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Synthesis, characterization and comparative biodistribution study of a new series of p-Iodine-125 benzamides as potential melanoma imaging agents
Nuclear Medicine and Biology 28 (2001) 799 – 808
Synthesis, characterization and comparative biodistribution study of a new series of p-Iodine-125 benzamides as potential melanoma imaging agents Nicole Moinsa,*, Janine Papona, He´le`ne Seguinb, Daniel Gardetteb, Marie-France Moreaua, Pierre Labarrea, Martine Baylea, Josette Michelota, Jean-Claude Gramainb, Jean-Claude Madelmonta, Annie Veyrea b
a INSERM U484, 63005 Clermont-Ferrand, France UMR SEESIB 6504, Universite Blaise Pascal, 63177 Aubiere, France
Received 23 February 2001; received in revised form 4 April 2001; accepted 3 May 2001
Abstract Iodobenzamides are reported to possess some affinity for melanoma. In order to identify the compound having the most appropriate pharmacokinetic properties as a potential melanoma imaging agent, thirteen new [125I]radioiodobenzamides with a butylene amide-amine spacer and various substituents on the terminal amino group were investigated. Their synthesis, radioiodination and biodistribution in B16 melanoma bearing C57BL6 mice are described and compared to [125I] labeled N-(2-diethylaminoethyl)-4-iodobenzamide ([125I]BZA), our reference compound. Changes in the terminal amino constituents induced modifications of lipophilicity, tumor uptake and organ distribution. The dimethylaminobutyl iodobenzamide appeared to be the most promising radiopharmaceutical imaging agent for the detection of melanoma and its metastases. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Melanoma scintigraphy; Iodine-125-benzamides; Radioactivity biodistribution analysis; Murine melanoma model
1. Introduction Over recent decades, the incidence of malignant melanoma (MM) is increasing at an annual rate of about 7% in most countries, more rapidly in some parts of Australia, and faster than for any other tumors [15]. Malignant melanoma is a tumor with a high potential of metastasis spreading, often early in the disease development but sometimes after a long disease-free period, in particular for the uveal form [19]. Until now, no treatment has demonstrated any significant efficacy although hope arises from immunological and gene therapy under investigation [5]. The early detection of metastases is always an important diagnostic goal and a follow-up is also clinically relevant even for patients without detected proliferation at the surgical removal time of the * Corresponding author. INSERM U484, Rue Montalembert, BP 184, 63005 Clermont-Ferrand cedex, France. Tel.: ⫹(33) 4 73 15 08 00; fax: ⫹(33) 4 73 15 08 01. E-mail address: [email protected] (N. Moins).
primary lesion. So, a noninvasive test of high specificity and precision for the detection of melanoma cells is suitable and scintigraphic imaging with a specific radiotracer would be of large interest. We have been the first to propose a series of iodobenzamide derivatives showing an affinity for melanoma tissue [11,12]. Preclinical studies reported a selective uptake in melanoma tumors induced in mice, in murine B16 tumors, as well as in human tumors transplanted into athymic mice [4,8]. A phase II scintigraphic clinical trial evaluating iodine-123 labeled N-(2-diethylaminoethyl)-4-iodobenzamide ([123I]-BZA) as an imaging agent of primary melanomas and metastases conducted on 110 patients resulted in a diagnostic sensitivity of 81% and a specificity of 100% [9]. The interest of this compound for the management of patients with melanoma has been confirmed [1,2,16]. The only result of the trial suggesting improvements may be required was the time with the best tumoral definition: 18 –24 hr after [123I]BZA administration. For cost and dosimetry reasons, we sought an agent that would provide quality images sooner after injection. With this view, a first
0969-8051/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 1 ) 0 0 2 4 2 - 6
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N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
Fig. 1. General structural formula of the radioiodinated benzamides 1–13. Substituents are listed in Table 1.
series of twelve [125I]BZA derivatives has been assessed in melanoma bearing mice [13]. Good results were obtained with para-iodobenzamide structures, and a lengthening of amide-amine spacer appeared favorable. In order to evaluate systematically a chemical series, we chose to synthesize and study thirteen para-iodobenzamides with a chain length of four methylene groups and varying amino alkyl substituents (Fig. 1 and Table 1).
2. Materials and methods 2.1. Materials All commercially available chemicals were of analytical grade and used without further purification. Sodium [125I]iodide was supplied by CIS bio international (Saclay, France) as a no-carrier added solution in reductant-free 0.1N aqueous sodium hydroxide. The melting points (Mp) were determined on a Reichert hot stage apparatus and are uncorrected. Chromatography refers to flash column chromatography on Merck Kieselgel 60 (230 – 400 mesh) or on Table 1 n-Octanol/phosphate buffer partition coefficient (log P) and 50% lethal dose of 4-iodobenzamide derivatives Compound BZA 1 2 3 4 5 6 7 8 9 10 11 12 13
R1* C2H5
R2* C2H5
H H H CH3 H C2H5 H n-C3H7 H i-C3H7 H n-C4H9 CH3 CH3 C2H5 C2H5 n-C3H7 n-C3H7 i-C3H7 i-C3H7 n-C4H9 n-C4H9 ⫺(CH2)4⫺ ⫺(CH2)5⫺
log P
LD50 (mmol/kg)
1.34
0.25
0.02 0.01 0.18 0.70 0.52 1.16 0.58 0.69 1.48 0.82 1.10 0.70 1.10
0.49 0.38 0.34 0.23 0.25 0.16 0.38 0.19 0.09 0.11 0.05 0.18 0.17
BZA: N-2-diethylaminoethyl-4-iodobenzamide. * See Fig. 1 for structure. LD50: determined in OF1 mice after i.v. administration.
Acros neutral Aluminum oxide (50 –200 ) as specified. TLC analyses were performed on Merck 60 F254 silica gel plates and spots were visualized with ultraviolet light (254 nm) and exposition of the plates to iodine vapors. Infrared spectra were recorded on a Perkin-Elmer 881 spectrometer. NMR spectra were performed on an AC 400 spectrometer (Bruker, Wissemburg, France) operating at 1H and 13C frequencies of 400.13 MHz and 100.61 MHz, respectively, in CDCl3 as solvent. Chemical shift data are reported in parts per million and in the following order for 1H data: multiplicity (s ⫽ singlet; bs ⫽ broad singlet; d ⫽ doublet; t ⫽ triplet; q ⫽ quadruplet; qt ⫽ quintuplet; st ⫽ sextuplet; m ⫽ multiplet); number of protons; proton assignment; coupling constant. Elemental analyses were determined at the Service Central d’Analyze (CNRS, Vernaison, France). Mass spectra were realized on a micromass VG 305 and Hewlett-Packard HP5989B spectrometers at the University Blaise Pascal (Clermont-Ferrand, France). 2.2. Synthesis Unlabeled iodobenzamides 1–13 were prepared in good yields in a simple one-step reaction by condensation of 4-iodobenzoyl chloride or 4-nitrophenyl 4-iodobenzoate with the already reported N–substituted aminobutylamines [17]. N-(4-aminobutyl)-4-iodobenzamide 1. 4-Aminobutylamine (0.5 mL; 0.48 g; 5.4 mmol), in solution in tetrahydrofuran (10 mL), was added to a solution of 4-iodobenzoate of 4-nitrophenol (1.0 g; 2.7 mmol) in THF (10 mL). The mixture was then stirred and refluxed for 30 min. After evaporation of the solvent, the residue was dissolved in methylene chloride (25 mL). The organic layer was washed with 1N NaOH (2 x 15 mL), then with water (15 mL) and dried over magnesium sulfate. 1 was obtained after purification by chromatography on a silica gel column (MeOH/ NH4OH) (0.49 g; 57%); Mp 110 –111°C; HCl salt mp 245–248°C. IR (CCl4) 1660 cm-1. 1H NMR of the free base ␦ 1.42 ppm (bs, 2H, NH2); 1.55 (qt, 2H, H-3⬘, J ⫽ 7.0 Hz); 1.68 (qt, 2H, H-2⬘, J ⫽ 7.0 Hz); 2.77 (m, 2H, H-4⬘); 3.45 (m, 2H, H-1⬘); 7.23 (bs, 1H, CONH); 7.53 (dd, 2H, aromatic H, J ⫽ 8.5 Hz); 7.76 (dd, 2H, aromatic H, J ⫽ 8.5 Hz). 13C NMR 27.1 ppm (C-3⬘); 30.9 (C-2⬘); 40.1 (C-4⬘); 41.7 (C1⬘); 98.1 (C-4); 128.7 (C-3, C-5); 134.4 (C-1); 137.7 (C-2, C-6); 166.8 (CO). MS: m/z 318 ([M⫹], 22). Elemental analysis: theoretical (%) C 41.51, H 4.75, N 8.81. I 39.90; Found (%) C 41.84, H 4.89, N 8.72; I 39.55. 2.2.1. General procedure for the preparation of iodobenzamides 2–6 The solution of the amine (10 mmol) in anhydrous tetrahydrofuran (20 mL) was added to a solution of the 4-iodobenzoate of 4-nitrophenol (10 mmol; 3.7 g) in THF (20 mL). The reaction mixture was stirred for ten hours at room temperature. After evaporation, the crude mixture was dissolved in methylene chloride (75 mL) and washed with 1N
N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
NaOH. The organic phase was then washed with water, dried over MgSO4 and the solvent removed. Purification by chromatography on neutral alumina column or by crystallization led to the desired iodobenzamides. N-(4-methylaminobutyl)-4-iodobenzamide 2. 2 was obtained from 4-iodobenzoate of 4-nitrophenol and N-methyl4-aminobutylamine after purification by chromatography on neutral alumina (AcOEt/MeOH 8/2) as white needles (2.26 g; 68%); Mp 60 – 61°C; HCl salt mp 245°C. IR (CCl4) 1655 cm-1. 1H NMR of the free base ␦ 1.50 ppm (bs, 1H, NHCH3); 1.60 (qt, 2H, H-3⬘, J ⫽ 6 Hz); 1.70 (qt, 2H, H-2⬘, J ⫽ 6.0 Hz); 2.40 (s, 3H, CH3); 2.60 (t, 2H, H-4⬘, J ⫽ 6.0 Hz); 3.40 (m, 2H, H-1⬘); 7.50 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 7.70 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 8.00 (bs, 1H, CONH); 13C NMR 27.4 ppm (C-3⬘); 27.7 (C-2⬘); 36.5 (CH3); 40.1 (C-1⬘); 51.6 (C-4⬘); 98.0 (C-4); 128.7 (C-3, C-5); 134.5 (C-1); 137.5 (C-2, C-6); 166.7 (CO). MS: m/z 332 ([M⫹], 8). Elemental analysis: theoretical (%) C 43.37, H 5.16, N 8.43; Found (%) C 42.95, H 5.08, N 8.13. N-(4-ethylaminobutyl)-4-iodobenzamide 3. 3 was purified by chromatography on neutral alumina (AcOEt/MeOH 9/1) and obtained as white crystals (1.94 g; 56%); Mp 64 – 65°C; HCl salt mp 209 –211°C. IR (CCl4) 1655 cm-1. 1 H NMR of the free base ␦ 0.85 ppm (t, 3H, CH3, J ⫽ 7.0 Hz); 1.60 (m, 5H, H-2⬘, H-3⬘, NHCH2CH3); 2.51 (m, 4H, H-4⬘, CH2CH3); 3.40 (m, 2H, H-1⬘); 7.49 (dd, 2H, aromatic H, J ⫽ 8.4 Hz); 7.55 (bs, 1H, CONH); 7.72 (dd, 2H, aromatic H, J ⫽ 8.4 Hz). 13C NMR 15.2 ppm (CH3); 27.4 (C-3⬘); 27.9 (C-2⬘); 40.1 (C-1⬘); 44.2 (CH2CH3); 49.2 (C4⬘); 98.0 (C-4); 128.7 (C-3, C-5); 134.5 (C-1); 137.6 (C-2, C-6); 166.8 (CO). MS: m/z 346, ([M⫹], 2). Elemental analysis: theoretical (%) C 45.08, H 5.53, N 8.09; Found (%) C 44.42, H 5.60, N 7.85. N-(4-propylaminobutyl)-4-iodobenzamide 4. 4 was purified by chromatography on neutral alumina (AcOEt/MeOH 9/1) and obtained as white crystals (2.16 g; 60%); Mp 73–74°C; HCl salt mp 219 –220°C. IR (CCl4) 1660 cm-1. 1H NMR of the free base ␦ 0.90 ppm (t, 3H, CH3, J ⫽ 7.4 Hz); 1.30 (bs, 1H, NHPr); 1.50 (st, 2H, CH2CH2CH3, J ⫽ 7.4 Hz); 1.60 (qt, 2H, H-3⬘; J ⫽ 6.6 Hz); 1.70 (qt, 2H, H-2⬘, J ⫽ 6.6 Hz); 2.55 (t, 2H, CH2CH2CH3, J ⫽ 7.4 Hz); 2.65 (t, 2H, H-4⬘, J ⫽ 6.6 Hz); 3.45 (m, 2H, H-1⬘); 7.50 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 7.55 (bs, 1H, CONH); 7.75 (dd, 2H, aromatic H, J ⫽ 8.3 Hz). 13C NMR 11.7 ppm (CH3); 22.9 (CH2CH3); 27.3 (C-2⬘); 27.7 (C-3⬘); 40.0 (C-1⬘); 49.2 (C-4⬘); 51.8 (CH2CH2CH3); 97.0 (C-4); 128.7 (C-3, C-5); 134.4 (C-1); 137.4 (C-2, C-6); 166.8 (CO). MS : m/z 360 ([M⫹], 16). Elemental analysis: theoretical (%) C 46.66, H 5.88, N 7.78, I 35.24; Found (%) C 45.92, H 5.87, N 7.55, I 34.41. N-(4-isopropylaminobutyl)-4-iodobenzamide 5. 5 was purified by chromatography on neutral alumina (AcOEt/ MeOH 9/1) and obtained as white crystals (1.87 g; 52%); Mp 76 –78°C; HCl salt mp 180 –182°C. IR (CCl4) 1665 cm-1. 1H NMR of the free base ␦ 1.00 ppm (d, 6H, CH3, J ⫽ 6.3 Hz); 1.30 (bs, 1H, NHiPr); 1.57 (qt, 2H, H-3⬘, J ⫽ 6.7
801
Hz); 1.65 (qt, 2H, H-2⬘, J ⫽ 6.7 Hz); 2.50 (t, 2H, H-4⬘; J ⫽ 6.7 Hz); 2.75 (ht, 1H, CH, J ⫽ 6.3 Hz); 3.50 (m, 2H, H-1); 7.25 (bs, 1H, CONH); 7.50 (dd, 2H, aromatic H, J ⫽ 8.5 Hz); 7.75 (dd, 2H, aromatic H, J ⫽ 8.5 Hz). 13C NMR 22.8 ppm (CH3); 27.3 (C-2⬘); 27.9 (C-3⬘); 39.9 (C-1⬘); 46.7 (C-4⬘); 48.9 (CH); 97.7 (C-4); 128.7 (C-3, C-5); 134.1 (C-1); 137.6 (C-2, C-6); 166.2 (CO). MS: m/z 360 ([M⫹], 4). Elemental analysis: theoretical (%) C 46.66, H 5.88, N 7.78, I 35.24; Found (%) C 46.42, H 5.91, N 7.56, I 34.45. N-(4-butylaminobutyl)-4-iodobenzamide 6. 6 was purified by chromatography on neutral alumina (AcOEt/MeOH 95/5) and obtained as white crystals (2.51 g; 67%); Mp 75–76°C; HCl salt mp 230 –235°C. IR (CCl4) 1655 cm-1. 1H NMR of the free base ␦ 0.90 ppm (t, 3H, CH3, J ⫽ 7.2 Hz); 1.30 (st, 2H, H-8⬘, J ⫽ 7.2 Hz); 1.40 (qt, 2H, H-7⬘, J ⫽ 7.2 Hz); 1.65 (m, 5H, H-2⬘; H-3⬘, NHBu); 2.55(m, 2H, H-4⬘); 2.65 (t, 2H, H-6⬘, J ⫽ 7.2 Hz); 3.40 (m, 2H, H-1⬘); 7.45 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 7.65 (bs, 1H, CONH); 7.70 (dd, 2H, aromatic H, J ⫽ 8.3 Hz). 13C NMR 14.0 ppm (C-9⬘); 20.5 (C-8⬘); 27.3 (C-3⬘); 27.5 (C-2⬘); 31.9 (C-7⬘); 40.0 (C-1⬘); 49.2 (C-4⬘); 49.7 (C-6⬘); 98.0 (C-4); 128.7 (C-3, C-5); 134.4 (C-1); 137.6 (C-2, C-6); 166.9 (CO). MS: m/z 374 ([M⫹], 100). Elemental analysis: theoretical (%) C 48.12, H 6.19, N 7.49; Found (%) C 47.45, H 6.19, N 7.34. 2.2.2. General procedure for the preparation of iodobenzamides 7–13. The solution of the amine (10 mmol) and triethylamine (10 mmol) in anhydrous methylene chloride (20 mL) was added drop wise to a solution of 4-iodobenzoyl chloride (10 mmol, 2.66 g). The mixture was stirred under nitrogen atmosphere at room temperature for 12 h and poured into 100 mL ice-water. The aqueous phase was extracted three times with CH2Cl2 (100 mL each time); the organic phase was dried over anhydrous MgSO4 and the solvent removed. The crude mixture was then purified by chromatography on neutral alumina column. N-(4-dimethylaminobutyl)-4-iodobenzamide 7. The reaction of 4-dimethylaminobutylamine and 4-iodobenzoyl chloride gave 7 after purification on neutral alumina column chromatography (AcOEt/MeOH 9/1) (1.83g; 53%); Mp 64 – 65°C; HCl salt mp 190 –192°C. IR (CCl4) 1660 cm-1. 1H NMR of the free base ␦ 1.60 ppm (qt, 2H, H-3⬘, J ⫽ 6.5 Hz); 1.65 (qt, 2H, H-2⬘, J ⫽ 6.5Hz); 2.20 (s, 6H, CH3); 2.35 (t, 2H, H-4⬘, J ⫽ 6.5 Hz); 3.40 (m, 2H, H-1⬘); 7.50 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 7.72 (dd, 2H, aromatic H, J ⫽ 8.3 Hz), 8.20 (bs, 1H, CONH). 13C NMR 24.9 ppm (C-3⬘); 27.0 (C-2⬘); 39.7 (C-1⬘); 44.8 (CH3); 58.8 (C-4⬘); 97.7 (C-4); 128.5 (C-3, C-5); 134.2 (C-1); 137.2 (C-2, C-6); 166.6 (CO). MS: m/z 346 ([M⫹], 8). Elemental analysis: theoretical (%) C 45.08, H 5.53, N 8.09, I 36.67; Found (%) C 45.28, H 5.46, N 7.87, I 36.44. N-(4-diethylaminobutyl)-4-iodobenzamide 8. The reaction of 4-diethylaminobutylamine and 4-iodobenzoyl chloride gave 8 after purification on neutral alumina column chromatography (AcOEt/MeOH 95/5) (2.17 g; 58%); Mp
802
N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
55–57°C; HCl salt mp 178 –180°C. IR (CCl4) 1665 cm-1. 1H NMR of the free base ␦ 1.00 ppm (t, 6H, CH2CH3, J ⫽ 7.1 Hz); 1.58 (qt, 2H, H-3⬘, J ⫽ 6.8 Hz); 1.66 (qt, 2H, H-2⬘, J ⫽ 6.8 Hz); 2.45 (t, 2H, H-4⬘, J ⫽ 6.8 Hz); 2.50 (q, 4H, CH2CH3, J ⫽ 7.1 Hz); 3.40 (m, 2H, H-1); 7.49 (dd, 2H, aromatic H, J ⫽ 8.5 Hz); 7.55 (bs, 1H, CONH), 7.75 (dd, 2H, aromatic H, J ⫽ 8.5 Hz). 13C NMR 11.0 ppm (CH3); 24.9 (C-3⬘); 27.6 (C-2⬘); 40.0 (C-4⬘); 46.6 (CH2CH3); 52.3 (C-1⬘); 98.0 (C-4); 128.8 (C-3, C-5); 134.5 (C-1); 137.5 (C-2, C-6); 166.9 (CO). MS: m/z 374 ([M⫹], 2). Elemental analysis: theoretical (%) C 48.13, H 6.19, N 7.49; Found (%) C 48.29, H 6.35, N 7.67. N-(4-dipropylaminobutyl)-4-iodobenzamide 9. The reaction of 4-dipropylaminobutylamine and 4-iodobenzoyl chloride gave 9 after purification on neutral alumina column chromatography (AcOEt) (2.73 g; 68%); Mp 70 –71°C; HCl salt mp 159 –160°C. IR (CCl4) 1670 cm-1. 1H NMR of the free base ␦ 0.80 ppm (t, 6H, CH3, J ⫽ 7.3 Hz); 1,40 (m, 4H, CH2CH2CH3); 1,50 (m, 2H, H-3⬘); 1.60 (m, 2H, H-2⬘); 2.30 (m, 4H, CH2CH2CH3); 2.40 (t, 2H, H-4⬘, J ⫽ 6.8 Hz); 3.40 (m, 2H, H-1⬘); 7.25 (bs, 1H, CONH); 7.50 (dd, 2H, aromatic H, J ⫽ 8.4 Hz); 7.72 (dd, 2H, aromatic H, J ⫽ 8.4 Hz). 13C NMR 11.9 ppm (CH3); 19.6 (CH2-CH3); 24.9 (C-3⬘); 27.5 (C-2⬘); 39.9 (C-1⬘); 53.5 (C-4⬘); 56.0 (CH2 CH2CH3); 98.0 (C-4); 128.7 (C-3, C-5); 134.5 (C-1); 137.5 (C-2, C-6); 166.9 (CO). MS: m/z 402 ([M⫹], 10). Elemental analysis: theoretical (%) C 50.75, H 6.77, N 6.96, I 31.56; Found (%) C 50.78, H 6.83, N 6.80, I 31.25. N-(4-diisopropylaminobutyl)-4-iodobenzamide 10. The reaction of 4-diisopropylaminobutyl-amine and 4-iodobenzoyl chloride gave 10 after purification on neutral alumina column chromatography (AcOEt/MeOH 95/5) (2.61 g; 65%); Mp 69 –71°C; HCl salt mp 135°C. IR (CCl4) 1675 cm-1. 1H NMR of the free base ␦ 1.15 ppm (d, 12H, CH3, J ⫽ 6.6 Hz); 1.42 (qt, 2H, H-3⬘, J ⫽ 7.0 Hz); 1.55 (qt, 2H, H-2⬘, J ⫽ 7.0 Hz); 2.37 (t, 2H, H-4⬘, J ⫽ 7.0 Hz); 2.95 (ht, 2H, CH, J ⫽ 6.6 Hz); 3.37 (m, 2H, H-1⬘); 6.75 (bs, 1H, CONH); 7.45 (dd, 2H, aromatic H, J ⫽ 8.4 Hz); 7.69 (dd, 2H, aromatic H, J ⫽ 8.4 Hz). 13C NMR 20.6 ppm (CH3); 27.3 (C-3⬘); 28.5 (C-2⬘); 40.2 (C-3⬘); 44.7 (C-4⬘); 48.4 (CH); 98.0 (C-4); 128.6 (C-3, C-5); 134.3 (C-1); 137.5 (C-2, C-6); 166.7 (CO). Elemental analysis: theoretical (%) C 50.75, H 6.77, N 6.96; Found (%) C 50.87, H 6.70, N 6.77. N-(4-dibutylaminobutyl)-4-iodobenzamide 11. The reaction of 4-dibutylaminobutylamine and 4-iodobenzoyl chloride gave 11 after purification on neutral alumina column chromatography (AcOEt/Hexane 3/7) (2.80 g; 65%); Mp 93–95°C; HCl salt mp 128 –130°C. IR (CCl4) 1650 cm-1. 1H NMR of the free base ␦ 0.90 ppm (t, 6H, CH3, J ⫽ 7.5 Hz); 1.30 (st, 4H, CH2CH3, J ⫽ 7.5 Hz); 1.70 (m, 6H, H-3⬘, CH2CH2CH2); 1.80 (m, 2H, H-2⬘); 2.95 (m, 6H, H-4⬘, NCH2CH2CH2CH3); 3.45 (m, 2H, H-1⬘); 7.70 (m, 4H, aromatic H); 8.25 (bs, 1H, CONH). 13C NMR 14.1 ppm (C-9⬘, C-13⬘), 20.0 (C-8⬘, C-12⬘); 21.0 (C-3⬘); 24.9 (C-7⬘, C-11⬘); 26.0 (C-2⬘); 38.1 (C-1⬘); 52.1 (C-6⬘, C-10⬘); 52.5 (C-4⬘); 98.3 (C-4); 129.3 (C-3, C-5); 133.6 (C-1); 137.5 (C-2, C-6);
166.9 (CO). Elemental analysis: theoretical (%) C 53.02, H 7.26, N 6.51; Found (%) C 53.20, H 7.36, N 6.79. N-(4-pyrrolidinobutyl)-4-iodobenzamide 12. The reaction of 4-pyrrolidinobutylamine and 4-iodobenzoyl chloride gave 12 after purification on neutral alumina column chromatography (AcOEt/MeOH 95/5) (1.93 g; 52%); Mp 89 – 91°C; HCl salt mp 202–203°C. IR (CCl4) 1665 cm-1. 1H NMR of the free base ␦ 1.70 ppm (m, 2H, H-2, H-3⬘); 1.80 (m, 4H, H-7⬘, H-8⬘); 2.65 (m, 6H, H-4⬘, H-6⬘, H-9⬘); 3.45 (m, 2H, H-1⬘); 7.50 (dd, 2H, aromatic H, J ⫽ 8.5 Hz); 7.75 (dd, 2H, aromatic H, J ⫽ 8.5 Hz), 7.90 (bs, 1H, CONH). 13C NMR 23.4 ppm (C-7⬘, C-8⬘), 26.0 (C-2⬘); 27.2 (C-3⬘); 39.7 (C-1⬘); 54.0 (C-6⬘, C-9⬘); 55.6 (C-4⬘); 98.1 (C-4); 128.8 (C-3, C-5); 134.5 (C-1); 137.6 (C-2, C-6); 166.9 (CO). Elemental analysis: theoretical (%) C 48.39, H 5.69, N 7.53; Found (%) C 48.59, H 5.81, N 7.67. N-(4-piperidinobutyl)-4-iodobenzamide 13. The reaction of 4-piperidinobutylamine and 4-iodobenzoyl chloride gave 13 after purification on neutral alumina column chromatography (AcOEt/MeOH 95/5), (2.74 g; 71%); Mp 120 –122°C; HCl salt mp 188 –189°C. IR (CCl4) 1665 cm-1. 1H NMR of the free base ␦ 1.40 ppm (m, 2H, H-8); 1.50 (qt, 4H, H-7⬘, H-9⬘, J ⫽ 6.8 Hz); 1.60 (m, 4H, H-2⬘, H-3⬘); 2.30 (m, 6H, H-4⬘, H-6⬘, H-10⬘); 3.40 (m, 2H, H-1⬘); 7.18 (bs, 1H, CONH); 7.47 (dd, 2H, aromatic H, J ⫽ 8.4 Hz); 7.73 (dd, 2H, aromatic H, J ⫽ 8.4 Hz). 13C NMR 24.4 ppm (C-8⬘); 24.6 (C-2⬘); 25.8 (C-7⬘, C-9⬘); 27.4 (C-3⬘); 40.1 (C-1⬘); 54.6 (C-6⬘, C-10⬘); 58.7 (C-4⬘); 98.0 (C-4); 128.8 (C-3, C-5); 134.5 (C-1); 137.6 (C-2, C-6); 166.9 (CO) MS: m/z 386 ([M⫹], 59). Elemental analysis: theoretical (%) C 49.75, H 6.01, N 7.25; Found (%) C 49.87, H 6.20, N 7.47. 2.3. [125I] Radiolabeling Radioiodination was performed by isotopic exchange with sodium [125I]iodide in citrate buffer with copper sulfate used as a catalyst accordingly to already published procedures [13]. The labeled compounds were identified by TLC comparison with the corresponding non-radioactive samples. The chromatograms were scanned with a Berthold LB 2832 linear analyzer. 2.4. pKa measurements pKa of each molecule (HCl salt) has been determined by an acid/base assay with 0.1 N aqueous sodium hydroxide. 2.5. Partition coefficient measurements For each molecule, the partition coefficient between noctanol and phosphate buffer was determined. The measurement was performed by shaking 2 mL of [125I] benzamide solution (50 M in phosphate buffer solution, PBS, pH 7.4) with 2 mL n-octanol. The activity of each phase was counted. P was calculated as the ratio of activities (n-
N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
octanol/buffer) and its logarithm was determined to express lipophilicity. 2.6. Animal studies All experiments were carried out in compliance with French laws governing animal experimentation. 2.6.1. Toxicity Acute toxicity (LD50) was determined in male OF1 mice (Iffa Credo, Les Oncins, France) after intravenous administration. The non-radioactive iodobenzamides were dissolved in saline (0.9% NaCl). The LD50 value determination was performed with six different doses varying by a geometrical progression, each of them being administered to six animals. 2.6.2. Biodistribution The biodistribution of radioiodinated benzamides was studied in C57BL6 male mice, bearing the B16 murine melanoma. B16 melanoma cells were originally obtained from ICIG (Villejuif, France) and maintained as a monolayer in tissue culture minimum essential medium (MEM, Gibco) supplemented with 10% fetal calf serum and antibiotics, and passaged by trypsination. Early passages were frozen and stored in liquid nitrogen, and for transplantation an aliquot was grown in a monolayer culture to confluence and cells were trypsinized and washed with phosphate buffer saline (PBS). They were resuspended in PBS and each mouse received 0.1 mL subcutaneously (5 x 105 cells) on the left flank. Ten days later, the tumors became palpable with a percentage of tumor take equal to 98 –100%. 2.6.2.1. Experimental procedure. The [125I]benzamide was administered intravenously via a tail vein (0.1 mol, 0.74 – 0.92 MBq/animal) in ten animals for each compound. Two injected mice were sacrified by quickly freezing in liquid nitrogen after ether anesthesia, at different times after administration (1, 3, 6, 24 or 72 hours). Frozen animals were cryosectioned using the technique described by Ullberg [18]. Slices of 40 m were obtained using a Reichert-Jung cryopolycut (Leica Instrument, Rueil Malmaison, France) at -22°C and dehydrated for 48 hr in the cryochamber. 2.6.2.2. Detection and quantification of radioactivity. Radioactivity contained in slices was analyzed using an AMBIS 4000 detector (Scanalytics, CSPI, San Diego, CA) which is a computer-controlled multi-wire proportional counter previously described [6] and validated for evaluation in mice of iodinated agents [7]. A two dimensional image of the slice was displayed on the computer screen for analysis, and quantification of different organs was made after contouring suitable zones. Measurements were performed after a 1000 min acquisition time. The radioactivity per unit area (net cpm/mm2) was converted into radioactive
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concentration (kBq/g) and expressed as percentage of injected dose/g of tissue (%ID/g). The tumor (T) uptake was compared to those of other tissues and ratios (T/organ) were determined illustrating the image contrast. For two animals, urine and feces have been collected until 72 hr and counted to determine the cumulative urinary and fecal excretions.
3. Results 3.1. Chemistry All iodobenzamides 1–13 (Table 1) were synthesized, according to two different procedures, from already described aminobutyl amines prepared from readily available starting materials [17]. The primary amino group of N-alkylaminobutylamines was selectively acylated on treating with 4-nitrophenyl 4iodobenzoate at room temperature for 10 hours, in good yields, to lead to iodobenzamides 1– 6 bearing a mono or a disubstituted amino function. On the other hand, iodobenzamides 7–13, with a tertiary amino group, were prepared by reacting the required N,N-dialkylaminobutyl amines with p-iodobenzoyl chloride at room temperature for 2 hours. All synthesized iodobenzamides 1–13 exhibited expected IR, 1 H and 13C NMR data. Iodobenzamides 1–13 were converted, for pre-clinical use, into hydrochlorides after treatment by an anhydrous hydrogen chloride solution in diethyl ether. 3.2. [125I] Radiolabeling Radioiodination was achieved by Cu (II) assisted isotopic exchange. Radiochemical yields based on TLC of exchange reaction mixture varied from 88 to 95%. After purification by passing the crude mixture through silicic acid cartridge (ExtrelutR20, Merck), the [125I]-labeled compounds were shown by TLC to be free of significant chemical and radiochemical impurities. 3.3. pKa measurements pKa values ranged from 10.2 to 11.7, indicating an almost complete protonation of amino groups at physiological pH. 3.4. Partition coefficients No significant lipophilicity has been measured for compounds 1 and 2. The partition coefficient value of iodobenzamides 2–10 increased with the number and the length of substituents (Table 1). This variation was regular for the iodobenzamides 2– 6, with a secondary amino group. However, for compounds 7–13 with a tertiary amino group, such a variation was only observed for compounds 7 to 9, with a
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Table 2 Uptake of [125I]benzamide derivatives in tumor at various times after injection in B16 melanoma bearing mice (%ID/g)* Compound
1 hr
3 hr
6 hr
24 hr
72 hr
BZA
9.5 ⫾ 1.9
9.1 ⫾ 1.6
7.7 ⫾ 3.3
3.7 ⫾ 1.2
0.8 ⫾ 0.3
1 2 3 4 5 6 7 8 9 10 11 12 13
0.6 ⫾ 0.2 1.1 ⫾ 0.3 2.7 ⫾ 0.8 6.4 ⫾ 1.2 5.0 ⫾ 1.3 5.5 ⫾ 1.4 7.8 ⫾ 1.6 6.3 ⫾ 3.7 8.9 ⫾ 1.6 3.8 ⫾ 0.5 2.6 ⫾ 0.3 4.3 ⫾ 0.7 5.2 ⫾ 0.5
0.5 ⫾ 0.1 0.9 ⫾ 0.4 1.6 ⫾ 0.3 5.7 ⫾ 1.0 2.8 ⫾ 1.2 9.6 ⫾ 4.0 7.1 ⫾ 1.9 5.2 ⫾ 1.9 11.2 ⫾ 1.9 6.3 ⫾ 1.4 5.1 ⫾ 1.3 4.5 ⫾ 0.9 2.9 ⫾ 1.1
0.3 ⫾ 0.0 0.7 ⫾ 0.2 2.0 ⫾ 0.9 3.6 ⫾ 0.9 2.5 ⫾ 0.9 6.2 ⫾ 1.0 6.8 ⫾ 2.5 4.7 ⫾ 1.4 7.6 ⫾ 1.6 5.2 ⫾ 1.2 7.1 ⫾ 1.0 1.3 ⫾ 0.2 3.0 ⫾ 0.8
— 0.8 ⫾ 0.1 1.0 ⫾ 0.5 2.4 ⫾ 1.3 2.1 ⫾ 0.8 5.3 ⫾ 1.5 3.0 ⫾ 2.5 3.3 ⫾ 1.0 4.6 ⫾ 1.0 2.6 ⫾ 0.7 3.8 ⫾ 1.8 2.0 ⫾ 0.7 1.3 ⫾ 0.3
— — — 0.4 ⫾ 0.1 1.3 ⫾ 0.4 3.3 ⫾ 0.8 1.0 ⫾ 0.3 — 3.2 ⫾ 0.6 0.8 ⫾ 0.3 1.1 ⫾ 0.3 0.5 ⫾ 0.2 1.7 ⫾ 0.5
* Mean ⫾ SD (two mice for each compound and each time). Absence of value: organ concentration was equal to background value.
maximal value of 1.48 for benzamide 9. Surprisingly, the log P value of benzamide 11 was notably lower than that of benzamide 9. 3.5. Animal studies 3.5.1. Toxicity LD50 values determined in mice as a parameter of acute toxicity were summarized in Table 1. Compared to the BZA value (0.25 mmol/kg), the LD50 values were of the same order except for benzamides 9 or 11 (dipropyl or dibutyl derivatives) which appeared to be the more toxic compounds. It must be noticed that iodobenzamides 1– 6 bearing a secondary amino group were generally less toxic than iodobenzamides 7–13 with a tertiary one. Moreover, in each series of compounds 1– 6 and 7–13, the toxicity increased with the length of the amino substituent. Nevertheless, the mass injected in the biodistribution studies was much lower than toxic ones, and even with the most toxic benzamide 11, the LD50 value equal to 0.9 mol/mouse, was at least 10 times the injected dose. 3.5.2. Biodistribution For the thirteen [125 I]-labeled compounds, the tissue distribution was evaluated in melanoma bearing mice. In fact, total radioactivity was measured and if metabolites may be involved, only the total of [125I] containing moieties was estimated. The values of tumoral fixation (%ID/g) of the benzamides are summarized in Table 2 and compared with our reference compound, [125I]BZA. For all the studied molecules, a tumoral accumulation was observed (⬎2.5%ID/g at 1 hour p.i.) except for benzamides 1 and 2, the two compounds with a log P value close to zero, which exhibited the lowest concentrations in tumor and other tissues. However, the values were distributed in a large range. For the monobutyl 6 and the dipropyl 9 derivatives, the
tumoral concentration appeared at 3 hours p.i. at least equal or higher than with BZA. This uptake remained higher in melanoma than in the other organs as illustrated on Fig. 2, and was particularly more persistent than with BZA since 72 hours after injection, benzamides 6 and 9 were still concentrated in tumors (Table 2). Benzamide 7, which still exhibited a tumoral concentration similar to BZA after 3 and 72 hours, showed a rapid clearance from the other organs in which tracer concentration was already very low at 3 hours. For all compounds, the higher radioactive concentration was found in the uvea with values ⬎5%ID/g, 1 hour after the injection, except for benzamides 1 or 2. With compounds 3–13, the blood activity ranged from 0.5 to 2.3%ID/g 1 hour after the injection and as BZA, a rapid clearance was observed. These eleven compounds were able to cross the blood brain barrier. However, the brain concentration values were low ranging from 0.1 to 1.4%ID/g at 1 hour p.i., and decreased very quickly. Differences in iodobenzamide excretions have been noted, but the urinary elimination was the main route for most of the compounds (Table 3). Among the remarkable molecules (6, 7, 9) benzamide 6 appeared with the slowest urinary excretion and concomitantly the highest fecal excretion. On the contrary, benzamide 7 exhibited a low fecal excretion. As lung, liver or brain are known to develop melanoma metastases, the ratios between concentrations of benzamide in tumor and these organs have been calculated and are summarized in Table 4. From 1 hour after the injection, the tumor:brain ratios were high due to the low values in the brain and notably higher than BZA values. The highest tumor:lung or tumor:liver ratios were observed for benzamide 7 which appeared to be the most selective of the tumoral tissue. The 1 hour values were already equivalent to the ones of BZA at 3 hours, and the 3 hour values were
N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
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Fig. 2. Concentrations of [125I]benzamide derivatives in selected tissues 3 hours after i.v. administration in C57BL6 mice-bearing B16 melanoma. Comparison with [125I]BZA (% ID/g: mean ⫾ S.D., two mice).
about 3– 4 times higher. With benzamides 6 or 9, the most concentrated in tumor at that time, the selectivity was maintained compared to BZA and even slightly improved for benzamide 9. 4. Discussion The radioiodinated benzamide BZA was the first compound of this chemical class successfully developed as Table 3 Urinary and fecal excretions* of radioactivity after [125I]benzamide derivative injection in B16 melanoma bearing mice Urinary %ID 24hr/72hr
Fecal %ID 24hr/72hr
BZA
81 / 83
4/5
1 2 3 4 5 6 7 8 9 10 11 12 13
76 / 81 67 / 68 88 / 89 74 / 75 45 / 45 57 / 59 76 / 77 65 / 66 63 / 66 62 / 62 56 / 58 54 / 54 30 / 31
1/2 2/3 2/2 5/5 2/2 13 / 13 2/2 5/6 6/7 13 / 14 4/5 14 / 14 21 / 22
* Cumulative excretions (two mice for each compound).
radiotracer agent for the imaging of melanoma and its metastases [8,9]. In order to propose a more efficient derivative with even better pharmacokinetic characteristics, different structural variations have been realized by changing the position of radioiodine on the phenyl ring, the length of the amide-amine spacer (two or three methylene groups) or the substituents borne by the terminal amino group. The derivatives obtained have been investigated by studying their biodistribution in melanoma-bearing mice [3,8,10,13,14]. While no compound showed major enhancement of characteristics compared to BZA, variations have been elicited Table 4 Melanoma/organ ratios of [125I]benzamide derivative uptake in B16 melanoma bearing mice 3 hours after the injection Compound
Lung 1hr/3hr
Liver 1hr/3hr
BZA
1.7 / 4.5
1.6 / 2.8
3 4 5 6 7 8 9 10 11 12 13
1.2 / 1.9 1.1 / 4.1 1.4 / 2.8 0.9 / 3.1 4.8 / 12.8 1.0 / 5.7 1.4 / 6.2 1.3 / 6.2 0.4 / 2.2 1.5 / — 2.7 / —
1.5 / 1.8 1.3 / 2.5 1.4 / 1.7 1.0 / 2.5 3.5 / 12.3 0.9 / 3.1 1.3 / 3.1 0.9 / 2.3 0.3 / 1.1 1.1 / 2.1 1.0 / 1.3
Brain 1hr/3hr 6.21 / 11.0 13.6 / 19.6 12.5 / 27.3 22.7 / 15.8 15.2 / 22.3 5.4 / 21.6 7.2 / — 17.0 / 41.6 26.9 / 44.9 4.5 / 19.5 8.5 / 24.8 8.4 / —
Absence of value: very high ratio, the organ concentration being equal to background value.
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Fig. 3. Tumoral concentrations of [125I]benzamide derivatives 3 hours after i.v. administration in C57BL6 mice-bearing B16 melanoma, compared to n-octanol/buffer partition coefficient for N-alkylated (1– 6; A) and N, N-dialkylated (7–13; B) series, respectively.
with some promising evolution. Due to encouraging results observed when the amide-amine spacer was extended from two to three methylene groups [13], the present work reports synthesis and biodistribution study in melanoma-bearing mice of a series of thirteen radioiodobenzamides characterized by a four methylene linear spacer between amide and
amine. The substituents carried by the secondary or tertiary amino function were either linear, branched or cyclic. The synthetic procedures used allowed us a gram scale production. The thirteen new iodobenzamides described in our study showed a tumoral affinity for all but compounds 1 and 2, specially high for benzamides 6, 7 and 9.
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The biological behavior of a chemical compound, at least its background-tissue retention is usually reported to be related to its lipophilicity. The variations of tumoral fixation and n-octanol/buffer partition coefficient are compared in Fig. 3 for N-alkylated (A) and N,N-dialkylated (B) series, respectively. A relation between these two parameters was obvious for the two series : benzamides 1 and 2, with the lowest lipophilicity, failed to concentrate in any tissue, while the compounds 6 and 9, with the highest lipophilicity showed the best tumoral concentrations of the tracer. For the N,N-dialkylated series, the decrease in lipophilicity observed from the compound 9 onwards, was related to a decrease in melanoma uptake. These results evidenced the role of lipophilicity in the biological behavior for compounds of an homogenous chemical series. The highest melanoma uptake was observed for benzamide 9 with log P ⫽ 1.48. Nicholl et al. [14], with related compounds, in a similar study, have already described the best conditions for the log P values range : 1.30 –1.45. Moreover, these authors observed a reduced uptake for very lipophilic or hydrophilic compounds, with the exception of N-N-(3-diethylaminopropyl)-4-iodobenzamide which showed high uptake although its log P is only 0.75. Therefore, log P is not the only characteristic required to explain the melanoma concentration and remains questionable in predicting the in vivo behavior. Nevertheless, in our study, the two parameters exhibited a very close variation, at least for linear substituents. However, the low log P value (0.58) of benzamide 7 may justify a lower labeling of nontarget tissues and organs, and therefore, high tumor/organ ratio values. The challenge of this work was to find malignant melanoma agents which would exhibit an improved pharmacological behavior with respect to BZA. A large variety of biodistribution was displayed among the thirteen iodobenzamides of our study. Contrary to some results reported in the literature, variations of the amine substituents appeared to have a significant impact. At least, three compounds exhibited original promising data. Two of them, the mono n-butyl 6 and the dipropyl 9 derivatives demonstrated an affinity for the tumor at least equal to BZA with an equivalent specificity for short times but a very slow tumoral clearance. Such pharmacokinetic data showing a durable and specific labeling of the tumoral site may be of potential interest for use in internal radiotherapy. The dimethyl derivative 7 was also comparable to BZA concerning the melanoma affinity but a more rapid clearance from nontarget tissues conferred potentially improved melanoma imaging on this structure. The high melanoma:nontarget organ ratios at 3 hours p.i. allowed us to consider it as the best radiopharmaceutical candidate for diagnosis. Note that a tendency for such a behavior has already been reported for N, N-(3-dimethylaminopropyl)-4-iodobenzamide [13]. Thus, compared to previous results obtained with the iodobenzamide series [13], increasing the amide-amine spacer length until butylene group appeared efficient in view of the application to melanoma diagnosis. Further
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attempts to synthesize other iodobenzamides are under investigation. In conclusion, thirteen new iodine-125-benzamides with a butyl amide-amine spacer and varying aminoalkyl substituents have been successfully synthesized and evaluated as imaging agents in melanoma-bearing mice. Different pharmacokinetic profiles were observed although an affinity for melanoma was obvious for all but the unsubstituted and the monomethyl molecules. Overall, the tumoral uptake appeared closely related to the n-octanol/buffer partition coefficient value. Three compounds were distinguished : the mono n-butyl and the dipropyl derivatives which concentrated strongly in the tumor with a high specificity and an exceptionally long retention time, and the dimethyl iodobenzamide which displayed, three hours after the injection, remarkable melanoma/non target tissue ratios associated with a good melanoma affinity, making it a promising new radiopharmaceutical for the scintigraphic detection of malignant melanoma and metastases.
Acknowledgments The authors thank CIS bio international for providing sodium [125I]iodide and their support in all the benzamide development.
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[8] J.M. Michelot, M.F. Moreau, P.G. Labarre, J.C. Madelmont, A.J. Veyre, J.M. Papon, D.F. Parry, J.F. Bonafous, J.Y. Boire, G.G. Desplanches, S.J. Bertrand, G. Meyniel, Synthesis and evaluation of new iodine-125 radiopharmaceuticals as potential tracers for malignant melanoma, J. Nucl. Med. 32 (1991) 1573–1580. [9] J.M. Michelot, M.F. Moreau, A.J. Veyre, J.F. Bonafous, F.J. Bacin, J.C. Madelmont, F. Bussiere, P.A. Souteyrand, L.P. Mauclaire, F.M. Chossat, J.M. Papon, P.G. Labarre, P. Kauffmann, R.J. Plagne, Phase II scintigraphic clinical trial of malignant melanoma and metastases with iodine-123-N-(2-diethylaminoethyl)-4-iodobenzamide, J. Nucl. Med. 34 (1993) 1260 –1266. [10] A. Mohammed, C. Nicholl, U. Titsch, M. Eisenhut, Radioiodinated N-(alkylaminoalkyl)-substituted 4-methoxy-, 4-hydroxy-, and 4-aminobenzamides: biological investigations for the improvement of melanoma-imaging agents, Nucl. Med. Biol. 24 (1997) 373–380. [11] M.F. Moreau, J.M. Michelot, A. Veyre, J.C. Madelmont, D. Godeneche, P. Labarre, D. Parry, G. Meyniel, 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 (1989) n° P.V. 890 1898. [12] M.F. Moreau, J.M. Michelot, A. Veyre, J.C. Madelmont, D. Godeneche, P. Labarre, D. Parry, G. Meyniel, Agents for diagnosing and treating melanomas. Aromatic halogenated derivatives usable as such agents and their preparation, European Patent (1993) n° 909038 –572–2107.
[13] M.F. Moreau, J. Michelot, J. Papon, M. Bayle, P. Labarre, J.C. Madelmont, D. Parry, J.Y. Boire, N. Moins, H. Seguin, A. Veyre, L. Mauclaire, Synthesis, radiolabeling, and preliminary evaluation in mice of some (N-diethylaminoethyl)-4-iodobenzamide derivatives as melanoma imaging agents, Nucl. Med. Biol. 22 (1995) 737– 747. [14] C. Nicholl, A. Mohammed, W.E. Hull, B. Bubeck, M. Eisenhut, Pharmacokinetics of iodine-123-IMBA for melanoma imaging, J. Nucl. Med. 38 (1997) 127–133. [15] R.A. Reisfeld, S. Ferrone, Conference report. International conference on biology of melanoma, Melanoma Research 6 (1996) 447– 456. [16] S. Rodot, J. Darcourt, F. Bussiere, J.P. Lacour, O. Migneco, A. Thyss, J.M. Michelot, J.F. Bonafous, M. Schneider, M. Barety, D. Beckhechi, J.P. Ortonne, A radiolabelled iodobenzamide for malignant melanoma staging, Melanoma Research 4 (1994) 307–312. [17] H. Seguin, D. Gardette, M.F. Moreau, J.C. Madelmont, J.C. Gramain, A general method for the synthesis of N,N-dialkylaminobutylamines, Synthetic Commun. 28 (1998) 4257– 4272. [18] S. Ullberg, The technique of whole body autoradiography cryosectioning of large specimens, Sci. Tools LKB Instr. J. (1977) 1–32. [19] E. Wo¨ ll, A. Bedikian, S.S. Legha, Uveal melanoma: natural history and treatment options for metastatic disease, Melanoma Research 9 (1999) 575–581.
Synthesis, characterization and comparative biodistribution study of a new series of p-Iodine-125 benzamides as potential melanoma imaging agents Nicole Moinsa,*, Janine Papona, He´le`ne Seguinb, Daniel Gardetteb, Marie-France Moreaua, Pierre Labarrea, Martine Baylea, Josette Michelota, Jean-Claude Gramainb, Jean-Claude Madelmonta, Annie Veyrea b
a INSERM U484, 63005 Clermont-Ferrand, France UMR SEESIB 6504, Universite Blaise Pascal, 63177 Aubiere, France
Received 23 February 2001; received in revised form 4 April 2001; accepted 3 May 2001
Abstract Iodobenzamides are reported to possess some affinity for melanoma. In order to identify the compound having the most appropriate pharmacokinetic properties as a potential melanoma imaging agent, thirteen new [125I]radioiodobenzamides with a butylene amide-amine spacer and various substituents on the terminal amino group were investigated. Their synthesis, radioiodination and biodistribution in B16 melanoma bearing C57BL6 mice are described and compared to [125I] labeled N-(2-diethylaminoethyl)-4-iodobenzamide ([125I]BZA), our reference compound. Changes in the terminal amino constituents induced modifications of lipophilicity, tumor uptake and organ distribution. The dimethylaminobutyl iodobenzamide appeared to be the most promising radiopharmaceutical imaging agent for the detection of melanoma and its metastases. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Melanoma scintigraphy; Iodine-125-benzamides; Radioactivity biodistribution analysis; Murine melanoma model
1. Introduction Over recent decades, the incidence of malignant melanoma (MM) is increasing at an annual rate of about 7% in most countries, more rapidly in some parts of Australia, and faster than for any other tumors [15]. Malignant melanoma is a tumor with a high potential of metastasis spreading, often early in the disease development but sometimes after a long disease-free period, in particular for the uveal form [19]. Until now, no treatment has demonstrated any significant efficacy although hope arises from immunological and gene therapy under investigation [5]. The early detection of metastases is always an important diagnostic goal and a follow-up is also clinically relevant even for patients without detected proliferation at the surgical removal time of the * Corresponding author. INSERM U484, Rue Montalembert, BP 184, 63005 Clermont-Ferrand cedex, France. Tel.: ⫹(33) 4 73 15 08 00; fax: ⫹(33) 4 73 15 08 01. E-mail address: [email protected] (N. Moins).
primary lesion. So, a noninvasive test of high specificity and precision for the detection of melanoma cells is suitable and scintigraphic imaging with a specific radiotracer would be of large interest. We have been the first to propose a series of iodobenzamide derivatives showing an affinity for melanoma tissue [11,12]. Preclinical studies reported a selective uptake in melanoma tumors induced in mice, in murine B16 tumors, as well as in human tumors transplanted into athymic mice [4,8]. A phase II scintigraphic clinical trial evaluating iodine-123 labeled N-(2-diethylaminoethyl)-4-iodobenzamide ([123I]-BZA) as an imaging agent of primary melanomas and metastases conducted on 110 patients resulted in a diagnostic sensitivity of 81% and a specificity of 100% [9]. The interest of this compound for the management of patients with melanoma has been confirmed [1,2,16]. The only result of the trial suggesting improvements may be required was the time with the best tumoral definition: 18 –24 hr after [123I]BZA administration. For cost and dosimetry reasons, we sought an agent that would provide quality images sooner after injection. With this view, a first
0969-8051/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 1 ) 0 0 2 4 2 - 6
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N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
Fig. 1. General structural formula of the radioiodinated benzamides 1–13. Substituents are listed in Table 1.
series of twelve [125I]BZA derivatives has been assessed in melanoma bearing mice [13]. Good results were obtained with para-iodobenzamide structures, and a lengthening of amide-amine spacer appeared favorable. In order to evaluate systematically a chemical series, we chose to synthesize and study thirteen para-iodobenzamides with a chain length of four methylene groups and varying amino alkyl substituents (Fig. 1 and Table 1).
2. Materials and methods 2.1. Materials All commercially available chemicals were of analytical grade and used without further purification. Sodium [125I]iodide was supplied by CIS bio international (Saclay, France) as a no-carrier added solution in reductant-free 0.1N aqueous sodium hydroxide. The melting points (Mp) were determined on a Reichert hot stage apparatus and are uncorrected. Chromatography refers to flash column chromatography on Merck Kieselgel 60 (230 – 400 mesh) or on Table 1 n-Octanol/phosphate buffer partition coefficient (log P) and 50% lethal dose of 4-iodobenzamide derivatives Compound BZA 1 2 3 4 5 6 7 8 9 10 11 12 13
R1* C2H5
R2* C2H5
H H H CH3 H C2H5 H n-C3H7 H i-C3H7 H n-C4H9 CH3 CH3 C2H5 C2H5 n-C3H7 n-C3H7 i-C3H7 i-C3H7 n-C4H9 n-C4H9 ⫺(CH2)4⫺ ⫺(CH2)5⫺
log P
LD50 (mmol/kg)
1.34
0.25
0.02 0.01 0.18 0.70 0.52 1.16 0.58 0.69 1.48 0.82 1.10 0.70 1.10
0.49 0.38 0.34 0.23 0.25 0.16 0.38 0.19 0.09 0.11 0.05 0.18 0.17
BZA: N-2-diethylaminoethyl-4-iodobenzamide. * See Fig. 1 for structure. LD50: determined in OF1 mice after i.v. administration.
Acros neutral Aluminum oxide (50 –200 ) as specified. TLC analyses were performed on Merck 60 F254 silica gel plates and spots were visualized with ultraviolet light (254 nm) and exposition of the plates to iodine vapors. Infrared spectra were recorded on a Perkin-Elmer 881 spectrometer. NMR spectra were performed on an AC 400 spectrometer (Bruker, Wissemburg, France) operating at 1H and 13C frequencies of 400.13 MHz and 100.61 MHz, respectively, in CDCl3 as solvent. Chemical shift data are reported in parts per million and in the following order for 1H data: multiplicity (s ⫽ singlet; bs ⫽ broad singlet; d ⫽ doublet; t ⫽ triplet; q ⫽ quadruplet; qt ⫽ quintuplet; st ⫽ sextuplet; m ⫽ multiplet); number of protons; proton assignment; coupling constant. Elemental analyses were determined at the Service Central d’Analyze (CNRS, Vernaison, France). Mass spectra were realized on a micromass VG 305 and Hewlett-Packard HP5989B spectrometers at the University Blaise Pascal (Clermont-Ferrand, France). 2.2. Synthesis Unlabeled iodobenzamides 1–13 were prepared in good yields in a simple one-step reaction by condensation of 4-iodobenzoyl chloride or 4-nitrophenyl 4-iodobenzoate with the already reported N–substituted aminobutylamines [17]. N-(4-aminobutyl)-4-iodobenzamide 1. 4-Aminobutylamine (0.5 mL; 0.48 g; 5.4 mmol), in solution in tetrahydrofuran (10 mL), was added to a solution of 4-iodobenzoate of 4-nitrophenol (1.0 g; 2.7 mmol) in THF (10 mL). The mixture was then stirred and refluxed for 30 min. After evaporation of the solvent, the residue was dissolved in methylene chloride (25 mL). The organic layer was washed with 1N NaOH (2 x 15 mL), then with water (15 mL) and dried over magnesium sulfate. 1 was obtained after purification by chromatography on a silica gel column (MeOH/ NH4OH) (0.49 g; 57%); Mp 110 –111°C; HCl salt mp 245–248°C. IR (CCl4) 1660 cm-1. 1H NMR of the free base ␦ 1.42 ppm (bs, 2H, NH2); 1.55 (qt, 2H, H-3⬘, J ⫽ 7.0 Hz); 1.68 (qt, 2H, H-2⬘, J ⫽ 7.0 Hz); 2.77 (m, 2H, H-4⬘); 3.45 (m, 2H, H-1⬘); 7.23 (bs, 1H, CONH); 7.53 (dd, 2H, aromatic H, J ⫽ 8.5 Hz); 7.76 (dd, 2H, aromatic H, J ⫽ 8.5 Hz). 13C NMR 27.1 ppm (C-3⬘); 30.9 (C-2⬘); 40.1 (C-4⬘); 41.7 (C1⬘); 98.1 (C-4); 128.7 (C-3, C-5); 134.4 (C-1); 137.7 (C-2, C-6); 166.8 (CO). MS: m/z 318 ([M⫹], 22). Elemental analysis: theoretical (%) C 41.51, H 4.75, N 8.81. I 39.90; Found (%) C 41.84, H 4.89, N 8.72; I 39.55. 2.2.1. General procedure for the preparation of iodobenzamides 2–6 The solution of the amine (10 mmol) in anhydrous tetrahydrofuran (20 mL) was added to a solution of the 4-iodobenzoate of 4-nitrophenol (10 mmol; 3.7 g) in THF (20 mL). The reaction mixture was stirred for ten hours at room temperature. After evaporation, the crude mixture was dissolved in methylene chloride (75 mL) and washed with 1N
N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
NaOH. The organic phase was then washed with water, dried over MgSO4 and the solvent removed. Purification by chromatography on neutral alumina column or by crystallization led to the desired iodobenzamides. N-(4-methylaminobutyl)-4-iodobenzamide 2. 2 was obtained from 4-iodobenzoate of 4-nitrophenol and N-methyl4-aminobutylamine after purification by chromatography on neutral alumina (AcOEt/MeOH 8/2) as white needles (2.26 g; 68%); Mp 60 – 61°C; HCl salt mp 245°C. IR (CCl4) 1655 cm-1. 1H NMR of the free base ␦ 1.50 ppm (bs, 1H, NHCH3); 1.60 (qt, 2H, H-3⬘, J ⫽ 6 Hz); 1.70 (qt, 2H, H-2⬘, J ⫽ 6.0 Hz); 2.40 (s, 3H, CH3); 2.60 (t, 2H, H-4⬘, J ⫽ 6.0 Hz); 3.40 (m, 2H, H-1⬘); 7.50 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 7.70 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 8.00 (bs, 1H, CONH); 13C NMR 27.4 ppm (C-3⬘); 27.7 (C-2⬘); 36.5 (CH3); 40.1 (C-1⬘); 51.6 (C-4⬘); 98.0 (C-4); 128.7 (C-3, C-5); 134.5 (C-1); 137.5 (C-2, C-6); 166.7 (CO). MS: m/z 332 ([M⫹], 8). Elemental analysis: theoretical (%) C 43.37, H 5.16, N 8.43; Found (%) C 42.95, H 5.08, N 8.13. N-(4-ethylaminobutyl)-4-iodobenzamide 3. 3 was purified by chromatography on neutral alumina (AcOEt/MeOH 9/1) and obtained as white crystals (1.94 g; 56%); Mp 64 – 65°C; HCl salt mp 209 –211°C. IR (CCl4) 1655 cm-1. 1 H NMR of the free base ␦ 0.85 ppm (t, 3H, CH3, J ⫽ 7.0 Hz); 1.60 (m, 5H, H-2⬘, H-3⬘, NHCH2CH3); 2.51 (m, 4H, H-4⬘, CH2CH3); 3.40 (m, 2H, H-1⬘); 7.49 (dd, 2H, aromatic H, J ⫽ 8.4 Hz); 7.55 (bs, 1H, CONH); 7.72 (dd, 2H, aromatic H, J ⫽ 8.4 Hz). 13C NMR 15.2 ppm (CH3); 27.4 (C-3⬘); 27.9 (C-2⬘); 40.1 (C-1⬘); 44.2 (CH2CH3); 49.2 (C4⬘); 98.0 (C-4); 128.7 (C-3, C-5); 134.5 (C-1); 137.6 (C-2, C-6); 166.8 (CO). MS: m/z 346, ([M⫹], 2). Elemental analysis: theoretical (%) C 45.08, H 5.53, N 8.09; Found (%) C 44.42, H 5.60, N 7.85. N-(4-propylaminobutyl)-4-iodobenzamide 4. 4 was purified by chromatography on neutral alumina (AcOEt/MeOH 9/1) and obtained as white crystals (2.16 g; 60%); Mp 73–74°C; HCl salt mp 219 –220°C. IR (CCl4) 1660 cm-1. 1H NMR of the free base ␦ 0.90 ppm (t, 3H, CH3, J ⫽ 7.4 Hz); 1.30 (bs, 1H, NHPr); 1.50 (st, 2H, CH2CH2CH3, J ⫽ 7.4 Hz); 1.60 (qt, 2H, H-3⬘; J ⫽ 6.6 Hz); 1.70 (qt, 2H, H-2⬘, J ⫽ 6.6 Hz); 2.55 (t, 2H, CH2CH2CH3, J ⫽ 7.4 Hz); 2.65 (t, 2H, H-4⬘, J ⫽ 6.6 Hz); 3.45 (m, 2H, H-1⬘); 7.50 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 7.55 (bs, 1H, CONH); 7.75 (dd, 2H, aromatic H, J ⫽ 8.3 Hz). 13C NMR 11.7 ppm (CH3); 22.9 (CH2CH3); 27.3 (C-2⬘); 27.7 (C-3⬘); 40.0 (C-1⬘); 49.2 (C-4⬘); 51.8 (CH2CH2CH3); 97.0 (C-4); 128.7 (C-3, C-5); 134.4 (C-1); 137.4 (C-2, C-6); 166.8 (CO). MS : m/z 360 ([M⫹], 16). Elemental analysis: theoretical (%) C 46.66, H 5.88, N 7.78, I 35.24; Found (%) C 45.92, H 5.87, N 7.55, I 34.41. N-(4-isopropylaminobutyl)-4-iodobenzamide 5. 5 was purified by chromatography on neutral alumina (AcOEt/ MeOH 9/1) and obtained as white crystals (1.87 g; 52%); Mp 76 –78°C; HCl salt mp 180 –182°C. IR (CCl4) 1665 cm-1. 1H NMR of the free base ␦ 1.00 ppm (d, 6H, CH3, J ⫽ 6.3 Hz); 1.30 (bs, 1H, NHiPr); 1.57 (qt, 2H, H-3⬘, J ⫽ 6.7
801
Hz); 1.65 (qt, 2H, H-2⬘, J ⫽ 6.7 Hz); 2.50 (t, 2H, H-4⬘; J ⫽ 6.7 Hz); 2.75 (ht, 1H, CH, J ⫽ 6.3 Hz); 3.50 (m, 2H, H-1); 7.25 (bs, 1H, CONH); 7.50 (dd, 2H, aromatic H, J ⫽ 8.5 Hz); 7.75 (dd, 2H, aromatic H, J ⫽ 8.5 Hz). 13C NMR 22.8 ppm (CH3); 27.3 (C-2⬘); 27.9 (C-3⬘); 39.9 (C-1⬘); 46.7 (C-4⬘); 48.9 (CH); 97.7 (C-4); 128.7 (C-3, C-5); 134.1 (C-1); 137.6 (C-2, C-6); 166.2 (CO). MS: m/z 360 ([M⫹], 4). Elemental analysis: theoretical (%) C 46.66, H 5.88, N 7.78, I 35.24; Found (%) C 46.42, H 5.91, N 7.56, I 34.45. N-(4-butylaminobutyl)-4-iodobenzamide 6. 6 was purified by chromatography on neutral alumina (AcOEt/MeOH 95/5) and obtained as white crystals (2.51 g; 67%); Mp 75–76°C; HCl salt mp 230 –235°C. IR (CCl4) 1655 cm-1. 1H NMR of the free base ␦ 0.90 ppm (t, 3H, CH3, J ⫽ 7.2 Hz); 1.30 (st, 2H, H-8⬘, J ⫽ 7.2 Hz); 1.40 (qt, 2H, H-7⬘, J ⫽ 7.2 Hz); 1.65 (m, 5H, H-2⬘; H-3⬘, NHBu); 2.55(m, 2H, H-4⬘); 2.65 (t, 2H, H-6⬘, J ⫽ 7.2 Hz); 3.40 (m, 2H, H-1⬘); 7.45 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 7.65 (bs, 1H, CONH); 7.70 (dd, 2H, aromatic H, J ⫽ 8.3 Hz). 13C NMR 14.0 ppm (C-9⬘); 20.5 (C-8⬘); 27.3 (C-3⬘); 27.5 (C-2⬘); 31.9 (C-7⬘); 40.0 (C-1⬘); 49.2 (C-4⬘); 49.7 (C-6⬘); 98.0 (C-4); 128.7 (C-3, C-5); 134.4 (C-1); 137.6 (C-2, C-6); 166.9 (CO). MS: m/z 374 ([M⫹], 100). Elemental analysis: theoretical (%) C 48.12, H 6.19, N 7.49; Found (%) C 47.45, H 6.19, N 7.34. 2.2.2. General procedure for the preparation of iodobenzamides 7–13. The solution of the amine (10 mmol) and triethylamine (10 mmol) in anhydrous methylene chloride (20 mL) was added drop wise to a solution of 4-iodobenzoyl chloride (10 mmol, 2.66 g). The mixture was stirred under nitrogen atmosphere at room temperature for 12 h and poured into 100 mL ice-water. The aqueous phase was extracted three times with CH2Cl2 (100 mL each time); the organic phase was dried over anhydrous MgSO4 and the solvent removed. The crude mixture was then purified by chromatography on neutral alumina column. N-(4-dimethylaminobutyl)-4-iodobenzamide 7. The reaction of 4-dimethylaminobutylamine and 4-iodobenzoyl chloride gave 7 after purification on neutral alumina column chromatography (AcOEt/MeOH 9/1) (1.83g; 53%); Mp 64 – 65°C; HCl salt mp 190 –192°C. IR (CCl4) 1660 cm-1. 1H NMR of the free base ␦ 1.60 ppm (qt, 2H, H-3⬘, J ⫽ 6.5 Hz); 1.65 (qt, 2H, H-2⬘, J ⫽ 6.5Hz); 2.20 (s, 6H, CH3); 2.35 (t, 2H, H-4⬘, J ⫽ 6.5 Hz); 3.40 (m, 2H, H-1⬘); 7.50 (dd, 2H, aromatic H, J ⫽ 8.3 Hz); 7.72 (dd, 2H, aromatic H, J ⫽ 8.3 Hz), 8.20 (bs, 1H, CONH). 13C NMR 24.9 ppm (C-3⬘); 27.0 (C-2⬘); 39.7 (C-1⬘); 44.8 (CH3); 58.8 (C-4⬘); 97.7 (C-4); 128.5 (C-3, C-5); 134.2 (C-1); 137.2 (C-2, C-6); 166.6 (CO). MS: m/z 346 ([M⫹], 8). Elemental analysis: theoretical (%) C 45.08, H 5.53, N 8.09, I 36.67; Found (%) C 45.28, H 5.46, N 7.87, I 36.44. N-(4-diethylaminobutyl)-4-iodobenzamide 8. The reaction of 4-diethylaminobutylamine and 4-iodobenzoyl chloride gave 8 after purification on neutral alumina column chromatography (AcOEt/MeOH 95/5) (2.17 g; 58%); Mp
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55–57°C; HCl salt mp 178 –180°C. IR (CCl4) 1665 cm-1. 1H NMR of the free base ␦ 1.00 ppm (t, 6H, CH2CH3, J ⫽ 7.1 Hz); 1.58 (qt, 2H, H-3⬘, J ⫽ 6.8 Hz); 1.66 (qt, 2H, H-2⬘, J ⫽ 6.8 Hz); 2.45 (t, 2H, H-4⬘, J ⫽ 6.8 Hz); 2.50 (q, 4H, CH2CH3, J ⫽ 7.1 Hz); 3.40 (m, 2H, H-1); 7.49 (dd, 2H, aromatic H, J ⫽ 8.5 Hz); 7.55 (bs, 1H, CONH), 7.75 (dd, 2H, aromatic H, J ⫽ 8.5 Hz). 13C NMR 11.0 ppm (CH3); 24.9 (C-3⬘); 27.6 (C-2⬘); 40.0 (C-4⬘); 46.6 (CH2CH3); 52.3 (C-1⬘); 98.0 (C-4); 128.8 (C-3, C-5); 134.5 (C-1); 137.5 (C-2, C-6); 166.9 (CO). MS: m/z 374 ([M⫹], 2). Elemental analysis: theoretical (%) C 48.13, H 6.19, N 7.49; Found (%) C 48.29, H 6.35, N 7.67. N-(4-dipropylaminobutyl)-4-iodobenzamide 9. The reaction of 4-dipropylaminobutylamine and 4-iodobenzoyl chloride gave 9 after purification on neutral alumina column chromatography (AcOEt) (2.73 g; 68%); Mp 70 –71°C; HCl salt mp 159 –160°C. IR (CCl4) 1670 cm-1. 1H NMR of the free base ␦ 0.80 ppm (t, 6H, CH3, J ⫽ 7.3 Hz); 1,40 (m, 4H, CH2CH2CH3); 1,50 (m, 2H, H-3⬘); 1.60 (m, 2H, H-2⬘); 2.30 (m, 4H, CH2CH2CH3); 2.40 (t, 2H, H-4⬘, J ⫽ 6.8 Hz); 3.40 (m, 2H, H-1⬘); 7.25 (bs, 1H, CONH); 7.50 (dd, 2H, aromatic H, J ⫽ 8.4 Hz); 7.72 (dd, 2H, aromatic H, J ⫽ 8.4 Hz). 13C NMR 11.9 ppm (CH3); 19.6 (CH2-CH3); 24.9 (C-3⬘); 27.5 (C-2⬘); 39.9 (C-1⬘); 53.5 (C-4⬘); 56.0 (CH2 CH2CH3); 98.0 (C-4); 128.7 (C-3, C-5); 134.5 (C-1); 137.5 (C-2, C-6); 166.9 (CO). MS: m/z 402 ([M⫹], 10). Elemental analysis: theoretical (%) C 50.75, H 6.77, N 6.96, I 31.56; Found (%) C 50.78, H 6.83, N 6.80, I 31.25. N-(4-diisopropylaminobutyl)-4-iodobenzamide 10. The reaction of 4-diisopropylaminobutyl-amine and 4-iodobenzoyl chloride gave 10 after purification on neutral alumina column chromatography (AcOEt/MeOH 95/5) (2.61 g; 65%); Mp 69 –71°C; HCl salt mp 135°C. IR (CCl4) 1675 cm-1. 1H NMR of the free base ␦ 1.15 ppm (d, 12H, CH3, J ⫽ 6.6 Hz); 1.42 (qt, 2H, H-3⬘, J ⫽ 7.0 Hz); 1.55 (qt, 2H, H-2⬘, J ⫽ 7.0 Hz); 2.37 (t, 2H, H-4⬘, J ⫽ 7.0 Hz); 2.95 (ht, 2H, CH, J ⫽ 6.6 Hz); 3.37 (m, 2H, H-1⬘); 6.75 (bs, 1H, CONH); 7.45 (dd, 2H, aromatic H, J ⫽ 8.4 Hz); 7.69 (dd, 2H, aromatic H, J ⫽ 8.4 Hz). 13C NMR 20.6 ppm (CH3); 27.3 (C-3⬘); 28.5 (C-2⬘); 40.2 (C-3⬘); 44.7 (C-4⬘); 48.4 (CH); 98.0 (C-4); 128.6 (C-3, C-5); 134.3 (C-1); 137.5 (C-2, C-6); 166.7 (CO). Elemental analysis: theoretical (%) C 50.75, H 6.77, N 6.96; Found (%) C 50.87, H 6.70, N 6.77. N-(4-dibutylaminobutyl)-4-iodobenzamide 11. The reaction of 4-dibutylaminobutylamine and 4-iodobenzoyl chloride gave 11 after purification on neutral alumina column chromatography (AcOEt/Hexane 3/7) (2.80 g; 65%); Mp 93–95°C; HCl salt mp 128 –130°C. IR (CCl4) 1650 cm-1. 1H NMR of the free base ␦ 0.90 ppm (t, 6H, CH3, J ⫽ 7.5 Hz); 1.30 (st, 4H, CH2CH3, J ⫽ 7.5 Hz); 1.70 (m, 6H, H-3⬘, CH2CH2CH2); 1.80 (m, 2H, H-2⬘); 2.95 (m, 6H, H-4⬘, NCH2CH2CH2CH3); 3.45 (m, 2H, H-1⬘); 7.70 (m, 4H, aromatic H); 8.25 (bs, 1H, CONH). 13C NMR 14.1 ppm (C-9⬘, C-13⬘), 20.0 (C-8⬘, C-12⬘); 21.0 (C-3⬘); 24.9 (C-7⬘, C-11⬘); 26.0 (C-2⬘); 38.1 (C-1⬘); 52.1 (C-6⬘, C-10⬘); 52.5 (C-4⬘); 98.3 (C-4); 129.3 (C-3, C-5); 133.6 (C-1); 137.5 (C-2, C-6);
166.9 (CO). Elemental analysis: theoretical (%) C 53.02, H 7.26, N 6.51; Found (%) C 53.20, H 7.36, N 6.79. N-(4-pyrrolidinobutyl)-4-iodobenzamide 12. The reaction of 4-pyrrolidinobutylamine and 4-iodobenzoyl chloride gave 12 after purification on neutral alumina column chromatography (AcOEt/MeOH 95/5) (1.93 g; 52%); Mp 89 – 91°C; HCl salt mp 202–203°C. IR (CCl4) 1665 cm-1. 1H NMR of the free base ␦ 1.70 ppm (m, 2H, H-2, H-3⬘); 1.80 (m, 4H, H-7⬘, H-8⬘); 2.65 (m, 6H, H-4⬘, H-6⬘, H-9⬘); 3.45 (m, 2H, H-1⬘); 7.50 (dd, 2H, aromatic H, J ⫽ 8.5 Hz); 7.75 (dd, 2H, aromatic H, J ⫽ 8.5 Hz), 7.90 (bs, 1H, CONH). 13C NMR 23.4 ppm (C-7⬘, C-8⬘), 26.0 (C-2⬘); 27.2 (C-3⬘); 39.7 (C-1⬘); 54.0 (C-6⬘, C-9⬘); 55.6 (C-4⬘); 98.1 (C-4); 128.8 (C-3, C-5); 134.5 (C-1); 137.6 (C-2, C-6); 166.9 (CO). Elemental analysis: theoretical (%) C 48.39, H 5.69, N 7.53; Found (%) C 48.59, H 5.81, N 7.67. N-(4-piperidinobutyl)-4-iodobenzamide 13. The reaction of 4-piperidinobutylamine and 4-iodobenzoyl chloride gave 13 after purification on neutral alumina column chromatography (AcOEt/MeOH 95/5), (2.74 g; 71%); Mp 120 –122°C; HCl salt mp 188 –189°C. IR (CCl4) 1665 cm-1. 1H NMR of the free base ␦ 1.40 ppm (m, 2H, H-8); 1.50 (qt, 4H, H-7⬘, H-9⬘, J ⫽ 6.8 Hz); 1.60 (m, 4H, H-2⬘, H-3⬘); 2.30 (m, 6H, H-4⬘, H-6⬘, H-10⬘); 3.40 (m, 2H, H-1⬘); 7.18 (bs, 1H, CONH); 7.47 (dd, 2H, aromatic H, J ⫽ 8.4 Hz); 7.73 (dd, 2H, aromatic H, J ⫽ 8.4 Hz). 13C NMR 24.4 ppm (C-8⬘); 24.6 (C-2⬘); 25.8 (C-7⬘, C-9⬘); 27.4 (C-3⬘); 40.1 (C-1⬘); 54.6 (C-6⬘, C-10⬘); 58.7 (C-4⬘); 98.0 (C-4); 128.8 (C-3, C-5); 134.5 (C-1); 137.6 (C-2, C-6); 166.9 (CO) MS: m/z 386 ([M⫹], 59). Elemental analysis: theoretical (%) C 49.75, H 6.01, N 7.25; Found (%) C 49.87, H 6.20, N 7.47. 2.3. [125I] Radiolabeling Radioiodination was performed by isotopic exchange with sodium [125I]iodide in citrate buffer with copper sulfate used as a catalyst accordingly to already published procedures [13]. The labeled compounds were identified by TLC comparison with the corresponding non-radioactive samples. The chromatograms were scanned with a Berthold LB 2832 linear analyzer. 2.4. pKa measurements pKa of each molecule (HCl salt) has been determined by an acid/base assay with 0.1 N aqueous sodium hydroxide. 2.5. Partition coefficient measurements For each molecule, the partition coefficient between noctanol and phosphate buffer was determined. The measurement was performed by shaking 2 mL of [125I] benzamide solution (50 M in phosphate buffer solution, PBS, pH 7.4) with 2 mL n-octanol. The activity of each phase was counted. P was calculated as the ratio of activities (n-
N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
octanol/buffer) and its logarithm was determined to express lipophilicity. 2.6. Animal studies All experiments were carried out in compliance with French laws governing animal experimentation. 2.6.1. Toxicity Acute toxicity (LD50) was determined in male OF1 mice (Iffa Credo, Les Oncins, France) after intravenous administration. The non-radioactive iodobenzamides were dissolved in saline (0.9% NaCl). The LD50 value determination was performed with six different doses varying by a geometrical progression, each of them being administered to six animals. 2.6.2. Biodistribution The biodistribution of radioiodinated benzamides was studied in C57BL6 male mice, bearing the B16 murine melanoma. B16 melanoma cells were originally obtained from ICIG (Villejuif, France) and maintained as a monolayer in tissue culture minimum essential medium (MEM, Gibco) supplemented with 10% fetal calf serum and antibiotics, and passaged by trypsination. Early passages were frozen and stored in liquid nitrogen, and for transplantation an aliquot was grown in a monolayer culture to confluence and cells were trypsinized and washed with phosphate buffer saline (PBS). They were resuspended in PBS and each mouse received 0.1 mL subcutaneously (5 x 105 cells) on the left flank. Ten days later, the tumors became palpable with a percentage of tumor take equal to 98 –100%. 2.6.2.1. Experimental procedure. The [125I]benzamide was administered intravenously via a tail vein (0.1 mol, 0.74 – 0.92 MBq/animal) in ten animals for each compound. Two injected mice were sacrified by quickly freezing in liquid nitrogen after ether anesthesia, at different times after administration (1, 3, 6, 24 or 72 hours). Frozen animals were cryosectioned using the technique described by Ullberg [18]. Slices of 40 m were obtained using a Reichert-Jung cryopolycut (Leica Instrument, Rueil Malmaison, France) at -22°C and dehydrated for 48 hr in the cryochamber. 2.6.2.2. Detection and quantification of radioactivity. Radioactivity contained in slices was analyzed using an AMBIS 4000 detector (Scanalytics, CSPI, San Diego, CA) which is a computer-controlled multi-wire proportional counter previously described [6] and validated for evaluation in mice of iodinated agents [7]. A two dimensional image of the slice was displayed on the computer screen for analysis, and quantification of different organs was made after contouring suitable zones. Measurements were performed after a 1000 min acquisition time. The radioactivity per unit area (net cpm/mm2) was converted into radioactive
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concentration (kBq/g) and expressed as percentage of injected dose/g of tissue (%ID/g). The tumor (T) uptake was compared to those of other tissues and ratios (T/organ) were determined illustrating the image contrast. For two animals, urine and feces have been collected until 72 hr and counted to determine the cumulative urinary and fecal excretions.
3. Results 3.1. Chemistry All iodobenzamides 1–13 (Table 1) were synthesized, according to two different procedures, from already described aminobutyl amines prepared from readily available starting materials [17]. The primary amino group of N-alkylaminobutylamines was selectively acylated on treating with 4-nitrophenyl 4iodobenzoate at room temperature for 10 hours, in good yields, to lead to iodobenzamides 1– 6 bearing a mono or a disubstituted amino function. On the other hand, iodobenzamides 7–13, with a tertiary amino group, were prepared by reacting the required N,N-dialkylaminobutyl amines with p-iodobenzoyl chloride at room temperature for 2 hours. All synthesized iodobenzamides 1–13 exhibited expected IR, 1 H and 13C NMR data. Iodobenzamides 1–13 were converted, for pre-clinical use, into hydrochlorides after treatment by an anhydrous hydrogen chloride solution in diethyl ether. 3.2. [125I] Radiolabeling Radioiodination was achieved by Cu (II) assisted isotopic exchange. Radiochemical yields based on TLC of exchange reaction mixture varied from 88 to 95%. After purification by passing the crude mixture through silicic acid cartridge (ExtrelutR20, Merck), the [125I]-labeled compounds were shown by TLC to be free of significant chemical and radiochemical impurities. 3.3. pKa measurements pKa values ranged from 10.2 to 11.7, indicating an almost complete protonation of amino groups at physiological pH. 3.4. Partition coefficients No significant lipophilicity has been measured for compounds 1 and 2. The partition coefficient value of iodobenzamides 2–10 increased with the number and the length of substituents (Table 1). This variation was regular for the iodobenzamides 2– 6, with a secondary amino group. However, for compounds 7–13 with a tertiary amino group, such a variation was only observed for compounds 7 to 9, with a
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Table 2 Uptake of [125I]benzamide derivatives in tumor at various times after injection in B16 melanoma bearing mice (%ID/g)* Compound
1 hr
3 hr
6 hr
24 hr
72 hr
BZA
9.5 ⫾ 1.9
9.1 ⫾ 1.6
7.7 ⫾ 3.3
3.7 ⫾ 1.2
0.8 ⫾ 0.3
1 2 3 4 5 6 7 8 9 10 11 12 13
0.6 ⫾ 0.2 1.1 ⫾ 0.3 2.7 ⫾ 0.8 6.4 ⫾ 1.2 5.0 ⫾ 1.3 5.5 ⫾ 1.4 7.8 ⫾ 1.6 6.3 ⫾ 3.7 8.9 ⫾ 1.6 3.8 ⫾ 0.5 2.6 ⫾ 0.3 4.3 ⫾ 0.7 5.2 ⫾ 0.5
0.5 ⫾ 0.1 0.9 ⫾ 0.4 1.6 ⫾ 0.3 5.7 ⫾ 1.0 2.8 ⫾ 1.2 9.6 ⫾ 4.0 7.1 ⫾ 1.9 5.2 ⫾ 1.9 11.2 ⫾ 1.9 6.3 ⫾ 1.4 5.1 ⫾ 1.3 4.5 ⫾ 0.9 2.9 ⫾ 1.1
0.3 ⫾ 0.0 0.7 ⫾ 0.2 2.0 ⫾ 0.9 3.6 ⫾ 0.9 2.5 ⫾ 0.9 6.2 ⫾ 1.0 6.8 ⫾ 2.5 4.7 ⫾ 1.4 7.6 ⫾ 1.6 5.2 ⫾ 1.2 7.1 ⫾ 1.0 1.3 ⫾ 0.2 3.0 ⫾ 0.8
— 0.8 ⫾ 0.1 1.0 ⫾ 0.5 2.4 ⫾ 1.3 2.1 ⫾ 0.8 5.3 ⫾ 1.5 3.0 ⫾ 2.5 3.3 ⫾ 1.0 4.6 ⫾ 1.0 2.6 ⫾ 0.7 3.8 ⫾ 1.8 2.0 ⫾ 0.7 1.3 ⫾ 0.3
— — — 0.4 ⫾ 0.1 1.3 ⫾ 0.4 3.3 ⫾ 0.8 1.0 ⫾ 0.3 — 3.2 ⫾ 0.6 0.8 ⫾ 0.3 1.1 ⫾ 0.3 0.5 ⫾ 0.2 1.7 ⫾ 0.5
* Mean ⫾ SD (two mice for each compound and each time). Absence of value: organ concentration was equal to background value.
maximal value of 1.48 for benzamide 9. Surprisingly, the log P value of benzamide 11 was notably lower than that of benzamide 9. 3.5. Animal studies 3.5.1. Toxicity LD50 values determined in mice as a parameter of acute toxicity were summarized in Table 1. Compared to the BZA value (0.25 mmol/kg), the LD50 values were of the same order except for benzamides 9 or 11 (dipropyl or dibutyl derivatives) which appeared to be the more toxic compounds. It must be noticed that iodobenzamides 1– 6 bearing a secondary amino group were generally less toxic than iodobenzamides 7–13 with a tertiary one. Moreover, in each series of compounds 1– 6 and 7–13, the toxicity increased with the length of the amino substituent. Nevertheless, the mass injected in the biodistribution studies was much lower than toxic ones, and even with the most toxic benzamide 11, the LD50 value equal to 0.9 mol/mouse, was at least 10 times the injected dose. 3.5.2. Biodistribution For the thirteen [125 I]-labeled compounds, the tissue distribution was evaluated in melanoma bearing mice. In fact, total radioactivity was measured and if metabolites may be involved, only the total of [125I] containing moieties was estimated. The values of tumoral fixation (%ID/g) of the benzamides are summarized in Table 2 and compared with our reference compound, [125I]BZA. For all the studied molecules, a tumoral accumulation was observed (⬎2.5%ID/g at 1 hour p.i.) except for benzamides 1 and 2, the two compounds with a log P value close to zero, which exhibited the lowest concentrations in tumor and other tissues. However, the values were distributed in a large range. For the monobutyl 6 and the dipropyl 9 derivatives, the
tumoral concentration appeared at 3 hours p.i. at least equal or higher than with BZA. This uptake remained higher in melanoma than in the other organs as illustrated on Fig. 2, and was particularly more persistent than with BZA since 72 hours after injection, benzamides 6 and 9 were still concentrated in tumors (Table 2). Benzamide 7, which still exhibited a tumoral concentration similar to BZA after 3 and 72 hours, showed a rapid clearance from the other organs in which tracer concentration was already very low at 3 hours. For all compounds, the higher radioactive concentration was found in the uvea with values ⬎5%ID/g, 1 hour after the injection, except for benzamides 1 or 2. With compounds 3–13, the blood activity ranged from 0.5 to 2.3%ID/g 1 hour after the injection and as BZA, a rapid clearance was observed. These eleven compounds were able to cross the blood brain barrier. However, the brain concentration values were low ranging from 0.1 to 1.4%ID/g at 1 hour p.i., and decreased very quickly. Differences in iodobenzamide excretions have been noted, but the urinary elimination was the main route for most of the compounds (Table 3). Among the remarkable molecules (6, 7, 9) benzamide 6 appeared with the slowest urinary excretion and concomitantly the highest fecal excretion. On the contrary, benzamide 7 exhibited a low fecal excretion. As lung, liver or brain are known to develop melanoma metastases, the ratios between concentrations of benzamide in tumor and these organs have been calculated and are summarized in Table 4. From 1 hour after the injection, the tumor:brain ratios were high due to the low values in the brain and notably higher than BZA values. The highest tumor:lung or tumor:liver ratios were observed for benzamide 7 which appeared to be the most selective of the tumoral tissue. The 1 hour values were already equivalent to the ones of BZA at 3 hours, and the 3 hour values were
N.Moins et al. / Nuclear Medicine and Biology 28 (2001) 799 – 808
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Fig. 2. Concentrations of [125I]benzamide derivatives in selected tissues 3 hours after i.v. administration in C57BL6 mice-bearing B16 melanoma. Comparison with [125I]BZA (% ID/g: mean ⫾ S.D., two mice).
about 3– 4 times higher. With benzamides 6 or 9, the most concentrated in tumor at that time, the selectivity was maintained compared to BZA and even slightly improved for benzamide 9. 4. Discussion The radioiodinated benzamide BZA was the first compound of this chemical class successfully developed as Table 3 Urinary and fecal excretions* of radioactivity after [125I]benzamide derivative injection in B16 melanoma bearing mice Urinary %ID 24hr/72hr
Fecal %ID 24hr/72hr
BZA
81 / 83
4/5
1 2 3 4 5 6 7 8 9 10 11 12 13
76 / 81 67 / 68 88 / 89 74 / 75 45 / 45 57 / 59 76 / 77 65 / 66 63 / 66 62 / 62 56 / 58 54 / 54 30 / 31
1/2 2/3 2/2 5/5 2/2 13 / 13 2/2 5/6 6/7 13 / 14 4/5 14 / 14 21 / 22
* Cumulative excretions (two mice for each compound).
radiotracer agent for the imaging of melanoma and its metastases [8,9]. In order to propose a more efficient derivative with even better pharmacokinetic characteristics, different structural variations have been realized by changing the position of radioiodine on the phenyl ring, the length of the amide-amine spacer (two or three methylene groups) or the substituents borne by the terminal amino group. The derivatives obtained have been investigated by studying their biodistribution in melanoma-bearing mice [3,8,10,13,14]. While no compound showed major enhancement of characteristics compared to BZA, variations have been elicited Table 4 Melanoma/organ ratios of [125I]benzamide derivative uptake in B16 melanoma bearing mice 3 hours after the injection Compound
Lung 1hr/3hr
Liver 1hr/3hr
BZA
1.7 / 4.5
1.6 / 2.8
3 4 5 6 7 8 9 10 11 12 13
1.2 / 1.9 1.1 / 4.1 1.4 / 2.8 0.9 / 3.1 4.8 / 12.8 1.0 / 5.7 1.4 / 6.2 1.3 / 6.2 0.4 / 2.2 1.5 / — 2.7 / —
1.5 / 1.8 1.3 / 2.5 1.4 / 1.7 1.0 / 2.5 3.5 / 12.3 0.9 / 3.1 1.3 / 3.1 0.9 / 2.3 0.3 / 1.1 1.1 / 2.1 1.0 / 1.3
Brain 1hr/3hr 6.21 / 11.0 13.6 / 19.6 12.5 / 27.3 22.7 / 15.8 15.2 / 22.3 5.4 / 21.6 7.2 / — 17.0 / 41.6 26.9 / 44.9 4.5 / 19.5 8.5 / 24.8 8.4 / —
Absence of value: very high ratio, the organ concentration being equal to background value.
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Fig. 3. Tumoral concentrations of [125I]benzamide derivatives 3 hours after i.v. administration in C57BL6 mice-bearing B16 melanoma, compared to n-octanol/buffer partition coefficient for N-alkylated (1– 6; A) and N, N-dialkylated (7–13; B) series, respectively.
with some promising evolution. Due to encouraging results observed when the amide-amine spacer was extended from two to three methylene groups [13], the present work reports synthesis and biodistribution study in melanoma-bearing mice of a series of thirteen radioiodobenzamides characterized by a four methylene linear spacer between amide and
amine. The substituents carried by the secondary or tertiary amino function were either linear, branched or cyclic. The synthetic procedures used allowed us a gram scale production. The thirteen new iodobenzamides described in our study showed a tumoral affinity for all but compounds 1 and 2, specially high for benzamides 6, 7 and 9.
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The biological behavior of a chemical compound, at least its background-tissue retention is usually reported to be related to its lipophilicity. The variations of tumoral fixation and n-octanol/buffer partition coefficient are compared in Fig. 3 for N-alkylated (A) and N,N-dialkylated (B) series, respectively. A relation between these two parameters was obvious for the two series : benzamides 1 and 2, with the lowest lipophilicity, failed to concentrate in any tissue, while the compounds 6 and 9, with the highest lipophilicity showed the best tumoral concentrations of the tracer. For the N,N-dialkylated series, the decrease in lipophilicity observed from the compound 9 onwards, was related to a decrease in melanoma uptake. These results evidenced the role of lipophilicity in the biological behavior for compounds of an homogenous chemical series. The highest melanoma uptake was observed for benzamide 9 with log P ⫽ 1.48. Nicholl et al. [14], with related compounds, in a similar study, have already described the best conditions for the log P values range : 1.30 –1.45. Moreover, these authors observed a reduced uptake for very lipophilic or hydrophilic compounds, with the exception of N-N-(3-diethylaminopropyl)-4-iodobenzamide which showed high uptake although its log P is only 0.75. Therefore, log P is not the only characteristic required to explain the melanoma concentration and remains questionable in predicting the in vivo behavior. Nevertheless, in our study, the two parameters exhibited a very close variation, at least for linear substituents. However, the low log P value (0.58) of benzamide 7 may justify a lower labeling of nontarget tissues and organs, and therefore, high tumor/organ ratio values. The challenge of this work was to find malignant melanoma agents which would exhibit an improved pharmacological behavior with respect to BZA. A large variety of biodistribution was displayed among the thirteen iodobenzamides of our study. Contrary to some results reported in the literature, variations of the amine substituents appeared to have a significant impact. At least, three compounds exhibited original promising data. Two of them, the mono n-butyl 6 and the dipropyl 9 derivatives demonstrated an affinity for the tumor at least equal to BZA with an equivalent specificity for short times but a very slow tumoral clearance. Such pharmacokinetic data showing a durable and specific labeling of the tumoral site may be of potential interest for use in internal radiotherapy. The dimethyl derivative 7 was also comparable to BZA concerning the melanoma affinity but a more rapid clearance from nontarget tissues conferred potentially improved melanoma imaging on this structure. The high melanoma:nontarget organ ratios at 3 hours p.i. allowed us to consider it as the best radiopharmaceutical candidate for diagnosis. Note that a tendency for such a behavior has already been reported for N, N-(3-dimethylaminopropyl)-4-iodobenzamide [13]. Thus, compared to previous results obtained with the iodobenzamide series [13], increasing the amide-amine spacer length until butylene group appeared efficient in view of the application to melanoma diagnosis. Further
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attempts to synthesize other iodobenzamides are under investigation. In conclusion, thirteen new iodine-125-benzamides with a butyl amide-amine spacer and varying aminoalkyl substituents have been successfully synthesized and evaluated as imaging agents in melanoma-bearing mice. Different pharmacokinetic profiles were observed although an affinity for melanoma was obvious for all but the unsubstituted and the monomethyl molecules. Overall, the tumoral uptake appeared closely related to the n-octanol/buffer partition coefficient value. Three compounds were distinguished : the mono n-butyl and the dipropyl derivatives which concentrated strongly in the tumor with a high specificity and an exceptionally long retention time, and the dimethyl iodobenzamide which displayed, three hours after the injection, remarkable melanoma/non target tissue ratios associated with a good melanoma affinity, making it a promising new radiopharmaceutical for the scintigraphic detection of malignant melanoma and metastases.
Acknowledgments The authors thank CIS bio international for providing sodium [125I]iodide and their support in all the benzamide development.
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