FIELD OF THE INVENTION The present invention relates to'8F-labeled thia fatty acids, and to methods of making and using the same, in particular, to methods of using such fatty acids as a tracer compound in positron emission tomography (PET).

BACKGROUND OF THE INVENTION A radioiodinated 4-thia fatty acid analog has been previously reported (Gildehaus et al, J Nucl Med 38: 124P, 1997, abstract).'8F- labeled fatty acids are known generally from U. S. Patent No. 4,323,547 (incorporated hereinto by reference) as useful in PET studies of myocardial metabolism. More recently,'8F-labeled 6-thia fatty acid (14F6THA) has been synthesized and evaluated (DeGrado, J Lab Compd Radiopharm 24: 989-995,1991; DeGrado et al, J Nucl Med 32: 1888-1896, 1991, each incorporated hereinto fully by reference). Although 14F6THA tracks beta-oxidation of palmitate in a number of conditions, it was found to be insensitive to inhibition of beta-oxidation in myocardium in conditions of hypoxia with normal blood flow. Retention of tracer in

hypoxic myocardium likely reflects retention of metabolic intermediates that precede beta-oxidation (long chain acyl-CoA, acyl-carnitine, and/or esterified lipids).

SUMMARY OF THE INVENTION The present invention is based on the discovery that sulfur heteroatom substitution at the C4 position, instead of the C6 position, of '8F-labeled fatty acids yields a tracer that is retained in proportion to the beta-oxidation rates in normoxic and hypoxic mammalian tissue, particularly normoxic and hypoxic myocardium. Most preferably, the invention is embodied in an ['8F] fluoro-4-thia-fatty acid having a chain length of between 8 to 20 carbon atoms, and may be saturated or at least partially unsaturated (i. e., contain one or more double bonds).

The'8F-labeled 4-thia fatty acids of this invention find particular utility in the noninvasive assessment of regional beta-oxidation rates using PET techniques which may allow early detection of abnormalities in the myocardium that might presage irreversible tissue injury.

Although not wishing to be bound to any particular theory, it is surmised that the 4-thia intermediates that precede beta-oxidation are poorly retained in the myocardium, possibly due to facile hydrolysis of the CoA and/or carnitine esters.

The ['8F] fluoro-4-thia-fatty acids according to this invention are most conveniently synthesized by subjecting a hydrolyzable ester precursor of a 4-thia-fatty acid having a readily substitutable group at the terminal carbon or an odd-numbered carbon from the terminal carbon to '8F substitution conditions. Thereafter, the'8F-substituted hydrolyzable

ester precursor of the 4-thia fatty acid may be subjected to hydrolysis conditions to form the corresponding ['8F] fluoro4-thia-fatty acid. Most preferably, the readily substitutabte group is selected from bromo, iodo, tosylate, benzenesulfonylate and the like, while the group that makes the precursor readily hydrolyzable may be benzyl, methyl and the like. By way of example, methyl 16-bromo-4-thia-hexadecanoate is a synthetic precursor of 16- ['8F] fluoro4-thia-hexadecanoic acid. Bromo and iodo esters are preferable since their respective acids are easily separated from the radioactive product fatty acid.

The precursor is synthesized by conventional organic synthesis techniques. For example, methyl 16-bromo-4-thia-hexadecanoate is synthesized by reaction of methyl 3-mercaptopropionate with 1,12- dibromododecane in acetonitrile in the presence of potassium carbonate.

The product ester is separated from reactants and other reaction products by silica gel liquid chromatography (hexane/ether 3: 1, Rf=0.6). Labeling at the e)-3 position requires two additional synthetic steps preceding the condensation reaction with methyl 3-mercaptopropionate, namely oxidation of co-bromo- (1) alcohol to the m-bromo (1) aldehyde in dichloromethane using pyridinium chlorochromate followed by reaction of the aldehyde with propyl magnesium chloride in ether. The resultant (co- 3)-alcool is then condensed with methyl 3-mercaptopropionate to yield the hydroxyester. The hydroxyester is converted to the corresponding tosyloxyester by reaction with Ts-CI in pyridine. Finally, the tosyloxyester is converted to the bromide, for example, by reaction with LiBr in acetone.

Liquid chromatography is used at each step to isolate the products.'8F labeling may then conventionally be carried out as described in the literature cited above.

A further understanding of this invention will be obtained from the following non-limiting Examples.

EXAMPLES In the Examples which follow, four'8F-labeled thia fatty acids were synthesized and evaluated: (1) 10- [BF] Fluoro-4-thia-decanoic acid (10F4TDA), (2) 16- ['8F] Fluoro-4-thia-hexadecanoic acid (16F4THA, FTP), (3) 13 (R, 5)- ['3F] Fiuoro4-thia-hexadecanoic acid (13F4THA), and for comparative purposes (4) 17-['8F] Fluoro-6-thia-heptadecanoic acid (17F6THA).

Chemicals were of analytical grade. Dry acetonitrile was obtained commercially (Pierce, Rockford, IL).'H-NMR spectra were recorde with a Varian Unity 500 MHz spectrometer using CDCI3 as solvent (Me4Si, 0.00 ppm). R, values refer to thin layer chromatography (TLC) performed on silica gel with the solvent system noted. Routine column chromatography was performed under normal pressure with silica gel (100-200 mesh) and the solvent system noted.

(1) Synthesis of Methyl 16-iodo-4-thia-hexadecanoate 1,12-diiodododecane (2.66 g, 6.3 mmol) was dissolve in 20 mL acetonitrile. Methyl 3-mercaptopropionate (0.76 g, 6.3 mmol) and K2CO3 (1.1 g, 8 mmol) were added and the mixture was allowed to react at room temperature for 20 h. The mixture was acidified with ice-diluted HCI and the solution was extracted twice with ether (20 mL). The combined ether fractions were washed successively with dilute NaHC03, water and brine, dried (MgSO4 and evaporated under reduced pressure. The product 1 (0.84 g, 32% yield) was isolated by column chromatography (hexane/ethyl

acetate 9: 1) and recrystallized in MeOH. TLC (hexane/ethyl acetate 9: 1) Rf = 0.4. Melting point = 33°C.'H-NMR 8 1.4 (m, 20H, CH2), 2.52 (t, 2H, C (2) H2), 2.61 (t, 2H, C (5) H2), 2,79 (t, 2H, C (3) H2), 3.20 (t, 2H, C (16) H2), 3.71 (s, 3H,-COO-CH3).

(2) Synthesis of 16-Fluoro-4-thia-hexadecanoic acid Fluorination of 1 (0.2g, 0.5 mmol) was performed by addition of tetrabutylammonium fluoride (TBAF) (4 mmol as 1 M THF solution). The mixture was allowed to react at room temperature for 20 h. The resulting fluoro-ester was hydrolyzed by addition of 1 ml 1 N KOH and 1 ml ethanol for 2 h at room temperature. The product 2 (0.05 g, 34% yield) was isolated by column chromatography (hexane/ether/acetic acid 3: 1: 0.1) and recrystallized in hexane, TLC (hexane/ether/acetic acid 3: 1: 0.1) Rf = 0.25. Melting point = 59°C,'H-NMR 8 1.4 (m, 20H, CH2), 2.55 (t, 2H, C (2) H2), 2.65 (t, 2H, C (5) H2), 2.80 (t, 2H, C (3) H2), 4.44 (dt, 2H, C (16) H2JHH=6 2 Hz).

(3) Synthesis of Methyl 17-tosyloxv-6-thia-heptadecanoate To a solution of 11-bromo-1-undecanol (10g, 40 mmol) in DMSO (80 mL) was added thiourea (3.64g, 48 mmol), and the mixture was allowed to react at room temperature for 21 hr. The mixture was extracted twice with hexane (20 mL) to remove the unreacted bromo alcool. To the DMSO fraction was added 2N KOH (50 mL) and the mixture was heated at 80 °C for 5 min, releasing the thiol 11-mercapto- undecanol. The mixture was acidified (HCI) and extracted twice with ether (40 ml). The combined ether phases were washed successively with water and brine, dried over MgS04, and evaporated under reduced pressure. The resulting thiol (-7.2g) was caused to react with methyl 5-

bromo-pentanoate (7g, 37 mmol) according to the procedure for compound 1. Crystallization of the product in hexane yielded 5.5g (18 mmol 48% yield from the thiol) of methyl 17-hydroxy-6-thia- heptadecanoate. TLC (hexane/ether 1: 1) showed a single product at Rf=0.3. Without further characterization, the hydroxy-ester (4g, 12 mmol) was caused to react with Ts-CI (2.7g, 12 mmol) and pyridine (14 mmol) in CHCI2 (40 mL) at 5 °C for 4 hr. The mixture was acidified with ice-diluted HCI, and the organic layer separated, dried (MgSo4) and evaporated under reduced pressure. The product 3 (2.4 g, 34% yield) was isolated by column chromatography (hexane/ethyl acetate 7: 3) and recrystallized in hexane. TLC (hexane/ethyl acetate 7: 3) Rf = 0.6. Melting point = 54 °C.'H-NMR 8 1.3 (m, 22H, CH2), 2.38 (t. 2H, C (2) H2), 2.5 (m, 7H, C (5) H2, C (7) H2, (tosyl) CH3), 3.7 (s, 3H,-COO-CH3), 4.0 (t, 2H, C (17) H2), 7.6 (m, 4H, aryl).

(4) Synthesis of 17-Fluoro-6-thia-heptadecanoic acid Compound 3 (1 g, 1.8 mmol) wascaused to react with TBAF (2-5 mmol) according to the procedure for compound 2. The resultant fluoro- ester was hydrolyzed in KOH/ethanol as previously described, and crystallized twice in ethanol/water to yield 0.4 g (70% yield) product 4.

Melting point = 54 °C.'H-NMR 8 1.3 (m, 22H, CH2), 2.38 (t, 2H, C (2) H2), 2.5 (m, 4H, C (5) H2, C (7) H2), 4.44 (dt, 2H, C (17) H2, JHF= 47.3, JHH = 6.2 Hz).

(5) Synthesis of Methyl 10-bromo4-thia-decanoate 1,6-dibromo-hexane (1.6 g. 13 mmol) and methyl 3-mercapto- propionate (13 mmol) were caused to react according to the procedure for compound 1. The product 5 (1.3 g, 35% yield) was isolated as an oil by

column chromatography (hexane/ether 1: 1). TLC (hexane/ether 1: 1) R, = 0.6.'H-NMR 6 1.4 (m, 12H, CH2), 2.52 (t, 2H, C (2) H2), 2.61 (t, 2H, C (5) H2), 2.8 (t, 2H, C (3) H2), 3.40 (t, 2H, C (10) H2), 3.7 (s, 3H, COO-CH3).

(6) Synthesis of 10-Fluoro4-thia-decanoic acid Compound 5 (0.5 g, 1,8 mmol) was caused to react with TBAF (3 mmol) according to the procedure for compound 2. The resultant fluoro- ester was hydrolyzed in KOH/ethanol as previously described, and isolated as an oil by HPLC (Table 1) to yield 0.14 g (37% yield) product 6.

'H-NMR 5 1.3 (m, 8H, CH2), 2.55 (t, 2H, C (2) H2), 2,65 (t, 2H, C (5) H2), 2.80 (t, 2H, C (3) H2), 4.44 (dt, 2H, C (16) H2 JHF=47.5 Hz, JHH= 6.2 Hz).

(7) Methyl 13 (R. S)-hydroxy-4-thia-hexadecanoate To a solution of 12-bromo-3-dodecanol (8.78 g, 33.1 mmol) in acetonitrile (80 mL) was added methyl 3-mercaptopropionate (3-98 g.

33.1 mmol) and K2CO3 (5,5 g, 40 mmol). The mixture was allowed to react at room temperature for 20 hr. The mixture was acidified with ice- diluted HCI and the solution was extracted twice with ether (40 mL). The combined ether fractions were washed successively with dilute NaHCO3, water and brine, dried (MgS04) and evaporated under reduced pressure.

The product 7 (4.4 g, 44% yield) was isolated by column chromatography (hexane/ether 1: 1) and recrystallized in hexane. TLC (hexane/ether 1: 1) Rf= 0.4. Melting point = 38.8 °C'H-NMR ã 0.93 (t, 3H, C (16) H2), 1.4 (m, 18H, CH2), 2.52 (t, 2H, C (2) H2), 2.61 (t, 2H, C (5) H2), 2.79 (t, 2H, C (3) H2), 3.60 (m, 1H, C (13) H), 3.70 (s, 3H,-COO-CH3).

(8) Methyl 12 (R. S)-benzenesulfonyloxy-4-thia-hexadecanoate Compound 7 (0.83 g, 2.7 mmol) was caused to react with benzenesulfonylchloride (0.45 ml, 3.5 mmol) and pyridine (3.5 mmol) in CHCI2 (20 mL) at 5 °C for 20 hr. The mixture was ice-diluted HCI, and the organic layer separated, dried (MgSO4 and evaporated under reduced pressure. The product 8 (1.0 g. 83% yield) was isolated as an oil by column chromatography (hexane/ether 1: 1). TLC (hexane/ether 1: 1) R, = 0.5.'H-NMR 5 0.82 (t, 3H, C (16) H2), 1.4 (m, 18H, CH2), 2.52 (t, 2H, C (2) H2), 2.62 (t, 2H, C (5) H2), 2.78 (t, 2H, C (3) H23.70 (s, 3H,-COO-CH3), 4.60 (m, 1H, C (13) H), 7.7 (m, 5H, aryl).

(9) 13 (R. S)-Fluoro-4-thia-hexadecanoic acid Compound 11 (0.5 g, 1.1 mmol) was caused to react with TBAF (2.5 mmol) according to the procedure for compound 2. The resultant fluoro-ester was hydrolyzed in KOH/ethanol as previously described, and crystallized twice in hexane to yield product 12 (0.12 g, 44% yield). TLC (hexane/ether/AcOH 1: 2: 0.03) Rf = 0.6 Melting point = 61 °C.'H-NMR å 0.94 (t, 3H, C (16) H2), 1.4 (m, 18H, CH2), 2.52 (t, 2H, C (2) H2), 2.62 (t, 2H, C (5) H2), 2.78 (t, 2H, C (3) H2), 4.47 (double septuplet, 1H, C (13) H, JHF=49.3 Hz JHH=3.8 Hz).

'8F-labeling procedure The precursors for 16F4THA, 13F4THA, 1 OF4THA, and 17F6THA were compounds 1,8,5, and 3, respectively. To a 2 ml glass vial was added Kryptofix 2.2.2 (10 mg), acetonitrile (0.5 ml) and 20 ut of a 9% K2CO3 solution in water. ['8F] Fluoride, produced via proton bombardment of H2'80 (>95 atom %), was then added, the vessel placed in an aluminum heating block at 90 °C, and the solvent evaporated under a

stream of helium or nitrogen. The residue was further dried by azeotropic distillation with acetonitrile (2 x 0.3 ml). A solution of the precursor (-2 mg) in acetonitrile (0.5 ml) was added, the vial was sealed and returned to the heating block. Reaction time was 15 min. The via was briefly cooled by placing in ice-water. The incorporation of ['8F] fluoride was monitored by radio-TLC (hexane/ethyl acetate 3: 1). Rf values were 0.0 and >0.4 for ['°F] fluoride and ['8F] fluoro-ester, respectively, Subsequent hydrolysis of the resulting ['8F] fluoro-ester was performed in the same vessel by the addition of 0.2 ml 0.2N KOH and continued heating at 90 °C for 4 min. The mixture was cooled, acidified with concentrated acetic acid (25 pI), filtered, and applied to the preparative HPLC column (Table 1). The ['8F] fluoro-fatty acid fraction was collected, evaporated to dryness, formulated In isotonic NaCI solution (for long-chain fatty acid analogs, 1-2% albumin was present), and filtered through a 0.22 um filter (Millex-GS).

Table 1. Semi-preparative reverse phase HPLC capacity factors (k') of fatty acid analogs (Nucleosil, C-18 (10A), 250 x 10 mm, flow =4.3 ml/min, mobile phase is MeOH/H2O/AcOH X: Y: 0.5, X+Y=99.5) Compound Mobile Phase k' (%MeOH) 16-lodo-4-thia-hexadecanoic acid 90 5. 1 16-Fluoro-4-thia-hexadecanoic acid (2, 16F4THA, FTP) 90 3.2 13-Benzenesulfonyloxy-4-thia-hexadecanoic acid 90 2. 7 13-Fluoro-4-thia-hexadecanoic acid (9, 13F4THA) 90 3.5 17-Tosyloxy-6-thia-heptadecanoic acid 90 3.0 17-Fluoro-4-thiaheptadecanoic acid (4, 17F6THA 90 3. 5 10-Bromo-4-thia-decanoic acid 70 4.3 1 0-Fluoro-4-thia-decanoic acid (6, 1 OF4TDA) 70 2.7

Biological Studies Model 4-thia analogs (16- [eF] fluoro-4-thia-hexadecanoic acid and 12-['8F] fluoro-4-thia-dodecanoic['8F] fluoro-4-thia-dodecanoic acid) were examined as tracers in isolated rat hearts. The perfusate was Krebs-Hensleit buffer with 1 % albumin, 0.15mM palmitate, and 5 mM glucose. Sprague Dawley rats (200-225 g) were fed ad libetum. The hearts were excised from pentobarbital-anesthetized animals and perused at 7 ml/min in Langendorff retrograde fashion. The perfusate was gassed with either 95% oxygen (normoxic) or 35% oxygen (hypoxic) gas mixture. The gases comprising the remaining fraction were nitrogen and carbon dioxide (5% for both). The tracer was administered as a bolus or as a pulse infusion into the isolated rat hearts. Pulse infusion allowed absoute quantification of'8F accumulation rates that reflect the metabolic rate of the tracer in the heart. Clearance rates are measured during the washout phase of these experiments. Beta-oxidation rates of 9,10 [3H] palmitate were measured by collection of venous effluent samples and separation of titrated water.

Results showed good correlation of beta-oxidation rates with uptake rates of the model 4-thia analogs (r>0.86) for both normoxic and hypoxic conditions. In contrast, the 6-thia analog (17F6THA) showed poor correlation with beta-oxidation rates in the same conditions.

PET imaging studies were performed with 16F4THA in patients with ischemic cardiomyopathy. Conventional nuclear medicine perfusion scans were obtained in the same subjects. The PET scans were of superior quality to the conventional nuclear scans and showed different information form the perfusion images, possibly reflecting greater sensitivity to ischemia.

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.