Cysteine Derivatized ^99mTc-Labelled Fatty Acids as β-Oxidation Markers

Abstract

With the aim of developing ^99mTc-labeled fatty acids intended for myocardial metabolism imaging we report herein the synthesis and characterization of two novel derivatives of undecanonoic and hexadecanonoic acid that have been functionalized at the ω-site by cysteine through the formation of a thioether bond (Cys–FA11 and Cys–FA16). Equimolar amounts of each ligand and the [NEt₄]₂[Re(CO)₃Br₃] precursor generated the respective hexacoordinated neutral complexes in which the ligand coordinated to the metal through the SNO donor system of cysteine. The rhenium complexes were characterized by elemental analysis, IR and NMR spectroscopies. The analogous technetium-99m complexes, ^99mTc–Cys–FA11 and ^99mTc–Cys–FA16 were prepared by incubation of the ligand with the precursor [^99mTc(CO)₃(H₂O)₃]⁺ (radiochemical yield ≥98%). Their structure was established by comparative HPLC techniques. In vivo studies in mice showed high initial heart uptake for both ^99mTc complexes (7.4 ± 0.53 and 7.07 ± 0.73 percentage of injected dose (%ID)/g at 1 min post injection. Rapid clearance (0.60 ± 0.02 %ID/g) was observed for ^99mTc–Cys–FA11 while the clearance of the longer fatty acid ^99mTc–Cys–FA16 was slower (2.31 ± 0.09 %ID/g at 15 min p.i.). Metabolite analysis study indicated that complexes were catabolized through the β-oxidation process.

1. Introduction

Fatty acids (FAs) are the main source of energy in normal myocardium providing up to 90% of the energy needed [1]. Once inside the myocardium, where they are transported by passive diffusion or by protein transporters [2], they can either be catabolized through β-oxidation which degrades them by removing two-carbon units, or they can be stored in the form of triglycerides for future use as energy supplies [3]. Under ischaemic conditions oxidation of fatty acids is suppressed and glycolysis and glycogen breakdown are used as an energy source [4]. Therefore, the metabolism of fatty acids and its suppression during ischaemia is a sensitive marker for the normal function of the heart. As a result, the use of radiolabelled fatty acids is considered a valuable diagnostic tool for the detection of ischaemic heart disease, cardiomyopathies and other heart conditions that tamper with cardiac metabolism using either positron emission tomography (PET) or single photon emission computed tomography (SPECT).

¹¹C-Labelled fatty acids are considered the true PET tracers of FAs, however their complicated metabolic profile has prevented wide application [5,6]. The alternatively ¹⁸F-labelled fatty acids have presented kinetic differences compared to their unlabelled counterparts that hampered their development [7,8]. Moreover, a major disadvantage for the application of PET tracers is that an on-site cyclotron is required to produce ¹¹C- and ¹⁸F-labelled fatty acids [9].

Regarding SPECT radiotracers, one of the earliest and most promising radiotracers of fatty acid metabolism was the 15-(p-iodophenyl) pentadecanoic acid (IPPA), which accumulated rapidly in the heart and was cleared with a kinetic profile that resembled that of ¹¹C-palmitate [10,11,12]. However, due to the rapid washout, quantification of β-oxidation was not possible and image quality was poor. This led to the development of branched-chain analogues of IPPA (Scheme 1), such as the ¹²³I-beta-methyl-p-iodophenylpentadecanoic acid (BMIPP) [13,14,15] which only partially undergoes β-oxidation and is mainly retained in the triglyceride pool. Thus, retention time is increased and image quality is vastly improved [10] rendering BMIPP the radiopharmaceutical of choice for the assessment of cardiac β-oxidation. However, as is the case for all radio-iodinated compounds that must be supplied in a ready to use dosage form, there are logistic problems associated with the production cost and transportation of BMIPP.

The generator produced ^99mTc is the alternative SPECT radiotracer due to its desirable characteristics (t_1/2: 6 h, photon energy of 140 keV, widespread availability, low cost). The development of ^99mTc-labelled fatty acids suitable for evaluation of myocardial function and metabolism has been pursued by various research groups since 1975 [16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44]. In all these approaches a chelating moiety has been incorporated into a fatty acid (Scheme 2). Ideally, the moiety should not interfere with the biological properties of the fatty acid allowing recognition by membrane and intracellular proteins [36]. However, the biodistribution studies of the ^99mTc-labelled fatty acids indicated mediocre myocardial uptake, rapid myocardial washout and poor heart/blood and heart/liver ratios when compared to the advantageous biodistribution patterns of the established radio-iodinated fatty acid tracers [16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44].

As the chelating moiety used in the synthesis of the ^99mTc-labelled fatty acids plays a prominent role in their biodistribution profile, we report herein the synthesis and characterization of two novel derivatives of undecanoic and hexadecanoic acid with cysteine (Cys–FA11 and Cys–FA16 respectively) which acts as a chelator for the M(CO)₃⁺ core (M = Tc and Re) (Scheme 3). The synthesis, characterization, biodistribution in mice, stability and metabolic studies of their tricarbonyl ^99mTc(CO)₃⁺ complexes, ^99mTc–Cys–FA11 and ^99mTc–Cys–FA16 (Scheme 4), is also reported. For characterization purposes the synthesis of the corresponding Re(CO)₃⁺ complexes Re–Cys–FA11 and Re–Cys–FA16 was effected. Finally, in order to assist in the evaluation of the metabolism data, the analogous complexes with cysteine joined to valeric acid (Cys–Val) were also studied.

2. Results and Discussion

The cysteine conjugated fatty acid derivatives Cys–Val, Cys–FA11 and Cys–FA16 (Scheme 1) were synthesized by reacting cysteine with the corresponding ω-bromo fatty acid in alkaline environment and were characterized by elemental analysis, and IR and NMR spectroscopies.

The synthesis of non-radioactive Re(I)-complexes, Re–Cys–Val, Re–Cys–FA11 and Re–Cys–FA16, was achieved by ligand exchange reaction using the organometallic Re tricarbonyl precursor [Net₄]₂[ReBr₃(CO)₃] and equimolar quantities of ligands, as shown in Scheme 3. HPLC analysis of reaction mixtures showed in each case the formation of a single peak with retention times 13.6, 19.5 and 22.3 min for Re–Cys–Val, Re–Cys–FA11 and Re–Cys–FA16 respectively. Complexes were purified with recrystallization and were characterized by elemental analysis and IR and NMR spectroscopies. All complexes were stable in the solid state and in solution as shown by NMR and HPLC for a period of months.

The infrared spectra of all rhenium complexes show strong bands at 1872–2027 cm⁻¹ which were attributed to symmetrical and asymmetrical stretches of the C≡O groups of the fac-[Re(CO)₃]⁺ core [45,46]. Characteristic double peaks in the region 2848–2922 cm⁻¹ were attributed to C–C stretching of the methylene groups. The complexes also showed strong bands at 1703–1708 cm⁻¹ which were due to C=O stretching of their uncoordinated carboxylate groups, while the CO stretch of their coordinated carboxylates appeared in the 1614–1649 cm⁻¹ range.

NMR spectra of all ligands and rhenium complexes were obtained in DMSO-d₆, at 25 °C and assignment was affected through the combined analysis of ¹H–¹H and ¹H–¹³C correlation spectra (Figures S1–S6). The full ¹H and ¹³C assignments appear in

Table 1. ¹H NMR chemical shift assignments for ligands Cys–Val, Cys–FA11, Cys–FA16 and complexes. Re–Cys–Val, Re–Cys–FA11, Re–Cys–FA16 in DMSO-d₆ at 25 °C. The numbering of the atoms is given in Scheme 1.

	Cys–Val	Re–Cys–Val		Cys–FA11	Re–Cys–FA11		Cys–FA16	Re–Cys–FA16
H-2	2.20	2.25	H-2	2.18	2.17	H-2	2.28	2.27
H-3	1.57	1.63	H-3	1.47	1.48	H-3	1.50	1.49
H-4	1.52	1.69	H4, H4a–H4c	1.24	1.25	H4, H-4a–H-4h	1.23	1.25
H-5	2.54	2.90	H-4d	1.24	1.39	H-4i	1.23	1.36
H-6	2.71, 2.97	2.72, 2.89	H-4e	1.32	1.60	H-4j	1.29	1.63
H-7	3.28	4.09	H-4f	1.49	1.50	H-4k	1.49	1.49
NH2	not visible	6.09, 4.91	H-5	2.54	2.51	H-5	2.53	2.49
OH	not visible	12.01	H-6	2.95, 2.81	2.98, 2.70	H-6	2.98, 2.84	2.87, 2.68
			H-7	3.30	4.10	H-7	3.29	4.09
			NH2	not visible	6.14, 4.95	OH	8.54	11.83
			OH	not visible	11.82	NH2	-	6.13, 4.95

and

Table 2. ¹³C NMR chemical shift assignments for ligands Cys–Val, Cys–FA11, Cys–FA16 and complexes. Re–Cys–Val, Re–Cys–FA11, Re–Cys–FA16 in DMSO-d₆ at 25 °C. The numbering of the atoms is given in Scheme 1.

	Cys–Val	Re–Cys–Val		Cys–FA11	Re–Cys–FA11		Cys–FA16	Re–Cys–FA16
C-1	174.92	173.96	C-1	174.44	174.37	C-1	173.39	173.32
C-2	33.65	32.87	C-2	33.61	33.54	C-2	33.50	33.18
C-3	23.65	23.21	C-3	24.40	24.35	C-3	24.12	24.33
C-4	28.28	27.07	C4, C4a–C4d	28.41–28.79	28.23–28.59	C-4, C-4a–C-4h	28.92–28.35	28.89–28.56
C-5	30.54	32.04	C-4e	28.05	27.69	C-4i	″	28.34
C-6	33.33	36.22	C-4f	28.58	28.51	C-4j	″	27.73
C-7	53.58	57.62	C-5	30.99	31.66	C-4l	28.81	28.76
C-8	168.77	177.13	C-6	31.13	32.22	C-5	31.15	31.59
C≡O	-	193.36, 195.35	C-7	52.28	56.81	C-6	31.16	32.30
			C-8	168.39	176.96	C-7	52.25	56.98
			C≡O	-	193.23, 195.29	C-8	168.58	177.16
						C≡O	-	195.45, 193.20

″: values are the same as above (28.92–28.35).

while the numbering of the atoms is given in Scheme 3. At room temperature, ¹H and ¹³C peaks appear broad apparently reflecting the existence in solution of multiple conformations in slow equilibrium.

In the Cys–Val ligand, assignment of the valeric acid moiety is straightforward starting from the H-2 methylene protons adjacent to the valeric acid carboxylate group which appear as a distinct, well-separated from the rest, triplet peak. In the Cys–FA11 and Cys–FA16 ligands, the H-2 methylene protons, the neighbouring H-3, as well as protons H-5 adjacent to the cysteine sulphur, were easily distinguished, however, extensive peak overlap precluded the specific assignment of most of the middle methylene carbons and protons of the fatty acid chains.

Upon coordination, downfield shifts of 0.8 ppm were noted in all complexes for the H-7 alpha-proton of cysteine, while coordination did not significantly affect the chemical shifts of the H-6 beta-protons, as previously noted in other cysteine complexes with the tricarbonyl core [46]. In the ¹³C spectra, downfield shifts were noted upon coordination for the cysteine C-7 alpha-carbon by 4.0–4.7 ppm, and the C-8 carboxylate carbon by approximately 8.5 ppm, while smaller downfield shifts were noted for the cysteine C-6 beta-carbon (1–3 ppm) and the C-5 carbon adjacent to the cysteine sulphur (0.5–1.5 ppm). In addition, upon coordination the amine protons of cysteine that were invisible in the spectra of the ligands, became visible appearing as two distinct broad peaks at approximately 6.1 ppm and 4.9 ppm, the great chemical shift separation apparently reflecting the asymmetry of the complexes.

The technetium-99m complexes (Scheme 4) were synthesized in high yield and radiochemical purity (>95%) by ligand substitution reaction using the precursor fac-[^99mTc(H₂O)₃(CO)₃] and ligand concentration 10⁻³ M. The identity of the technetium-99m complexes was established by HPLC comparison of their retention time to that of the analogous well characterized rhenium complexes by applying parallel radiometric and photometric detection (Figure 1).

Lipophilicity studies showed that all complexes were of medium lipophilicity. As expected, complex ^99mTc–Cys–FA16 with the hexadecanoic acid was more lipophilic (Log Do/w 2.24) than complex ^99mTc–Cys–FA11 with the undecanoic acid (Log Do/w 1.018). In vitro stability against transchelation and/or decomposition as determined by the histidine/cysteine challenge study, demonstrated that all complexes were stable against the presence of the two strong tricarbonyl ^99mTc core competitors since after 6 h incubation all complexes remained completely intact.

Human plasma stability studies were performed in order to determine whether plasma proteins bound the radiolabelled complexes and also whether the complexes remained stable after incubation in a biological medium. Results revealed that both complexes remained intact under the employed conditions (≥98%) and were also characterized by low plasma binding, being 38.2% ± 3% for complex ^99mTc–Cys–FA11 and 29.2% ± 4.3% for complex ^99mTc–Cys–FA16 after 2 h incubation (

Table 3. In vitro protein binding and plasma stability of ^99mTc–Cys–FA11 and ^99mTc–Cys–FA16 at 37 °C.

99mTc Complexes	Incubation Time	Protein Binding	Stability of 99mTc Complexes
99mTc–Cys–FA11	15 min	16.2% ± 1.1%	≥98%
	2 h	38.2% ± 3%	≥98%
99mTc–Cys–FA16	15 min	13.8% ± 0.6%	≥98%
	2 h	29.2% ± 4.3%	≥98%

).

In vivo evaluation of the ^99mTc complexes was carried out in male Swiss Albino mice and the data are presented in Figure 2. The biodistribution study was performed 1 and 15 min after a rapid bolus intravenous injection via the tail vein. Rapid blood clearance was observed for both complexes. The absence of radiation in the stomach and in the spleen indicates the in vivo stability of the complexes in terms of re-oxidation to pertechnetate and TcO₂ respectively. The main excretion route was via the urinary tract for both complexes, and secondary through the hepatobiliary system. The initial heart uptake was similar in both complexes, although slightly lower for the hexadecanoic fatty acid derivative (7.40 ± 0.53 %ID/g for complex ^99mTc–Cys–FA11 and 7.07 ± 0.73 %ID/g for complex ^99mTc–Cys–FA16). This finding is consistent with literature reports that correlate myocardial uptake with the length of the fatty acids chain [29,35]. The initial heart uptake values in this study were lower compared to the ¹²³I-IPPA in clinical use (9.51 %ID/g) [37], however, it is comparable to the values of other ^99mTc-labelled fatty acid analogues in mice [27,29,31,37,38,41,42,43,44].

The cardiac retention observed for ^99mTc–Cys–FA16 (2.31 ± 0.09 %ID/g) was higher than that observed for ^99mTc–Cys–FA11 (0.60 ± 0.02 %ID/g) at 15 min post injection, possibly due to its longer aliphatic side chain that prolongs metabolism. Despite the favourable profile of the ^99mTc complexes (high radiochemical yield, stability, heart uptake), the key ratios (heart/blood, heart/liver, heart/lungs) were below one for both complexes at all time points studied, a fact that hampers their application as heart metabolism agents without structural modifications.

Metabolite analysis using urine and liver samples was carried out for both radiocomplexes (Figure 3). Urine samples were analysed after a simple filtration through membrane filters, while liver samples after a process involving homogenization with a mixture of chloroform, methanol and sodium hydroxide, centrifugation and filtration [47]. Most of the radioactivity was extracted in the aqueous fractions (considerably less radioactivity remained in the organic fractions and practically none at the residual pellets formed after centrifugation) and consequently these fractions were analysed by RP-HPLC. Analysis showed the formation of a multitude of radioactivity peaks for both complexes in liver as well as in urine samples. This multitude of radiometabolites was also observed in metabolite analysis of radio-iodinated fatty acid analogues [48,49,50,51]. The radiometabolites were more polar than their paternal complexes, as indicated by their retention times, a fact consistent with a catabolisation process through the aliphatic chain of radiolabelled fatty acid analogues. The ^99mTc–Cys–FA11 complex, when catabolized through β-oxidation, should give products with an odd number of carbon atoms in the aliphatic chain, while the ^99mTc–Cys–FA16 complex, should give products with an even number of carbon atoms. RP-HPLC analysis of ^99mTc–Cys–FA11 showed the formation of four main metabolites in liver as well as in urine samples. An assumption would be that these are complexes with three, five, seven and nine carbon atoms in the side chain. This is supported by the fact that the retention time of the metabolite with five carbon atoms in the side chain is exactly the same as the retention time of the well characterized ^99mTc–Cys–Val. RP-HPLC analysis of ^99mTc–Cys–FA16 showed the formation of seven peaks in liver samples that may belong to seven metabolites with two, four, six, eight, 10, 12 and 14 carbon atoms in the side chain. In urine samples two additional metabolites were observed which may be attributed to the participation of the complex in additional biochemical processes.

3. Experimental Section

3.1. Materials and Methods

All laboratory chemicals were reagent grade and were used without further purification. Solvents for high-performance liquid chromatography (HPLC) were HPLC-grade and were filtered through membrane filters (0.22 μm, Millipore, Milford, MA, USA) and degassed by a helium flux before and during use.

IR spectra were recorded on a Nicolet 6700 ATR-IR from Thermo Scientific (Waltham, MA, USA) in the region 4000–500 cm⁻¹. The NMR spectra were recorded in DMSO-d₆, at 25 °C on a Bruker 500 MHz Avance DRX spectrometer (Brucker Biospin, Billerica, MA, USA) using tetramethylsilane (TMS) as internal standard. Elemental analyses for C, H and N were conducted on a Perkin-Elmer 2400 automatic elemental analyser (Perkin Elmer Ltd, Beaconsfield, England). HPLC analysis was performed on a Waters 600 chromatography system coupled to both a Waters 2487 Dual λ absorbance detector and a Gabi gamma detector from Raytest (Elysia-Raytest, Straubenhardt, Germany). Separations were achieved on a Nucleosil C18 (10 μm, 250 mm × 4 mm) column eluted with a binary gradient system at a 1 mL min⁻¹ flow rate. Mobile phase A was methanol containing 0.1% trifluoroacetic acid, while mobile phase B was water containing 0.1% trifluoroacetic acid. The elution gradient was 0–1 min 5% A (95% B), followed by a linear gradient to 95% A (5% B) in 15 min; this composition was held for another 15 min. The precursor [Et₄N]₂[ReBr₃(CO)₃] was synthesized according to a literature method [52]. Na^99mTcO₄ was obtained in physiological saline as a commercial ⁹⁹Mo/^99mTc generator eluate (Mallinckrodt Medical B.V., Petten, The Netherlands). The radioactive precursor fac-[^99mTc(H₂O)₃(CO)₃]⁺ was prepared using a kit containing 5.5 mg of NaBH₄, 4 mg of Na₂CO₃, and 20 mg of Na–K tartrate, purged with CO gas, prior to addition of Na^99mTcO₄, as described in the literature [53].

3.2. Synthesis of 5-((2-Amino-2-Carboxyethyl) Thio) Pentanoic Acid, Cys–Val

Cysteine hydrochloride (1.57 g, 10 mmol) and 5-bromovaleric acid (1.81 g, 10 mmol) were dissolved in 25 mL of 2.5 N aqueous NaOH solution under N₂. The solution was stirred at RT for 24 h, followed by pH adjustment to 5 with glacial acetic acid. The solid formed was collected and recrystallized from methanol [54]. Yield 75%; mp: 209–212 °C. The ¹H and ¹³C NMR data are given in Table 1 and Table 2 respectively. IR: 2915, 2850, 1702 cm⁻¹; elemental analysis: C₈H₁₅NO₄S. Calc: C: 43.43, H: 6.83, N: 6.33. Found: C: 43.28, H: 6.95, N: 6.23.

3.3. Synthesis of 11-((2-Amino-2-Carboxyethyl) Thio) Undecanoic Acid, Cys–FA11

Cysteine hydrochloride (1.57 g, 10 mmol) and (2.65 g, 10 mmol) 11-bromoundecanoic acid were dissolved in 25 mL of 2.5 N aqueous NaOH solution under N₂. The solution was stirred at RT for 24 h, followed by pH adjustment to 5 with glacial acetic acid. The solid formed was collected, triturated with hot aqueous methanol (20%) and collected by filtration. Yield 66%; mp: 199–200 °C. The ¹H and ¹³C NMR data are given in Table 1 and Table 2 respectively. IR (cm⁻¹): 2916, 2850, 1704; elemental analysis: C₁₄H₂₇NO₄S. Calc: C: 55.05, H: 8.91, N: 4.59. Found: C: 55.20, H: 8.99, N: 4.34.

3.4. Synthesis of 16-((2-Amino-2-Carboxyethyl) Thio) Hexadecanoic Acid, Cys–FA16

Cysteine (600 mg, 4.95 mmol), 16-bromohexadecanoic acid (500 mg, 1.49 mmol), 10 mL 5.4 M sodium methoxide in methanol, and 10 drops of DMSO were added to 170 mL of methanol under N₂. The solution was stirred at RT for 2.5 h followed by the evaporation of the solvent. In the remaining solid, water was added and the pH of the solution was adjusted to pH 5 by the addition of glacial acetic acid. The solid formed was collected by filtration and dried. Yield 64%; m.p.: 215–218 °C. The ¹H and ¹³C NMR data are given in Table 1 and Table 2 respectively. IR: 2915, 2850, 1703 cm⁻¹; elemental analysis: C₁₉H₃₇NO₄S. Calc: C: 60.76, H: 9.93, N: 3.73. Found: C: 60.61, H: 10.28, N: 3.61.

3.5. Synthesis of Rhenium Complexes Re–Cys–Val

General method: A mixture of [NEt₄]₂[ReBr₃(CO)₃] (0.2 mmol) and the corresponding ligand (Cys–Val, Cys–FA11, Cys–FA16, 0.2 mmol) dissolved in 5 mL acetonitrile and 1 mL 0.2 N NaOH (0.2 mmol) was refluxed for 2 h. Upon standing at room temperature for 48 h, a pale yellowish product precipitated, collected by filtration and recrystallized from a mixture of methanol/diethylether.

Re–Cys–Val: white powder. Yield 69%, t_R = 13.6 min, IR: 2926, 2851, 2023, 1889, 1705, 1627 cm⁻¹. The ¹H and ¹³C NMR data are given in Table 1 and Table 2 respectively. Elemental analysis: C₁₁H₁₄NO₇ReS. Calc: C: 26.94, H: 2.88, N: 2.86. Found: C: 26.78, H: 2.97, N: 2.65.

Re–Cys–FA11: white powder. Yield 58%, t_R = 19.5 min; IR: 2922, 2852, 2022, 1878, 1703, 1614 cm⁻¹. The ¹H and ¹³C NMR data are given in Table 1 and Table 2 respectively. Elemental analysis: C₁₇H₂₆NO₇ReS. Calc: C: 35.53, H: 4.56, N: 2.44. Found: C: 35.15, H: 4.79, N: 2.78.

Re–Cys–FA16: yellowish powder. Yield 63%, t_R = 22.3 min, IR: 2915, 2848, 2027, 1872, 1708, 1649 cm⁻¹. The ¹H and ¹³C NMR data are given in Table 1 and Table 2 respectively. Elemental analysis: C₂₂H₃₆NO₇ReS. Calc: C: 40.98, H: 5.63, N: 2.17. Found C: 40.67, H: 5.74, N: 1.93.

3.6. Synthesis of ^99mTc Complexes

A freshly prepared solution (400 μL) of the fac-[^99mTc(CO)₃(H₂O)₃]⁺ precursor (pH 8) were added to a vial containing 100 μL of a 5 × 10⁻³ M aqueous alkaline (pH 8) solution of the cysteine derivatized acids Cys–Val, Cys–FA11 and Cys–FA16. The vial was sealed, flushed with N₂ and heated for 30 min at 75 °C. In each case, HPLC analysis demonstrated the formation of a single complex with radiochemical yield >95%. ^99mTc–Cys–Val: t_R = 13.9 min, ^99mTc–Cys–FA11: t_R = 19.8 min and ^99mTc–Cys–FA16: t_R = 22.7 min. Assignment of the structure was based on comparative HPLC studies using the corresponding Re complex as reference.

For all the experiments described below the technetium complexes were isolated by HPLC in order to remove excess ligand and small amounts of radiochemical impurities.

3.7. In Vitro Stability Studies of ^99mTc Complexes

Aliquots of 400 μL of the isolated ^99mTc complexes, ^99mTc–Cys–FA11 and ^99mTc–Cys–FA16, were added to 100 μL of a 5 × 10⁻² M histidine or 5 × 10⁻² M cysteine solution in Phosphate Buffer Saline pH 7.4, respectively (final concentration of cysteine and histidine was 10⁻² M). The samples were incubated for 1, 3 and 6 h at 37 °C and aliquots were removed and analysed by HPLC.

3.8. Plasma Stability

Human blood (~5 mL) was collected in heparinized polypropylene tubes and was centrifuged at 2000 rpm (4 °C) for 15 min and the supernatant (plasma) was collected. Aliquots of the HPLC-purified ^99mTc complexes (100 μL) were incubated at 37 °C with plasma (900 μL). After 15 and 120 min, fractions (450 μL) were withdrawn, mixed with EtOH in a 2:1 EtOH/aliquot v/v in order to precipitate plasma proteins, and centrifuged at 14,000 rpm for 30 min. Supernatants were filtered and analysed by reverse phase (RP) HPLC.

3.9. Determination of Distribution Coefficient (Log Do/w)

For the evaluation of the lipophilicity of the compounds, the distribution coefficient of the ^99mTc complexes, ^99mTc–Cys–FA11 and ^99mTc–Cys–FA16, was determined by the shake-flask method. HPLC-purified ^99mTc complexes (10 μL) were mixed with 1.5 mL of 1-octanol and 1.5 mL phosphate buffer (0.125 M, pH 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and finally centrifuged at 5000 rpm for 5 min. Three weighed samples from the octanol and aqueous layers were then counted in a gamma counter and the cpm/mL was calculated. Partition coefficient (Log Do/w) was calculated by dividing the cpm/mL (octanol) to cpm/mL (buffer).

3.10. Animal Distribution Studies

Our experimental animal facility is registered according to the Greek Presidential Decree 56/2013 (Reg. Number: EL 25 BIO 022), in accordance to the European Directive 2010/63 which is harmonized with national legislation, on the protection of animals used for scientific purposes. All applicable national guidelines for the care and use of animals were followed. The study protocol was approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (Protocol Number: 1606/11-04-2018).

Two groups, each of five Swiss Albino mice (male, 25 ± 3 g), were injected with the HPLC purified ^99mTc complexes, ^99mTc–Cys–FA11 and ^99mTc–Cys–FA16, (0.1 mL, 1–2 μCi) via the tail vein. The animals were sacrificed by cardiectomy under slight ether anaesthesia at predetermined times (1 and 15 min). The organs of interest were excised, weighed and counted in an automatic gamma counter. Bladder and excreted urine were not weighed. The stomach and intestines were not emptied of food contents prior to radioactivity measurements. The percentage of injected dose per organ (%ID/organ) was calculated by comparison of sample radioactivity to standard solutions containing 10% of the injected dose. The calculation for blood and muscle was based on measured activity, sample weight and body composition data (considering that blood and muscle comprise 7% and 43% of body weight). The percentage of injected dose per gram (%ID/g) was calculated by dividing the %ID/organ by the weight of the organ or tissue. All the animal experiments were carried out in compliance with the relevant national laws relating to the conduct of animal experimentation.

3.11. Metabolites Study

Swiss albino mice were injected with the HPLC purified ^99mTc complexes, ^99mTc–Cys–FA11 and ^99mTc–Cys–FA16, (0.1 mL, 1–2 mCi) via the tail vein, and liver tissue and urine samples were collected at 15 min post-injection. Liver samples were homogenized in a 2:1:1 mixture of CHCl₃, CH₃OH, and 0.001 N NaOH in an automatic homogenizer (Ultra Turrax T25, Janke and Kunkel, IKA-Labortechnik, Staufen, Germany) for 5 min and centrifuged at 14,000 rpm for 30 min [38,47]. The organic and aqueous fractions and residual tissue pellets were counted. Aqueous fractions were filtered through a membrane filter (Millipore, 0.22 μm) and were subsequently analysed by RP-HPLC. Urine samples were analysed by RP-HPLC, after filtration of membrane filters (Millipore, 0.22 μm). The membrane filters were also counted after filtration.

4. Conclusions

In conclusion, the tridentate NSO cysteine ligand was conjugated with an undecanoic and a hexadecanoic acid, to produce two derivatives which coordinated with the fac-[M(CO)₃]⁺ (M = Re, ^99mTc) core through the amine nitrogen, the thioether sulphur and the carboxylate oxygen of the cysteine moiety, forming neutral complexes of medium lipophilicity. The ^99mTc complexes were stable against transchelation in the presence of cysteine and histidine. The complexes also displayed stability in plasma, and a relatively low binding with plasma proteins. Both complexes displayed heart uptake comparable to that of other ^99mTc-labeled fatty acids. Metabolite analysis indicated that complexes were catabolised through the β-oxidation process. In agreement with the involvement of the complexes in β-oxidation metabolic cycles, the heart retention of complex ^99mTc–Cys–FA16 bearing the longer fatty acid chain was longer compared to ^99mTc–Cys–FA11. Even though the key biodistribution ratios displayed by the complexes precludes their use for the assessment of fatty acid metabolism in the heart, they could serve as a base for structural modifications. In addition, they could be further evaluated as β-oxidation markers in other organs, like the liver.