A Potential Melanoma Tracer: Synthesis, Radiolabeling, and

technetium core25 ([TcdO]3+) and the nitridotechnetium core26 ([TctN]2+) (Chart 1). The synthesis of a new ligand based on the attachment of the BAT s...
0 downloads 0 Views 186KB Size
Download PDF
190

J. Med. Chem. 2000, 43, 190-198

A Potential Melanoma Tracer: Synthesis, Radiolabeling, and Biodistribution in Mice of a New Nitridotechnetium Bis(aminothiol) Derivative Pharmacomodulated by a N-(Diethylaminoethyl)benzamide Philippe Auzeloux,*,† Janine Papon,† El Mostapha Azim,† Miche`le Borel,† Roberto Pasqualini,‡ Annie Veyre,† and Jean-Claude Madelmont*,† INSERM Unite´ 484, rue Montalembert, BP 184, 63005 Clermont-Ferrand, France, and Cis Bio International, BP 32, 91192 Gif-sur-Yvette Cedex, France Received August 17, 1999

Radioiodobenzamides are the best-known agents under study for the diagnosis of cutaneous melanoma and its metastases. We report the synthesis of a new BAT derivative radiopharmaceutical in which radioiodine is replaced by 99m-technetium. The cyclic intermediary methyl 4-[3-(4,4,7,7-tetramethyl-5,6-dithia-2,9-diazacyclodecyl)-2-oxapropyl]benzoate (5) occurred in two different conformations identified by spectroscopic analysis. The final BAT ligand was radiolabeled using the nitridotechnetium core by a ligand-exchange reaction. Two different complexes were purified. After macroscopic 99-technetium synthesis, syn and anti isomers were identified. The global radiochemical yield was over 80%. The biodistribution of these two complexes was evaluated in mice bearing murine B16 melanoma. Extensive liver and kidney uptake was observed, but the benzamide tropism for the tumor was partially preserved. Introduction

Chart 1

The incidence of cutaneous malignant melanoma has been increasing in fair-skinned populations faster than that of any other single cancer, particularly in Australia, North America, and Northern Europe.1-6 As this tumor is also characterized by extensive metastasis, early detection of the primary disease and its metastases is of paramount importance. N-Alkyl-p-iodobenzamides have proved useful for the scintigraphic detection of malignant melanoma and metastases, and they constitute a new class of radiopharmaceuticals.7-10 A phase II clinical trial of [123I]N-(2-diethylaminoethyl)-4-iodobenzamide (123I-BZA; Chart 1) has been successfully completed.11,12 The benzamide uptake mechanism is not yet fully understood and is controversial.13,14 First, many benzamides are known to have a nanomolar affinity for the σ-1 binding site.15,16 This kind of receptor has been found in different normal organs such as the liver, kidney, and brain and also in a wide variety of human tumor cells including malignant melanoma.17 Second, the intracellular melanin seems to be a good target for the radioiodinated benzamides; I-BZA is localized specifically on the pigmented cells, the melanosomes, and in the cytoplasm of tumor cells.18 Concurrently, the uptake of the benzamide on murine melanoma cells (B16/C3) has been correlated with the intracellular melanin synthesis.19,20 The effect of the specific activity of the 131I-BZA on its biodistribution in mice has also been investigated.21 An increase in the melanoma uptake was observed when the specific activity of the radioiodinated * Authors for correspondence. Tel: (33) 04 73 26 56 85. Fax: (33) 04 73 27 36 29. E-mail: [email protected] or [email protected]. † INSERM Unite ´ 484. ‡ Cis Bio International.

benzamide was decreased. These results suggest a nonreceptor binding uptake mechanism. Owing to its physical characteristics (t1/2 ) 6 h and γ ) 140 keV) and accessibility (99mTc is produced from a 99Mo generator system) 99m-technetium has become the most commonly used radionuclide for single photon emission computed tomography (SPECT) applications.22,23 For scintigraphic applications, the replacement of radioiodine 123I by 99m-technetium had to be developed. However, the metallic nature of technetium compelled us to use a ligand structure to form a stable complex. The conjugate approach consists of attaching a ligand to small technetium-labeled pharmaceutical molecules. Recent results using this approach have been encouraging.22,24 In our study a bis(aminoethanethiol) (BAT) derivative ligand was chosen, first because of the high stability of the corresponding 99mTc complex and second because of the possibility of complexing both the oxotechnetium core25 ([TcdO]3+) and the nitridotechnetium core26 ([TctN]2+) (Chart 1). The synthesis of a new ligand based on the attachment of the BAT structure to N-(diethylaminoethyl)benzamide is reported here. 99mTc and 99Tc radiolabeling, followed by biodistribution

10.1021/jm981089a CCC: $19.00 © 2000 American Chemical Society Published on Web 12/30/1999

A Potential Melanoma Tracer

Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2 191

Scheme 1

of the nitridotechnetium complexes on mice bearing murine B16 melanoma, is also discussed. Results and Discussion Synthesis of Ligand. Ethyl 3,3,10,10-tetramethyl1,2-dithia-5,8-diazacyclodeca-4,8-diene-6-carboxylate (1) was chosen as precursor. This C-alkylated 10-membered ring diimine was synthesized as previously described.27 The first step was the selective reduction of the ester group (Scheme 1), achieved using lithium aluminum hydride in cold ether (0 °C). The corresponding alcohol 2 was obtained in nearly quantitative yield (94%) with no reduction of the two imine functions. Sodium hydride and methyl 4-(bromomethyl)benzoate were used to introduce the aromatic ring link by an ether function (compound 3). The reactivity of the primary alcoholate

was found to be very weak as indicated by the low kinetics of substitution. Also, replacement of the bromo by the chloro derivative left the unreacted starting compounds. Two routes, A and B, leading to the same molecule 6 were investigated. For route A we first introduced the corresponding diamine to obtain the desired benzamide 4. Trimethylaluminum was used to convert the ester into an amide because of its high yield and easier purification.28 The second step was a gentle reduction of the two imine functions. It has already been reported that the use of sodium borohydride leads to a bicyclic imidazolidine resulting from a transannular cyclization.29,30 Sodium cyanoborohydride has been proposed as a mild reducing agent.31 This reaction took place in acid conditions, and reduction of 4 yielded the cyclic diamine 6. However, the NMR characterization

192

Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2

Auzeloux et al.

Figure 1. Partial 1H NMR spectrum of compound 5 in CDCl3.

of this compound was difficult. To make a complete NMR assignment and confirm the chemical structure, we took the second route B. Compound 5 was first obtained by similar reduction of 3 using sodium cyanoborohydride. In a second step, the ester 5 was converted into benzamide 6 with N,N-diethylethylenediamine and trimethylaluminum in nearly quantitative yield. Both 1H and 13C NMR spectra revealed two components, both consistent with the expected cyclic diamine structure. For instance, the four -CH3 signals of the BAT structure appeared as eight singlets (in both 1H and 13C spectra). The two proposed correlations for each component on the 1H and COSY spectra are given in Figures 1 and 2. A constant major/minor component ratio was observed (55/45 at 296 K). This might be explained in either of two ways. First, protonation of the nitrogen atoms may occur, owing to the acidity of the deuteriochloroform. This explanation is supported by the fact that the addition of trifluoroacetic acid to the sample measurably changed the ratio of the two components in favor of the major form. However, the two NMR forms were also observed in DMSO-d6. Second, the two components may be chemical conformations of the same molecule, resulting in two possible intramolecular hydrogen bonds. Unfortunately, the spectral resolution obtained in DMSO-d6 was not high enough compared with that obtained in deuteriochloroform. However, by increasing the temperature of the DMSO-d6 sample, an alteration in the spectrum in favor of the major product was obtained. A similar spectrum was recorded for compound 6. Table 1 gives the NMR data obtained for 5 (two forms) and 6 (two forms) (BAT variable area only). The last reaction, the selective disulfide bond reduction, yielded the desired ligand. Usual reducing agents such as lithium aluminum hydride used for BAT synthesis32,33 could not be employed. The use of dithiothreitol (DTT), a selective reducing compound,34 afforded the desired ligand, but the yields were very low and the purification was difficult due to thiol reactivity (the final compound reacted quickly to

reform the starting cyclic compound). A better result was obtained using tris(2-carboxyethyl)phosphine (TCEP), a hydrosoluble phosphine. In acid media, this agent is able to reduce the disulfur bond (Scheme 1). By this reaction pathway, the final dithiol 7 was isolated in 84% yield. 99mTc Labeling. 99mTc radiolabeling of ligand 7 was performed in two steps by an already published procedure:35-37 First the [TctN]VI nitridotechnetium core was obtained by acid reduction of the [99mTc]pertechnetate(VII) solution using triphenylphosphine in the presence of N-methyl-S-methyldithiocarbazate (MDTCZ, the nitrogen donor). The ligand-exchange reaction then successfully took place in basic conditions (Scheme 2). Two isomers (syn and anti, approximately 1:1 ratio) resulting from the nitrogen-technetium bond position were separated by thin-layer chromatography (Figure 3) and HPLC (Figure 4). The radiochemical yield was >40% for each complex, and the radiochemical purity obtained was >92%. No significant degradation was noted in 24 h. The partition coefficient was measured in octanol and pH 7.4 Tris buffer. Both complexes were hydrophilic, the syn isomer (first eluted) showing the higher log P (-0.30) and the anti one being slightly more hydrophilic (-0.45). 99Tc Labeling. We prepared analogous 99Tc complexes to verify the chemical structure of the two 99mTc isomers. The synthesis was achieved using the literature procedure (Scheme 2).38 Compound 7 was successfully radiolabeled with 36-37% radiochemical yield for each isomer. The same eluting profile as the 99mTc-labeled molecule was obtained by TLC (alumina, CH2Cl2/MeOH, 94/6). The syn and anti complexes were purified by preparative chromatography, and final products could not be crystallized. NMR experiments were performed (1H, 13C, COSY, and HETCOR experiments) on a 200MHz instrument. The complete characterization of the TcO-bis(aminothio)phenylpiperidine complexes (TcOBAT-PPP) by NMR and X-ray crystallographic experiments has been published elsewhere.39 Both syn and anti isomeric complexes were identified. NMR similari-

A Potential Melanoma Tracer

Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2 193

Figure 2. Partial COSY spectrum of compound 5: major product correlations (upper) and minor product correlations (lower).

194

Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2

Table 1. 1H NMR Partial Data for Compounds 5 and 6 compd

δ (ppm)

integrat

mult

assign

5 (minor)

1.21 1.23 1.25 1.40 2.53 2.59 2.65 2.71

1.35 1.35 1.35 1.35 0.45 0.45 0.45 0.45

s s s s d m d dd

7-H 17-H 18-H 8-H 5-H 3-H 2-H 3′-H

2.90 3.03 3.22 3.47

0.45 0.45 0.45 0.90

d m d d

5′-H 4-H 2′-H 9-H

1.19 1.24 1.32 1.41 2.36 2.57 2.59 2.80 2.82 3.05 3.06 3.40

1.65 1.65 1.65 1.65 0.55 0.55 0.55 0.55 0.55 0.55 0.55 1.10

s s s s d m m d m m m m

7-H 17-H 18-H 8-H 2-H 3-H 5-H 2′-H 3′-H 5′-H 4-H 9-H

1.17 1.19 1.21 1.36 2.49 2.54 2.61 2.66 2.84 2.99 3.16 3.42

1.35 1.35 1.35 1.35 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.90

s s s s d m m m d m d m

7-H 20-H 21-H 8-H 5-H 3-H 2-H 3′-H 5′-H 4-H 2′-H 9-H

1.15 1.19 1.28 1.36 2.29 2.50 2.53 2.76 2.77 3.01 3.03 3.40

1.65 1.65 1.65 1.65 0.55 0.55 0.55 0.55 0.55 0.55 0.55 1.10

s s s s d m m m d m m m

20-H 7-H 8-H 21-H 2-H 3-H 5-H 3′-H 2′-H 5′-H 4-H 9-H

5 (major)

6 (minor)

6 (major)

J (Hz)

12.8 (5-5′) 12.6 (2-2′) 12.6 (3′-3), 5.1 (3′-4) 12.8 (5′-5) 12.6 (2′-2) 5.9

12.6 (2-2′) 12.6 (2′-2)

12.6 (5-5′)

12.6 (5′-5) 12.2 (2′-2)

12.1 (2-2′)

12.1 (2′-2)

Scheme 2

Auzeloux et al.

noted a -0.84 ppm shift difference for the 4-1H signal between the syn and anti isomers (respectively -1.14 ppm for the two TcO-BAT-PPP isomers). The shift difference for the 11-13C signal was +5.6 ppm (respectively +9.2 ppm). The infrared spectra indicated an absorption of the TctN core at 1068 cm-1 for the syn isomer and respectively 1069 cm-1 for the anti isomer. These values agree with the reported usual general ν[TctN] infrared absorption and also with values for nitridotechnetium chelate complexes with N2S2 ligands.40-42 Both compounds were stored for several weeks with no noticeable degradation, showing the excellent stability of the nitridotechnetium bis(aminoethane)thiol complex. Biological Results. The tissue biodistribution of the syn isomer 8 and the syn plus anti mixture in the tissues of mice bearing the B16 murine melanoma tumor was determined to evaluate the affinity of the molecules for cancerous lesions. The results in Table 2 are expressed as percent injected dose/gram (% ID/g). The biodistribution of the syn plus anti mixture was similar to that of the syn isomer alone. High concentrations of radioactivity in liver and kidney were noted 5 min postinjection. A rapid clearance was observed for blood and all the tissues. The tumor uptake ranged from 2.63% ID/g at 5 min to 0.43% ID/g at 1 h, also showing a rapid clearance. Owing to their hydrophilic character, the complexes were unable to cross the blood-brain barrier. Table 3 shows a biodistribution comparison of the syn complex 8 isomer, 125I-BZA, and TcN-BAT.10,26 The BAT nitridotechnetium complex was known to have no particular specificity for tumor tissues as indicated by a weak uptake of the B16 and by low tumor/organs ratios.26 For the TcN-BZA-BAT complex, tumor uptake was less than for 125I-BZA. Those different levels of tumor uptake could be due in part to the less lipophilicity of the technetium complex in comparison with 125I-BZA. Actually, best results with iodobenzamides were obtained with a partition coefficient between 1 and 1.5.10 Another possibility might be a modification of the final molecule biokinetics due to the introduction of the technetium chelate. A reduction of the binding to the melanoma by unknown mechanism(s) cannot be excluded. Nevertheless, the tumor/blood ratios suggest that the initial iodobenzamide tropism for the tumor was partially preserved. Conclusion

ties were found for the two oxotechnetium isomers described and our nitridotechnetium complexes. We

In this study, the synthesis, radiolabeling, and biological evaluation of a nitridotechnetium bis(aminoethanethiol) conjugated to a benzamide structure are reported. The complex formation was successfully achieved using the ligand-exchange reaction to afford two isomers, syn and anti, in high yield (g40% for each compound). These two complexes were characterized using high half-time isotope 99-technetium chelation. The high stability of the TcN-BZA-BAT complex enabled us to carry out a study in mice to map the biodistribution and assess the validity of this complex as a melanoma tracer scintigraphic agent. A partial tropism for melanoma was observed. The weak tumor uptake could be explained in part by its hydrophilicity, it being

A Potential Melanoma Tracer

Figure 3. TLC of syn and anti complexes 8: (a) pertechnetate; (b) syn (upper) and anti (lower) isomers; (c) nitridotechnetium intermediary.

Figure 4. HPLC profiles: (a) UV; (b) RA for syn plus anti mixture; (c) syn complex; (d) anti complex.

well-known that iodobenzamides show a typically high tumor uptake for a log P value between 1.0 and 1.5.13 The study has now been extended using the oxotechnetium core to investigate its effect on biological properties (manuscript in preparation). Experimental Section General. Chemistry: All reagents and solvents were from commercial suppliers and used with no further purification.

Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2 195 All reaction solvents were anhydrous. TLC used precoated silica gel plates (SDS, plastic sheet 60 F254, layer thickness 0.25 mm) and aluminum oxide plates (Merck, plastic sheet 60 F254, neutral type E, layer thickness 0.20 mm). Mediumpressure chromatography was on silica gel (SDS, Chromatogel 60A, 40-60 µm). Solvent mixtures are given as volume-tovolume ratios (v:v). Melting points were determined with a Reichert-Jung Kofler apparatus. Infrared spectra were recorded in KBr pellets on a FT Vector 22 Bruker instrument (ν expressed in cm-1). Proton and carbon magnetic resonance spectra (1H and 13C NMR) in CDCl3 were recorded on a Bruker AM 200 (4.7 T) or DRX 500 (11.7 T) instrument. Chemical shifts (δ) are reported in parts per million relative to the internal standard (CH3)4Si or using deuterated chloroform (δ ) 7.26 ppm for 1H NMR and δ ) 77.0 ppm for 13C NMR). Radiochemistry: [99mTc]Sodium pertechnetate as no-carrier-added solution was purchased from the Jean Perrin Cancer Hospital (Clermont-Ferrand, France). All solvents were degassed under argon before use. TLC radioactive spots were scanned and recorded by an AMBIS 4000 detector (a computercontrolled multiwire proportional counter). HPLC purification was performed on a Shimadzu HPLC system (LC6A pump, SCL6B system controller, and CR5A integrator) equipped with a semipreparative reverse-phase column (Merck, Licroprep RP 18, 12 × 200 mm), connected to a Shimadzu SPD6AV UV spectrophotometric detector (254 nm) in series with a Raytest NaI (Tl) gamma detector. NH499TcO4 was obtained from Cis Bio International and purified before use as previously reported.43 Tetraphenylarsonium tetrachloronitridotechnetate [99TcNCl4AsPh4] was prepared by the published procedure in the same yield.38 Synthesis. 6-Hydroxymethyl-3,3,10,10-tetramethyl-1,2dithia-5,8-diazacyclodeca-4,8-diene (2). LiAlH4 in ether (20.0 mL of 1 M solution, 20.0 mmol) was added to a 0 °C solution of 1 (6.05 g, 20.0 mmol) in dry ether (50 mL). The mixture was stirred for 24 h and the reaction was quenched with water (0.76 mL), 15% aq NaOH (0.76 mL), and water (0.76 mL). The white solid obtained was filtered over Celite and dried (MgSO4). The solvent was evaporated under reduced pressure to give desired alcohol 2 as a white solid (4.91 g, 18.8 mmol, 94% yield): Rf 0.47 (silica, CH2Cl2/EtOH 90:10); mp 129 °C; 1H NMR (200 MHz) δ 1.37, 1.42, 1.46 (s, 12H, CH3), 2.20 (br, 1H, OH), 3.06 (dd, J ) 10.0, 9.5 Hz, 1H, 3-CH2), 3.44 (m, 1H, 4-CH), 3.87 (d, J ) 4.7 Hz, 2H, CH2OH), 4.09 (ddd, J ) 9.5, 3.4, 1.4 Hz, 1H, 3′-CH2), 6.88 (d, J ) 1.3 Hz, 1H, 2-CH), 7.01 (s, 1H, 5-CH); 13C NMR (50 MHz) δ 21.2-24.4, 24.6 (4 × CH3), 52.7, 53.0 (1-C and 6-C), 63.3 (3-CH2), 64.3 (CH2OH), 73.0 (4-CH), 167.7 (5-CHdN), 168.1 (2-CHdN); IR ν 3300, 2970-2920, 1630, 1045. Anal. (C11H20N2OS2) C, H, N. Methyl 4-[3-(4,4,7,7-Tetramethyl-5,6-dithia-2,9-diazacyclodeca-2,8-dienyl)-2-oxapropyl]benzoate (3). In a threenecked round-bottomed flask under argon atmosphere was added NaH (524 mg, 13.0 mmol) in THF (20 mL); 2.84 g of alcohol 2 (10.9 mmol) in THF (5 mL) was added dropwise to this suspension. The mixture was stirred for 30 min and methyl 4-(bromomethyl)benzoate (2.80 g, 12.0 mmol) in THF (5 mL) was added dropwise. The reaction was left overnight at 60 °C. The resulting mixture was quenched with water (80 mL) and extracted with ether (3 × 80 mL). The combined organic layers were washed with saturated NaCl solution, dried (MgSO4), and concentrated to give the pure ester 3 (4.35 g, 10.6 mmol): colorless oil; yield 97%; Rf 0.62 (silica, CH2Cl2/ EtOH 90:10); 1H NMR (200 MHz) δ 1.33, 1.42 (s, 12H, 2 × C(CH3)2), 2.89 (t, J ) 9.9 Hz, 1H, 3-CH2), 3.51 (m, 1H, 4-CH), 3.75 (m, 2H, 9-CH2), 3.89 (s, 3H, 16-CH3), 4.20 (ddd, J ) 9.9, 3.5, 1.4 Hz, 1H, 3-CH2), 4.61 (s, 2H, 10-CH2), 6.87 (d, J ) 1.4 Hz, 1H, 2-CH), 6.96 (s, 1H, 5-CH), 7.37 (d, J ) 8.3 Hz, 2H, 12-CH), 7.98 (d, J ) 8.3 Hz, 2H, 13-CH); 13C NMR (50 MHz) δ 21.2-24.4, 24.6 (2 × C(CH3)2), 52.0 (16-CH3), 52.7 and 52.9 (2C, 2 × C(CH3)2), 63.8 (3-CH2), 70.8, 72.3 and 72.4 (4-CH, CH2OCH2), 126.9 (12-C), 129.2 (14-C), 129.6 (13-C), 143.7 (11C), 166.8 (5-CH), 168.1 (2-CH). Anal. (C20H28N2O3S2) C, H, N. N-Diethylaminoethyl-4-[3-(4,4,7,7-tetramethyl5,6-dithia-2,9-diazacyclodeca-2,8-dienyl)-2-oxapropyl]benz-

196

Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2

Table 2. Biodistribution of

99mTc-Labeled

Auzeloux et al.

Complex 8 in Micea % ID/g syn + anti

syn

a

tissue

5 min

1h

6h

5 min

1h

6h

blood liver kidney lung muscle brain eyes heart tumor

4.72 ( 0.32 25.23 ( 0.32 19.52 ( 0.71 6.26 ( 0.39 1.02 ( 0.15 0.16 ( 0.01 0.80 ( 0.10 1.90 ( 0.20 2.63 ( 0.18

0.14 ( 0.01 3.95 ( 0.40 2.55 ( 0.58 2.03 ( 0.24 0.04 ( 0.00 0.02 ( 0.00 0.20 ( 0.01 0.12 ( 0.01 0.43 ( 0.09

0.04 ( 0.00 1.97 ( 0.02 1.30 ( 0.17 1.21 ( 0.27 0.02 ( 0.00 0.01 ( 0.00 0.14 ( 0.01 0.06 ( 0.00 0.14 ( 0.02

5.11 ( 0.50 28.47 ( 5.52 17.54 ( 1.57 4.52 ( 0.49 0.88 ( 0.16 0.12 ( 0.01 0.71 ( 0.07 1.82 ( 0.20 3.55 ( 0.63

0.32 ( 0.05 6.16 ( 0.78 3.58 ( 0.61 1.26 ( 0.10 0.07 ( 0.01 0.02 ( 0.00 0.26 ( 0.01 0.24 ( 0.06 0.43 ( 0.03

0.11 ( 0.00 5.59 ( 1.14 2.80 ( 1.11 0.81 ( 0.10 0.05 ( 0.00 0.01 ( 0.00 0.26 ( 0.02 0.19 ( 0.07 0.18 ( 0.03

Tumor weight: 185 ( 121 mg.

Table 3. Biodistributions in Mice: Comparison between 99mTc-8 (Syn), 125I-BZA, and 99mTcN-BAT time after inject (h)

tumor (% ID/g)

TcN-BZA-BAT

1 3 6 24

0.43 ( 0.16 0.31 ( 0.10 0.14 ( 0.04 0.13 ( 0.15

3.07 ( 0.94 6.49 ( 2.82 4.97 ( 0.46 2.80 ( 0.41

0.12 ( 0.06 0.13 ( 0.04 0.07 ( 0.02 0.16 ( 0.18

125I-BZA

1 3 6 24

6.75 ( 0.67 3.85 ( 0.37 3.53 ( 0.31 0.79 ( 0.09

6.49 ( 0.65 7.26 ( 1.09 16.8 ( 1.50 39.5 ( 5.50

1.12 ( 0.10 1.05 ( 0.19 3.71 ( 0.35 4.94 ( 0.70

TcN-BAT

1 3 6 24

0.75 ( 0.18 0.20 ( 0.03 0.11 ( 0.01 0.04 ( 0.01

0.72 ( 0.09 0.69 ( 0.09 1.05 ( 0.19 0.68 ( 0.10

0.18 ( 0.07 0.10 ( 0.04 0.10 ( 0.03 0.06 ( 0.02

compd

tumor/blood tumor/liver ratio ratio

amide (4). N,N-Diethylethylenediamine (220 mg, 1.89 mmol) was diluted with CH2Cl2 (10 mL) in a three-necked, roundbottomed flask equipped with a reflux condenser under argon atmosphere. The solution was stirred and cooled in an ice bath at 0 °C and trimethylaluminum in hexane (1.13 mL, 2 M solution, 2.26 mmol) was slowly added. Fifteen minutes after complete addition, the cooling bath was removed and ester 3 (643 mg, 1.57 mmol) in CH2Cl2 (5 mL) was added. The resulting solution was heated under reflux for 40 h, cooled to room temperature, and slowly hydrolyzed with water to prevent foam formation. The mixture was extracted three times with CH2Cl2 and the organic layers were combined and dried (MgSO4). The solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography (aluminum oxide, ethyl acetate/EtOH 95:5) to afford 475 mg of benzamide 4 (0.96 mmol): yellowish oil; yield 61%; TLC Rf 0.30 (aluminum oxide, ethyl acetate/EtOH 95:5); 1H NMR (200 MHz) δ 1.05 (t, J ) 7.1 Hz, 6H, 19-CH3), 1.36, 1.45 (s, 12H, 2 × C(CH3)2), 2.58 (q, J ) 7.1 Hz, 4H, 18-H), 2.67 (t, J ) 5.1 Hz, 2H, 17-H), 2.90 (t, J ) 9.9 Hz, 1H, 3-H), 3.45 (m, 1H, 4-H), 3.50 (m, 4H, 9-H and 16-H), 4.22 (ddd, J ) 9.9, 3.3, 1.5 Hz, 3′-H), 4.61 (s, 2H, 10-H), 6.89 (d, J ) 1.4 Hz, 1H, 2-H), 6.98 (m, 2H, NH and 5-H); 7.39 (d, J ) 8.3 Hz, 2H, 12-H), 7.76 (d, J ) 8.3 Hz, 2H, 13-H); 13C NMR δ 10.1 (19-C), 21.0, 22.0, 24.2, 24.4 (2 × C(CH3)2), 36.1 (16-C), 47.4 (18-C), 51.9 (17-C), 52.5, 52.7 (1-C and 6-C), 63.8 (3-C), 71.7, 72.0, 72.3 (4-C, 9-C and 10-C), 127.0, 127.1 (12-C and 13-C), 132.9 (14C), 141.9 (11-C), 166.6 (5-CHdN), 167.0 (CdO), 167.8 (2-CHd N). Anal. (C25H40N4O2S2) C, H, N. Methyl 4-[3-(4,4,7,7-Tetramethyl-5,6-dithia-2,9-diazacyclodecyl)-2-oxapropyl]benzoate (5). Compound 3 (3.74 g, 9.15 mmol) in absolute ethanol (20 mL) was placed in a three-necked round-bottomed flask under argon atmosphere. The solution was stirred and cooled in an ice bath at 0 °C and NaBH3CN (687 mg, 10.9 mmol) in ethanol (5 mL) was slowly added. Ether/HCl (2 M, 10 mL) was added and the mixture was stirred 4 h at room temperature. The solution was quenched with water (5 mL) and 1 M aq NaOH (20 mL). The organic solvents were evaporated under reduced pressure. Three CH2Cl2 extractions afforded crude 5 (3.05 g), purified

by flash chromatography, ethyl acetate/EtOH/Et3N 93:5:2 (2.14 g, 5.19 mmol, 57% yield): colorless oil; TLC Rf 0.63 (aluminum oxide, CH2Cl2/MeOH 95:5); IR ν 3340, 3260, 2950-2850, 1710, 1600, 1460, 1420, 1270, 1100; EI/MS m/z 116.1 (100), 149.1 (40), 262.2 (19), 412.3 (M+, 14); major form: 1H NMR (500 MHz) δ 1.19, 1.41 (s, 6H, 7-H and 8-H), 1.24, 1.32 (s, 6H, 17-H and 18-H), 2.36 (d, J ) 12.6 Hz, 1H, 2-H), 2.57 (m, 1H, 3-H), 2.59 (m, 1H, 5-H), 2.80 (m, 1H, 2′-H), 2.82 (m, 1H and 3′-H), 3.05 (m, 1H, 5′-H), 3.06 (m, 1H, 4-H), 3.36-3.43 (m, 2H, 9-H), 3.90 (s, 3H, 16-H), 4.56 (s, 2H, 10-H), 7.37 (d, J ) 8.2 Hz, 2H, 12H), 8.00 (d, J ) 8.2 Hz, 2H, 13-H); 13C NMR δ 25.0, 28.7 (17C, 18-C), 26.8, 27.5 (7-C, 8-C), 47.7 (3-C), 51.6, 51.7 (1-C and 6-C), 52.1 (16-C), 56.1 (4-C), 72.6 (10-C), 72.9 (9-C), 127.1 (12C), 129.5 (14-C), 129.7 (13-C), 143.7 (11-C), 166.9 (15-CdO); minor form: 1H NMR (500 MHz) δ 1.21, 1.40 (s, 6H, 7-H and 8-H), 1.23, 1.25 (s, 6H, 17-H and 18-H), 2.53 (d, J ) 12.8 Hz, 1H, 5-H), 2.59 (m, 1H, 3-H), 2.65 (d, J ) 12.6 Hz, 1H, 2-H), 2.71 (dd, J ) 12.6, 5.1 Hz, 1H, 3′-H), 2.90 (d, J ) 12.8 Hz, 1H, 5′-H), 3.03 (mu, 1H, 4-H), 3.22 (d, J ) 12.6 Hz, 1H, 2′-H), 3.47 (d, J ) 5.9 Hz, 2H, 9-H), 3.90 (s, 3H, 16-H), 4.56 (s, 2H, 10-H), 7.37 (d, J ) 8.2 Hz, 2H, 12-H), 8.00 (d, J ) 8.2 Hz, 2H, 13-H); 13C NMR δ 24.1, 29.4 (17-C and 18-C), 27.1, 27.8 (7-C and 8-C), 50.2 (1C, 3-C), 50.7 (1-C), 52.0 (6-C), 52.1 (16-C), 53.8 (5-C), 55.6 (4-C), 63.2 (2-C), 72.6 (10-C), 73.5 (9-C), 127.2 (12-C), 129.4 (14-C), 129.7 (13-C), 143.5 (11-C), 166.9 (15-CdO). Anal. (C20H32N2O3S2) H, N, O; C: calcd, 58.22; found, 57.77. N-Diethylaminoethyl-4-[3-(4,4,7,7-tetramethyl-5,6dithia-2,9-diazacyclodecyl)-2-oxapropyl]benzamide (6). From 4: As synthesis of 5 from 3: 1.30 g, 2.61 mmol, 94% yield. From 5: As synthesis of 4 from 3, followed by chromatographic purification on aluminum oxide, eluting with CH2Cl2/ MeOH 95:5: 1.04 g, 2.09 mmol, 95% yield; yellowish oil; TLC Rf 0.35 (aluminum oxide, CH2Cl2/MeOH 95:5); IR ν 3400-3100, 2950-2800, 1630, 1540, 1380, 1350, 1050; major form: 1H NMR (500 MHz) δ 1.00 (t, J ) 7.1 Hz, 6H, 19-H), 1.15, 1.36 (s, 6H, 20-H and 21-H), 1.19, 1.28 (s, 6H, 7-H and 8-H), 2.0 (br, 2H, NH), 2.29 (d, J ) 12.1 Hz, 1H, 2-H), 2.50 (m, 1H, 3-H), 2.53 (m, 5H, 5-H and 18-H), 2.60 (m, 2H, 17-H), 2.76 (m, 1H, 3′-H), 2.77 (m, 1H, 2′-H), 3.01 (m, 1H, 5′-H), 3.03 (m, 1H, 4-H), 3.35 (m, 2H, 9-H), 3.45 (m, 2H, 16-H), 4.50 (s, 2H, 10-H), 7.06 (m, 1H, CONH), 7.33 (dd, J ) 8.1, 3.8 Hz, 2H, 12-H), 7.73 (d, J ) 8.1 Hz, 2H, 13-H); 13C NMR δ 12.0 (19-C), 24.9, 28.5 (7-C and 8-C), 26.6, 27.4 (20-C and 21-C), 37.3 (16-C), 46.7 (18-C), 48.0 (3-C), 51.2 (17-C), 51.5 (1-C and 6-C), 55.6 (4-C), 73.0 (9-C and 10-C), 127.2 (13-C), 127.7 (12-C), 134.7 (14-C), 140.5 (11C), 166.7 (1C, CdO); minor form: 1H NMR (500 MHz) δ 1.00 (t, J ) 7.1 Hz, 19-H), 1.17, 1.36 (s, 7-H and 8-H), 1.19, 1.21 (s, 6H, 20-H and 21-H), 2.0 (br, 2H, NH), 2.49 (d, J ) 12.6 Hz, 1H, 5-H), 2.53 (q, J ) 7.1 Hz, 4H, 18-H), 2.54 (m, 1H, 3-H), 2.61 (m, 3H, 2-H and 17-H), 2.66 (m, 1H, 3′-H), 2.84 (d, J ) 12.6 Hz, 5′-H), 2.99 (m, 1H, 4-H), 3.16 (d, J ) 12.2 Hz, 1H, 2′-H), 3.42 (m, 2H, 9-H), 3.45 (m, 2H, 16-H), 4.50 (s, 2H, 10H), 7.06 (m, 1H, CONH), 7.33 (dd, J ) 8.1, 3.8 Hz, 2H, 12-H), 7.73 (d, J ) 8.1 Hz, 2H, 13-H); 13C NMR δ 12.0 (19-C), 23.9, 29.4 (20-C and 21-C), 27.2, 27.6 (7-C and 8-C), 37.3 (16-C), 46.7 (18-C), 50.0 (1-C and 3-C), 51.2 (17-C), 52.0 (6-C), 52.3 (5-C), 55.6 (4-C), 63.5 (2-C), 73.0 (9-C and 10-C), 127.2 (13-C),

A Potential Melanoma Tracer 127.7 (12-C), 134.7 (14-C), 140.5 (11-C), 166.7 (15-CdO). Anal. (C25H44N4O2S2) H, N, O; C: calcd, 60.44; found, 59.12. N-Diethylaminoethyl-4-[8-methyl-3-(3-methyl-3-thio-1azabutyl)-8-thio-2,6-oxoazanonyl]benzamide (7). In a reaction vial were placed 6 (249 mg, 0.500 mmol) and tris(2carboxyethyl)phosphine hydrochloride (TCEP; 216 mg, 0.750 mmol) in ethanol (3.0 mL) and water (3.0 mL) under argon atmosphere. The pH was adjusted to 4 with hydrochloric acid. The resulting mixture was stirred at 50 °C for 5 days, by which time no starting disulfide compound was present (TLC). The resulting yellow solution was cooled to room temperature, readjusted to pH 8 with NaOH solution, and extracted under argon atmosphere with CHCl3. The organic layer was dried (MgSO4), filtered, and evaporated to give 210 mg of 7 (0.42 mmol, 84% yield): TLC 0.55 (aluminum oxide, CH2Cl2/MeOH 96:04); IR ν 3300, 2970-2820, 2510, 1651, 1540, 1455, 1305, 1100; 1H NMR (200 MHz) δ 1.12 (t, J ) 7.1 Hz, 6H, 19-H), 1.36 (s, 12H, 2 × C(CH3)2), 2.09 (br, 2H, 2 × NH), 2.53-2.85 (mu, 15H, 2-H, 3-H, 4-H, 5-H, 17-H, 18-H and 2 × SH), 3.52 (d, J ) 5.0 Hz, 2H, 9-H), 3.59 (m, 2H, 16-H), 4.56 (s, 2H, 10H), 7.40 (m, 3H, CONH and 12-H), 7.82 (d, J ) 8.3 Hz, 2H, 13-H); 13C NMR δ 11.8 (19-C), 30.4, 30.6 (2 × C(CH3)2), 37.1 (16-C), 46.9 (18-C), 51.4 (17-C), 51.7 (3-C), 45.5 (1-C and 6-C), 57.7 (4-C), 61.2 (5-C), 63.7 (2-C), 71.9 (10-C), 72.7 (9-C), 127.1 (13-C), 127.4 (12-C), 134.0 (14-C), 141.9 (11-C), 167.0 (15-Cd O). The corresponding hydrochloride was obtained by stirring 7 in Et2O/HCl as a very hygroscopic salt (185 mg, 0.30 mmol, 61% total yield). Radiolabeling with 99mTc. 700 µL of 99mTcO4Na solution (0.37 to 0.74 GBq) was placed in a vial containing N-methylS-methyldithiocarbazate (MDTCZ; 1 mg, 7.3 µmol), triphenylphosphine (1 mg, 3.8 µmol), HCl(aq) (100 µL, 1 M), and water (0.6 mL) under argon atmosphere. The resulting mixture was heated to 70 °C for 30 min and then cooled to room temperature. The pH was adjusted by adding 100 µL of NaOH (1 M) and 900 µL of NaHCO3 buffer (pH 9.4); 1.0 mL of this solution was placed in a second reaction vial containing the ligand (1.0 mg, 1.6 µmol) and 1.0 mL of 95 °C ethanol under argon atmosphere. The resulting solution was heated for another 30 min to 70 °C and the solvents were evaporated under reduced pressure. The residue was dissolved in 1.0 mL of CH2Cl2 before HPLC (5 mL/min). The stereomer retention times were 24.8 and 37.2 min (respectively 36% and 28% yield, radiochemical purity > 92%): syn isomer: TLC 0.34 (aluminum oxide, MeOH/CH2Cl2 6:94), log P ) -0.30; anti isomer: TLC 0.21 (aluminum oxide, MeOH/CH2Cl2 6:94), log P ) -0.45. The HPLC solvents were evaporated under reduced pressure and the residue was dissolved in Tri buffer (3.7 MBq/mL final activity) for biological studies. Radiolabeling with 99Tc. Tetraphenylarsonium tetrachloronitridotechnetate(VI) (47.7 mg, 74.6 µmol) was added to a solution of base ligand 7 (105 mg, 211 µmol) in MeOH (1.7 mL) and MeCN (2.0 mL) under argon atmosphere. This brown mixture rapidly turned yellow. The reaction was stopped when no further evolution was noted by TLC (less than 1 h). The yellow residue collected after evaporation to dryness was purified by chromatography on aluminum oxide gel, eluting with 0-2% MeOH/CH2Cl2: syn isomer: 9.8 mg, 16 µmol, 21% yield (contain 11% of anti); TLC 0.35 (aluminum oxide, MeOH/ CH2Cl2 6:94); IR ν 3450-3200, 2960-2850, 1650, 1540, 1457, 1261, 1090, 1068 (TctN), 1020, 802; 1H NMR (200 MHz, CD3OD) δ 1.19 (t, J ) 7.2 Hz, 6H, 2 × CH2CH3), 1.30, 1.38 (s, 12H, 4 × CH3), 2.15 (d, J ) 11.0 Hz, 2H, 2-H, 5-H), 2.74 (d, J ) 11.0 Hz, 1H, 5′-H), 2.81 (d, J ) 11.0 Hz, 1H, 2′-H), 2.88 (m, J ) 13.0 Hz, 1H, 4-H), 2.92 (m, 1H, 3-H), 3.05 (q, J ) 7.2 Hz, 4H, 2 × CH2CH3), 3.10 (m, 2H, CH2NEt2), 3.40 (dd, J ) 13.0, 7.8 Hz, 1H, 3′-H), 3.60 (t, J ) 6.7 Hz, 2H, CONHCH2), 3.65 (m, 2H, 9-H), 4.56 (s, 2H, 10-H), 7.42 (d, J ) 8.2 Hz, 2H, Harom), 7.68 (d, J ) 8.2 Hz, 2H, Harom); 13C NMR (50 MHz, CD3OD) δ 9.9 (2C, 19-C), 29.9-30.1 (4C, 7-C, 8-C, 20-C, 21-C), 36.9 (1C, 16-C), 48.9 (2C, 18-C), 52.4 (2C, 1-C, 6-C), 52.8 (1C, 17-C), 56.9, 67.9, 69.2 (3C, 2-C, 3-C, 5-C), 64.4 (1C, 4-C), 67.7 (1C, 9-C), 73.7 (1C, 10-C), 128.7, 129.1, 135.2, 143.4 (6C, Carom), 168.2 (1C, CdO); anti isomer: 6.9 mg, 11 µmol, 15% yield (contain

Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2 197 28% of syn); TLC 0.21 (same conditions); IR ν 3450-3200, 2960-2850, 1650, 1540, 1457, 1261, 1090, 1069 (TctN), 1020, 802; 1H NMR (200 MHz, CD3OD) δ 1.15 (t, J ) 7.3 Hz, 6H, 2 × CH2CH3), 1.31-1.39 (s, 12H, 4 × CH3), 2.15 (m, 2H, 2-H, 5-H); 2.48 (t, J ) 13.0 Hz, 1H, 3-H), 2.65 (d, J ) 10.8 Hz, 1H, 5′-H), 2.86 (d, J ) 11.0 Hz, 1H, 2′-H), 2.97 (q, J ) 7.3 Hz, 4H, 2 × CH2CH3), 3.05 (m, 2H, CH2NEt2), 3.08 (m, 1H, 3-H), 3.56 (t, J ) 6.2 Hz, 2H, CONHCH2), 3.65 (m, 2H, 9-H), 3.72 (m, 1H, 4-H), 4.52 (s, 2H, 10-H), 7.39 (d, J ) 8.7 Hz, 2H, Harom), 7.77 (d, J ) 8.7 Hz, 2H, Harom); 13C NMR (50 MHz, CD3OD) δ 10.1 (2C, 19-C), 28.8-30.4 (4C, 7-C, 8-C, 20-C, 21-C), 37.1 (1C, 16-C), 48.8 (2C, 18-C), 52.1 (2C, 1-C, 6-C), 52.8 (1C, 17-C), 54.9, 67.9, 69.8 (3C, 2-C, 3-C, 5-C), 62.1 (1C, 9-C), 63.8 (1C, 4-C), 73.5 (1C, 10-C), 128.6, 129.0, 135.2, 143.4 (6C, Carom), 169.0 (1C, CdO). Biological Evaluation. Tumor uptake was studied in C57BL/6 J1 co male mice bearing the B16 murine melanoma. Transplantable B16 mouse melanotic melanoma was originally obtained from ICIG (Villejuif, France); 5 × 105 viable cells were injected subcutaneously. Ten days later, the tumors became palpable. Following the intravenous injection in the tail vein of 0.74 MBq 99mTc-labeled complex, mice (n ) 3) were sacrificed by exsanguination after set time intervals of 5 min, 15 min, 1, 3, 6, and 24 h. Aliquots of different tissues were weighed and radioactivity was measured. Samples were counted in a γ-counter (Packard Autogamma A 5530). The fractional accumulation of radioactivity in the tissue was expressed as % injected dose/gram of tissue (% ID/g).

Acknowledgment. This work was supported by a grant from the Association pour la Recherche sur le Cancer (ARC). We also thank Cis Bio International for its financial support. References (1) Osterlind, A. Epidemiology on malignant melanoma in Europe. Rev. Oncol. 1992, 5, 903. (2) Burton, R. C.; Coates, M. S.; Hersey, P.; Roberts, G.; Chetty, P. P.; Chen, S.; Hayes, M. H.; Howe, C. G.; Armstrong, B. K. An analysis of a melanoma epidemic. Int. J. Cancer 1993, 55, 765770. (3) Hill, C.; Koscielny, S.; Doyon, F.; Benhamou, E. Evolution de la mortalite´ par cancer en France 1950-1990; Les e´ditions INSERM: Paris, 1993. (4) Mackie, R. M. The march of melanoma. Clin. Exp. Dermatol. 1993, 18, 2. (5) Boyle, P.; Maisonneuve, P.; Dore´, J. F. Epidemiology of malignant melanoma. Br. Med. Bull. 1995, 51, 523-547. (6) Hill, C.; Doyon, F. La mortalite´ par cancer en France. Med. Sci. 1997, 13, 172-175. (7) Michelot, J. M.; Moreau, M. F. C.; Labarre, P. G.; Madelmont, J. C.; Veyre, A. J.; Papon, J. M.; Parry, D. F.; Bonafous, J. F.; Boire, J. Y. P.; Desplanches, G. G.; Bertrand, S. J.; Meyniel, G. Synthesis and evaluation of new iodine-125 radiopharmaceuticals as potential tracers for malignant melanoma. J. Nucl. Med. 1991, 32, 1573-1580. (8) Moreau, M. F.; Michelot, J.; Veyre, A.; Madelmont, J. C.; Godene`che, D.; Labarre, P.; Parry, D.; Meyniel, G. Agents pour le diagnostic et le traitement des me´lanomes, de´rive´s haloge´ne´s aromatiques utilisables comme de tels agents et leur pre´paration. French patent 890 1898, Feb 14, 1989. (9) Moreau, M. F.; Madelmont, J. C.; Michelot, J.; Labarre, P.; Veyre, A.; Papon, J.; Bayle, M.; Boire, J. Y.; Desplanches, G.; Meyniel, G. New 125I-radiopharmaceuticals for diagnosis and treatment for malignant melanoma. Eur. J. Nucl. Med. 1993, 18, 538. (10) Moreau, M. F.; Michelot, J.; Papon, J.; Bayle, M.; Labarre, P.; Madelmont, J. C.; Parry, D.; Boire, J. Y.; Seguin, H.; Veyre, A.; Mauclaire, L. Synthesis, radiolabeling, and preliminary evaluation in mice of some (N-diethylaminoethyl)-4-iodobenzamide derivatives as melanoma imaging agents. Nucl. Med. Biol. 1995, 22, 737-747. (11) Michelot, J.; Veyre, A.; Bonafous, J.; Moreau, M. F.; Madelmont, J. C.; Papon, J.; Labarre, P.; Bacin, F.; Kauffman, P.; Plagne, R. Imaging of malignant melanoma and metastases with 123IBZA. Melanoma Res. 1993, 3, 83. (12) Michelot, J. M.; Moreau, M. F. C.; Veyre, A. J.; Bonafous, J. F.; Bacin, F. J.; Madelmont, J. C.; Bussiere, F.; Souteyrand, P. A.; Mauclaire, L. P.; Chossat, F. M.; Papon, J. M.; Labarre, P. G.; Kauffman, Ph.; Plagne, R. J. Phase II scintigraphic clinic trial of malignant melanoma and metastases with iodine-123-N-(2diethylaminoethyl-4-iodobenzamide). J. Nucl. Med. 1993, 34, 1260-1266.

198

Journal of Medicinal Chemistry, 2000, Vol. 43, No. 2

(13) Nicholl, C.; Mohammed, A.; Hull, W. E.; Bubeck, B.; Eisenhut, M. Pharmacokinetics of iodine-123-IMBA for melanoma imaging. J. Nucl. Med. 1997, 38, 127-133. (14) John, C. S.; Lim, B. B.; Vilner, B. J.; Geyer, B. C.; Bowen, W. D. Substitued halogenated arylsulfonamides: a new class of σ receptor binding tumor imaging agents. J. Med. Chem. 1998, 41, 2445-2450. (15) John, C. S.; Bowen, W. D.; Saga, T.; Kinuya, S.; Vilner, B. J.; Baumgold, J.; Paik, C. H.; Reba, R. C.; Neuman, R. D.; Varma, V. M.; McAfee, J. G. A malignant melanoma imaging agent: synthesis, characterization, in vitro binding and biodistribution of iodine-125-(2-piperidinylaminoethyl)4-iodobenzamide. J. Nucl. Med. 1993, 34, 2169-2175. (16) Efange, S. M.; Michelson, R. H.; Knusel, B.; Hefti, F.; Boudreau, R. J.; Thomas, J. R.; Tennison, J. R. Synthesis and biological evaluation of radioiodinated N-2-(4-piperidyl)ethyl benzamides. Nucl. Med. Biol. 1993, 20, 527-538. (17) Vilner, B. J.; John, C. S.; Bowen, W. D. Sigma-1 and sigma-2 receptors are expressed in a wide variety of human and rodent tumor cell lines. Cancer Res. 1995, 55, 408-413. (18) Chehade, F.; Michelot, J.; Hindie, E.; Papon, J.; DelabriolleVaylet, C.; Zhang, L.; Escaig, F.; Moreau, M. F.; Veyre, A. Localization of N-(2-diethylaminoethyl)-4-iodobenzamide in the pigmented mouse eye: a microanalytical study. Cell. Mol. Biol. 1996, 42, 343-350. (19) Coenen, H. H.; Brandau, W.; Dittman, H.; Dutschka, K.; Niehoff, T.; Pulawski, P.; Zo¨lzer, P.; Sciuk, J.; Streffer, C. Evaluation of melanoma seeking N-(dialkylamino)-alkyl-[123, 131I]iodobenzamides by animal and cell-culture studies. J. Labelled Compd. Radiopharm. 1995, 37, 260-262. (20) Dittman, H.; Coenen, H. H.; Zo¨lzer, P.; Dutschka, K.; Brandau, W.; Streffer, C. In vitro studies on the cellular uptake of melanoma imaging aminoalkyl-iodobenzamide derivatives (ABA). Nucl. Med. Biol. 1999, 26, 51-56. (21) Nicholl, C.; Mohammed, A.; Eisenhut, M. Dialkylaminoalkyl-4iodobenzamides: Influence of specific activity and substituents on melanoma uptake and biodistribution. J. Labelled Compd. Radiopharm. 1995, 37, 277-279. (22) Jurisson, S. S.; Lydon, J. D. Potential technetium small molecule radiopharmaceuticals. Chem. Rev. 1999, 99, 2205-2218. (23) Liu, S.; Edwards, D. S. 99mTc-labeled small peptides as diagnostic radiopharmaceuticals. Chem. Rev. 1999, 99, 2235-2268. (24) Hom, R. K.; Katzenellengogen, J. A. Technetium-99m-labeled receptor-specific small-molecule radiopharmaceuticals: recent developments and encouraging results. Nucl. Med. Biol. 1997, 24, 485-498. (25) Epps, L. A.; Kramer, A. V.; Burns, H. D.; Zemyan, S. E.; Dannals, R. F.; Goldfarb, H.; Ravert, H. Synthesis and characterization of a neutral oxotechnetium(V)diaminothiol complex. J. Nucl. Med. 1983, 24, P10. (26) Borel, M.; Rapp, M.; Pasqualini, R.; Madelmont, J. C.; Godene`che, D.; Veyre, A. Synthesis of potential 99mTc nitrido tumor imaging disposition in mice. Appl. Radiat. Isot. 1992, 43, 425-436. (27) Kung, H. F.; Yu, C.-C.; Billings, J.; Molnar, M.; Blau, M. Synthesis of new bis(aminoethanethiol) (BAT) derivatives: possible ligands for 99mTc brain agents. J. Med. Chem. 1985, 28, 1280-1284. (28) Lipton, M. F.; Basha, A.; Weinreb, M. Conversion of esters to amides with dimethylaluminium amides: N,N-dimethylcyclohexanecarboxamide. Organic syntheses; John Wiley & Sons: New York, 1988; Vol. 6, pp 492-495. (29) Joshua, A. V.; Scott, J. R.; Sondhi, S. M.; Ball, R. G.; Lown, J. W. Transannular cyclisation of 1,2-dithia-5,8-diazacyclodeca-4,8dienes during borohydride reduction. J. Org. Chem. 1987, 52, 2447-2451. (30) Lever, S. Z.; Sun, S.-Y.; Kaltovich, F.; Scheffel, U.; Goldfarb, H.; Mahmood, A.; Baidoo, K. E.; Wagner, H. N. Jr. Pulmonary accumulation of neutral alkyl-DADT-technetium complexes: Structure-biodistribution relationships. J. Nucl. Med. 1988, 5, 789.

Auzeloux et al. (31) Shiba, K.; Mori, H.; Matsuda, H.; Tsuji, S.; Kinuya, K.; Hisada, K.; Synthesis of technetium-99m labeled diaminodithiol for bifunctional chelating agent. Appl. Radiat. Isot. 1991, 42, 11591164. (32) Kung, H. F.; Liu, B. L.; Pan, S. Kinetic study of ligand exchange reaction between 99mTc-glucoheptonate and N-benzyl-N-methylpiperazinyl-bis-(aminoethanethiol) (BPA-BAT). Appl. Radiat. Isot. 1989, 40, 677-681. (33) Mach, R. H.; Kung, H. F.; Guo, Y. Z.; Yu, C. C.; Subramanyam, V.; Calabrese, J. C. Synthesis, characterization and biodistribution of neutral and lipid-soluble 99mTc-PAT-HM and 99mTcTMR for brain imaging. Nucl. Med. Biol. 1989, 16, 829-837. (34) Eisenhut, M.; Brandau, W.; Miβfeldt, M. Synthesis and in vivo testing of a bromobutyl substituted 1,2-dithia-5,9-diazacycloundecane: a versatile precursor for new 99mTc-bis (aminoethanethiol) complexes. Nucl. Med. Biol. 1989, 16, 805-811. (35) Duatti, A.; Marchi, A.; Pasqualini, R. A new method for the preparation of Tc-99m radiopharmaceuticals containing the Tct N multiple bond. Eighth international symposium on radiopharmaceutical chemistry abstracts. J. Labelled Compds. Radiopharm. 1991, 30, 13. (36) Pasqualini, R.; Comazzi, V.; Bellande, E.; Duatti, A.; Marchi, A. A new efficient method for the preparation of 99mTc-radiopharmaceuticals containing TctN multiple bond. Appl. Radiat. Isot. 1992, 43, 1329-1333. (37) Marchi, A.; Marvelli, L.; Rossi, R.; Poncara`, P.; Uccelli, L.; Giganti, M.; Study on the role of N-methyl, S-methyl dithiocarbazate in the formation of the 99mTctN multiple bond. In Technetium and Rhenium in Chemistry and Nuclear Medicine; Nicolini, M., Bandoli, G., Mazzi, U., Eds.; SGE Ditoriali: Padova, Italy, 1995; Vol. 4, pp 113-116. (38) Baldas, J.; Boas, J. F.; Bonnyman, J.; Williams, G. A. Studies of technetium complexes. Part 6. The preparation, characterisation, and electron spin resonance spectra of salts of tetrachloroand tetrabromo-nitridotechnetate(vi): crystal structure of tetraphenyl-arsonium tetrachloro-nitridotechnetate(vi). J. Chem. Soc., Dalton Trans. 1984, 2395-2400. (39) Francesconi, L. C.; Yang, Y. Y.; Kung, M.-P.; Zhang, X. X.; Billings, J.; Guo, Y. Z.; Kung, H. F. Technetium-99m N,N′-bis(2-mercapto-2-methylpropyl)-2-aminobenzylamine: technetium99m complexes of a novel bis(aminoethanethiol) ligand. J. Med. Chem. 1994, 37, 3282-3288. (40) Colmanet, S. F.; Williams, G. A. Structural aspects of technetium nitrido complexes. In Technetium and Rhenium in Chemistry and Nuclear Medicine; Nicolini, M., Bandoli, G., Mazzi, U., Eds.; Cortinal International-Verona Raven Press: New York, 1990; Vol. 3, pp 55-62. (41) Duatti, A.; Marchi, A.; Pasqualini, R. Formation of the TcdN multiple bond from the reaction of ammonium pertechnetate with S-methyl dithiocarbazate and its application to the preparation of technetium-99m radiopharmaceuticals. J. Chem. Soc., Dalton Trans. 1990, 3729-3733. (42) Marchi, A.; Marvelli, L.; Rossi, R.; Magon, L.; Bertolasi, V.; Ferretti, V.; Gilli, P. Nitrido- and oxo-technetium(v) chelate complexes with N2S2 ligands: Synthesis and crystal structures. J. Chem. Soc., Dalton Trans. 1992, 1485-1492. (43) Stepniak-Biniakiewicz, D.; Chen, B.; Deutsch, E. A new, general synthetic route to multidentate N,S ligands for use in technetium-99m radiopharmaceuticals. Preparation of diamino disulfur, diamino dithiol, and tripodal N3S3 prototypes. Comparative biodistributions of [99mTc-DADS]- analogues which contain 5,5,5membered chelate ring systems. J. Med. Chem. 1992, 35, 274-279.

JM981089A