Peripheral Benzodiazepine Receptor Ligand−Melphalan Conjugates

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Bioconjugate Chem. 2003, 14, 830−839

Peripheral Benzodiazepine Receptor Ligand-Melphalan Conjugates for Potential Selective Drug Delivery to Brain Tumors Giuseppe Trapani,*,† Valentino Laquintana,† Andrea Latrofa,† Jianguo Ma,‡ Karin Reed,‡ Mariangela Serra,§ Giovanni Biggio,§ Gaetano Liso,† and James M. Gallo‡ Dipartimento Farmaco-Chimico, Facolta` di Farmacia, Universita` degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy, Dipartimento di Biologia Sperimentale, Sezione di Neuroscienze, Universita` di Cagliari, Cittadella Universitaria Monserrato, SS 554 Km 4.5 Monserrato (Cagliari), Italy, and Department of Pharmacology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111. Received February 18, 2003

To gain insight into the strategy to target PBR ligand-drug conjugates to brain tumors, novel N-imidazopyridinacetyl-melphalan conjugates and the corresponding ethyl esters have been prepared and evaluated for their cytotoxicity in melphalan-sensitive human (SF126, SF188) and rat (RG-2) glioma cell lines. These conjugates exhibited PBR binding affinity with IC50 values ranging from 57 and 2614 nM. By a computational approach it can be predicted that these conjugates possess significant brain penetration. The stability of the conjugates in 0.05 M phosphate buffer at pH 7.4 and, in some cases, in dilute human serum solution was determined. All the ethyl ester derivatives were stable in 0.05 M phosphate buffer at pH 7.4 and their half-lives exceeded 28 h. Conversely, under the same conditions, the corresponding acids were found to undergo a fast cleavage within a few minutes. HPLCMS analysis of the mixture from degradation in buffer and physiological medium of the representative cases allowed the identification of their main degradation products. A plausible degradation pathway accounting for the available experimental data is presented.

INTRODUCTION

The peripheral benzodiazepine receptors (PBRs)1 have been identified in various peripheral tissues as well as in glial cells in the brain (1, 2). They are pharmacologically distinct from the central benzodiazepine receptors (CBRs) which are associated with GABAA receptors and mediate classical sedative, anxiolytic, and anticonvulsant properties of benzodiazepines. From a structural point of view, PBRs are composed of at least three distinct protein subunits: an 18-kDa subunit containing binding site for isoquinolines, a 32-kDa subunit that functions as a voltage-dependent anion channel and binds benzodiazepines, and a 30-kDa subunit that functions as an adenine nucleotide carrier and also binds benzodiazepines (3). PBRs exhibit a high affinity for 4′-chlorodiazepam (Ro 5-4864), isoquinolines (PK 11195), indoleacetamides (FGIN1-27), pyrrolobenzoxazepines and phenoxyphenylacetamides (Figure 1) (4-8). Although the functions of PBRs are still not fully clarified, growing evidence suggests that these receptors are implicated in the regulation of cell proliferation, Ca2+

Figure 1. Chemical structures of the most high affinity and selectivity ligands for PBRs.

* To whom correspondence should be addressed. Phone (039) 080-5442764. Fax (039) 0805442724. E-mail: trapani@ farmchim.uniba.it. † Universita ` degli Studi di Bari. § Universita ` di Cagliari. ‡ Fox Chase Cancer Center. 1 Abbreviations: PBRs, peripheral benzodiazepine receptors; CBRs, central benzodiazepine receptors; PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide; MEL, melphalan; IC50, concentration causing 50% inhibition; log BB, log Cbrain/Cblood; NCI, National Cancer Institute; CLOG P, calculated log octanol/water partition coefficient; BBB, blood-brain barrier; SRB, sulpharodamine B; PSA, polar surface area; PBS, phosphate buffer solution.

flow, cellular respiration, and cellular immunity (9-12). Moreover, these receptors are abundant in steroidogenic tissues, in which their activation promotes cholesterol transport and neurosteroid biosynthesis (13). Besides their involvement in steroidogenesis, overwhelming experimental evidence indicates that PBR ligands are overexpressed in brain tumors compared to normal brain (14, 15). Therefore, it seems likely that these receptors could serve as a target to selectively increase anticancer drug delivery utilizing an appropriate PBR ligandanticancer drug conjugate. This attractive strategy for PBR-mediated selective targeting of antineoplastic agents has recently been explored with a benzodiazepine-

10.1021/bc034023p CCC: $25.00 © 2003 American Chemical Society Published on Web 06/24/2003

PBR Ligand−Melphalan Conjugates

melphalan conjugate possessing appreciable cytotoxicity in the human melphalan-resistant brain tumors cell lines (16). More recently, PBR ligand-gemcitabine conjugates, characterized by the PBR isoquinoline ligand PK 11195, have been synthesized, and a 2-fold increase in tumor target selectivity for a member of this series compared to gemcitabine alone has been demonstrated (17). We have recently shown that some 2-phenylimidazo[1,2-a]pyridine acetamides are potent and selective ligands for PBR and stimulate steroidogenesis in both the brain and periphery (18-20). Our data demonstrate that substitution at 8-position on the imidazopyridine moiety is a key factor for improving affinity and selectivity toward peripheral binding sites. In fact, members of the 8-substituted imidazopyridines show PBR selectivity >103-105 and are among the most selective ligands tested so far. The substitutions at 8-position and at the para-position of the phenyl ring at C(2) with a chlorine atom are crucial for high affinity and selectivity. The model compound of this new class of PBR ligands, CB 34 (Figure 1), selectively stimulated the synthesis of neuroactive steroids in rat brains with great efficacy (20) and has recently been labeled (21). The aim of the present work was to synthesize PBR ligand-melphalan (PBR-MEL) conjugates based on the 8-substituted imidazopyridines able to bind to PBRs in vitro and to evaluate their cytotoxicity in melphalansensitive glioma cell lines. Thus, further insight on the feasibility of the PBR ligand-drug conjugate strategy to target brain tumor could be gained. EXPERIMENTAL PROCEDURES

Apparatus. Melting points were determined in open capillary tubes with a Bu¨chi apparatus and are uncorrected. IR spectra were obtained on a Perkin-Elmer Spectrum one system spectrophotometer (KBr pellets for solid). Optical rotations were measured at 20 °C on a Perkin-Elmer Polarimeter 341 (10 cm cell). 1H and 13C NMR spectra were determined on a Varian VX Mercury instrument operating at 300 MHz. Chemical shifts are given in δ values downfield from Me4Si as internal standard. Mass spectra were recorded on a HewlettPackard 5995c GC-MS low resolution spectrometer. The mass spectra of conjugates 14-21 were obtained using an Agilent 1100 LC-MSD trap system VL instrument using methanol/ammonium formate 7 mM 9:1 (v/v). All compounds showed appropriate IR, 1H NMR, and mass spectra. Elemental analyses were performed on a HewlettPackard 185 C, H, N analyzer and agreed with theoretical values within (0.40%. High-performance liquid chromatography (HPLC) analyses were performed with a Waters Associates Model 600 pump equipped with a Waters 990 variable wavelength UV detector and a 20 µL loop injection valve. For kinetic studies on ester PBR-MEL conjugates 14-17, a reversed phase Symmetry C18 (25 cm × 3.9 mm; 5 µm particles) column in conjunction with a precolumn (Sentry Guard Symmetry C18, 20 × 3.9 mm) was eluted with mixtures of methanol and deionized water 80/20 (v/v). The volume injected amounted was 20 µL. The flow rate of 0.8 mL/min was maintained, and the column effluent was monitored continuously at 254 nm. Quantification of the compounds was carried out by measuring the peak areas or peak heights in relation to those of standards chromatographed under the same conditions. For kinetic studies on acid PBR-MEL conjugates 18-21, a reversed phase Symmetry C18 (25 cm × 3.9 mm; 5 µm particles) column in conjunction with a

Bioconjugate Chem., Vol. 14, No. 4, 2003 831

precolumn (Sentry Guard Symmetry C18, 20 × 3.9 mm) was eluted with mixtures of methanol and acetate buffer pH 4.7 80/20 (v/v). The volume injected amounted was 20 µL. The flow rate of 0.8 mL/min was maintained and the column effluent was monitored continuously at 254 nm. Stability studies were carried out at a controlled temperature of 37 °C ((0.2 °C) in a water bath. The release of melphalan from conjugate 14 was evaluated by using the reported HPLC conditions, except for the composition of elution mixture (methanol/acetate buffer, pH 4.7, 60/40 (v/v)). The lipophilicity indexes of conjugates 14-21, expressed as log k′ ) log(tR - t0)/t0, were obtained eluting these compounds on a reversed phase Symmetry C18 (25 cm × 3.9 mm; 5 µm particles) with mixtures of methanol and acetate buffer pH 4.7 80/20 (v/v), flux 0.8 mL/min. TLC analyses were performed on silica gel plate 60 F254 (Merck). Silica gel 60 (Merck 70-230 mesh) was used for column chromatography. All the following reactions were performed under a nitrogen atmosphere. Materials. The starting 2-aminopyridine compounds 4, melphalan, i.e., p-bis(2-dichloroethyl)amino-L-phenylalanine, ethyl 1,2-dihydro-2-ethoxy-1-quinolinecarboxylate (EEDQ), N-hydroxysuccinimide (HO-Suc), dicyclohexylcarbodiimide (DCC), 1,1′-carbonyldiimidazole (CDI), and triethylamine (TEA) were purchased from SigmaAldrich (Italy). The preparation of the ethyl 3-bromo-3benzoylpropionate compounds 3 has previously been reported (18, 19). Melphalan ethyl ester hydrochloride (13‚HCl) was prepared as follows. A stirred solution of melphalan in ethanol (20 mL) was saturated with HCl gas and heated at 60 °C for 2 h. Excess HCl gas was removed under a stream of nitrogen, and then the solvent was evaporated under reduced pressure. The residue was washed several times with anhydrous ethyl ether and then dried under vacuum to provide 13‚HCl in quantitative yield. Treatment of 13‚HCl with Na2CO3 solution and successive extractions with ethyl ether gave compound 13. This compound was also obtained from Alkeran tablets (5 mg, GlaxoWellcome) according to the following procedure. Twenty five Alkeran tablets were triturated in a glass mortar, and the resulting powder was suspended in 1 L of ethanol. Hydrogen chloride gas was bubbled through the stirred ethanolic mixture and heated at 60 °C over 2 h. The resulting mixture was filtered (Whatman filter paper) and the filtrate concentrated to a small volume and then treated with water. The so obtained mixture, cooled with an ice bath, was made alkaline with dilute Na2CO3 solution and extracted several times with ethyl ether. The organic phase, washed with water and brine, dried (Na2SO4), and concentrated under reduced pressure to give a residue which was purified by silica gel column chromatography [light petroleum ether/ethyl acetate 1/1 (v/v) as eluent)]. Compound 13 (90% yield) was obtained as an oil and characterized by spectral data and elemental analysis. IR(KBr) 3378, 1723, 1615 cm-1; 1H NMR (CDCl3) δ: 1.26 (t, J ) 6.7 Hz, 3H, CH3), 1.76 (bs, 2H, NH2), 2.7-2.8 (m, 1H, PhCH), 2.9-3.0 (m, 1H, PhCH), 3.6-3.8 (m, 9H, N(CH2CH2Cl)2 + CHCO), 4.18 (q, J ) 6.7 Hz, 2H, CH2OCO), 6.62 (d, J ) 8 Hz, 2H, Ar), 7.07 (d, J ) 8 Hz, 2H, Ar); MS m/z 332 (M+, 3), 230 (base). Anal. (C15H22Cl2N2O2) C, H, N. Reagents used for the preparation of the buffers were of analytical grade. Fresh deionized water from all glass apparatus was used in the preparation of all the solutions. HPLC mobile phase was prepared from HPLCgrade methanol. Lyophilized human serum was obtained from Sigma-Aldrich (Italy).

832 Bioconjugate Chem., Vol. 14, No. 4, 2003

General Procedure for the Preparation of Ethyl 2-Phenylimidazo[1,2-a]pyridine-3-carboxylates 5-8. To a solution of the suitably substituted 2-aminopyridine 4 (6.2 mmol) in DMF (50 mL) was added the appropriate bromoketoester 3 (6.8 mmol). The mixture was refluxed under stirring for 6-24 h. The progress of the reaction was monitored by TLC. Then the solvent was evaporated under reduced pressure, and the resulting residue was dissolved in CHCl3 (20 mL), washed with 5% NaHCO3, and dried (Na2SO4). Evaporation of the solvent gave a residue which was purified by silica gel column chromatography [light petroleum ether/ethyl acetate 8/2 (v/v) as eluent)]. Ethyl 2-(4-Chlorophenyl)-6,8-dichloroimidazo[1,2a]pyridine-3-acetate (5). This compound has been previously described (21). Ethyl 2-Phenyl-6,8-dichloroimidazo[1,2-a]pyridine3-acetate (6). IR (KBr) 1726 cm-1; 1H NMR (CDCl3) δ: 1.29 (t, J ) 7.1 Hz, 3H, CH3), 4.01 (s, 2H, CH2CO), 4.24 (q, J ) 7.1 Hz, 2H, CH2OCO), 7.32 (d, J ) 1.7 Hz, 1H, Ar), 7.4-7.6 (m, 3H, Ar), 7.81 (d, J ) 6.8 Hz, 2H, Ar), 8.14 (d, J ) 1.7 Hz, 1H, Ar); MS m/z 348 (M+, 38) 275 (base); Anal. (C17H14 Cl2N2O2) C, H, N. Ethyl 2-(4-Chlorophenyl)-8-chloroimidazo[1,2-a]pyridine-3-acetate (7). IR (KBr) 1728 cm-1; 1H NMR (CDCl3) δ: 1.27 (t, J ) 7.1 Hz, 3H, CH3), 4.00 (s, 2H, CH2CO), 4.22 (q, J ) 7.1 Hz, 2H, CH2OCO), 6.82 (t, J ) 7 Hz, 1H, Ar), 7.32 (d, J ) 7 Hz, 1H, Ar), 7.44 (d, J ) 8.4 Hz, 2H, Ar), 7.79 (d, J ) 8.4 Hz, 2H, Ar), 8.08 (d, J ) 7 Hz, 1H, Ar); MS m/z 348 (M+, 29) 275 (base); Anal. (C17H14Cl2N2O2) C, H, N. Ethyl 2-(4-Chlorophenyl)-6-methyl-8-bromoimidazo[1,2-a]pyridine-3-acetate (8). IR (KBr) 1723 cm-1; 1 H NMR (CDCl3) δ: 1.28 (t, J ) 7.1 Hz, 3H, CH3), 2.36 (s, 3H, CH3-Ar), 3.96 (s, 2H, CH2CO), 4.22 (q, J ) 7.1 Hz, 2H, CH2OCO), 7.38 (d, J ) 1.5 Hz, 1H, Ar), 7.42 (d, J ) 8.5 Hz, 2H, Ar), 7.78 (d, J ) 8.5 Hz, 2H, Ar), 7.87 (d, J ) 1.5 Hz, 1H, Ar); MS m/z 408 (M+, 25) 335 (base); Anal. (C18H16BrClN2O2) C, H, N. General Procedure for the Preparation of (Imidazo[1,2-a]pyridin-3-yl)acetic Acids 9-12. To a solution of the appropriate ethyl ester 5-8 (2.3 mmol) in 95% ethanol (20 mL) was dropwise added NaOH 1 N (2 mL). The mixture was stirred at room temperature and under a nitrogen atmosphere for 4 h. Then, the solvent was evaporated under reduced pressure, and the residue was taken up with water and extracted five times with CHCl3 (30 mL portions). The cooled water phase was acidified with dilute HCl. The resulting precipitate corresponded to the essentially pure carboxylic acid, which was isolated by filtration. [2-(4-Chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]acetic Acid (9). This compound has been previously described (21). [2-Phenyl-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]acetic Acid (10). IR (KBr) 3441, 1698 cm-1; 1H NMR (DMSO-d6) δ: 4.13 (s, 2H, CH2CO), 7.3-7.5 (m, 3H, Ar), 7.64 (d, J ) 1.6 Hz, 1H, Ar), 7.73 (d, J ) 7.1 Hz, 2H, Ar), 8.74 (d, J ) 1.6 Hz, 1H, Ar); MS m/z 276 (base); Anal. (C15H10Cl2N2O2) C, H, N. [2-(4-Chlorophenyl)-8-chloroimidazo[1,2-a]pyridin3-yl)]acetic Acid (11). IR (KBr) 3414, 1716 cm-1; 1H NMR (DMSO-d6) δ: 4.17 (s, 2H, CH2CO), 6.97 (t, J ) 7 Hz, 1H, Ar), 7.50 (d, J ) 7 Hz, 1H, Ar), 7.56 (d, J ) 8.5 Hz, 2H, Ar), 7.78 (d, J ) 8.5 Hz, 2H, Ar), 8.43 (d, J ) 7 Hz, 1H, Ar); Anal. (C15H10Cl2N2O2) C, H, N. [2-(4-Chlorophenyl)-6-methyl-8-bromoimidazo[1,2a]pyridin-3-yl)]acetic Acid (12). IR (KBr) 3422, 1734 cm-1; 1H NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3-Ar), 4.11

Trapani et al.

(s, 2H, CH2CO), 7.5-7.6 (m, 3H, Ar), 7.76 (d, J ) 8.4 Hz, 2H, Ar), 8.29 (s, 1H, Ar); Anal. (C16H12N2BrClO2) C, H, N. General Procedure for Preparation of the Conjugates 14-17. Method A. To a stirred solution of the required imidazo[1,2-a]pyridine-3-acetic acid 9-12 (0.93 mmol) in anhydrous THF (20 mL) was at first added EEDQ (1.2 mmol) and melphalan ethyl ester 13 (0.9 mmol) and after 15 min, TEA (1.3 mmol) dropwise. Stirring was prolonged at room temperature for 6-12 h and then the mixture poured into 20 mL of water and extracted with ethyl acetate (3 × 30 mL). The organic layer was separated, washed with water and brine, and dried over Na2SO4. Solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [light petroleum ether/ethyl acetate 8/2 (v/v) as eluent)] to give the corresponding conjugate 14-17. Method B. To a stirred suspension of the suitably substituted 2-phenylimidazo[1,2-a]pyridine-3-acetic acid 10 (12) (1.0 mmol) in anhydrous CH2Cl2 (20 mL) were added HO-Suc (0.9 mmol) and DCC (0.9 mmol) at room temperature, and stirring was prolonged overnight. Then, ethyl ether was added and the resulting dicyclohexylurea precipitate removed by filtration. The filtrate was concentrated and the residue taken up with anhydrous THF (20 mL). To the resulting mixture was added melphalan ethyl ester 13 (1.0 mmol), and the stirring was prolonged overnight. Then, the reaction mixture was washed with water, made alkaline with NaHCO3 solution, and extracted with ethyl acetate (3 × 30 mL). The organic phase was washed with water and brine and dried over Na2SO4. Solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography [light petroleum ether/ethyl acetate 8/2 (v/ v) as eluent)] to give the corresponding melphalan conjugate 15 (17). Method C. A solution of the acid 9 (0.3 g, 0.85 mmol) and CDI (0.2 g, 1.3 mmol) in anhydrous THF (20 mL) was stirred at room temperature. Then, melphalan ethyl ester hydrochloride 13‚HCl (0.31 g, 0.84 mmol) was added, and the stirring was prolonged for 3 h. Then the reaction mixture was washed with water, extracted with CHCl3, and dried over Na2SO4. Solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography [light petroleum ether/ ethyl acetate 8/2 (v/v) as eluent)] to give compound 14. N-(p-Bis(2-chloroethyl)amino-L-phenylalanine ethyl ester) [2-(4-Chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]acetamide (14). IR (KBr): 3340, 1727, and 1642 cm-1; 1H NMR (CDCl3) δ: 1.27 (t, J ) 7.1 Hz, 3H, CH3), 2.8-3.0 (m, 2H, CH2Ar), 3.5-3.7 (m, 8H, N(CH2CH2Cl)2), 3.84 (d, J ) 17.8 Hz, 1H, CH2CON), 3.92 (d, J ) 17.8 Hz, 1H, CH2CON), 4.21 (q, J ) 7.1 Hz, 2H, CH2OCO), 4.7-4.8 (m, 1H, CHCOO), 5.98 (d, J ) 7.3 Hz,1H, NHCO), 6.45 (d, J ) 8.5 Hz, 2H, Ar of melphalan moiety), 6.73 (d, J ) 8.5 Hz, 2H, Ar of melphalan moiety), 7.3-7.4 (m, 3H, Ar), 7.57 (d, J ) 8.4 Hz, 2H, Ar), 8.12 (s, 1H, Ar); 13C NMR (CDCl3): δ: 171.3, 167.4, 145.5, 144.9, 141.1, 134.8, 131.3, 130.3, 130.1, 129.1, 125.7, 123.9, 123.6, 121.1, 120.5, 116.4, 112.1, 62.0, 53.5, 40.5, 36.3, 32.4, 14.4; MS (ESI) m/z 671.0 [M + H]+. Anal. (C30H29Cl5N4O3) C, H, N. N-(p-Bis(2-chloroethyl)amino-L-phenylalanine ethyl ester) (2-Phenyl-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)acetamide (15). IR (KBr) 3405, 1737, and 1661 cm-1;1H NMR (CDCl3) δ: 1.26 (t, J ) 7.1 Hz, 3H, CH3), 2.9-3.0 (m, 2H, CH2Ar), 3.5-3.7 (m, 8H, N(CH2CH2Cl)2), 3.88 (d, J ) 17.2 Hz, 1H, CH2CON), 3.96 (d, J ) 17.2

PBR Ligand−Melphalan Conjugates

Hz, 1H, CH2CON), 4.19 (q, J ) 7.1 Hz, 2H, CH2OCO), 4.7-4.8 (m, 1H, CHCO), 5.95 (d, J ) 7.3 Hz, 1H, NHCO), 6.41 (d, J ) 8.8 Hz, 2H, Ar of melphalan moiety), 6.71 (d, J ) 8.8 Hz, 2H, Ar of melphalan moiety), 7.36 (d, J ) 1.8 Hz, 1H, Ar), 7.4-7.5 (m, 3H, Ar), 7.6-7.7 (m, 2H, Ar), 8.11 (d, J ) 1.8 Hz, 1H, Ar). MS (ESI) m/z 637.1 [M + H] +. Anal. (C30H30Cl4N4O3) C, H, N. N-(p-Bis(2-chloroethyl)amino-L-phenylalanine ethyl ester) [2-(4-Chlorophenyl)-8-chloroimidazo[1,2a]pyridin-3-yl)]acetamide (16). IR (KBr): 3410, 1737, and 1658 cm-1; 1H NMR (CDCl3) δ: 1.25 (t, J ) 7.1 Hz, 3H, CH3), 2.8-2.3 (m, 2H, CH2-Ar), 3.5-3.7 (m, 8H, N(CH2CH2Cl)2), 3.9-4.0 (m, 2H, CH2CON), 4.17 (q, J ) 7.1 Hz, 2H, CH2OCO), 4.7-4.8 (m, 1H, CHCO), 6.15 (br s, 1H, NHCO), 6.41 (d, J ) 8.4 Hz, 2H, Ar of melphalan moiety), 6.69 (d, J ) 8.4 Hz, 2H, Ar of melphalan moiety), 6.87 (t, J ) 7 Hz, 1H, Ar); 7.3-7.4 (m, 3H, Ar), 7.58 (d, J ) 8.4 Hz, 2H, Ar), 8.06 (d, J ) 7 Hz, 1H, Ar); 13C NMR (CDCl3) δ: 171.3, 167.5, 145.4, 135.1, 130.4, 130.3, 129.1, 126.2, 124.2, 123.6, 122.6, 116.4, 113.7, 112.2, 61.8, 53.7, 53.6, 40.6, 36.3, 32.2, 29.9, 14.3; MS (ESI) m/z 637.1 [M + H] +. Anal. (C30H30Cl4N4O3) C, H, N. N-(p-Bis(2-chloroethyl)amino-L-phenylalanine ethyl ester) [2-(4-Chlorophenyl)-6-methyl-8-bromoimidazo[1,2-a]pyridin-3-yl)]acetamide (17). IR (KBr) 3327, 1737, and 1626 cm-1; 1H NMR (CDCl3) δ: 1.25 (t, J ) 7.1 Hz, 3H, CH3), 2.42 (s, 3H, CH3Ar), 2.9-3.1 (m, 2H, CH2-Ar), 3.5-3.7 (m, 8H, N(CH2CH2Cl)2), 3.9-4.0 (m, 2H, CH2CON), 4.15 (q, J ) 7.1 Hz, 2H, CH2OCO), 4.7-4.8 (m, 1H, CHCO), 6.43 (d, J ) 8.5 Hz, 2H, Ar of melphalan moiety), 6.76 (d, J ) 8.5 Hz, 2H, Ar of melphalan moiety), 7.29 (d, J ) 8.1 Hz, 2H, Ar), 7.5-7.6 (m, 3H, Ar), 8.10 (br s, 1H, Ar). MS (ESI) m/z 695.0 [M + H] +. Anal. (C31H32BrCl3N4O3) C, H, N. General Procedure for Preparation of N-(p-Bis(2-chloroethyl)amino-L-phenylalanine) [2-Phenylimidazo[1,2-a]pyridin-3-yl)]acetamides (18-21). To a cooled solution of the appropriate compound 14-17 (0.2 mmol) in ethanol (10 mL) was added NaOH 1 N (0.2 mL). The reaction mixture was kept under stirring at 0 °C overnight and then concentrated. The resulting residue taken up with water was acidified with HCl 0.1 N until pH 3 and extracted several times with CHCl3 (30 mL). The organic layer was dried over Na2SO4, and by solvent evaporation under reduced pressure the desired compound 18-21, respectively, was obtained. N-(p-Bis(2-chloroethyl)amino-L-phenylalanine) [2-(4-Chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]acetamide (18). IR (KBr) 3452, 1716, 1640, 1615 cm-1; 1H NMR (DMSO-d6) δ: 2.7-2.9 (m, 1H, CH2Ar), 2.9-3.1 (m, 1H, CH2Ar), 3.62 (s, 8H, N(CH2CH2Cl)2), 4.02 (s, 2H, CH2CON), 4.3-4.5 (m, 1H, CHCO), 6.56 (d, J ) 8.4 Hz, 2H, Ar of melphalan moiety), 7.04 (d, J ) 8.4 Hz, 2H, Ar of melphalan moiety), 7.45 (d, J ) 8.4 Hz, 2H, Ar), 7.65 (s, 1H, Ar), 7.82 (d, J ) 8.4 Hz, 2H, Ar), 8.68 (s, 1H, Ar); MS (ESI) m/z 641.1 [M - H] -. Anal. (C28H25Cl5N4O3) C, H, N. N-(p-Bis(2-chloroethyl)amino-L-phenylalanine) (2Phenyl-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)acetamide (19). IR (KBr) 3298, 1716, 1638, 1615 cm-1; 1H NMR (DMSO-d6): δ 2.7-2.9 (m, 1H, CH--Ar), 2.9-3.0 (m, 1H, CH--Ar), 3.62 (s, 8H, N(CH2CH2Cl)2 ), 4.03 (s, 2H, CH2CON), 4.3-4.4 (m, 1H, CHCO), 6.52 (d, J ) 8.2 Hz, 2H, Ar of melphalan moiety), 7.00 (d, J ) 8.2 Hz, 2H, Ar of melphalan moiety), 7.3-7.5 (m, 3H, Ar), 7.62 (s 1H, Ar), 7.80 (d, J ) 8.0 Hz, 2H, Ar), 8.63 (s, 1H, Ar); MS (ESI) m/z 607.1 [M - H] -. Anal. (C28H26Cl4N4O3) C, H, N.

Bioconjugate Chem., Vol. 14, No. 4, 2003 833

N-(p-Bis(2-chloroethyl)amino-L-phenylalanine) [2-(4-Chlorophenyl)-8-chloroimidazo[1,2-a]pyridin3-yl)]acetamide (20). IR (KBr) 3296, 1711, 1644, 1615 cm-1; 1H NMR (DMSO-d6) δ: 2.7-2.8 (m, 1H, CH2Ar), 2.9-3.0 (m, 1H, CH2Ar), 3.66 (s, 8H, N(CH2CH2Cl)2), 4.00 (s, 2H, CH2CON), 4.3-4.4 (m, 1H, CHCO), 6.60 (d, J ) 8.5 Hz, 2H, Ar of melphalan moiety), 6.86 (t, J ) 7 Hz 1H, Ar), 7.06 (d, J ) 8.5 Hz, 2H, Ar of melphalan moiety), 7.46 (d, J ) 8.5 Hz, 2H, Ar), 7.80 (d, J ) 8.5 Hz, 2H, Ar), 8.23 (d, J ) 7 Hz, 1H, Ar), 8.72 (d, J ) 7 Hz, 1H, Ar); 13C NMR (DMSO-d6) δ: 173.6 168.6, 145.5, 135.4, 130.7, 130.5, 129.1, 126.1, 124.8, 124.6, 121.8, 118.3, 112.4, 112.3, 54.4, 53.0, 41.5, 36.4, 31.2; MS (ESI) m/z 607.1 [M - H] -. Anal. (C28H26Cl4N4O3) C, H, N. N-(p-Bis(2-chloroethyl)amino-L-phenylalanine) [2-(4-Chlorophenyl)-6-methyl-8-bromo-imidazo[1,2a]pyridin-3-yl)]acetamide (21). IR (KBr) 3303, 1716, 1642, 1615 cm-1; 1H NMR (DMSO-d6) δ 2.7-2.9 (m, 1H, CH--Ar), 2.9-3.0 (m, 1H, CH--Ar), 3.65 (s, 8H, N(CH2CH2Cl)2 ), 3.96 (s, 2H, CH2CON), 4.3-4.5 (m, 1H, CHCO), 6.57 (d, J ) 8.5 Hz, 2H, Ar of melphalan moiety), 7.05 (d, J ) 8.5 Hz, 2H, Ar of melphalan moiety), 7.45 (d, J ) 8.6 Hz, 2H, Ar), 7.53 (s, 1H, Ar), 7.79 (d, J ) 8.5 Hz, 2H, Ar), 8.20 (s, 1H, Ar); MS (ESI) m/z 665.1 [M - H]-. Anal. (C29H28BrCl3N4O3) C, H, N. Computational Calculations of Physicochemical and Penetration Characteristics of the Melphalan Conjugates 14-21. Lipophilicity and log Cbrain/Cblood (log BB) values were estimated by using CLOG P (v. 4, BioByte Corp) and MAREA (v. 1.4 Department of Pharmacy, Uppsala, Sweden) softwares, respectively, and are reported in Table 2. Stability in Buffered Solution. The stability of the melphalan conjugates 14-21 was studied at pH 7.4 in 0.05 M phosphate buffer at 37 ( 0.2 °C in a water bath. The reaction was carried out by adding 500 µL of a stock solution of the conjugates (5 mg in 25 mL of DMSO) to 5 mL of the buffer solution preheated at 37 °C. The final concentration of the compounds was about 3 × 10-5 M. The resulting solutions were vortexed and maintained in a water bath at constant temperature of 37 ( 0.2 °C. Aliquots of 20 µL were removed at appropriate intervals and either immediately analyzed or frozen at -20 °C until analyzed by HPLC. Each experiment was repeated in triplicate. Pseudo-first-order rate constants for degradation of the derivatives were determined from the slopes of linear plots of the logarithms of residual melphalan conjugate against time. Stability in Physiological Medium. The stability in physiological medium of conjugates 14, 18, 19, and 20 was studied at 37 °C in 0.05 M phosphate buffer and 0.14 M NaCl at pH 7.4, containing 50% v/v of human serum. The reaction was carried out by adding 100 µL of the stock solution of compound in DMSO (7.6 mg/25 mL for 14 and 5 mg/10 mL for 18, 19, and 20) to 1.6 mL of preheated serum solution, and the mixture was maintained in water bath at 37 ( 0.2 °C. Aliquots of 100 µL were withdrawn at appropriate intervals and added to 500 µL of cold acetonitrile in order to deproteinize the serum (the final concentration of compounds being 5.3 × 10-6 M for 14 and 9 × 10-6 M for 18, 19, and 20). After mixing and centrifugation for 10 min at 4000 rpm, 20 µL of the clear supernatant was analyzed by HPLC. Each experiment was repeated in duplicate. Pseudo-first-order rate constants for degradation of the derivatives were determined from the slopes of linear plots of the logarithms of remaining melphalan conjugate against time. Structural Identification of Degradation Products of 14 and 18 in Buffer and in Physiological

834 Bioconjugate Chem., Vol. 14, No. 4, 2003

Medium. Compound14 was allowed to react under conditions above-reported for chemical stability except for the use of 250 µL of stock solution of the conjugate (1 mg/5 mL of DMSO). After 24 h, to the reaction mixture was added 5 mL of CHCl3, and it was vortexed and centrifugated. The organic phase was separated, dried over Na2SO4, evaporated under a stream of nitrogen, taken up with methanol (500 µL), purified by reversed phase HPLC, and characterized by ESI(negative mode)MS. The same procedure was followed for compound 18 except for 2 mL of CHCl3 was added after 120 min of incubation. Compound 14 was also allowed to react under conditions above-reported for stability in physiological medium. After 120 min, an aliquot of the reaction mixture (200 µL) was added to 500 µL of acetonitrile. To the resulting mixture was added water (300 µL), and it was vortexed and centrifugated. The supernantant (400 µL) was separated, treated with water (300 µL) and CHCl3 (500 µL), and then vortexed. An aliquot of 500 µL of the chloroformic phase was dried over Na2SO4, evaporated under a stream of nitrogen, purified by reversed phase HPLC, and characterized by ESI (negative mode)MS. The same procedure was followed for compound 18. For these studies, a reversed phase Phenomenex Luna C18 (15 cm × 3 mm; 5 µm particles) column in conjunction with a precolumn module was eluted in gradient HPLC conditions. The mobile phase used was the following: eluent A, methanol:deionized water:acetic acid (60:39:1, v:v:v); eluent B, methanol:deionized water:acetic acid (90: 9:1, v:v:v) (injection volume 20 µL). Gradient 0-5 min eluent A, 5-20 min from 100% of eluent A to 100% of eluent B; 20-40 min 100% of eluent B; 40-45 min from 100% of eluent B to 100% of eluent A. Preliminary experimental work included the HPLC-ESI-MS analysis under identical experimental conditions of commercial melphalan [retention time (tR) 6.2 min] and the pure acids 9 (tR 5.1 min, m/z 337) and 18 (tR 19 min, m/z 640). Biological Methods. Materials. Adult male or female Sprague-Dawley CD rats (Charles River, Como, Italy) with body masses of 200-250 g at the beginning of the experiments were maintained under an artificial 12-h-light/dark cycle (light on 08.00 to 20.00 h) at a constant temperature of 23 ( 2 °C and 65% humidity. Food and water were freely available, and the animals were acclimated for >7 days before use. Experiments were performed between 08.00 and 14.00 h. Animal care and handling througouth the experimental procedure were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC). The experimental protocol were approved by the Animal Ethical Committee of the University of Cagliari. In Vitro Receptor Binding Assays. After sacrifice the brain was rapidly removed, the cerebral cortex was dissected, and tissues were stored at -80 °C until assay. [3H]Flunitrazepam Binding. The tissues were thawed and homogenized with a Polytron PT 10 in 50 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4) and centrifuged twice at 20 000g for 10 min. The pellet was reconstituted in 50 volumes of Tris-HCl buffer and was used for the binding assay. Aliquots of 400 µL of tissue homogenate (0.4-0.5 mg of protein) were incubated in the presence of [3H]flunitrazepam at a final concentration of 0.5 nM, in a total incubation volume of 1000 µL. The drugs were added in 100 µL aliquots. After a 60 min incubation at 0 °C, the assay was terminated by rapid filtration through glass-fiber filter strips (Whatman GF/ B). The filters were rinsed with 2- to 4-mL portions of ice-cold Tris-HCl buffer as described above. Radioactivity bound to the filters was quantitated by liquid scintillation

Trapani et al.

spectrometry. Nonspecific binding was determined as binding in the presence of 5 µM diazepam and represented about 10% of total binding. [3H]PK 11195 Binding. The tissues were thawed and homogenized in 50 volumes of Dulbecco’s phosphatebuffered saline (PBS) pH 7.4 at 4 °C with a Polytron PT 10 (setting 5, for 20 s). The homogenate was centrifuged at 40 000g for 30 min, and the pellet was resuspended in 50 volumes of PBS and recentrifuged. The new pellet was resuspended in 20 volumes of PBS and used for the assay. [3H]PK 11195 binding was determined in a final volume of 1000 µL of tissue homogenate (0.15-0.20 mg protein), 100 µL of [3H]PK 11195 (spec act. 85.5 Ci/mmol, New England Nuclear) at final assay concentration of 1 nM, 5 µL of drug solution or solvent, and 795 µL of PBS buffer (pH 7.4 at 25 °C). Incubations (25 °C) were initiated by addition of membranes and were terminated 90 min later by rapid filtration through glass-fiber filter strips (Whatman GF/B), which were rinsed with five 4 mL volumes of ice-cold PBS buffer using a Cell Harvaster filtration manifold (Brandel). Filter bound radioactivity was quantified by liquid scintillation spectrometry. Nonspecific binding was defined as binding in the presence of 10 µM unlabeled PK 11195 (Sigma). Cytotoxicity Assays in Human SF126, SF188, and Rat RG-2 Glioma Cells. The standard sulforhodamine B assay, used in the NCI screen (22), was used to determine cytotoxicity in the monolayer cell lines, SF126, SF188, and RG-2. All conjugates were dissolved in DMSO except for conjugate 16, which was dissolved in ethanol at initial concentrations of 3000 µg/mL, and except for conjugate 19, which was prepared at 1500 µg/mL. Drug solutions were diluted with medium to the desired concentrations, and then 100 µL aliquots were added to the cells that were in 200 µL of medium. Following a 3-fold serial dilution, the cells were incubated for 96 h. After the four-day incubation, the cells were fixed in 10% trichloroacetic acid (TCA) at 4 °C for 1 h and stained with 4% SRB (dissolved in 1% of acetic acid) after 5× washing with water. Bound SRB was dissolved in 10 mM Trisbuffer after washing away the unbound SRB. The absorption at 570 nm was measured in a microplate reader, and cell survival was calculated as an absorption ratio compared to control. RESULTS AND DISCUSSION

Synthetic Procedures. As shown in Scheme 2, the new PBR ligand-melphalan conjugates 14-17 were prepared by condensation of the imidazopyridineacetic acids 9-12 with melphalan ethyl ester 13. The condensation was successfully achieved by using ethyl 1,2dihydro-2-ethoxy-1-quinolinecarboxylate (EEDQ) as a dehydrating agent (Method A), or DCC and HO-Suc (Method B). Method C allowed the preparation of conjugate 14 by reaction of the imidazopyridineacetic acid 9 with melphalan ethyl ester hydrochloride 13‚HCl in anhydrous THF and in the presence of CDI. The preparation of conjugate 14 was also accomplished by using Method A, but a slightly lower yield was obtained. Treatment of conjugates 14-17 with NaOH in ethanol afforded the second series of target molecules, acids 1821, respectively. Compounds 9-12, in turn, were synthesized by methods previously reported by us (Scheme 1). Briefly, condensation of suitably substituted 2-aminopyridines 4 with the appropriate bromoketo esters 3 followed by alkaline hydrolysis gave the desired compounds 9-12 (Scheme 1). Compounds 3, in turn, were prepared by reaction of esters 2 with bromine in carbon

PBR Ligand−Melphalan Conjugates Scheme 1a

Bioconjugate Chem., Vol. 14, No. 4, 2003 835 Table 1. Structure and Physical Properties of Compounds 5-12, 14-21 compd

X

Y

Z

mp (°C)

5 6 7 8 9 10 11 12 14

Cl Cl H CH3 Cl Cl H CH3 Cl

Cl Cl Cl Br Cl Cl Cl Br Cl

Cl H Cl Cl Cl H Cl Cl Cl

c 108-110 156-158 173-175 c 245-247 224-226 230-232 172-174

15

Cl

Cl

H

79-81

16 17

H CH3

Cl Br

Cl Cl

70-72 69-71

18 19 20 21

Cl Cl H CH3

Cl Cl Cl Br

Cl H Cl Cl

220 dec 206 dec 198 dec 196-198 dec

yielda (%)

[R]20Db

35 30 33 70 75 59 31 (A) 38 (C) 38 (A) 30 (B) 55 (A) 41 (A) 32 (B) 61 40 57 57

+24.6 +14.5 +6.7 +6.9

a The method used is reported in parentheses. b c ) 5 mg/mL in CHCl3. c Reference 21.

a Reagents: (a) EtOH/HCl gas; (b) Br /CCl ; (c) DMF, reflux; 2 4 (d) NaOH/EtOH.

Scheme 2a

a Reagents: (a) Method A: EEDQ,THF; Method B: HO-Suc/ DCC; Method C: CDI (b) NaOH/EtOH.

tetrachloride. Physical data for new compounds are reported in Table 1. Lipophilicity and Computational Approach To Estimate the Blood-Brain Barrier (BBB) Penetration of Compounds 14-21. To be useful, a drugconjugate targeted to the brain should have pharmacokinetic and pharmacodynamic properties that include favorable membrane permeability, intrinsic binding affinity, and conversion to a cytotoxic moiety. Membrane

permeability is related to the drug-conjugate lipophilicity which is reflected by its partition coefficient (log P). The lipophilicity of compounds 14-21 was assessed, both calculating their 1-octanol/water partition coefficients, using CLOGP software, based on the fragmental method of Hansch and Leo, and measuring their retention times in RP-HPLC (Table 2). From a quantitative viewpoint, a significant linear correlation was found between the calculated log P values and capacity factors log k′ (n ) 8, r2 ) 0.74, s ) 0.13). This positive correlation indicates there are no significant differences in the calculated and measured lipophilicity values. Therefore, in the following calculations, we used only the lipophilicity data obtained from the CLOGP software. Log P values of 2.5 or higher were considered optimal for BBB penetration. As can be seen from Table 2, all the compounds (14-21) possessed log P values higher than 2.5 (i.e., in the range 6.36-7.74) and appeared to be sufficiently lipophilic to cross the BBB. To gain further information on the BBB penetration properties of compounds 14-21 by passive transport, a computational study was performed. Recently, several attempts to correlate BBB penetration with physicochemical parameters have been reviewed (23). It has been clarified that the octanol-water partition coefficient (log P) is an important factor, although by itself correlates poorly with the log Cbrain/Cblood (log BB). This ratio represents a useful measure of the degree of BBB penetration. Experimental values of log BB published to date cover the range from about -2.00 to +1.00. In particular, compounds with log BB greater than 0.3 cross the BBB readily, while compounds with log BB < -1.0 are only poorly distributed to the brain. In addition to log P, the importance of a molecular size descriptor has been shown (24) as well as the need to include a descriptor relating to hydrogen bond formation (23). To estimate the BBB penetration of compounds 14-21, we used the Clark model. This model relates log BB to polar surface area (PSA, defined as the area in Å2 contributed by nitrogen and oxygen atoms, plus the area of the hydrogen atoms attached to these heteroatoms) and calculated log P (CLOG P) according to the equation: log BB ) -0.0148((0.001)PSA + 0.152((0.036)CLOG P + 0.139((0.073) (25). For compounds 14-21, we calculated a PSA in the range 62.4-72.4 Å2 by using the MAREA computer program. As can be seen from the data obtained by this computa-

836 Bioconjugate Chem., Vol. 14, No. 4, 2003

Trapani et al.

Table 2. Calculated Lipophilicity, Penetration of BBB, and Chemical and Enzymatic Stability of Compounds 14-21

compd

CLOG Pa

PSAb (Å2)

log BBc

14 15 16 17 18 19 20 21 melphalan

7.74 7.03 7.03 6.97 7.08 6.36 6.36 7.01 -0.21

62.4 62.1 63.9 62.1 71.0 71.7 72.1 72.5

0.39 0.29 0.26 0.28 0.16 0.04 0.04 0.13

RP-HPLC log k′ d 0.72 0.52 0.48 0.64 0.35 0.08 0.12 0.28 -0.58

t1/2e(h) in phosphate buffer 0.05 M (pH 7.4) 28 44 34 76 0.8 0.67 0.77 1

t1/2e (min) in human serum 47

120 138 55

a Estimated according to CLOG P software. b Calculated according to MAREA software. c Calculated according to Clark’s model (25). The capacity factors (k′) of each melphalan conjugate has been calculated by the equation: k′ ) (tR - t0)/t0, where tR is the observed retention time of the solute and t0 is the column dead time. e Based on the loss of starting material.

d

Table 3. Affinity for Rat Cerebrocortical CBR and PBR and Cytotoxicity to SF 126, SF 188, and RG-2 Glioma Cells of Compounds 14-21 compd 14 15 16 17 18 19 20 21 PK 11195 melphalan

IC50 (nM)a CBR cerebral PBR cortex b b b b b b b b 24250 e

181 ( 8 521 ( 21 1040 ( 57 2614 ( 188 57 ( 2 261 ( 15 1081 ( 88 2179 ( 196 1.06 ( 0.06 e

SIc >20 000 >9500 >4800 >1900 >87 000 >19 000 >4600 >2200 22 877 e

IC50 (µg/mL)d SF 126 cells

IC50 (µg/mL)d SF 188 cells

IC50 (µg/mL)d RG-2 cells

26.12 ( 8.95 10.33 ( 1.35 6.75 ( 3.45 30.97 ( 14.89 84.32 ( 15.00 64.16 ( 13.52 82.29 ( 17.87 82.58 ( 14.12

51.43 ( 16.70 5.01 ( 1.49 2.94 ( 0.23 7.79 ( 1.35 56.11 ( 4.93 28.37 ( 1.65 28.45 ( 2.03 39.23 ( 7.8

31.33 ( 1.46 9.36 ( 1.48 9.75 ( 2.27 24.83 ( 2.10 27.42 ( 2.10 18.34 ( 2.19 25.03 ( 4.64 29.23 ( 3.65

0.92 ( 0.30

6.03 ( 1.05

3.3

Data are means ( SD of three separate experiments performed in duplicate. b No displacement up to 1 mM. c SI: selectivity index ) IC50(CBR)/IC50(PBR); data are calculated considering arbitrarily the IC50(CBR) value of the conjugates as 5 mM. d Data are means ( SD (from three to five different measurements). PBR density or Bmax (pmol/mg protein] values were for each cell line; SF126: 41( 3.3, SF188: 22.1 ( 1.4, RG-2: 33 ( 3.1. e No receptor binding affinity. a

Figure 2. Plots of the hydrolysis of the conjugates 14-21 at pH 7.4 in 0.05 M phosphate buffer at 37 ( 0.2 °C. 14 (9), 15 (2) 16 (1), 17 ([), 18 (O), 19 (0), 20 (4), and 21 (3).

tional approach and reported in Table 2, it can be predicted that compounds 14-21 possess significant brain penetration. Stability in Buffer and Physiological Medium. The stability of the derivatives 14-21 in buffer was determined in 0.05 M phosphate buffer at pH 7.4 as well as for selected compounds (14, 18, 19, and 20) in dilute (50%) human serum solution at 37 °C. All the esters derivatives (14-17) were stable in 0.05 M phosphate buffer at pH 7.4, and their half-lives exceeded 28 h (Table 2). Conversely, under the same conditions, acids 18-21 were found to undergo fast cleavage within a few minutes (half-lives in the range 0.67-1 h based on the loss of starting material, Table 2 and Figure 2). On the other hand, compounds 14, 18, 19, and 20 were found unstable in serum (half-lives in the range of 47120 min based on the disappearance of starting material, Table 2 and Figure 2). It is noteworthy that ester 14 was stable in buffered solution and unstable in human serum.

One possible explanation accounting for this finding is that ester 14 may be susceptible to enzyme (esterase)catalyzed hydrolysis in serum, leading to compound 18 which, in turn, is rapidly cleaved in physiological medium. To gain insight into the degradation pathway of ester 14, its main degradation products in buffer and physiological medium were characterized by LC-mass spectrometry (LC-MS). Thus, LC-MS analysis of the mixture from degradation of ester 14 in buffer after 24 h showed the presence of small amounts of degradation products of the melphalan moiety, occurring as dihydroxymelphalan (22) (tR 24.3 min, m/z 633), methoxymelphalan (23) (tR 25.4 min, m/z 667), and chloroethylaminomelphalan (24) (tR 22.5 min, m/z 606) derivatives, in addition to relevant quantities of the unchanged starting material 14 (tR 25.7 min, m/z 669). It is interesting to note that the formation of degradation products 22-24 can be easily rationalized taking into account the degradation of the parent nitrogen mustard (26, 27). On the other hand, LC-MS analysis of the mixture from degradation of compound 14 in physiological medium after 2 h showed the presence of acids 9 (tR 5.1 min, m/z 335) and 18 (tR 19 min, m/z 639) and melphalan degradation products together with the starting material (tR 25.5 min, m/z 669). Therefore, it can be concluded that, in physiological medium, 14 undergoes cleavage at the amide bond level. The LC-MS analysis carried out on the mixture from degradation of compound 18 in buffer showed the presence of acid 9 (tR 5.1 min, m/z 335) and a degradation product (25) of the melphalan moiety of 18 (tR 21.6 min, m/z 576), aside from the starting material. The corresponding LC-MS analysis of 18 in human serum demonstrated the presence of compounds arising from the cleavage of the amide bond.

PBR Ligand−Melphalan Conjugates

Bioconjugate Chem., Vol. 14, No. 4, 2003 837

Scheme 3

On the basis of the available experimental data, a reasonable degradation pathway may be put forward, and it is shown in Scheme 3. Compound 14 in 0.05 M phosphate buffer at pH 7.4 is stable enough, undergoing

a slow decomposition to give small amounts of degradation products involving the melphalan moiety. Under these conditions, both the ester and amide function of 14 are stable enough. Conversely, in human serum, 14

Figure 3. Inhibition of [3H]PK 11195 binding to rat cerebrocortical membranes by PK 11195 (9) and by the conjugates 14 (b), 15 (O), 18 (1), and 19 (3). The data are means of triplicate of a representative experiment and are expressed as percentage of specific [3H]PK 11195 binding apparent in the absence of drugs.

838 Bioconjugate Chem., Vol. 14, No. 4, 2003

is susceptible to esterase catalysis, yielding acid 18 which next is cleaved at the amide linkage, leading to acid 9. The suggestion that enzymatic hydrolysis at the ester group is faster than amide cleavage is derived from the fact that the amide bond is known to be quite stable in vivo. Quite probably, the degradation of esters 15-17 and acids 19-21 proceeds through a pathway similar to that of the ester 14 and acid 18, respectively. The fact that the acid conjugates 18-21 are less resistant than the corresponding esters 14-17 to degradation in buffer can be likely ascribed to the presence, in the former conjugates, of the carboxylic group which could promote amide cleavage. Affinities of Imidazopyridine Derivatives for Peripheral and Central Benzodiazepine Receptors. The affinities of the conjugates 14-21 for CBR and PBR were evaluated by measuring their ability to compete with [3H]flunitrazepam and [3H]PK 11195 binding, respectively, to membrane preparations from rat cerebral cortex. Their affinities were compared with those of unlabeled PK 11195. The measured binding affinities for CBR and PBR expressed as IC50 values as well as their ratios, as a measure of the in vitro selectivity, are shown in Table 3. The analysis of the binding affinities of the entire set of compounds 14-21 indicated that conjugates 18, 14, 19, and 15 possess, in the order given, the highest affinity for PBR (Figure 3). Moreover, the selectivity index of 18 is greater than the reference compound (PK 11195) while the selectivity indexes of conjugates 14 and 19 are comparable with that of PK 11195. The best PBR binding affinity and selectivity was observed for conjugate 18, whereas no binding affinity for CBR and PBR was found for melphalan as such (Table 3). The greater affinity exhibited by 18 and 14 in comparison with 19 and 15, respectively, is consistent with previous reports where the relevant influence of the substitution at the para-position of the phenyl ring at C(2) with a chlorine atom on binding affinity and selectivity was already demonstrated (18,19). Cytotoxicity. Cytotoxicity assays of the melphalan conjugates 14-21 were conducted in rat and human brain tumor cell lines using an SRB assay (22) with the results shown in Table 3. It can be seen that the ester conjugates 14-17 were more cytotoxic than the corresponding acids 18-21 but, in all cases, less toxic than melphalan alone. However, the ester conjugates 15 and 16 possessed cytotoxicity comparable to that of the parent drug. In conclusion, synthetic routes toward imidazopyridinePBR ligand-melphalan conjugates have been developed, and some compounds displayed suitable in vitro receptor affinity and cytotoxicity against glioma cell lines. The combined data of affinity, selectivity, and cytotoxicity support the further evaluation of conjugates 14, 15, 18, and 19. As these compounds are likely to exhibit favorable BBB penetration, their utility in a preclinical brain tumor model is warranted. Moreover, based on a comparison of affinities between these new types of PBRMEL conjugates and a previously reported benzodiazepine-melphalan conjugate (16), conjugates 14, 18, and 19 show improved binding characteristics and warrant further evaluation. ACKNOWLEDGMENT

This work was supported by a grant from Ministero dell′Universita` e della Ricerca Scientifica e Tecnologica (MIUR). We thank Mr Giovanni Dipinto for skillful

Trapani et al.

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