°C and thereafter cooled to ambient temperature. Trifluoroacetic acid/acetonitiϊle 10/90 v/v (50 μl) was added. The reaction mixture was diluted with acetonitrile/water (0.1 % TFA) 33/67 v/v (750 μl) and separated by a semipreparative HPLC(stationary phase: LiChrospher 60 Select B 5μ RP C-8; 250 x 10 mm, Chromatographic Service; mobile phase gradient: CH₃CN with 0,1% TFA and 0.1% TFA₅ 0.5 min. 50% CH₃CN with 0.1% TFA₅ 5 - 10 min. 50 - 80% CH₃CN with 0.1% TFA₅ 10 - 50 min. 80% CH₃CN with 0.1% TFA; flow: 4 ml/min; radio detector and UV-detector (220 nm)). The product containing fraction was collected via valve control,, diluted with water in a ratio of 1 :10 and concentrated via solid phase extraction (Sep-Pak light C- 18, Waters). After washing of the cartridge with 10 ml water, the product was eluted with 0.5 - 1 ml ethanol. The radiochemical yield ranged between 25 - 35 % with respect to the applied radioiodine.

Quality control

Quality control of the produced radio iodine labelled fatty acid was carried out by analytical radio HPLC (stationary phase: LiChrospher Select B 5μ, 250x4,6 mm, Chromatographic Service; mobile phase: CH₃CN/H₂O 80/20 v/v; flow: 1 ml/min; radio activity detector and TJV-detector (220 nm)). Product identification was carried out by comparison of the retention times of the radio labelled products with that of the respective "cold" standard compound. The radio chemical purity of the product was >95%.

Example 11

Uptake of [F18]-FSU 01 in LNCaP, PC3 and TRAMP cells. PC3 cells serve hereby as androgen independent negative control, whereas LNCap and TRAMP (Greenberg, N.M. et al., Proc Natl Acad Sci U S A₅ 92(8) (1995) p. 3439-43) cell lines are both androgen dependent, i.e. exhibiting an enzyme expression profile essentially corresponding to that of human prostate cancer cells in vivo.

5* 1O⁵ cells of each kind (LNCaP, PC3 und TRAMP cells) were plated in 12 well plates, respectively.

The cells were incubated for 24 h in the respective media according to the instructions provided by the German Collection of Microorganisms and Cell Cultures.

Addition of [F18]-FSU 01 in dilution series in respective medium (20 h-value~ 200 MBq /well -> final volume 200 μl on 2000 μl, 1 h- value ~ 10 MBq /well -> 100 μl on 2000 μl medium, 4 h- value ~ 25 MBq /well -> 100 μl on 2000 μl medium.

Incubation of the cells for Ih, 4h and 2Oh at 37⁰C. The medium was removed (tube 1: unbound FSU 01), followed by 2 times gentle rinsing of the cells with PBS (2x 1 ml) (tube 1: unbound FSU 01). Cells were resuspended in 1 ml NaOH and transferred in tube 2 (bound FSU 01). The wells were washed with 1 ml HCL (tube 3: activity sticked to the inner wall)

Determination by γ-counter:

1 h: washing step 10 μl taken from 4.1 ml (2.1 ml medium + 2 ml PBS) 4 h: washing step 10 μl taken from 4.1 ml (2.1 ml medium + 2 ml PBS) 20 h: washing step 10 μl taken from 4.2 ml (2.2 ml medium + 2 ml PBS) cell lysate 1ml (I ml NaOH) HCl washing step (1 ml HCL)

Activity (FSU 01) has been added at 1 and 20 h.

Values at 1 h were determined by removing 100 μl from each well and adding 900 μl PBS (activity per well: x ~ 10 MBq).

Values at 4 h were determined by removing 250 μl from each well and adding 750 μl PBS (activity per well: x - 25 MBq).

Values at 20 h were determined by removing 200 μl from the original solution and adding directly to each well (200 μl per well).

Cellular uptake if FSU 01 in % dosage per 500000 cells has been calculated after 2 h and 24 h incubation (cf. fig. 4). High enrichment of [F-18]FSU 01 in LNCaP and TRAMP carcinoma cells could be detected.

Example 12

Biodistribution of [F-18]-FSU 01 in TRAMP-mice

Determination of [F- 18] - FSLI 01 biodistribution with the γ-counter, immuno histology, at 30 and 60 min. after intravenous application.

Number of the animals: 10 SV40 Tg⁺ animals, 2 SV40 Tg^' animals Approx. 200 μl [F- 18] FSU 01 in isotonic NaCl were applied, (activity of the full and empty syringe as well as of the mouse has been measured in closable plastic bags). The mouse was sacrificed according to the appointments of the German ethic committee and the weight was determined. Tissue and organs (blood, heart, lungs, liver, spleen, kidney, large and small intestines, stomach, muscles, vertebral body, bones, genital tract, caudal) were removed. The weight of empty γ-counter tubes was determined, the tissue/organ included and the weight of the tubes with content determined. The caudal was additionally measured in the activimeter and the tubes were measured in the γ-counter.

Application scheme:

01 :00 Application mouse 3 (30 min for bio distribution, 25 MBq )

01:30 removal of organs of mouse 3 for bio distribution

01:45 Application mouse 4 (60 min for bio distribution, 25 MBq) 02:00 Application mouse 5 (30 min for bio distribution, 25 MBq)

02:30 removal of organs of mouse 5 for bio distribution

02:45 removal of organs of mouse 4 for bio distribution

03:00 Application mouse 6 (60 min for bio distribution, 25 MBq)

03:15 Application mouse 7 (30 min for bio distribution, 25 MBq) 03:45 removal of organs of mouse 7 for bio distribution

04:00 removal of organs of mouse 6 for bio distribution

04:15 Application mouse 8 (60 min for bio distribution, 25 MBq)

04:30 Application mouse 9 (30 min for bio distribution, 25 MBq)

05:00 removal of organs of mouse 9 for bio distribution 05:15 removal of organs of mouse 8 for bio distribution

05:30 Application mouse 10 (60 min for bio distribution, 25 MBq)

06:30 removal of organs of mouse 10 for bio distribution.

Tissue/organ uptake of FSU 01 was calculated 30 and 60 min after intravenous application of FSU 01 in a caudal vein (cf. fig. 6). A high uptake of [F-IS]FSU 01 in prostate primary tumors and lymph node metastasis of the prostate carcinoma may be derived therefrom. In particular, the ¹⁸F labeled alkylmalonic acid was enriched in the transgene TRAMP mouse model, for example, in prostate carcinoma and its metastasis in lymph nodes more than 10 -

20 fold in comparison to control tissue, such as periprostatic soft tissues, intestinal tissue, skeletal muscles or normal prostate tissue.

Claims

Claims

1. A method for the diagnosis of cancer in vivo, comprising: providing a prodrug of lipid metabolism to a mammal, said prodrug comprising a detectable label detecting and/or localizing the label by an imaging technique, wherein said prodrug is selected from

CO₂H CO₂H CO₂H

R^ JLXO₂H R^ λXONH₂ R^ λXONHR₁

wherein R represents an aliphatic, aromatic, heterocyclic, aralkylic, heteroaralkylic, or alicyclic substituent comprising a detectable label or a therapeutic residue. Rl and R2 are independently selected from lower alkyl, alkenyl or alkynyl, n = 1 - 8.

2. The method according to claim 1, wherein the cancer is selected from prostate carcinoma, colorectal carcinoma, breast cancer, lung tumors, tumors of the male or female genitourinary system, malignant melanoma, head and neck cancers, malignant lymphomas, neoplasias of the hematopoietic system and musculoskeletal tumors.

4. The method according to claim 1, wherein said prodrug further comprises a linker arranged between said prodrug and said detectable label.

5. The method according to claim 1, wherein said detectable label is a radioactive label or a label detectable by CT or MRT.

6. The method according to claim 1, wherein said imaging technique is selected from positron-emissions-tomography (PET), and positron-emissions-tomography/computer tomography (PET/CT).

7. A prodrug of lipid metabolism, wherein said prodrug is selected from

wherein R represents an aliphatic, aromatic, heterocyclic, aralkylic, heteroaralkylic, or alicyclic substituent comprising a detectable label or a therapeutic residue. Rl and R2 are independently selected from lower alkyl, alkenyl or alkynyl, n = 1 - 8.

8. The prodrug according to claim 7, wherein said prodrug further comprises a linker arranged between said prodrug and said detectable label or said therapeutic residue.

9. The prodrug according to claim 7, wherein said detectable label comprises a chemoluminescent, fluorescent, bioluminescent, radioactive label or a label detectable by CT or MRT.

10. The prodrug according to claim 7, which selected from alpha-methyl-carboxcylic acid.

11. The prodrug according to claim 7, wherein the aliphatic substituent is selected from C4 - C25 linear alkyl, C4 - Cl 5 branched alkyl or C4 - C30 alicyclic.

12. The prodrug according to claim 7, wherein the detectable marker is selected from the group consisting of ¹¹C₅ ¹³N, ¹⁸F, ³²P₉ ³⁵S₅ ⁶⁴Cu, ⁶²Cu₅ ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Br₅ ⁷⁶Br₅

⁷⁷Br₅ ⁸⁰⁰¹Br₅ ⁸⁶Y₅ ⁸⁹Sr₅ ⁸⁸Y₅ ⁹⁰Y₅ ^99mTc₅ ¹¹¹In₅ ¹²¹I₅ ¹²³I, ¹²⁴I₅ ¹²⁵I₅ ¹²⁷I₅ ¹³¹I, ¹⁵³Sm₅ ¹⁶⁵Dy; 1⁶⁹Er₅ ¹⁷⁷Lu₅ ¹⁷⁸Ta₅ ¹⁸⁶Re₅ ¹⁸⁸Re ^195mPt₅ ²¹¹At₅ ²¹³Bi, and ²²⁵Ac.

13. The prodrug according to claim 7, wherein the therapeutic residue is selected from the group consisting Of ³²P₅ ⁶⁷Cu, ⁸⁹Sr, ⁸⁸Y₅ ⁹⁰Y, ¹²³I₅ ¹²⁵I₅ ¹³¹I₅ ¹⁵³Sm, ¹⁶⁵Dy; ¹⁶⁹Er, ¹⁷⁷Lu,

¹⁷⁸Ta₅ ¹⁸⁶Re, ¹⁸⁸Re₅ ^195mPt, ²¹¹At₅ ²¹³Bi₅ and ²²⁵Ac.

14. The prodrug according to claim 7, wherein Rl and R2 are selected from H₅ Cl - C8 linear alkyl, C3 - C8 branched alkyl, C3 - C8 linear alkenyl, C4 - C8 branched alkenyl, C3 - C8 linear alkynyl or C4 - C8 branched alkynyl.

15. A method for the treatment of cancer, comprising: providing a prodrug for targeting of lipid metabolism to a mammal, said prodrug comprising a therapeutic residue administering said prodrug to a mammal, wherein said prodrug is selected from

CO_?H CO₂H CO₂H

R^ JLXO₂H R^ JLXONH₂ R^ JLXONHR₁

wherein R represents an aliphatic, aromatic, heterocyclic, aralkylic, heteroaralkylic, or alicyclic substituent comprising a detectable label or a therapeutic residue. Rl and R2 are independently selected from lower alkyl, alkenyl or alkynyl, n = 1 - 8.

16. The method for the treatment of cancer according to claim 15, wherein said prodrug further comprises a linker arranged between said prodrug and said therapeutic residue.

17. The method for the treatment of cancer according to claim 15, wherein said therapeutic residue is selected from the group consisting of ³²P, ⁶⁷Cu, ⁸⁹Sr, ⁸⁸Y, ⁹⁰Y, 1²³I, ¹²⁵I, ¹³¹I, ¹⁵³Sm, ¹⁶⁵Dy; ¹⁶⁹Er, ¹⁷⁷Lu, ¹⁷⁸Ta, ¹⁸⁶Re, ¹⁸⁸Re, ^195mPt, ²¹¹At₅ ²¹³Bi, and

²²⁵Ac.

18. The method for the treatment of cancer according to claim 15, wherein said therapeutic residue is ³²P, ⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y, ¹¹¹In₅ ¹²³I, ¹²⁵I₅ ¹³¹I, ¹⁵³Sm₅ ¹⁶⁵Dy₅ ¹⁶⁹Er₅ 1⁷⁷Lu₅ ¹⁷⁸Ta₅ ¹⁸⁶Re₅ ^19SmPt, ²¹¹At₅ ²¹²At₅ ²¹³Bi₅ or ²²⁵Ac.

19. The method for the treatment of cancer according to claim 15₅ wherein said prodrug is alpha-methyl-carboxylic acid.

20. The method for the treatment of cancer according to claim 15, wherein the aliphatic substituent is selected from C4 - C25 linear alkyl, C4 - C25 branched alkyl or C4 - C30 alicyclic.

21. The method for the treatment of cancer according to claim 15, wherein Rl and R2 are selected from Cl - C8 linear alkyl, C3 - C8 branched alkyl, C3 - C8 linear alkenyl, C4

- C8 branched alkenyl, C3 - C8 alkynyl, or C4 - C8 branched alkynyl.

22. The method for the treatment of cancer according to claim 15, wherein the cancer is selected from prostate carcinoma, colorectal carcinoma, breast cancer, lung tumors, tumors of the male or female genitourinary system, malignant melanoma, head and neck cancers, malignant lymphomas, neoplasias of the hematopoietic system and musculoskeletal tumors.

23. The method for the treatment of cancer according to claim 15, wherein the prodrug is administered by intravenous, subcutaneous, intramuscular or intracavitary injection.

24. The method for the treatment of cancer according to claim 15, wherein the prodrug is contained in a pharmaceutical composition.

25. The use of a prodrug of lipid metabolism according to claim 7 for the treatment and/or prevention of cancer.

26. The use of a prodrug of lipid metabolism according to claim 7 for the diagnosis of cancer.