Field of Invention

This invention relates to deuterated tyrosine derivatives labeled with ¹⁸ F or ¹⁹ F, methods of preparing such compounds, compositions comprising such compounds, kits thereof and uses of such compounds, compositions or kits for imaging proliferative diseases.

The invention relates to the subject matter referred to in the claims, i.e. deuterated tyrosine 10 derivatives of the formula (I), their use and their preparation processes.

Molecular i magi ng has the potential to detect disease progression or therapeutic effectiveness earlier than most conventional methods in the fields of oncology, neurology and cardiology. Of the several promising molecular imaging technologies having been developed as optical imaging and MRI, PET is of particular interest for drug development because of its high sensitivity and ability to provide quantitative and kinetic data.

Positron emitting isotopes include carbon, nitrogen, and oxygen. These isotopes can replace their non-radioactive counterparts in target compounds to produce tracers that function 20 biologically and are chemically identical to the original molecules for PET imaging. On the other hand, ¹⁸ F is the most convenient labeling isotope due to its relatively long half life (109.8 min) which permits the preparation of diagnostic tracers and subsequent study of biochemical processes. In addition, its high β+ yield and low β+ energy (635 keV) are also advantageous.

^ O

Due to its short 20 minutes half-life ¹¹ C containing radiotracers require an on-site cyclotron, whereas ¹⁸ F PET tracers, considering a half-life of 109 minutes, allow for off-site production and regional distribution.

30 The diagnosis of tumors using positron emission tomography (PET) exploits the fact that the tumor tissues proliferate rapidly in comparison to normal healthy tissues. The best known example for PET imaging of diseases is 2-[ ¹⁸ F]fiuorodeoxyglucose ([ ¹⁸ F]FDG), which is the most widely used PET radiopharmaceutical (J Nucl Med (1978), 19, 1154-1161). However, a number of pitfalls and artefacts have been ascribed to FDG imaging and more continue to

35 surface as the worldwide experience with FDG increases. The area most common for interpretative pitfalls with FDG is related to uptake in active skeletal muscle (Seminars in Nuclear Medicine, (2004), XXXIV, 2, pp.122-133). Many benign conditions can cause high accumulation of FDG creating the potential for false positive interpretation. Most of these artefacts are related to inflammatory, infective or granulomatous processes (Seminars in Nuclear Medicine, (2004), XXXIV, 2, pp.122-133, Seminars in N uclear Medicine, (2004), XXXIV, 1 , pp.56-69, (2004), J Nucl Med (2004), 45, pp. 695-700). Other tumors including mucosal associated lymphomas, small lymphocytic cell lymphoma, some neuroendocrine tumors, sclerotic bone metastases and renal cell carcinomas can be virtually inconspicuous due to low uptake or higher neighbouring background activity. Specifically related to PET-CT are pitfal ls associated with breathing pattern differences between modalities, despite dedicated combined scanners (Seminars in Nuclear Medicine, (2004), XXXIV, 2, pp.122- 133).

Radiolabeled amino acids have been explored for tumor imaging (Jager et a/. , J Nucl Med. , 2001 , 42(3), 432-45) to overcome the limitations seen for [ ¹⁸ F]FDG. Initially, naturally occurring amino acids were labeled with carbon-1 1 such [^C]valine, L-[ ^{1 1} C]leucine, L- [ ^{1 1} C]methionine, and structurally similar [ ¹⁸ F]analogues. After uptake of these mainly neutral amino acids into the tumor cells predominantly via sodium independent L-type amino acid transporters, the retention of these tracers within the tumor cell is mainly due to protein synthesis. Limitations of radiolabeled naturally occurring amino acids include metabolic degradation via several pathways resulting in multiple radiolabeled metabolites which obscure the analysis of tumor uptake of the mother compound . Ki netic stud ies have suggested that amino acid transport rather than protein synthesis better reflects tumor proliferation. Tyrosine derivatives have been of particular interest over the last decade with the introduction of 0-([ ⁸ F]fluoroethyl)-tyrosine ([ ¹⁸ F]FET) wh ich has been shown to be effective in imaging brain tumors but not for periphery tumors (Wester et aL , J. Nucl. Med. 1999, 40, 663, J, Nucl. Med. 1999, 40, 205 and J. Nucl. Med. 1999, 40, 1367). Other potentially interesting tyrosine derivatives include 3-[ ¹⁸ F]fluoro-a-methyi-tyrosine (inoue et aL , J. Nucl. Med. 1998, 39, 205), 0-([ ¹⁸ F]fluoromethyl)-tyrosine (ishiwata et aL , Nucl. Med. Biol. , 2004, 31 , 191 ), 0-([ ¹⁸ F]fluoropropyl)-tyrosine (Tang et a!. , Nucl. Med. Biol. , 2003, 30, 733), 0-([ ¹⁸ F]fluoropropenyl)-tyrosine (Arstad et a/., WO2007073200) and 0-([ ¹⁸ F]fluoroethyl)-a- methyi-tyrosine (Wang et aL , Bioorg. Med. Chem. Lett. , 2010, 20, 3482). These radiolabeled tyrosine derivatives all have their disadvantages, i.e. low yield and high mass dose due to the eiectrophilic radiofluorination method used for the production of 3-[ ¹⁸ F]fiuoro- -methyl- tyrosine. The other tyrosine derivatives either show poor uptake i n cel l cu ltu re , l ow accumulation in tumor bearing mice or relatively poor pharmacokinetics resulting in images with a high background. a-Methyl tyrosine derivatives have previously been disclosed (Tanaka et aL , US6,824,760 B2), however the example shown in this patent, 0-(3- fluoropropyl)-a-methyl-tyrosine does not show high uptake (0.454% I D at 30 min p.i) nor a fast washout (0.454% I D at 30 min p. i vs 0.069%/I D at 4 h p. i . ) in the 13.762 metastatic mammary breast rat tumors (US6, 824,760 B2). Another derivative, 0-([ ¹⁸ F]fluoroethyl)-a- methy!-tyrosine, disclosed within this patent has been recently published (Wang et a/., Bioorg. Med. Chem, Lett,, 2010, 20, 3482), where the uptake in U-138 MG human glioblastoma cells was shown to be 10-fold lower than the 'gold standard' FET. O- ([ ¹⁸ F]fiuoromethyl)-D-tyrosine (DFMT, Tsukada ef a/., J, NucL Med., 2006, 47, 679, Tsukada et al., Eur. J. Nucl. Med.Mol. Imaging, 2006, 33, 1017 and Urakami et a/., Nucl. Med, Biol., 2009, 36, 295) has recently been shown to give better images in tumor bearing mice. The reason for this could be that the D-isomer is recognised by the tumor but, in comparison to its L-isomer, not by other organs within the body and is excreted rapidly via the kidneys, thus enhancing the image quality (Tsukada et a!,, J. Nucl. Med., 2006, 47, 679). Unfortunately, the uptake of DFMT in tumor bearing mice is relatively low (2.57±0.547%ID/g at 1 h p.i, in HeLa xenografts).

The ali phatic ¹⁸ F-fluorination reaction is of great importance for ¹⁸ F-labeled radiopharmaceuticals which are used as in vivo imaging agents targeting and visualizing diseases, e.g. solid tumours or diseases of brain. A very important technical goal in using ¹⁸ F-labeled radiopharmaceuticals is the quick preparation and administration of the radioactive compound due to the fact that the ⁸ F isotopes have a short half-life of about only 1 10 minutes.

Numerous ¹⁸ F radiolabeled PET tracers are currently being investigated for a number of different diseases, a few of these tracers are highlighted below:

[ ¹⁸ F]FET (Gliomas): Floeth ei ai., J. Nuci. Med., 2008, 49(5), 730-737 and Vees et ai., Eur. J. Nuci. Med. Moi. imaging, 2009, 36(2), 182-193; [ ¹⁸ F]FLT (Ceil Proliferation): Buck et ai., Methods, 2009, 48(2), 205-215 and van Waarde et ai., Curr. Pharm. Des., 2008, 14(31 ), 3326-3339; [ ¹⁸ F]FGH (Prostate Cancer): Beheshti et ai., Moi. imaging. Bioi., 2009 , 11 (8), 446-454 and Husarik et ai., Eur. J. Nuci. Med. Moi. imaging, 2008, 35(2):253-263; [ ¹⁸ F]DF T (Tumor imaging): Urakami et ai., Nuci. Med. Bioi. , 2009, 36(3), 295-303 and Murayama et ai., J. Nuci. Med., 2009, 50(2), 290-295; [ ¹⁸ F]F eNER (Norepinephrine Transporter): Schou et ai., Synapse., 2004, 53, 57-67; [ ¹⁸ F]SPA-RQ (Brain Neurokinin Type- (NKi) receptor imaging): Hargreaves ef ai., J. Clin. Psychiatry, 2002, 63(Suppl 11 ), 18-24; [ ¹⁸ F]NR2B (N DA Receptor Imaging): Hamiil et ai., J. Labei. Compd. Radiopharm., 2005, 48, 1-10;

[ ¹⁸ F]FMDAA1 106 (Neuroinflammation): Zhang et ai., Bioorg. Med. Chem., 2005, 13, 181 1- 1818; [ ¹⁸ F]F PEP (Cannabinoid Subtype-1 Receptor): Donohue et ai., J. Med. Chem., 2008, 51 , 5833-5842.

A majority of these tracers have the ¹⁸ F radiolabei attached as a [ ¹⁸ F]fluoroalkyl group, i.e. fluoromethyl, fluoroethyl, fluoromethoxy, fluoroethoxy. A shorter [ ¹⁸ F]fluoroalkyl chain, e.g. fluoromethoxy, is typically associated with having a significantly decreased metabolic stability; this is often emphasized by high bone uptake during in vivo experiments as free [ ¹⁸ F]fluoride released accumulates preferentially in the bone. To increase the metabolic stability of these groups, one method is to replace the hydrogen atoms in the fluoroalkyl chain with deuterium. The reason behind this method is proposed with [ ⁸ F]FMDAA1 106 (Zhang et a!., Bioorg. Med. Chem., 2005, 13, 1811-1818); they propose that the carbon- hydrogen (C-H) bond of the [ ¹⁸ F]fluoromethy! moiety can be attacked by an enzyme and cleaved, this will be followed by the elimination of hydrofluoride from the same a-carbon atom to generate a carbene structure (Figure 2). This carbene will be stabilized by resonance with the adjacent oxygen atom, which further promotes the decomposition of the starting compound.

I n th e s a m e s tu d y (Zhang et a/., Bioorg. Med. Chem., 2005, 13, 1811-1818) [ ¹⁸ F]FDDAA1106, a deuterium-substituted analogue of [ ¹⁸ F]FMDAA1 106, was designed. The reasons for this were: 1) the deuterium atom has a similar and bioisoteric property to the hydrogen atom, deuterium substitution may only have minimal influence on the binding affinity; 2) deuterium substitution can be expected to reduce the rate of defluorination of the [ ¹⁸ F]fluoromethyi moiety since carbon-deuterium (C-D) bond is generally stronger to break than the C-H bond. This deuterium substitution strategy has been used in other cases to increase the metabolic stability, some examples are below:

D4-p ⁸ F]FCH (Prostate Cancer): Leyton et a/,, Cancer Res., 2009 , 69(19), 7721-7728;

[ ¹⁸ F]FMeNER-D ₂ (Norepinephrine Transporter): Schou et al., Synapse., 2004, 53, 57-67 and Takano et al., Eur. J. Nucl. Med. Mol. Imaging. , 2008, 35, 153-157; [ ¹⁸ F]NR2B-D ₂ (NMDA Receptor Imaging): Hamill et a/., J. Label. Compd. radiopharm,, 2005, 48, 1-10;

[ ¹⁸ F]FDDAA1106 (Neuroinf!ammation): Zhang et al., Bioorg. Med. Chem., 2005, 13, 181 1- 1818; [ ¹⁸ F]FMPEP-D ₂ (Cannabinoid Subtype-1 Receptor): Donohue et al., J. Med. Chem., 2008, 51 , 5833-5842. In all these cases the metabolic stability of the compound was found to be higher for the deuterium substituted analogues. However, none reported whether the stability of the deuterium substituted radiolabeled tracers gave increased hydrolytic stability. Only hydrolytic studies on non-deuterium substituted group, i.e. f ⁸ F]fluoromethoxy group has been reported. For example, [ ¹⁸ F]F e cN (Zessin et al., Nucl. Med. Biol., 2001 , 28, 857- 863) found that by altering the pH and solvents the hydrolytic stability could be increased significantly using pH 8 and 50% propylene glycol. However these conditions will not be applicable for all PET tracers especially those which are base sensitive.

There is a continued need for novel agents and methods for improving the hydrolytic stability of radiolabeled compounds. This present application discloses compounds, methods of synthesizing and using those compounds to improve the hydrolytic stability of radiolabeled compounds.

Despite the aforementioned advances in identifying DF T as a suitable tyrosine derivative for tumor imaging and specifically for the imaging of cancer, there remains a need for novel derivatives or agents with improved signal, increased hydrolytically stability and/or higher tumor uptake of the imaging agents within the tumor.

In contrast, compounds of the present invention feature a very surprisingly rapid and much higher accumulation in the tumors than their hydrogen analogues and are more stable in aqueous solutions over the same time period in comparison to DF T.

The invention relates to the subject matter referred to in the claims, i.e. tyrosine amino acid derivatives of the formula (I), (D-l), (D-la), (L-l) or (L-la), their use and their preparation processes.

Figure 1 : HPLC chromatogram of 0-([ ¹⁸ F]Fluoro[ ² H ₂ ]methyl)-D-tyrosine (D-DFMT). 1 a) Radiotrace, 1 b): UV-trace.

Figure 2: Cell uptake assay of DFMT and DDFMT in A549 and H460 cells

Figure 3: PET/CT-imaging of [18F]D-DFMT in NCI-H292-tumor bearing mice.

Figure 4: PET/CT-imaging of [18F]D-DFMT in A549-tumor bearing mice.

)escnptson

In a first aspect, the invention is directed to compounds of the formula (I)

(0

wherein

X is Fluorine atom (F);

Y is CHD or CD ₂ ; and

D stands for Deuterium

Formula I encompasses single isomers, diastereoisomers and enantiomers, mixtures thereof and pharmaceutically acceptable salts thereof.

Preferably, Fluorine atom (F) is an ¹⁸ F or ¹⁹ F isotope.

Preferably, Y is CD ₂ . In a first embodiment, the invention is directed to a compound of formula (I) wherein the Fluorine atom (F) is a ¹⁸ F isotope.

Preferably, Fluorine atom (F) is a ¹⁸ F isotope and Y is CD ₂ .

In a second embodiment, the invention is directed to a compound of formula (I) wherein the Fluorine atom (F) is a ¹⁹ F isotope.

Preferably, Fluorine atom (F) is a ¹⁹ F isotope and Y is CD ₂ .

In a third embo d to a compound of formula (D-l)

D- wherein

X is Fluorine atom (F);

Y is CHD, or CD _2, and

D stands for Deuterium

Preferably, Fluorine atom (F) is ¹⁸ F or ¹⁹ F isotope. More preferably, Fluorine atom (F) is ¹⁸ F isotope.

Preferably, Y i

( D-ia )

Preferably, compound of formula (D-l) or(D-la) is wherein Fluorine atom (F) is ¹⁸ F isotope and Y is CD _2.

In a fourth embodiment, the invention is directed to a compound of formula (L-l)

X is Fluorine atom (F);

Y is CHD, or CD _{2 ;} and

D stands for Deuterium.

Preferably, Fluorine atom (F) is ¹⁸ F or ¹⁹ F isotope. More preferably, Fluorine atom (F) is ¹⁸ F isotope.

Preferably, Y i

( L-la)

Preferably, compound of formula (L-i) or (L-la) is wherein Fluorine atom (F) is ¹⁸ F isotope and Y is CD _2.

The invention further refers to suitable salts of inorganic or organic acids, hydrates and solvates of the compounds of Formula (I), (L-l), (L-la), (D-l) or (D-la).

Embodiments and preferred features can be combined together and are within the scope of the invention.

Invention compounds are: L-tyrosine

(rac)

0-([ ¹⁸ F]Fluoro[ ² H ₂ ]methyl)-L-tyrosine

- tyrosine

osine

(rac)

sine

sine

Preferably, invention compounds are:

- tyrosine

0-(F!uoro[ ² H ₂ ]methyl)-D-tyrosine in a second aspect, the invention is directed to methods for obtaining compounds of formula (I). The method of the invention is an indirect fluoro-labeiing method, see scheme 2.

Sa N

(precursors for

Formuia Hi Formufa !

(precursors (F-18 labeled (novel F-18 labeled

for the synthesis of prosthetic group) PET imaging agent)

known F-18 labeled

prosthetic groups)

= novel compounds

Scheme 2: Indirect f!uoro radiolabeiing method

U nder the present invention, the methods are indirect labeling methods for obtaining compound of formula (I) as described above. Embodiment and preferred features described in first aspect are incorporated herein.

The indirect fluoro-labeiing method comprises the step

- Coupling compound of Formula (I I I) with compound of formula (IV) for obtaining compound of Formula (I)

wherein

X is Fluorine atom (F);

Y is CHD or CD ₂ ; and

D stands for Deuterium; and wherein compound of Formula (III) is a suitable F-18 or F-19 labeled prosthetic group and compound of formula (IV) is D- or L- Tyrosine or mixtures thereof and/or salts.

The indirect fiuoro-labeling method comprises the steps

Coupling compound of Formula (I I) with Fluorine atom (F) containing moiety for obtaining compound of Formula (III),

Coupling compound of Formula (III) with a compound of formula (IV) for obtaining compound of Formula (I)

wherein

X is Fluorine atom (F);

Y is CHD or CD ₂ ;

D stands for Deuterium , and

Optionally converting obtained compound into pharmaceutically acceptable salts of inorganic or organic acids thereof, hydrates, complexes, esters, amides, and solvates thereof,

wherein compound of Formula (II) is a suitable precursor for the synthesis of known F-18 or F-19 labeled prosthetic group, compound of Formula (IN) is a suitable F-18 or F-19 labeled prosthetic group and compound of formula (IV) is D- or L- Tyrosine or mixtures thereof and/or salts.

Preferably, the method is an indirect labeling method for obtaining compound of formula (I) comprising the steps

Coupling compound of Formula (II) with Fluorine atom (F) containing moiety wherein the Fluorine atom (F) containing moiety comprises ¹⁸ F isotope for obtaining compound of Formula (ill),

Coupling compound of Formula (III) with a compound of formula (IV) for obtaining compound of Formula (I), and

Optionally converting obtained compound into pharmaceutically acceptable salts of inorganic or organic acids thereof, hydrates, complexes, esters, amides, and solvates thereof, wherein compound of Formula (li) is a suitable precursor for the synthesis of known F-18 labeled prosthetic group, compound of Formula (III) is a suitable F-18 labeled prosthetic group and compound of formula (IV) is D- or L- Tyrosine or mixtures thereof and/or salts.

M ore preferably, the method is an indirect labeling method for obtaining 0-([ ¹⁸ F]Fluoro[

² H ₂ ]methyl)-D-tyrosine ([ ¹⁸ F]D-DFMT) comprising the steps

Coupling CD ₂ Br ₂ with K[ ¹⁸ F]F for obtaining ⁸ FCD ₂ Br,

Coupling ¹⁸ FCD ₂ Br with D-Tyrosine for obtaining [ ¹⁸ F]D-DFMT, and

Optionally converting obtained compound into pharmaceutically acceptable salts of inorganic or organic acids thereof, hydrates, complexes, esters, amides, and solvates thereof.

Preferably, the method is an indirect labeling method for obtaining compound of formula (I) comprising the steps.

Coupling compound of Formula (II) with Fluorine atom (F) containing moiety wherein the Fluorine atom (F) containing moiety comprises ¹⁹ F isotope for obtaining compound of Formula (111),

Coupling compound of Formula (III) with a compound of formula (IV) for obtaining compound of Formula I, and

Optionally converting obtained compound into pharmaceutically acceptable salts of inorganic or organic acids thereof, hydrates, complexes, esters, amides, and solvates thereof,

wherein compound of Formula (I I) is a suitable precursor for the synthesis of known F-19 labeled prosthetic group, compound of Formula (I I I) is a suitable F-19 labeled prosthetic group and compound of formula (IV) is D- or L- Tyrosine or mixtures thereof and/or salts.

Methods have been shown whereby the cold F19 compound is synthesized via the same synthetic routes used for the production of the hot F 1 8 compound (Iwata et al., J. Label. Compds. Radiopharm. , 2003, 46, 555-566 and Donohue et as. J , Med. Chem. , 2008, 51 , 5833- 5842).

Compound of formula (I) is preferably compound of formula (L-l), (L-ia), (D-l) or (D-ia) for all methods described above and more preferably (D-l) or (D-la). The reagents, solvents and conditions which can be used for this fluorination are common and well-known to the skilled person in the field. See, e.g., J. Fluorine Chem,, 27 (1985):177-191. Preferably, the solvents used in the present method is DMF, DMSO, acetonitrile, DMA, or mixture thereof, preferably the solvent is acetonitrile.

The reagents, solvents and conditions which can be used for the aikytion are common and well- known to the skilled person in the field. See, e.g., Wester et as, , J. Nucl. Med. 1999, 40, 663. Preferably, the solvents used in the present method is DMF, DMSO, acetonitrile, DMA, or mixture thereof, preferably the solvent is DMSO.

A method for obtaining compounds of formula (L-l), (L-ia), (D-l) or (D-la) wherein compounds of formula (L-l), (L-la), (D-l) or (D-la) are as disclosed above and wherein above embodiment and preferred features are herein enclosed.

Compounds of formula (I I) are well known suitable precursors for the synthesis of known F- 18 or F-19 labeled prosthetic groups (Zhang et aL , Bioog. Med. Chem. , 2005, 13, 181 1- 1818;).

Preferably, compounds of formula (II) is

R ¹ \ _v / ^R2

(II)

wherein

R1 is a leaving group selected from the group of halogen and sulfonate, wherein halogen is chloro, bromo or iodo, and sulfonate is mesylate, toysiate, trifiate or nosylate;

R2 is a leaving group selected from the group of halogen and sulfonate, wherein halogen is chloro, bromo or iodo, and sulfonate is mesylate, toysiate, trifiate or nosylate;

Y is CHD or CD ₂ and

D stands for Deuterium.

Non-limiting examples of compounds of Formula (II) known to those skilled in the art are:

(Eaborn and Stanczyk, J. Chem. Soc. Perkin Trans 2, 1991 , 471-473; Bothner-By et al., J.

Am. Chem. Soc, 1987, 109, 4180-4184; Takaya et al., J. Org. Chem., 1981 , 46, 2846-2854): deuterated dibromomethane (CD ₂ Br ₂ ), monodeuteriodibromomethane (CHDBr ₂ ), deuterated diiodomethane (CD ₂ I ₂ ), monodeuteriodiiodomethane (CHDI ₂ )

Preferably, compound of Formula (II) is deuterated dibromomethane (CD ₂ Br ₂ ). Compounds of formula (IN) are well known suitable F-18 or F-19 labeled prosthetic groups (Zhang et al. , Bioog. Med. Chem., 2005, 13, 181 1-1818; Donohue et a!., J. Med. Chem., 2008, 51 , 5833-5842).

Preferably, compounds of formula (III) is

^Y (ill)

wherein

R1 is a leaving group selected from the group of halogen and sulfonate, wherein halogen is chioro, bromo or iodo, and sulfonate is mesylate, toyslate, triflate or nosylate;

X is Fluorine atom (F), preferably Fluorine atom (F) is ¹⁸ F or ¹⁹ F isotope, more preferably ¹⁸ F isotope;

Y is CHD or CD ₂ and

D stands for Deuterium.

More preferably, compounds of formula (III) is

^Y (ill)

wherein

X is ¹⁹ F isotope; and

Y is CD ₂ .

More preferably, compounds of formula (III) is

R ¹ \ _ν · ^χ

(III)

wherein

X is ⁸ F isotope; and

Y is CD ₂ .

Non-limiting examples of compounds of Formula (ill) known to those skilled in the art are: deuterated bromof!uoromethane (FCD ₂ Br), deuterated bromo[ ¹⁸ F]fluoromethane (f ⁸ F]FCD ₂ Br), m o n od e u te riobromofluoromethane (FCHDBr), monodeuterio- bromo[ ¹⁸ F]fluoromethane ([ ¹⁸ F]FCHDBr), deuterated fluoroiodomethane (FCD ₂ I), deuterated [ ¹⁸ F]fiuoroiodomethane ([ ¹⁸ F]FCD ₂ I), monodeuteriofiuoroiodomethane (FCHDi), monodeuterio[ ¹⁸ F]fluoroiodomethane ([ ⁸ F]FCHDi), deuterated fiuoromethyl tosylate (FCD ₂ OTos).

Preferably, when Fluorine atom (F) is ¹⁸ F isotope, compounds of Formula (III) is deuterated bromo[ ¹⁸ F]fluoromethane ([ ¹⁸ F]FCD ₂ Br), monodeuterio-bromo[ ¹⁸ F]fluoromethane

f[ ¹⁸ F]FCHDBr), deuterated [ ¹⁸ F]fiuoroiodomethane f[ ¹⁸ F]FCD ₂ l),

monodeuterio[ ¹⁸ F]fluoroiodomethane (f ⁸ F]FCHDI), M o re preferably, compound of Formula (III) is deuieraied bromo[ ¹⁸ F]fluoromeihane ([ ¹⁸ F]FCD ₂ Br).

Preferably, when Fluorine atom (F) is ¹⁹ F isotope, compounds of Formula (III) is deuterated bromofluoromethane (FCD ₂ Br), monodeuteriobromofluoromethane (FCHDBr), deuterated fluoroiodomethane (FCD ₂ I), monodeuteriofluoroiodomethane (FCHDi), deuterated

fiuoromethyl tosyiate (FCD ₂ OTos).

Compounds of formula (IV) are well known D- or L- Tyrosine or mixtures thereof and/or salts thereof suitable as precursor for the indirect labelling.

Non-limiting examples of compounds of Formula (IV) known to those skilled in the art are:

L-Tyrosine, D-Tyrosine and mixtures thereof,

Salts of L-Tyrosine, D-Tyrosine and mixtures thereof.

Preferably, compound of Formula (IV) is D-Tyrosine.

Fluorine atom (F) containing moiety comprises preferably ¹⁸ F or ¹⁹ F.

More preferably, the Fluorine atom (F) containing moiety comprising ¹⁸ F can be chelated complexes known to those skilled in the art, e.g. 4,7, 13,18,21 ,24-Hexaoxa-1 ,10- diazabicycio[8.8.8]-hexacosane K ¹⁸ F (crown ether salt Kryptofix K ¹⁸ F), 18-crown-8 ether salt K ¹⁸ F, K ¹⁸ F, H ¹⁸ F, KH ¹⁸ F ₂ , Rb ¹⁸ F, Cs ¹⁸ F, Na ⁸ F, or tetraaikyiammonium salts of ¹⁸ F known to those skilled in the art, e,g.f ⁸ F] tetrabutylammonium fluoride, or tetraalkylphosphonium salts of ¹⁸ F known to those skilled in the art, e.g.[ ¹⁸ F] tetrabutylphosphonium fluoride. Most preferably, the Fluorine atom (F) containing moiety is Cs ¹⁸ F, K ¹⁸ F, H ¹⁸ F, or KH ¹⁸ F _2.

More preferably, Fluorine atom (F) containing moiety comprises ⁹ F. Even more preferably, the Fluorine atom (F) containing moiety is 4,7,13,16,21 ,24-Hexaoxa-1 ,10-diazabicyclo[8.8,8]- hexacosane KF (crownether salt Kryptofix KF), 1 ,4,7, 10, 13,16-hexaoxacyclooctadecane KF, KF, tetrabutylammonium fluoride, tetrabutylammonium dihydrogen trifluoride,

In a third aspect, the invention is directed to a composition comprising compounds of the formula (I), (L-l), (L-la), (D-l) or (D-la) or mixture thereof and pharmaceutically acceptable carrier or diluent.

The person skilled in the art is familiar with auxiliaries, vehicles, excipients, diluents, carriers or adjuvants which are suitable for the desired pharmaceutical formulations, preparations or compositions on account of his/her expert knowledge.

The administration of the compounds, pharmaceutical compositions or combinations according to the invention is performed in any of the generally accepted modes of administration available in the art. Intravenous deliveries are preferred. Generally, the pharmaceutical compositions according to the invention can be administered such that the dose of the active compound is in the range of 37 Bq (1 mCi) to 740 Bq (20 mCi). In particular, a dose in the range from 50 MBq to 370 MBq will be used.

I n a fourth aspect of this invention is directed to compounds of formula (I) as radiopharmaceutical in mammal. The compounds of formula (I) are radiopharmaceuticals for imaging proliferative diseases.

In other word, the invention is directed to the use of compounds of formula (I) for the manufacture of a radiopharmaceutical for imaging proliferative diseases in mammal.

Preferably, the compound of formula (I) is a compound of formula (L-l), (L-la), (D-l) or (D-la) wherein F is ¹⁸ F isotope.

Preferably, proliferative diseases are cancer characterised by the presence of tumor and/or metastases. Preferably, tumor is selected from the group of malignomas of the gastrointestinal or colorectal tract, liver carcinoma, pancreas carcinoma, kidney carcinoma, bladder carcinoma, thyroid carcinoma, prostrate carcinoma, prostrate tumor, endometrial carcinoma, ovary carcinoma, testes carcinoma, melanoma, small-cell and non-smail-ceil bronchial carcinoma, lung tumor, dysplastic oral mucosa carcinoma, invasive oral cancer; breast cancer, including hormone-dependent and hormone-independent breast cancer, squamous cell carcinoma, neurological cancer disorders including neuroblastoma, glioma, astrocytoma, osteosarcoma, meningioma, soft tissue sarcoma; haemangioma and endocrine tumors, including pituitary adenoma, chromocytoma, paraganglioma, haematological tumor disorders i ncluding lymphoma and leukaemias. Preferably, the tumor is a prostate carcinoma, prostate tumor or lung tumor.

Preferably, metastases are metastases of one of the tumors mentioned above. More preferably, metastases are metastases of a prostate carcinoma, prostate tumor or lung tumor.

Preferably, the invention compounds and use is for manufacturing a PET imaging tracer for imaging tumor in a mammal wherein the tumor is preferably prostate carcinoma/prostate tumor or lung tumor.

The radiopharmaceutical of present invention is Positron Emission Tomography PET suitable imaging tracer or MicroPET.

The imaging comprises the step of PET imaging and is optionally preceded or followed by a Computed Tomography (CT) imaging or Magnetic Resonance Tomography (MRT) imaging. The invention further provides a method for imaging proliferative diseases, the method comprising introducing into a patient a detectable quantity of a labeled compound of Formula (I), (L-l), (L-ia), (D-i) or (D-la) or pharmaceutically acceptable hydrate, solvate, ester, amide or prodrug thereof. Preferably, F is ¹⁸ F isotope.

The invention is also directed to a method for imaging or diagnosing of proliferative diseases comprising the steps:

Administrating to a mammal an effective amount of a compound comprising compounds of formula (I) (L-l), (L-la), (D-l) or (D-la),

Obtaining an image of the mammal and

Assessing the image.

The compounds of formula (I), (L-l), (L-la), (D-l) or (D-la) are herein defined as above and encompass all embodiments and preferred features herein enclosed.

In a fifth aspect, the invention is directed to the use of compounds of formula (I) for conducting biological assays and/or chromatographic identification. More preferably, the use relates to compounds of formula (I) wherein the fluorine isotope is ¹⁸ F or ¹⁹ F, more preferably

Compounds of formula (I), wherein the fluorine isotope is ¹⁹ F, and are useful as reference and/or measurement agent.

The compounds of formula (I) are herein defined as above and encompass all embodiments and preferred features herein enclosed.

In a sixth aspect, the present invention provides a kit comprising a sealed vial containing a predetermined quantity of

o the compounds of Formula II and;

o the compounds of Formula IV

and pharmaceutically acceptable salts of inorganic or organic acids thereof, hydrates, complexes, esters, amides, and solvates thereof. Optionally the kit comprises a

pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

Compounds of Formula II and IV are as described in second aspect.

I n a seventh aspect, the invention is directed to a method for monitoring tumor and/or metastases size by PET imaging a patient using compound of Formula (I). Ιΐ has been surprisingly found thai F-18-compounds of present invention are potential PET radio tracer for imaging tumor and/or metastases in mammal. Rapid accumulation and retention of invention compounds into tumor and/or metastases allows for effective delineation of tumor and/or metastases and measurement of the tumor and/or metastases size from the obtained PET images. For the monitoring of tumor and/or metastases size, patients are PET imaged with invention compounds at least two times within a suitable time interval.

Monitoring of tumor and/or metastases size by imaging patient with invention compounds and comparing the tumor and/or metastases size obtained .

Definitions

The terms used in the present invention are defined below but are not limiting the invention scope.

If chirai centers or other forms of isomeric centers are present in a compound according to the present invention, ail forms of such stereoisomers, including enantiomers and diastereoisomers, are intended to be covered herein. Compounds containing chirai centers may be used as racemic mixture or as an enantiomericaliy enriched mixture or as a diastereomeric mixture or as a diastereomerica!ly enriched mixture, or these isomeric mixtures may be separated using well-known techniques, and an individual stereoisomer maybe used alone. In cases wherein compounds may exist in tautomeric forms, such as keto-enoi tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.

In the context of the present invention, preferred salts are pharmaceutically acceptable salts of the compounds according to the invention. The invention also comprises salts which for their part are not suitable for pharmaceutical applications, but which can be used, for example, for isolating or purifying the compounds according to the invention.

Pharmaceutically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, for example salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesuifonic acid, ethanesuifonic acid, toiuenesulfonic acid, benzenesulfonic acid, naphthalene disuifonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maieic acid and benzoic acid.

Pharmaceutically acceptable salts of the compounds according to the invention also include salts of customary bases, such as, by way of example and by way of preference, alkali metal salts (for example sodium salts and potassium salts), alkaline earth metal salts (for example calcium salts and magnesium salts) and ammonium salts, derived from ammonia or organic amines having 1 to 16 carbon atoms, such as, by way of example and by way of preference, ethy!amine, diethy!amine, triethy!amine, ethy!diisopropylamine, monoethanolamine, dietha- nolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanoi, procaine, diben- zylamine, N-methylmorpholine, arginine, lysine, ethylenediamine and N-methylpiperidine.

The stereochemistry can be denoted in several ways. For the amino acids often D/L is used derived from the Fischer projection of the amino acid. Stereochemically D corresponds to the stereodescriptor "R" in the Cahn, i ngoid, Prelog System and L corresponds to the stereodescriptor "S" for all of the compounds of the invention.

Without further elaboration, it is believed that one skilled in the art can, using the preceeding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. 1 ⁸ F]DF T stands for Q-([ ¹⁸ F]Fluoromethy!)-D-tyrosine.

-([ ¹⁸ F]Fluoro[ ² H ₂ ]methyl)-D-tyrosine,

The entire disclosure[s] of all applications, patents and publications, cited herein are incorporated by reference herein.

The following examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Unless otherwise specified, when referring to the compounds of formula the present invention per se as well as to any pharmaceutical composition thereof the present invention includes ail of the hydrates, salts, and complexes.

General synthesis of F-18 compounds

The radiofluorination reaction can be carried out, for example in a typical reaction vessel (e.g. Wheaton vial) which is known to someone skilled in the art or in a microreactor. The reaction can be heated by typical methods, e.g. oil bath, heating block or microwave. The radiofluorination reactions are carried out in dimethylformamide with potassium carbonate as base and "kryptofix" as crown-ether. But also other solvents can be used which are well known to experts. These possible conditions include, but are not limited to: acetonitrile, dimethylsulfoxide, suifolane, dichloromethane, tetrahydrofuran, tertiary alcohols and o- dichlorobenzene as solvent and alkali metal with and without a suitable alkali metal chelating crown ether, tetraalkyi ammonium and tetraalkyi phosphonium carbonate as base. Water and/or alcohol can be involved in such a reaction as co-solvent. The radiofluorination reactions are conducted for one to 60 minutes. Preferred reaction times are five to 50 minutes. Further preferred reaction times are 10 to 40 min. This and other conditions for such radiofluorination are known to experts (Coenen, Fluorine-18 Labeling Methods: Features and Possibilities of Basic Reactions, (2006), in: Schubiger P. A., Friebe M., Lehmann L., (eds), PET-Chemistry - The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp.15- 50). The radiofluorination can be carried out in a "hot-cell" and/or by use of a module (Review: Krasikowa, Synthesis Modules and Automation in F-18 labeling (2006), in: Schubiger P. A., Friebe M., Lehmann L, (eds), PET-Chemistry - The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp. 289-316) which allows an automated or semi-automated synthesis.

Synthesis of F-19 compounds

Scheme 3a shows a method that could be used to synthesize of the racemic O- [ ¹⁹ F]fluoromethyl tyrosine 4 starting from 1 using methods known to those skilled in the art. The synthesis of 2 from 1 is known in the literature (Liu et a/., J. Med. Chem., 2004, 47, 1223-1233). The method for alkylating 2 to 3 is known for 0-(fluoromethyl)-tyrosine (J Labelled Compds. Radiopharm. 2003, 46, 555-566), similar methods can be applied here. The fluorobromodeuteriomeihane (Raymond, J. Phys. Chem., 1971 , 75, 3235) and fluorodeuteriomethyltosylate (Donohue et a/., J. Med. Chem., 2008, 51 , 5833-5842) are known. Methods for hydro!yzing carboxylic acid protecting groups like methyl esters and amine protecting groups like tert-butoxy carbonyl (Boc) groups are well precedented (Greene and Wuts, 'Protecting Groups in Organic Syntheses', third edition, pp. 369-453 and 494-653). The R and S isomers could be separated by methods known by those skilled in the art, i.e. chiral HPLC.

4{S) 4(R)

Scheme 3a: Synthesis of 0-(Fluoro[ ² H ₂ ]methyl)-DL-tyrosine 4, 4(S) and 4(R).

The synthesis could be carried out using the method described in Scheme 3b, similar methods are published whereby 3-chioro-O-methyl-L-tyrosine and 3-chloro-Q-methyl-D- tyrosine could be synthesized by the alkylation of 3-chioro-L-tyrosine or 3-chioro-D-tyrosine (Trimurtuiu et a/,, J. Am. Chem. Soc, 1994, 1 16, 4729-4737):

Synthesis of F-18 invention compounds

The ¹⁸ F-compounds were synthesized by reaction of precursors of type II with [ ¹⁸ F]fluoride to give ¹⁸ F labeled intermediates of type 111 which were then reacted with precursors of type IV to given the desired product of type \ as shown in Scheme 4,

CD ₂ Br ₂

Deuterated Dibromomethane

Formula M

K[«F]F, K ₂ C0 ₃

K,.

D-Tyrosine

Formula !

Formula IV

Scheme 4: Radiosynthesis of 0-([ ¹⁸ F]Fluoro[ ² H ₂ ]methy!)-D-tyrosine ([ ¹⁸ F]D-DFMT).

Experimental Section

Abbreviations

AcOH Acetic acid

nBuOH n-Butan-1-ol DMA Ν,Ν-Dimethyl acetamide

DMF Ν,Ν-Dimethyl formamide

DMSO Dimethyl sulfoxide

EtOH Ethanol

FCS fetal calf serum

GBq GigaBequerel

H Hour

HPLC High Pressure Liquid Chromatography

K ₂₂₂ Kryptofix 2.2.2

Min Minute

MBq MegaBequerel

MeOH Methanol

PBS Phosphate Buffered Saline

PET/CT Positron Emitting Tomography/Computed

Tomography

r.t. room temperature

SPE Solid Phase Extraction

TFA trifiuoroacetic acid

General: Ail solvents and chemicals were obtained from commercial sources and used without further purification. Anhydrous solvents and inert atmosphere (nitrogen or argon) were used if not stated otherwise. The preceding table lists the abbreviations used in this paragraph and in the Examples sections as far as they are not explained within the text body. The compounds and intermediates produced according to the methods of the invention may require purification, i.e. semi-preparative HPLC according to the preparative HPLC methods listed below.

The synthesis of 0-([ ¹⁸ F]fluoromethyl)-D-tyrosine (DFMT) was synthesized according to known literature procedures (Tsukada et a!. , J. Nucl. Med., 2006, 47, 679).

1. Chemistry experiment

Example 1 Q-([ ¹⁸ F]Fiuoro[ ² H ₂ ]methyi)-D-tyrosine (j/ ⁸ F]D-DFMT)

[ ¹⁸ F]Fluoride (2879 MBq) was immobilized on a preconditioned QMA (Waters) cartridge (preconditioned by washing the cartridge with 5 mL 0.5 M K ₂ C0 ₃ and 10 mL water), The [ ¹⁸ F]fiuoride was eiuted using a solution of K ₂ C0 ₃ (2,7 mg) in 50 μΙ_ water and K ₂₂₂ (15 mg) in 950 μ!_ acetonitrile. This solution was dried at 120°C with stirring under vacuum and with a nitrogen flow of 150 mL/min. Additional acetonitrile (1 mL) was added and the drying step was repeated. A solution of deuterated dibromomethane (CD ₂ Br ₂ ; 100 μΐ_) in acetonitrile (900 μΙ_) was added and heated at 130°C for 5 min. The reaction was cooled to 50°C and the [ ¹⁸ F]f!uorodeuteriomethy! bromide was distilled at 50°C with a nitrogen flow of 50 mL/min through 4 silica cartridges into a solution of D-tyrosine (3 mg), with 10% NaOH (13.5 μί) in DMSO (1 mL). This solution was heated at 110°C for 5 min and then cooled to 40°C. The reaction mixture was purified by HPLC (ACE 5 μ C18 250 x 10 mm; EtOH:AcOH:H ₂ 0 (100: 1 :900); flow 5 mL/min). The product peak was collected and concentrated to dryness. The dried product was re-dissolved in PBS. Starting from 2879 MBq [ ¹⁸ F]fluoride, 177 MBq (13 % d.c.) of [ ¹⁸ F]D-DFMT were obtained in 121 min (non-optimized).

Figure 1 shows the chromatogram of the final product D-DFMT 1 a): Radiotrace, 1 b): UV- trace.

Agilent 1 100

HPLC Column: Synergi Hydro 4 μ 250 x 4,8 mm

Eluents: A: Water + 0.1 % TFA (Agilent 7)

B: Acetonitrile + 0.1 % TFA

Gradient 00:00 min 15%B

15:00 15%B

Flow: 1 mL/min ample 2. Radiosynthesis of 0-([ ⁱ⁸ F]Fluoromethyl)-D-tyrosine (f ⁸ F]DFMT)

[ ¹⁸ F]Fluoride (3736 MBq) was immobiiized on a preconditioned QMA (Waters) cartridge (preconditioned by washing the cartridge with 5 ml_ 0.5 M K ₂ C0 ₃ and 10 mL water), The [ ¹⁸ F]f!uoride was eiuted using a solution of K ₂ C0 ₃ (2,7 mg) in 50 xL water and K ₂₂₂ ( 5 mg) in 950 μΐ_ acetonitrile. This solution was dried at 120°C with stirring under vacuum and with a nitrogen flow of 150 mL/min. Additional acetonitrile (1 mL) was added and the drying step was repeated. A solution of dibromomethane (CH ₂ Br ₂ ) 100 pL) in acetonitrile (900 μί) was added and heated at 130°C for 5 min. The reaction was cooled to 50°C and the [ ¹⁸ F]fluoromethy! bromide was distilled at 50°C with a nitrogen flow of 50 mL/min through 4 silica cartridges into a solution of D-tyrosine (3 mg), with 10% NaOH (13.5 xL) in DMSO (1 mL). This solution was heated at 1 10°C for 5 min and then cooled to 40°C. The reaction mixture was purified by HPLC (ACE 5 μ C18 250 x 10 mm; EtOH:AcOH: H ₂ 0 (100: 1 :900); flow 5 mL/min). The product peak was collected and concentrated to dryness. The dried product was re-dissolved in PBS. Starting from 3736 MBq [ ¹⁸ F]fluoride, 285 MBq (12 % d.c.) of [ ¹⁸ F]DFMT were obtained in 60 min (non-optimized).

Example 3. Hydrolytic stability [ ¹⁸ F]DFMT at Room Temperature

A solution of [ ¹⁸ F]DFMT in PBS was adjusted with 0.1 M HCI to pH 7 and pH 5. This solution was kept at room temperature and analyzed at different time-points using TLC TLC Silica gel 60 F254 plates Merck; nBuOH:AcOH: PBS (4: 1 :2)). The results are shown in Table 1.

Example 4. Hydrolytic stability [ ¹⁸ F]DFMT at 37°C

A solution of f ⁸ F]DFMT in PBS was adjusted with 0.1 M HCI to pH 7 and pH 5. This solution was heated at 37°C and analyzed at different time-points using TLC (TLC Silica gel 60 F254 plates Merck; nBuOH:AcOH: PBS (4: 1 :2)). The results are shown in Table 1 .

Example 5. Hydrolytic stability [ ¹⁸ F]D-DFMT at Room Temperature A solution of [ ¹⁸ F]D-DFMT in PBS was adjusted with 0.1 M HCI to pH 7 and pH 5. This solution was kept at room temperature and analyzed at different time-points using TLC (TLC Silica gel 60 F254 plates Merck; nBuOH:AcOH:PBS (4: 1 :2)). The results are shown in Table 1.

Example 8. Hydroiytic stability [ ¹⁸ F]D-DFMT at 37°C

A solution of f ⁸ F]D-DFMT in PBS was adjusted with 0.1 M HCI to pH 7 and pH 5. This solution was heated at 37°C and analyzed at different time-points using TLC (TLC Silica gel 60 F254 plates Merck; nBuOH:AcOH:PBS (4:1 :2)). The results are shown in Table 1.

Table 1. Hydroiytic stability of [ ¹⁸ F]DFMT and [ ¹⁸ F]D-DFMT in aqueous solutions at different pH and temperature.

These results ciearly show that the deuterium substituted analogue D-DFMT is more stable than DFMT.

2. Bio Experiment

Example 1 : Cell-uptake experiments

To assess the difference between [ ¹⁸ F]D-DFMT and f ⁸ F]D-FMT, we studied the uptake of the radiolabeled compounds into A549 and NCI-H460 human lung cancer cells. 80000 A549 cells or NCI-H460 cells were seeded per cavity of a 48 well incubation plate (Becton Dickinson; Cat. 353078) and incubated for 1 day in RPMi 1640 with GiutaMAX (Invitrogen; Cat. 31331) medium supplemented with 10% FCS in an incubator (37°C, 5% C0 ₂ ). Cells were washed once with PBS and then incubated for 10 - 30 min at 37°C in PBS with 1 Ci radioactive tracer ([ ¹⁸ F]D-DFMT, [ ¹⁸ F]D-FMT). After incubation, the ceils were washed once with cold PBS, lysed with 1 NaOH, and finally lysates were measured in a gamma counter. Surprisingly, the uptake of the [ ¹⁸ F]D-DFMT was higher than [ ¹⁸ F]D-FMT, [ ¹⁸ F]D-DFMT was highest with 6.8 % applied dose/10 ⁶ ceils in A549 cells after 30 min, while [ ¹⁸ F]D-FMT showed 5.3% applied dose/10 ⁶ cells in A549 ceils (see Figure 2). The NCI-H460 ceils showed [ ¹⁸ F]D-DF T uptake of 4.1 % applied dose/10 ⁶ cells after 30 min, while [ ¹⁸ F]D-F T showed 2.1 % applied dose/10 ⁶ cells in NCI-H460 ceils (see Figure 2).

Example 2: Biodistribution of [ ¹⁸ F]D-DFMT in NCi-H460-tumor bearing mice

Biodistribution and excretion studies were performed in female N RI (nu/nu) mice. For tumor induction tumor cells were inoculated subcutaneously according to a standard method with 5x10 ⁶ NCI-H460 lung tumor ceils into the right shoulder. At 1 week after tumor cell inoculation tracer injection and biodistribution study was performed. 6 μθί of [ ¹⁸ F]D-DF T were injected intravenously (3 animals per time point) via the tail vein into conscious animals. At 15, 30, 60, 120 and 240 min time points urine and feces were quantitatively collected. At the same time points, animals were sacrificed by decapitation and exsanguinations under isoflurane anesthesia and the following organs and tissues were removed for weighting and measurement of ¹⁸ F radioactivity using the gamma-counter: spleen, liver, kidney, lung, femur, heart, brain, fat, thyroid, muscle, skin, blood, tail, stomach (without content), intestine (with content), pancreas, adrenals and uterus.

In order to get the amount of total radioactivity administered in each animal (= 100%) 3 aiiquots of the injected solution were directly measured. The results of the biodistribution and excretion are reported as percentage of injected dose per gram of tissue (%ID/g) and tumor- to-organ-ratios (T T-ratio) were calculated (Table 2). [ ¹⁸ F]D-DFMT showed good uptake into the NCI-H460 tumor, which remained stable for 1 h before wash-out occurred. Pancreas was the only other organ showing [ ¹⁸ F]D-DFMT uptake. The elimination from the body was very rapid.

Table 2:

Example 3: Biodistribution of [ ¹⁸ F]D-DF T in NCI-H292-tumor bearing mice

Biodistribution and excretion studies were performed in female NMR! (nu/nu) mice bearing NCI-H292 iung tumors using [ ¹⁸ F]D-DF T as described in example 2. The results of the biodistribution and excretion are reported as percentage of injected dose per gram of tissue (%ID/g) and tumor-to-organ-ratios (T/T-ratio) were calculated (Table 3). [ ¹⁸ F]D-DF T showed very high uptake into the NCI-H292 tumor, which remained stable for 1 h before wash-out occurred. Pancreas was the only other organ showing [ ¹⁸ F]D-DF T uptake. The elimination from the body was very rapid.

Table 3: iimepolnt : 0,25 h 0,5 h 1,0 h 2,0 h 4,0 h

%Dose/a S.D. S.D. S.D. S.D. S.D.

?.p leen 2.S7 0.59 2.71 1 .53 1.72 0.74 0.82 0.14 0.54 0.17 liver 2.52 0.40 2.15 1 .02 1.16 0.25 0.57 0.08 0.58 0.24 kidney 5.95 1 .18 5.00 2.12 1.S9 0.21 0.82 0.10 0.75 0.29 lung 2.79 0.44 2.38 0.57 1.27 0.20 Θ.58 0.13 0.80 0.31 bone 1.73 0.15 1.85 0.54 1.23 0.25 1.11 0.14 1.75 0.43 heart 3.02 0.24 2.88 0.79 1.56 0.34 0.64 0.12 0.82 0 27 brain 0.88 0.18 1.22 0.476 1.01 0.41 0.81 0.08 0.49 0.21 fat 0.S5 0.31 0.05 S.88 2.62 0.31 0.13 0.14 0.02 thyroid 2.74 0.15 1.86 0.76 1.61 0.42 0.70 0.19 0.65 0.13 gallbladder 0.47 0.08 0.67 0.06 4.83 0.70 1.38 0.48 1.26 0.13 muscle 1.8S 0.10 2.52 0.439 1.50 0.23 0.78 0.10 0.55 0.23 tumor 14.77 1.72 15.16 4.59 12.34 4.61 4.31 1.05 4.14 1.57 skin 2.98 0.20 2.60 1 .29 1.65 0.40 0.71 0.06 0.8S 0.30 blood 2.S1 0 30 2.3S 1 01 1.32 0 26 0.60 0.08 0.62 0.27 tail 4.48 0.16 4,13 2.34 2.15 0.15 2.20 2.14 1 94 stomach 3.00 0.73 2.56 1 .68 1.39 0.19 0.78 0.11 0.S1 0.22 uterus 3.44 0.51 4.64 2.11 1.57 0.07 0.S1 0.18 0.68 0.14 ovaries 4.61 0.12 3.49 1 .04 1.51 0.09 1.03 0.22 0.86 0 38 intestine 2.04 0.46 1.85 0.86 0.S1 0.27 0.55 0 09 0.60 0.25 pankreas 22.55 3 43 16.21 5 77 8.06 3.22 5.34 1.48 5.25 2.62 ad renals 3.7S 0.85 4.15 3 39 1.55 0.49 0.90 0.15 0.61 0.20 summarv S.D. S.D. S.D. S.D. S.D. urine 26.31 9 16 34. S7 13 44 54.18 7 07 73.28 3.06 69.50 0.72 aeces - 0.01 0.01 1.77 2.77 1.58 1.05 1.13 1 .57

T/T-ratio S.D. S.D. S.D. S.D. S.D. sp ieeri 5.20 0.46 8.22 2.15 7.44 1 .74 6.93 0.56 7.6 1 .09 liver 5.91 0.60 7.39 1 .23 10.38 1 58 7.53 0.98 7.59 2.10 kidney 2.55 0.55 3.17 0.76 S.OS 1 .60 5.23 0.69 5.75 1 .50 lung 5.33 0.48 6.38 1 .03 9.47 1 .94 7.48 1.01 7.92 3 06 bone 3.55 1 .1 1 8.19 0.15 9.78 1 .55 3.93 1.13 2.33 0.39 brain 17.38 4 34 12.74 ! OS 12.35 0 43 7.01 1 .06 8.59 0.56 muscle 7.82 0.49 8.08 1 .79 S.14 2.18 5.46 0.66 7.85 1 .78 blood 5.2S 0.25 6.5S 1 .08 9.14 1 .56 7.17 1.37 7.08 2.14 intestine 7.41 1 38 8.82 1 34 13.37 1 53 7.98 1.98 7.23 2.11

Example 4: Biodistribution of [ ^1S F]D-DFMT in A549-tumor bearing mice

Biodistribution and excretion studies were performed in female N R! (nu/nu) mice bearing A549 lung tumors using [ ¹⁸ F]D-DF T as described in example 2. The results of the biodistribution and excretion are reported as percentage of injected dose per gram of tissue (%ID/g) and tumor-to-organ-ratios (T/T-ratio) were calculated (Table 3). [ ¹⁸ F]D-DFMT showed very high uptake into the A549 tumor, which remained stable for 1 h before wash-out occurred. Pancreas was the only other organ showing [ ¹⁸ F]D-DF T uptake. The elimination from the body was very rapid.

Table 4: timeposni : 0,25 h 0,5 h 1,0 h 2,0 h 4,0 h

%Dose/e? S.D. S.D. S.D. S.D. S.D. sp ieen 1.67 0.25 1.42 0.32 0.61 0.07 0.38 0.14 0.20 0.03 iiver 1.51 0.23 1.21 0.24 0.64 0.15 0.40 0.07 0.20 0.04 kidney 3.48 G.88 2.25 0.37 1.S3 1 .10 0.52 ο.ϊ ϊ ¹ 0.29 0.04 iung 1.80 0.21 1.31 0.18 0.73 0 17 0.40 0.10 0.24 0.05 bone 0.94 0.17 1.11 0.17 0.94 0.04 0.84 0.17 1.09 0 02 heart 2.03 0.16 1.59 0.20 0.85 0.19 0.41 0.12 0.28 0.0 : brain 0.40 G.03 0.63 0.1 0.55 0.18 0.33 0 09 0.17 0.03 fat 0.23 0.04 0.20 0.09 0.13 0.04 0.07 0.01 0.20 0.19 thyroid 1.25 0.17 1,43 0.17 0.75 0.21 0.48 0.05 0.31 0 09

^■ gallbladder 1.83 0 40 1.25 0 26 2.18 0.22 1.42 u,7 1.39 0.52 museie 1.00 0.07 1.71 0.10 0.80 0.16 0.40 0.06 0.20 0.04 tumor 3.05 0.34 3,59 0.57 2,01 0.73 0.99 0.33 0.58 0.14 skin 1.64 0 27 1.47 0 22 0.85 0.24 0.52 0.1 1 0.2S 0.05

Mood 1.71 0.21 1.26 0.25 0.73 0.21 0.41 0.10 0.25 0.04 fail 3.32 1 .12 2.53 0.23 3.05 0.65 1.37 0.53 1.99 0.67 stomach 1.80 0 23 1.70 C ,« 2 1.09 0.66 0.50 0.1 1 0.41 0.19 uterus 2.33 0.31 2.77 0.45 1.05 0.06 0.57 0.24 0.42 0.16 ovaries 2.63 0.41 2,18 0.26 0.85 0.17 0.6S 0.13 0.45 0 41 intestine 1.12 0 04 1.29 0 46 0.5S 0 17 0.3S 0.12 0.44 0.32 p nkreas 1 1.70 0.91 12.77 1 .14 4.69 0.23 3.53 0.98 2.85 0.16 ad renals 1.20 0.18 1.16 0.04 0.58 0.13 0,37 0.13 0.30 0 06 summarv S.D. S.D. S.D. S.D. S.D. urine 46.47 5.65 45.39 3.51 64.94 21 .09 69.70 10.01 78.71 1 52 aeces . 0.0G 0.00 0.02 0.01 4.87 7.49 2.39 1 .95

TiT-ratio S.D. S.D. S.D. S.D. S.D. spleen 1.33 0.08 2.55 0.17 3.27 0.80 2.60 0.10 2.93 0 82 iiver 2.04 0.26 2,99 0.23 3.10 0.40 2.46 0.38 2.88 0 15 kidney 0.90 0 15 ISO 0 04 1.27 0 67 1.88 0.25 2.02 0.54

!ung 1.70 0.18 2.75 0.26 2.71 0.39 2.47 0.27 2.43 0.54 bone 3.2S 0.24 3.23 0.14 2.13 0.74 1.17 0.22 0.53 0.12 brain 7.78 1 36 5.75 0 57 3.80 0.22 2.98 0.20 3.3S 0.36 muscle 3.0S 0 31 3.15 0 25 2.55 0 90 2.41 0.42 2.92 0.60 biood 1.78 0.04 2.87 0.24 2.73 0.22 2.40 0.31 2.35 0.48 intestine 2.71 0.28 3.01 1 .00 3.39 0.25 2.75 0.32 1.63 0.69

Example 5: Direct comparison of [ ^1S F]D-DF!VIT with [ ¹⁸ F]D-F!VIT i n a biodistribution experiment using NCI-H292-tumor bearing mice

Biodistribution and excretion studies were performed in female NMR! (nu/nu) mice bearing NCI-H292 lung tumors using [ ¹⁸ F]D-DFMT or [ ¹⁸ F]D-FMT as described in example 2. The time points used were 15, 30, 80, 120 and 240 min ([ ¹⁸ F]D-F T 180min instead of 240 min). The results of the biodistribution and excretion are reported as percentage of injected dose per gram of tissue (%ID/g) (Table 5) and tumor-to-organ-ratios (T/T-ratio) were calculated (Table 6). [ ¹⁸ F]D-DF T showed twice the uptake into the NCI-H292 tumor than [ ¹⁸ F]D-F T, while the other organs showed only a slight increase with [ ¹⁸ F]D-DF T vs. [ ¹⁸ F]D-FMT. This leads to higher and better tumor-to-organ ratios especially at earlier time points.

Table 5:

Table 8:

Timepoint : 0,25 h 0,5 h 1 ,0 h 2,0 h 3,0/4,0 h

T/T-raiio 3D. S.D. S.D. S.D. S.D. spleen D-D-FMT 5.20 0.46 6.22 2.15 7.44 1.74 6.93 0.56 7.60 1.09 spleen D-FMT 3.95 1.06 6.94 1.28 6.20 0.33 S.27 1 .1 1 5.76 0.79 liver D-D-FMT 5.91 0.60 7.39 1.23 10.38 1.58 7.53 0.98 7.59 2.10 liver D-FMT 3.76 1.01 6.74 0.41 6.77 1.47 6.74 0.55 6.31 0.49 kidney D-D-FMT 2.5S 0.55 3.17 0.76 6.09 1.60 5.23 0.69 5.75 1.50 kidney D-FMT 1.74 0.38 3.18 0.34 3.85 0.68 4.12 0.69 5.04 1.26 lung D-D-FMT 5.33 0.48 6.36 .03 9.47 1.94 7.46 1.01 7.92 3.06 lung D-FMT 2.88 0.54 6.39 0.55 6.16 0.76 6.24 1 .30 7.38 0.75 heart D-D-FMT 4.89 0.34 5.26 0.77 7.79 1.31 6.69 0.83 7.12 2.10 heart D-FMT 3.07 0.93 5.19 0.49 5.13 0.64 5.69 1 .01 6.50 0.34 brain D-D-FMT 17.38 4.34 12.74 1.08 12.35 0.43 7.01 1 .06 8.59 0.56 brain D-FMT 11.93 2.87 11.85 1.96 7.13 1.05 5.81 0.93 7.71 0.85 muscle D-D-FMT 7.82 0.49 6.06 1.79 8.14 2.18 5.46 0.66 7.85 1.78 muscle D-FMT 6.40 2.30 6.84 0.56 5.21 0.87 4.82 0.95 5.55 1.09 blood D-D-FMT 5.26 0.25 6.58 1.08 9.14 1.56 7.17 1 .37 7.08 2.14 blood D-FMT 3.80 1.03 6.33 0.30 6.19 0.74 5.93 1 .06 6.81 0.54

Example 6: Direct comparison of [ ¹⁸ F]D-DFMT with [ ¹⁸ F]D-FMT i n a biodistribution experiment using !MC!-H460-tumor bearing mice

Biodistribution and excretion studies were performed in femaie N R! (nu/nu) mice bearing NCI-H460 lung tumors using [ ¹⁸ F]D-DF T or [ ¹⁸ F]D-FMT as described in example 2. The time points used were 5, 30, 80, 120 and 240 min ([ ⁸ F]D-F T 180 min instead of 240 min). The results of the biodistribution and excretion are reported as percentage of injected dose per gram of tissue (% ! D/g) (Table 7) and tumor-to-organ-ratios (T/T-ratio) were calculated (Table 8). [ ¹⁸ F]D-DF T showed almost twice the uptake into the NCI-H460 tumor than [ ¹⁸ F]D-FMT, while the other organs showed only a slight increase with [ ¹⁸ F]D-DF T vs.

[ ¹⁸ F]D-FMT. This leads to higher and better tumor-to-organ ratios especially at earlier time points.

Table 7:

Timepoint : 0,J!S h t 2,0 h ^Q h i S.D. S.D. S.D. i S.D. S.D. spleen D-D-FMT 2.73 0.3-4 1.66 0.43 1.19 0.28 0.81 0.16 0.39 0.07 spleen D-FftTT 1.81 0.21 1.36 0.05 8.69 0.10 0.43 0.03 0.30 0.02 liver D-D-FSVST 229 0.53 1.56 0.26 1.00 0.21 0.64 0.12 0.31 0.05 liver D-FMT 1.92 0.05 0.96 0. 10 0.65 0.04 0.41 0.01 0.3Q 0.02 kidney D-D-FMT 5.35 0.71 2.66 0.22 1.75 0.32 1.34 0.76 0.44 0.05 kidney D-F!WT 5.78 2.71 2.18 0.40 1.03 0.16 0.S7 0.05 0.49 0.13 kmg D-D-FMi 2.89 0.52 1.81 0.55 1.10 0.1 1.04 0.56 0.98 1.18 lung D-FFviT 2.15 0.18 122 0.02 0.69 0.07 0.41 0.02 0.23 0.02 heart D-D-FIV!T 3.02 0.29 2.05 0,39 1.36 0.20 0.80 0.09 0.36 0.01 heart D-FW 2.32 0.1 4 1.75 0.13 8.94 0 ? 1 0.46 0.02 0.29 0.02

"tumor D-D-Ffy]T — IE— "W -HJ75~ ^{" "} TEST ^™ turner D-FfvST ΣΪ2 ^""" 0¾ ^" 6 ^{"" """} 0 ^" 2 ^" Γ ΣΪ4 ^""" 0 ^" 34 ^{""" ""'} ΤΪΒ ^{' '" """"} 0 ^" 35 ^""" __ ^""" 0 ^" 5 ^""'

Wood D-D-IF T 56 ^" IF ^" Ϊ Ϊ5

blood D-FMT 1.95 0.1 9 128 0.08 0.77 0.20 0.44 0.01 0.29 0.02 pankreas D-D-FMT 20.92 1.96 1 .30 1.74 8.35 3.40 6.24 2.26 3.10 0.16 pankreas D-FMT 13.23 0.38 8.45 0.78 4.83 Ί tp, 3.63 0.82 268 0.66

Nummary b. U. s.u. S.U. b.U. b.U. urine D-D-Fii/Π ^" 31 .55 7.79 38.35 18.04 51 .79 25.17 71.19 5.76 80.36 1.97 urine D-FKFF 38.71 5.96 53.72 12.06 64.01 6.61 79.40 2.22 84.49 5.13 faeces D-D-FMT - 0.01 0.00 0.32 0.55 0.18 0.29 ^r 0.51 0.45 faeces D-FMT - 0.05 0.08 0.01 0.01 0.19 0.28 0.22 0.17

Table 8:

Timepoint : 0,25 h 0,5 h 1 ,0 h 2,0 h 3,0/4,0 h

TIT-ra io i S.D. : S.D. S.D. i S.D. i S.D. spleen D-D-FMT 159 0.19 2.22 0.44 3.23 0.98 2.46 0.90 227 0.47 spleen D-FiVfi 1.21 0.63 174 0.10 3.12 0.49 3.05 1.03 3.15 0.26 liver D-D-FMT 1.94 0.42 2.31 0.32 3.79 0.82 3.02 0.57 282 0.81 liver D-FMT 1.11 0.52 2.47 0.20 3.26 0.34 3.12 0.83 3.18 0.66 kidney D-D-FMT 0.81 0.1 1 0.93 0.52 2.14 0.43 1.58 0.39 1.99 0.53 kidney D-FftlT 0.41 0.28 1.12 0.29 2.09 0.28 1.93 0.65 204 0.74 king D-D-FMT 1.52 0.25 2.03 0.30 3.37 0.50 2.02 0.41 203 1.56 iung D-FIVFF 1.01 0.51 1.95 0.20 3.12 0.48 3.08 0.79 4.05 0.78 heart D-D-FiVIT 1.43 0.15 1.75 0.15 2.74 0.47 2.42 0.63 242 0.39 heart D-Fi T 0.93 0.45 1.38 0.1 1 2.32 0.42 2.77 0.75 3.29 0.67 brain D-D-FMT 5.56 0.86 4.35 1.16 4.43 0.7S 3.31 1.02 3.37 0.40 brain D FMT 4.02 1.39 2.70 0.37 3.57 0.68 3.76 1.77 3.93 0.55 muscle D-D-FMT 2.12 0.47 2.14 0.50 2.27 0.40 2.33 0.84 230 0.40 miiscie D-FMT 1.49 0.68 1.68 0.35 2.28 0.2S 2.11 0.59 263 0.43 blood D-D-FMT 1.70 0.23 2.22 0.26 3.24 0.53 2.56 0.69 254 0.64 blood D-FMT 112 0.57 1.85 0.15 2.85 0.56 2.90 0.82 3.25 0.61

Example 7: PET/CT-imaging of [ ¹⁸ F]D-DFiVST in NCf-H292-tumor bearing mice

[ ¹⁸ F]D-DF T was imaged on a microPET/ CT (inveon, Siemens) in NCi-H292 tumor-bearing mice 55-65min after injection of 6.39 MBq [ ¹⁸ F]D-DFMT i.v.. High tumor-contrast was visible in NCI-H480 xenografts. Due to rapid renal clearance of this iigand very low background activity was observed except for pancreas uptake and urine in the bladder (Figure 3).

Example 8: PET/CT-imaging of [ ^1S F]D-DFMT in A549-tumor bearing mice

[ ¹⁸ F]D-DF T was imaged on a microPET/ CT (Inveon, Siemens) in H460 tumor-bearing mice 55-65min after injection of 8.43 MBq [ ¹⁸ F]D-DFMT i.v.. High tumor-contrast was visible in H460 xenografts. Due to rapid renal clearance of this Iigand very low background activity was observed except for pancreas uptake and urine in the bladder (Figure 4).