{F-19}-LABELED L-GLUTAMIC ACID AND L-GLUTAMINE DERIVATIVE (III) , USE THEREOF

AND METHOD FOR OBTAINING THEM

DESCRIPTION

FIELD OF INVENTION

The present invention relates to fluorinated glutamic acid (glutamate) and glutamine derivatives wherein the fluorine atom is 19F. The glutamic acid (glutamate) and glutamine derivatives are compound(s) of general Formula I, which encompasses all possible diastereoisomers and/or enantiomere derivatives or mixtures thereof. The compounds of the present invention are useful for therapy of diseases related to glutamine catabolism and the present invention further relates also to improved imaging agents useful for Fluorine-19 Magnetic Resonance Imaging ( ¹⁹ F MRI) and as reference compounds for the identification of the respective [F-18] derivatives.

BACKGROUND ART

Glutamate is a key molecule in cellular metabolism. In humans, dietary proteins are broken down by digestion into amino acids, which serves as metabolic fuel for other functional roles in the body. A key process in amino acid degradation is transamination, in which the amino group of an amino acid is transferred to an α-ketoacid, typically catalyzed by a transaminase. Glutamine has a variety of biochemical functions including: A substrate for DNA synthesis,

Major role in protein synthesis,

Primary source of fuel for enterocytes (cells lining the inside of the small intestine), Precursor for rapidly dividing immune cells, thus aiding in immune function, Regulation of acid-base balance in the kidney by producing ammonium. See : http://en.wikipedia.org/wiki/Glutamine and Daniel Larraya et al. Nutrients

Department, November-December 2002 ..Glutamine: This amino acid promises much - for mind, muscle and immunity. Can taking glutamine really help you? "

Medina et al. (Molecular and Cellular Biochemistry 113:1-15,1992) refer to the glutamine analog, L-glutamic acid gamma-mono-hydroxamate that has demonstrated high toxicity against tumor cells in culture and "in vivo" against leukemia and B16 melanoma. Medina et

al. suggest that glutaminase can be used for therapeutic use or that selective inhibition of glutamine transport by tumor cells maybe used for reduce tumor proliferation.

There is a clear need for alternative glutamine analogues that are involved in the treatment of proliferative diseases such as tumour and cancer.

It has been surprisingly found that the compounds of the invention are useful for MRI imaging as well as for therapeutic applications.

SUMMARY The present invention relates to fluorinated glutamic acid (glutamate) and glutamine derivatives wherein a 19F is incorporated. The compounds of the present invention are useful for MRI imaging and for treating proliferative diseases. Composition comprising compounds of the present invention are also disclosed. The invention relates also to kit comprising new fluorinated glutamic acid (glutamate) and glutamine derivatives.

DETAILLED INVENTION

In a first aspect, the present invention is directed to Compound(s) of general Formula I

wherein A is a) Hydroxyl, b) branched or unbranched C ₁ -C ₅ alkoxy, c) branched or unbranched Hydroxy C ₁ -C ₅ Alkoxy, d) branched or unbranched 0-C ₁ -C ₅ AIk^-(O-C ₁ -C ₄ 8^yI) _n -O-C ₁ -C ₄ alkyl, e) N(C ₁ -C ₅ AKyI) ₂ , f) NH ₂ , g) N(H)-L, h) O-L or i) O-Z,

G is

a) Hydroxyl, b) O-Z b) branched or unbranched 0-C ₁ -C ₅ Alkyl, c) branched or unbranched 0-C ₂ -C ₅ Alkenyl, d) branched or unbranched 0-Ci-C ₅ AlKyI-(O-C ₁ -C ₄ alkyl J _n -O-C ₁ -C ₄ alkyl or e) branched or unbranched 0-C ₂ -C ₅ Alkinyl,

and R ¹ and/or R ² , independently separate are a) Hydrogen, b) branched or unbranched ¹⁹ F-C ₁ -C ₁₀ Alkoxy, c) branched or unbranched ¹⁹ F-C ₁ -C ₁₀ Alkyl, d) branched or unbranched ¹⁹ F-C ₂ -C ₁₀ Alkenyl, e) branched or unbranched ¹⁹ F-C ₂ -C ₁₀ Alkinyl, f) substituted or unsubstituted ¹⁹ F-C ₆ -C ₁ O mono- or bicyclic Aryl, g) substituted or unsubstituted ¹⁹ F-alkyl-C ₆ -C ₁₀ mono- or bicyclic Aryl h) substituted or unsubstituted ¹⁹ F-C ₅ -Ci _O mono- or bicyclic Heteroaryl, i) substituted or unsubstituted ¹⁹ F-alkyl-C ₅ -d ₀ mono- or bicyclic Heteroaryl j) substituted or unsubstituted ¹⁹ F-C ₃ -C ₆ Cyclo-Alkyl, k) substituted or unsubstituted ¹⁹ F-aikyl-C ₃ -C ₆ Cyclo-Alkyl I) Hydroxyl, m) branched or unbranched C ₁ -C ₅ Alkyl or n) branched or unbranched C ₁ -C ₅ Alkoxy wherein alkyl is optionally interrupted or is replaced by O, S or N, with the proviso that one of the substituent R ¹ or R ² comprises ¹⁹ F atom and the other substituent comprises no ¹⁹ F atom,

L is a) branched or unbranched C ₁ -C ₅ Alkyl, b) branched or unbranched C ₂ -C ₅ Alkenyl, c) branched or unbranched C ₁ -C ₅ Alkyt— ( 0-Ci-C ₄ alkyl) _n -O-Ci-C ₄ alkyl or d) branched or unbranched C ₂ -C ₅ Alkinyl,

Z is a metal ion equivalent ,

n = 0, 1 , 2 or 3 and pharmaceutical salt, diastereomere and enantiomere thereof.

It's well known for a person skilled in the art, that the compounds of formula (I) of the invention are or may be in the form of zwitterions and/or salt at the physiological pH of 7.4.

In a preferred embodiment of compounds of Formula I, A is Hydroxyl, branched or unbranched C ₁ -C ₅ alkoxy or NH ₂ .

In a more preferred embodiment, A is ethoxy.

In a preferred embodiment of compounds of Formula I, G is Hydroxyl or branched or unbranched C ₁ -C ₅ alkoxy. In a more preferred embodiment, G is methoxy.

In a preferred embodiment of compounds of Formula I, R ¹ and/or R ² , independently from each other is a) Hydrogen, b) branched or unbranched ¹⁹ F-C ₂ -C ₁₀ Alkoxy, c) branched or unbranched ¹⁹ F-C ₁ -C ₁₀ Alkyl, d) branched or unbranched ¹⁹ F-C ₃ -C ₁₀ Alkenyl, e) branched or unbranched ¹⁹ F-C ₃ -C ₁₀ Alkinyl, f) substituted or unsubstituted ¹⁹ F-C ₆ -C ₁₀ mono- or bicyclic Aryl, g) substituted or unsubstituted ¹⁹ F-C ₅ -C ₁₀ mono- or bicyclic Heteroaryl, h) substituted or unsubstituted ¹⁹ F-C ₃ -C ₆ Cyclo-Alkyl, i) Hydroxyl, j) branched or unbranched C ₁ -C ₅ Alkyl or k) branched or unbranched C ₁ -C ₅ Alkoxy with the proviso that one of the substituent R ¹ or R ² comprises ¹⁹ F atom and the other substituent comprises no ¹⁹ F atom,

In a preferred embodiment of compounds of Formula I, R ¹ or R ² is a) branched or unbranched ¹⁹ F-C ₂ -C ₁₀ Alkoxy, a) branched or unbranched ¹⁹ F-C ₁ -C ₁₀ Alkyl, b) branched or unbranched ¹⁹ F-C ₃ -C ₁₀ Alkenyl, c) branched or unbranched ¹⁹ F-C ₃ -C ₁₀ Alkinyl.

In a preferred embodiment of compounds of Formula I, R ¹ or R ² is a) branched or unbranched ¹⁹ F-C ₂ -C ₅ Alkoxy, more preferably ¹⁹ F-C ₃ Alkoxy b) branched or unbranched ¹⁹ F-C ₁ -C ₅ Alkyl, more preferably ¹⁹ F-C ₃ Alkyl,

c) branched or unbranched ¹⁹ F-C ₃ -C ₅ Alkenyl, more preferably ¹⁹ F-C ₃ Alkenyl, d) branched or unbranched ¹⁹ F-C ₃ -C ₅ Alkinyl. more preferably ¹⁹ F-C ₃ Alkinyl.

In a more preferred embodiment of compounds of Formula I ₁ R ¹ or R ² Is a) branched or unbranched ¹⁹ F-C ₆ -C ₁₀ Alkoxy, more preferably ¹⁹ F-C ₆ Alkoxy, b) branched or unbranched ¹⁹ F-C ₆ -C ₁₀ Alkyl, more preferably ¹⁹ F-C ₆ Alkyl, c) branched or unbranched ¹⁹ F-C ₆ -C ₁₀ Alkenyl, more preferably ¹⁹ F-C ₆ Alkenyl, d) branched or unbranched ¹⁹ F-C ₆ -C ₁₀ Alkinyl, more preferably ¹⁹ F-C ₆ Alkinyl.

In a further preferred embodiment of compounds of Formula I ₁ R ¹ or R ² Is a) substituted or unsubstituted ¹⁹ F-C ₆ -C ₁₂ mono- or bicyclic Aryl, b) substituted or unsubstituted ¹⁹ F-C ₅ -C ₁₂ mono- or bicyclic Heteroaryl, c) substituted or unsubstituted ¹⁹ F-C ₃ -C ₆ Cyclo-Alkyl.

In a further preferred embodiment of compounds R ¹ is ¹⁹ F and R ² is hydrogen.

In a preferred embodiment, Z is selected from Mg ²⁺ , Ca ²⁺ , Na ⁺ und K ⁺ . In a more preferred embodiment Z is Na ⁺ .

In a preferred embodiment, n = O ₁ 1 or 2. In a more preferred embodiment, n = 0, 1.

The compounds of the present invention are derivatives of D- or L- glutamic acid / glutamine (position C ₂ ) and have in position C ₄ R- or S-configuration. In a preferred embodiment the compounds of Formula I are derivatives of L-glutamic acid / glutamine and have in position C ₄ S-configuration..

Of this group, a preferred subgroup of compounds is selected from the following:

a)

f)

g) k)

In a preferred embodiment of compounds of Formula I, the following compounds are disclaimed

4-[F- 19]fluoro-glutamate,

4-[F-19]fluoroethoxy-glutamate,

4-[F-19]fluoropropyl-glutamate and 4-[F-19]fluorobutyl-glutamate.

In a second aspect, the present invention is directed to one compound or more compounds of general Formula I and a pharmaceutical acceptable carrier.

In a third aspect, the present invention is directed to a method for obtaining compounds of formula I by reacting a non-fluorinated compound of formula I with fluoride or a fluorine containing moiety.

In a fourth aspect, the present invention is directed to a compound of formula I for use as a medicament.

In a preferred embodiment, the present invention relates to the use of compound of formula I for the manufacturing of a medicament for use as an inhibitor of proliferative diseases. Preferably, proliferative diseases are characterized by metastasis or tumor.

More preferably proliferative disease is a disease developing malignant tumour selected from malignant lymphoma, pharyngeal cancer, lung cancer, liver cancer, bladder tumour, rectal cancer, prostatic cancer, uterine cancer, ovarian cancer, breast cancer, brain tumour, and malignant melanoma.

Further the compound of formula I is to be administered orally, parenterally, rectally, or locally.

In a more preferred embodiment the medicament is for treating, preventing or alleviating proliferative diseases growth.

In a preferred embodiment, the present invention relates a method for treating proliferative diseases comprising administering to an individual in need thereof a therapeutically effective amount of compound of formula I as defined above.

In a fifth aspect, the present invention is directed to a compound of formula I for use as imaging agent.

In a preferred embodiment, the present invention relates to the use of compound of formula I for the manufacturing of an imaging agent for imaging proliferative diseases. Preferably, proliferative diseases are characterized by metastasis or tumor.

More preferably proliferative disease is a disease developing malignant tumour selected from malignant lymphoma, pharyngeal cancer, lung cancer, liver cancer, bladder tumour, rectal cancer, prostatic cancer, uterine cancer, ovarian cancer, breast cancer, brain tumour, and malignant melanoma.

Further the compound of formula I is to be administered orally, parenterally, rectally, or locally.

In a more preferred embodiment the imaging agent is a Magnetic Resonance Imaging (MRI) agent.

In a preferred embodiment, the present invention relates to a method for imaging proliferative diseases more preferably metastasis and tumor comprising administering to an individual in need thereof a therapeutically effective amount of compound of formula I.

In a sixth aspect, the present invention is directed to the use of the compounds of formula I as defined above for the identification of F-18-PET-Tracer. The compounds of formula I are useful as a competition agent for identifying new F-18-PET-Tracer.

EXAMPLES

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. The following schematic examples relates to the preparation of a compounds according to Formula I. The examples presented below are not to be understood as to limit the invention to the methods exemplified herein.

DEFINITIONS

The term "therapeutically effective amount" as used herein refers to that amount of a compound of the invention which, when administered to an individual in need thereof, is sufficient to effect treatment, as defined below, for metastasis. The amount which constitutes a "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age of the human to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

"Treating" or "treatment" as used herein refers to the treatment proliferative diseases and include:

(i) preventing the disease from recurring in an individual, in particular, when such individual is in need of further medicamentous treatment after a previous surgical or medicamentous therapy; (ii) inhibiting the disease, i.e., arresting its development; or

(iii) relieving the disease, i.e., causing regression of the disease.

The term "alkyl" as used herein refers to C ₁ to Ci ₀ straight or branched alkyl groups, e. g., methyl, ethyl, propyl, isopropyl, n-butyl, (-butyl, n-pentyl, neopentyl, heptyl, or decyl. Alkyl groups can be perfluorated or substituted by one to five substituents selected from the group consisting of halogen, hydroxyl, C ₁ -C ₄ alkoxy, or C ₆ -C ₁₂ aryl (which can be substituted by one to three halogen atoms). More preferably, alkyl is a C ₁ to C ₅ or C ₅ to C ₁₀ alkyl. In case alkyl is interrupted by O, S or N then Alkyl is a straight or branched alkyl group of C ₁ to C ₂₀ .

The term "alkenyl" as used herein refers to a straight or branched chain monovalent or divalent radical, containing at least one double bond and having from two to ten carbon atoms, e.g., ethenyl, prop-2-en-1-yl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.

The term "alkynyl" as used herein refers to a substituted or unsubstituted straight or branched chain monovalent or divalent radical, containing at least one triple bond and having from two to ten carbon atoms, e.g., ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-3-ynyl, and the like.

Alkenyl and alkynyl groups can be substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, alkoxy, -CO ₂ H, -CO ₂ Alkyl, -NH ₂ , -NO ₂ , -N ₃ , -CN, C ₁ - C ₂₀ acyl, or C ₁ -C ₂₀ acyloxy.

The term "aryl" as used herein refers to an aromatic carbocyclic or heterocyclic moiety containing five to 10 ring atoms, e.g., phenyl, naphthyl, furyl, thienyl, pyridyl, pyrazolyl, pyrimidinyl, oxazolyl, pyridazinyl, pyrazinyl, chinolyl, or thiazolyl. Aryl groups can be substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, alkoxy, -CO ₂ H, -CO ₂ Alkyl, -NH ₂ , Alkyl-NH _2l C ₁ -C ₂₀ alkyl-thiolanyl, -NO ₂ , -N ₃ , -CN, C ₁ -C _2O alkyl, C ₁ -C ₂₀ acyl, or C ₁ -C ₂₀ acyloxy. The heteroatoms can be oxidized, if this does not cause a loss of aromatic character, e. g., a pyridine moiety can be oxidized to give a pyridine N-oxide.

Whenever the term "substituted" is used, it is meant to indicate that one or more hydrogens on the atom indicated in the expression using "substituted" is replaced with a selection from the indicated group, provided that the indicated atom's normal valency is not exceeded, and that the substitution results in a chemically stable compound, i. e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a pharmaceutical composition. The substituent groups may be selected from halogen atoms, hydroxyl groups, nitro, (CrC^carbonyl, cyano, nitrile, trifluoromethyl, (C ₁ - C ₆ )sulfonyl, (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ JaIkOXy and (CrQOsulfanyl.

It's well known for a person skilled in the art, that the compounds of the invention, as applicable, are or may be in the form of zwitterions and/or salt at the physiological pH of 7.4.

Typically, an effective amount of an imaging agent formulation comprising the F magnetic resonance imaging agent and a pharmaceutically acceptable carrier is administered to the patient, and the patient, or a portion of the patient, is imaged. The term "effective amount", as used herein, denotes a non-toxic amount sufficient to enhance or alter the MRI image obtained, more particularly, an amount which permits better visualization of the organs and/or tissues being imaged. Preferably the patient is a mammal; most preferably the patient is a human. The ¹⁹ F magnetic resonance imaging agents of the present invention may be variously administered by any suitable route, including, for example, orally, for imaging of the upper gastrointestinal tract; rectally, for imaging of the lower gastrointestinal tract including the colon; nasally, for imaging of the nasal and communicating passages; vaginal, for imaging of the fallopian tubes and communicating passages; parenteral (including subcutaneous, intramuscular, intravenous, intradermal and pulmonary), for imaging of internal organs, tissues, tumours, and the like. It will be appreciated that the preferred route will vary with the

organs or tissues to be imaged. Preferred routes of administration include parenteral and oral, more preferably intravenous.

While it is possible for the imaging agent to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one imaging agent compound, together with one or more pharmaceutically acceptable carriers, such as diluents or excipients which may include, for example, fillers, extenders, wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature and mode of administration and the dosage forms. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. The pharmaceutical formulation may optionally include other diagnostic or therapeutic agents. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA. (latest edition).

Formulations of the present invention suitable for oral administration may be presented as an aqueous solution. Alternatively, formulations can be administered as capsules, cachets or tablets, each containing a predetermined amount of the imaging agent; powder; granules; or paste.

Formulations suitable for parenteral administration include aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to one or more tissues or organs. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules or tablets. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavouring agents.

The F magnetic resonance imaging agents of the present invention may also be presented for use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art. For effective F MRI, dosages of the ¹⁹ F magnetic resonance imaging agent will depend on the spin density, flow (diffusion and perfusion), susceptibility, and relaxivity (Tl and 12) of the imaging agent formulation. Dosages of ' F containing imaging agents may be conveniently calculated in milligrams of ¹⁹ F per kilogram of patient (abbreviated as mg F/kg). For example,

for parenteral administration, typical dosages may be from about 50 to about 1000 mg F/kg, more preferably from about 100 to about 500 mg F/kg. For methods of continuous administrations (e.g., intravenous), suitable rates of administration are known in the art. Typical rates of administration are about 0.5 to 5 mL of formulation per second, more preferably about 1-3 ml_/s. Imaging may begin before or after commencing administration, continue during administration, and may continue after administration. It will be appreciated that dosages, dosage volumes, formulation concentrations, rates of administration, and imaging protocols will be individualized to the particular patient and the examination sought, and may be determined by an experienced practitioner. Guidelines for selecting such parameters are known in the art (see, inter alia, Katzberg, 1992, supra).

The usefulness and efficiency of chemical compounds as contrast agents depends on their ability to exhibit a predictable and desirable biodistribution and metabolism in vivo. Their behaviour in vivo depends on parameters such as molecular weight, charge, osmolality, hydrophobicity, partition coefficient, susceptibility to metabolic breakdown, and tissue or organ targeting efficiency. In order to improve their solubility and/or biodistribution, many contrast agents are used in conjunction with delivery systems such as emulsions, liposomes, and microparticles.

Examples: Example 1

(2S,4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid a) (2S,4S)-4-allyl-2-tert-butoxycarbonylamino-pentane dioic acid dimethyl ester

11.01 g (40 mmol) of Boc-Glutamic acid dimethylester (Advanced Chemtech) were dissolved in 160 mL tetrahydrofurane (THF) and cooled to -70 ⁰ C. 88 ml_ (88 mmol) of a 1 M solution of Lithium-bis(trimethylsilyl)amide in THF was added drop wise over a time-period of 1 hr at -70 0C and was further stirred for 2 hr at -70 ⁰ C. Subsequently, 14.52 g (120 mmol) of allylbromide were added drop wise over 2 hr, then the cooling bath was removed and 200 mL of 2 N hydrochloric acid and 400 mL of ethylacetate were added. The organic phase was separated, washed neutral with water, dried over sodium sulphate, filtered and reduced in volume by evaporation. The crude material was chromatographed with hexane/ ethylacetate on silica gel. The obtained product fractions were combined and the solvents were evaporated to dryness. Yield: 3.3 g (26 %)

Elemental analysis: calc: C 57.13 H 7.99 N 4.44 found: C 56.97 H 8.12 N 4.30

b) (2S,4S)-2-tert-Butoxycarbonylamino-4-(3-hydroxypropyl)-penta n dioic acid dimethyl ester

3.15 g (10 mmol) of product 1a was dissolved in 50 mL THF and was cooled in an ice bath. 13.3 mL of 1 M Diboran/THF-complex in THF was added drop wise over a time period of. 20

min under a nitrogen flow and ice cooling. The mixture was stirred for 1 hr on ice, and over night at room temperature. Subsequently, 15 mL of 1 N sodium hydroxide followed by 13.3 ml_ of 30% aqueous hydrogen peroxide solution were added drop wise. The reaction mixture was diluted with water after 30 min, the THF was distilled off and the aqueous remainder was extracted with ethyl acetate. The organic phase was separated, washed neutral with water, dried over sodium sulphate, filtered and reduced in volume by evaporation on an evaporator. The crude product was purified by column chromatography using a gradient of hexane/ ethylacetate on silica gel. The product fractions were combined and the solvents were evaporated to dryness. Yield: 0.6 g (18 %)

Elemental analysis: calc: C 54.04 H 8.16 N 4.20 found: C 53.88 H 8.25 N 4.39

c) (2S,4S)-2-tert-Butoxycarbonylamino-4-(3-fluoropropyl)-pentan e dioic acid dimethyl ester

0.33 g (1 mmol) of hydroxyl compound 1b was dissolved in 15 mL of dichloro methane and cooled in an ice bath. The reaction mixture was stirred for 1 hr on the ice bath after addition of 0.32 g (2 mmol) Diethylaminosulphurtrifluoride (DAST). Then the mixture was washed with water and the organic phase was dried over sodium sulphate, filtered and reduced in volume by evaporation on an evaporator. The raw material was chromatographed in hexane/ ethyl acetate on silica gel. The product fractions were combined and the solvents were removed to dryness by evaporation. Yield: 25 mg (8 %)

Elemental analysis: calc: C 53.72 H 7.81 F 5.66 N 4.18 found: C 53.55 H 7.94 F 5.21 N 4.37

d) (2S,4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid

23.5 mg (0,07 mmol) of fluorinated compound 1d was dissolved in 2 ml_ THF ₁ supplemented with 1 mL of 1 N sodium hydroxide and stirred for 4 hr at room temperature. Subsequently, the mixture was reduced in volume to dryness. The remainder was re-dissolved in. 20 mL of 3 N HCI/ diethylether, stirred over night, reduced in volume by evaporation and re-distilled with diethylether repeatedly. The crude material was chromatographed with a water/ methanol-gradient on C18-silica gel. The product fractions were combined and reduced in volume to dryness. Yield: 4 mg (27 %)

Elemental analysis (calculated for water-free compound): calc: C 46.37 H 6.81 F 9.17 N 6.76 found: C 46.11 H 7.02 F 8.87 N 6.93

Example 2

Neutral sodium salt of (2S.4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid

a) (2S.4S)-4-Allyl-2-tert-butoxycarbonylamino-pentane dioic acid di-tert-butyl ester

26.96 g (75 mmol) of Boc-Glutamic acid di-f-butylester (Journal of Peptide Research (2001), 58, 338) was dissolved in 220 mL THF and cooled to -70 ⁰ C. 165 ml. (165 mmol) of a 1 M solution of lithium-bis(trimethylsilyl)amide in THF was added over a period of 2 h at -70 "C and were further stirred for 2 h at -70 ⁰ C. Then 27.22 g (225 mmol) of allyl bromide was added drop wise and after 2 h at -70 ^C C, the cooling bath was removed and 375 mL of 2 N hydrochloric acid and 1.25 L of ethyl acetate were added. The organic phase was separated, washed neutral with water, dried over sodium sulphate, filtered and reduced in volume by evaporation. The resulting crude material was chromatographed with hexane / ethyl acetate on silica gel. The product fractions obtained were combined and the solvents were evaporated to dryness. Yield: 15.9 g (53.1 %)

MS (ESIpos): m/z = 400 [M+H] ⁺

1H NMR (300 MHz, CHLOROFORM-cQ d ppm 1.32-1.58 (m, 27H) 1.81-1.92 (m, 2H) 2.25- 2.39 (m, 2H) 2.40-2.48 (m, 1 H), 4.10-4.18 (m, 1H) 4.85-4.92 (d, 1 H) 5.02-5.11 (m, 2H) 5.68- 5.77 (m, 1H)

b) (2S.4S)-2-tert-Butoxycarbonylamino-4-(3-hydroxypropyl)-penta ne dioic acid di-tert- butyl ester

15.58 g (39 mmol) of the compound described in Example 2a were dissolved in 200 mL of THF and cooled in an ice bath. 54.6 mL (54.6 mmol) of 1 M diborane / THF complex in THF was added drop wise over a period of 20 minutes under a flow of nitrogen with ice cooling. The mixture was stirred for 2 h on ice and overnight at room temperature. 58.5 mL (58.5 mmol) 1 N sodium hydroxide followed by 58.5 mL of 30 % aqueous hydrogen peroxide solution were then added drop wise again at 0 ⁰ C. After one hour at this temperature, the reaction mixture was diluted with water, the THF was distilled off and the aqueous remainder was extracted with ethyl acetate. The organic phase was separated, washed neutral with water, dried over sodium sulphate, filtered and the filtrate was evaporated to dryness. The resulting crude product was chromatographed with hexane / ethyl acetate on silica gel. The product fractions were combined and the solvents were evaporated to dryness. Yield : 8.5 g (52.2 %)

MS (ESIpos): m/z = 418 [M+H] ⁺ 1 H NMR (300 MHz, CHLOROFORM-d) d ppm 1.32-1.58 (m, 27H) 1.60-1.70 (m, 2H) 1.73- 1.94 (m, 4H) 2.05-2.12 (m, 1 H), 2.33-2.40 (m, 1 H) 3.58-3.68 (m, 2H) 4.15-4.22 (m, 1 H) 4.95- 5.03 (d, 1 H)

c) (2S.4S)-2-tert-Butoxycarbonylamino-4-(3-fluoropropyl)-pentan e dioic acid di-t-butyl ester

29.22 g (70 mmol) of the hydroxyl compound described in Example 2b was dissolved in 700 mL of THF, followed by addition of 42.5 g (420 mmol) of triethylamine. After addition of 25.14 mL (140 mmol) of perfluorobutane sulfonyl fluoride (Aldrich) and 22.57 g (140 mmol) of triethylamine / hydrogen fluoride (Aldrich), the reaction mixture was stirred for 65 h at room temperature, reduced in volume by evaporation and the resulting crude product was chromatographed with hexane / ethyl acetate on silica gel. The product fractions were combined and reduced to dryness by evaporation. Yield: 15.9 g (54.1 %)

MS (ESIpos): m/z = 420 [M+H] ⁺

1 H NMR (300 MHz, CHLOROFORM-c/) d ppm 1.40-1.55 (m, 27H) 1.60-1.95 (m, 6H) 2.33- 2.42 (m, 1H) 4.15-4.22 (m, 1H) 4.30-4.40 (m, 1 H) 4.48-4.55 (m, 1H) 4.85-4.90 (d, 1H)

d) Neutral sodium salt of (2S.4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid

15.52 g (37 mmol) of the compound described in Example 2c were cautiously dissolved in 110 mL of trifluoroacetic acid (foams!) and stirred for 3 days at room temperature. The reaction mixture was then evaporated to dryness and the resulting crude product was redistilled three times with diethyl ether and the residue dissolved in about 200 mL of water, adjusted to pH 2 with 20 mL of 1N hydrochloric acid, then washed successively with dichloromethane and ethyl acetate and the aqueous solution was adjusted to pH 7.4 with 1 N sodium hydroxide (about 65 mL). The solution was freeze-dried and then chromatographed with water / methanol on C18-silica gel and the resulting fractions were combined and reduced in volume by evaporation. Yield: 7.5 g (88 %)

MS (ESIpos): m/z = 208 [M+H] ⁺

1H NMR (300 MHz, methanol-c/) d ppm 1.62-1.87 (m, 5H) 2.11 (m, 1 H) 2.47-2.52 (m, 1 H)

3.45 (m, 1 H) 4.41 (m, 2H)

Example 3

Biological data

Biological effects of (2S,4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid, (2S,4S)-2-Amino- 4-[2-fluoro-5-(trifluoromethyl)benzyl]-pentane dioic acid and L-Glutamate were investigated in cytotoxicity assay with A549 cells (human Non Small Cell Lung Cancer) using standard Alamar Blue Assay (Invitrogen # DAL1025). A Dulbecco's modified Eagle's Medium (Sigma D0422), supplemented with Glutamine and 10% foetal calf serum (FCS) were used as incubation buffer. The cells were incubated for 48h with test compounds and investigated according to manufacturer's protocol. Control cells and cells incubated with L-Glutamate at 2 mM showed no differences in cell vitality. However, cell incubation with (2S,4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid

and (2S,4S)-2-Amino-4-[2-fluoro-5-(trifluoromethyl)benzyl]-penta ne dioic acid surprisingly showed both a strong decrease of the cell vitality. This effect was most pronounced with (2S,4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid.

For determination of the dose dependency, A549 cells were incubated with increasing concentrations of (2S,4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid ranging from 4 μM - 2 rtiM. Afterwards, cell vitality was measured with the Alamar Blue assay kit as described above.

It appears that the cell vitality is compromised in a dose dependent manner due to incubation with the test compound (2S,4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid. The EC50 is about 38 μM.

Figure 1 :

Comparison of cytotoxicity of (2S,4S)-2-Amino-4-(3-fluoropropyl)-pentane dioic acid and non- substituted L-Glutamate. A549 cells were incubated with the test compounds and investigated according to the Alamar Blue Assay manufacturer protocol.

Figure 2:

Dose dependency of the cytotoxicity potential of (2S,4S)-2-Amino-4-(3-ftuoropropyl)-pentane dioic acid in A459 cells.

Example 4

2-Amino-4-(6-fluorohexyl)-pentane dioic acid a) (2S.4S)-2-tert-Butoxycarbonylamino-4-(6-iodohexyl)-pentane dioic acid dimethyl ester

5.51 g (20 mmol) of Boc-L-Glutamic acid dimethylester (Advanced Chemtech) were dissolved in 60 mL of tetrahydrofuran (THF) and cooled to -70 ⁰ C. 44 mL (44 mmol) of 1 M solution of lithium-bis(trimethylsilyl)amide in THF was added drop wise over a period of 1 hour at -70 ⁰ C and stirring was continued over 2 h at -70 ⁰ C. Then 20.28 g (60 mmol) of 1,6- diiodohexane were added drop wise and, after 2 h at -70 ⁰ C, the cooling bath was removed and 100 mL of 2 N hydrochloric acid and 300 mL of ethyl acetate were added. The organic

phase was separated, washed neutral with water, dried over sodium sulphate, filtered and the filtrate was reduced in volume by evaporation. The resulting crude product was chromatographed with hexane / ethyl acetate on silica gel. The resulting fractions were combined and reduced in volume by evaporation. Yield: 0.2 g (2.1 %)

MS (ESIpos): m/z = 486 [M+H] ⁺

1 H NMR (300 MHz, CHLOROFORM-c/) d ppm 1.20-1.70 (m, 29H) 1.75-2.10 (m, 5H) 2.40- 2.50 (m, 1 H) 3.14-3.20 (m, 2H), 3.50-3.75 (m, 3H), 4.15-4.25 (2H) 4.32-4.42 (m, 1H) 5.00- 5.10 (d, 1H)

b) (2S.4S)-2-tert-Butoxycarbonylamino-4-(6-fluorohexyl)-pentane dioic acid dimethyl ester

A solution of 152 mg (1.12 mmol) of silver fluoride in 1.5 mL water was added to 0.49 g (1 mmol) of the compound described in Example 3a in 30 mL acetonitrile and stirred overnight at 40 ⁰ C. The resulting suspension was filtered, the solution was evaporated to dryness and the resulting crude product was chromatographed with hexane / ethyl acetate on silica gel. The resulting fractions were combined and reduced in volume by evaporation.. Yield: 132 mg (35 %)

MS (ESIpos): m/z = 378 [M+H] ⁺

1H NMR (300 MHz, CHLOROFORM-d) d ppm 1.20-1.70 (m, 29H) 1.75-2.10 (m, 5H) 2.40- 2.50 (m, 1 H) 3.14-3.20 (m, 2H), 3.50-3.75 (m, 3H), 4.15-4.25 (2H) 4.32-4.42 (m, 1 H) 5.00- 5.10 (d, 1 H)

c) (2S.4S)-2-Amino-4-(6-fluorohexyl)- pentane dioic acid

26.4 mg (0.07 mmol) of the compound described in Example 3b were dissolved in 2 mL of THF, followed by addition of 1 mL of 1 N sodium hydroxide and stirring for 4 h at room temperature. The reaction mixture was then evaporated to dryness and the resulting crude product was dissolved in about 20 ml of 3 N hydrogen chloride in diethyl ether, stirred overnight, evaporated to dryness and redistilled several times with diethyl ether. The resulting crude product was chromatographed with water / methanol on C18-silica gel. The resulting fractions were combined and reduced in volume by evaporation. Yield: 5.8 mg (33 %)

MS (ESIpos): m/z = 250 [M+H] ⁺

Example 5

a) (2S,4S)-2-tert-Butoxycarbonylamino-4-(2-fluoro-5-trifluorome thyl-benzyl)-pentane-dioic acid di-tert-butyl ester

2.7 g (7.5 mmol) of Boc-Glutamic acid di-t-butylester (Journal of Peptide Research (2001), 58, 338) was dissolved in 30 mL THF and cooled to -70 ⁰ C. 16.5 mL (16.5 mmol) of a 1 M solution of lithium-bis(trimethylsilyl)amide in THF was added over a period of 40 min at 70 ⁰ C and were further stirred for 2 h at -70 ⁰ C. Then 1.93 g (7.5 mmol) of 2-fluoro-5-trifluoromethyl benzyl bromide in 7 mL of THF was added dropwise and after 2 h at -70 ⁰ C, the cooling bath was removed and 37.5 mL of 2 N hydrochloric acid and 100 mL of dichloromethane were added. The organic phase was separated, washed neutral with water, dried over sodium sulphate, filtered and reduced in volume by evaporation. The resulting crude material was

chromatographed with hexane / ethyl acetate on silica gel. The product fractions obtained were combined and the solvents were evaporated to dryness. Yield: 2.3 g (57.3 %)

MS (ESIpos): m/z = 536 [M+H]+

1 H NMR (300 MHz, CHLOROFORM-d) d ppm 1.03-1.50 (m, 27H) 1.80-2.00 (m, 2H) 2.60- 3.10 (m, 3H) 4.05-4.30 (m, 1H) 4.85-4.95 (d, 1H) 7.05-7.15 (m, 1H) 7.40-7.55 (m, 2H)

b) Neutral sodium salt of (2S,4S)-2-Amino-4-(2-fluoro-5-trifluoromethyl-benzyl)-pentan e dioic acid

2.14 g (4 mmol) of the compound described in Example 5a were dissolved in 10 ml_ of THF. 50 ml. 2 N hydrochloric acid in diethyl ether were added and the mixture was stirred for 2 days at room temperature. The reaction mixture was then evaporated to dryness and the resulting crude product was redistilled three times with diethyl ether and the residue dissolved in about 10 ml_ of water and the aqueous solution was adjusted to pH 7.4 with 1 N sodium hydroxide. The solution was freeze-dried and then chromatographed with water / methanol on C18-silica gel and the resulting fractions were combined and reduced in volume by evaporation. Yield: 1.25 g (97 %)

MS (ESIpos): m/z = 324 [M+H]+

1 H NMR (300 MHz, D2O) d ppm 1.97-2.20 (m, 2H) 3.02-3.08 (m, 3H) 3.72-3.78 (m, 1H) 7.25-7.32 (m, 1 H) 7.62-7.68 (m, 2H)