A Radioiodinated MIBG–Octreotate Conjugate Exhibiting Enhanced

the radioiodinated target compound (MIBG-octreotate; [131I]12a) from a tin precursor were futile; however, it could be accomplished from a bromo precu...
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2122

Bioconjugate Chem. 2007, 18, 2122–2130

A Radioiodinated MIBG–Octreotate Conjugate Exhibiting Enhanced Uptake and Retention in SSTR2-Expressing Tumor Cells Ganesan Vaidyanathan,*,† Donna J. Affleck,† Joseph Norman,† Susan O’Dorisio,‡ and Michael R. Zalutsky† Department of Radiology, Duke University Medical Center, Durham, North Carolina, and Division of Pediatric Hematology/ Oncology, University of Iowa, Iowa City, Iowa. Received June 28, 2007; Revised Manuscript Received September 11, 2007

Several neuroendocrine tumors are known to express both the somatostatin receptor subtype 2 (SSTR2) and the norepinephrine transporter (NET), and radiopharmaceuticals directed toward both these targets such as MIBG and octreotide derivatives are routinely used in the clinic. To investigate the possibility of targeting both NET and SSTR2 conjointly, a conjugate of radioiodinated MIBG and octreotate was synthesized. Attempts to synthesize the radioiodinated target compound (MIBG-octreotate; [131I]12a) from a tin precursor were futile; however, it could be accomplished from a bromo precursor by exchange radioiodination in 3–36% (n ) 10) radiochemical yields. The total uptake of [131I]12a in SK-N-SH human neuroblastoma cells transfected to express SSTR2 (SKN-SHsst2) was similar to that for [125I]MIBG at all time points (34.9 ( 2.4% vs 43.8 ( 1.2% at 4 h; p < 0.05), while it was substantially lower (5.4 ( 0.3% vs 35.9 ( 1.2%) in the SH-SY5Y cell line, a subclone of SK-N-SH line that is known to express SSTR2. The NET blocker desipramine reduced the uptake of [131I]12a only to a small extent, further suggesting a limited role of NET in its binding and accumulation. Uptake of [131I]12a in SK-N-SHsst2 cells was 8–10-fold higher (p < 0.05) than that of [125I]I-Gluc-TOCA, an octreotide analogue, at all time points over a 4 h period and was reduced to about 20% by 10 µM octreotide demonstrating that the uptake of [131I]12a in this cell line is predominantly mediated by SSTR2. The intracellularly trapped radioactivity in SK-N-SHsst2 cells was substantially higher for [131I]12a compared to that for [125I]OIBG-octreotate, an isomeric congener of 12a. Because MIBG has more specific NET-mediated uptake than OIBG, this suggests at least a partial role for NET-mediated uptake of [131I]12a in this cell line. While further refinement in the structure of the conjugate—probably interposition of a flexible and/or cleavable linker between the MIBG and octreotate moieties—may be necessary to make it a substrate/ligand for both NET and SSTR2, this conjugate is demonstrated to be much superior than I-Gluc-TOCA with respect to the uptake in SSTR2-expressing cells.

INTRODUCTION Neuroblastoma (NB) and other neuroendocrine tumors accumulate meta-iodobenzylguanidine (MIBG), an analogue of the neurotransmitter norepinephrine, via the norepinephrine transporter (NET), which is also known as the uptake-1 pump (1, 2). MIBG, labeled with the β-particle-emitter 131I, has been used extensively in the treatment of NB (3). However, responses were not of long duration even when targeted [131I]MIBG therapy was combined with other modalities (4). Many neuroblastomas and other neuroendocrine tumors express somatostatin receptors as well (5–7), and a number of radiolabeled derivatives of octreotide, an octapeptide ligand for somatostatin receptor subtype 2, have been developed for diagnosis and targeted therapy of these tumors (8–11). The complementary role of [*I]MIBG and 111In-pentetreotide in the imaging of neuroendocrine tumors has been demonstrated (12–14). For imaging, while tumor foci of certain cancers have been visualized by both labeled radiopharmaceuticals, there are cases in which one of these radiopharmaceuticals and not the other is sequestered in the tumors. In general, 111In-labeled octreotide was found to be more sensitive; however, it was also less selective (15–20). For therapy, the combined use of [131I]MIBG and 90Y-labeled octreotide derivatives has been recommended (12, 21, 22). * Correspondence to Ganesan Vaidyanathan, Ph.D., Box 3808, Radiology, Duke University Medical Center, Durham, North Carolina 27710. Phone: (919) 684-7811. Fax: (919) 684-7122. E-mail: [email protected] † Duke University Medical Center. ‡ University of Iowa.

We hypothesized that a radiopharmaceutical derived by combining the structural elements of MIBG and octreotide may have an additive, if not synergistic, effect with respect to uptake and retention in a neuroendocrine tumor that expressed both somatostatin receptor and NET; in addition, it could limit false negative lesion detection because the hybrid radiopharmaceutical might be taken up by tumors via either mechanism. Another impetus for developing such a reagent is that it could overcome the fast washout of labeled MIBG from tumors, presumably also mediated by NET, that likely contributes to the mismatch between [131I]MIBG tumor pharmacokinetics and the 8.1-day half-life of 131I. Our working hypothesis was that a chimeric molecule containing both SSTR2 and NET binding elements could have higher tumor retention. Octreotide derivatives undergo endocytosis after receptor binding, translocating the receptor–ligand complex to lysosomes wherein they undergo extensive catabolism (23) with polar and charged catabolites generally being retained in lysosomes. Attempts have been made to exploit this property of octreotide derivatives in order to increase the tumor specificity and intracellular bioavailability of various pharmaceuticals by conjugating them with octreotide analogues. For example, oligonucleotides (24), peptide nucleic acids (25), as well as the chemotherapeutic agents doxorubicin (26) and paclitaxel (27) have been conjugated to somatostatin analogues for such a purpose. Recently, hybrid molecules derived by covalent conjugation of an octreotate (Thr-ol at the C-terminus in octreotide replaced with Thr; Chart 1) derivative and the Rvβ3-targeting RGD peptide also have been described (28, 29). In addition, simultaneous targeting of multiple receptors has become an emerging paradigm in drug discovery (30–35).

10.1021/bc700240r CCC: $37.00  2007 American Chemical Society Published on Web 11/03/2007

Radioiodinated MIBG–Octreotate Conjugate

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Chart 1. Chemical Structures of Octreotate, Iodo-Gluc-TOCA, MIBG-Octreotate, MBBG-Octreotate, and OIBG-Octreotate

Herein, we describe the synthesis of a conjugate of [131I]MIBG and octreotate and its evaluation in human tumor cell lines expressing both SSTR2 and NET. This chimeric conjugate demonstrated superior targeting to SSTR2 receptors compared to one of the best-known SSTR2 targeting peptides; however, unexpectedly, NET-mediated binding of the conjugate was only moderate.

EXPERIMENTAL PROCEDURES General. All chemicals were purchased from Sigma-Aldrich unless otherwise noted. Sodium [125I]iodide and sodium [131I]iodide with specific activities of 2200 Ci/mmol and 1200 Ci/mmol, respectively, were obtained from Perkin Elmer Life and Analytical Sciences (Boston, MA). N-Dde-Lys5-octreotate (9), N-(4-guanidinomethyl-3-[125I]iodobenzoyl)-D-Phe1-octreotate ([131I]OIBG-octreotate), and [*I]Iodo-Gluc-TOCA were synthesized as reported earlier (36). No-carrier-added [*I]MIBG was synthesized from a silicon precursor (37). Aluminum-backed sheets (Silica gel 60 F254) were used for analytical TLC, and normal-phase column chromatography was performed using silica gel 60, both obtained from EM Science (Gibbstown, NJ). Preparative thick-layer chromatography was performed using 20 × 20 cm, 1000 µm plates (Whatman, Clifton, NJ). Before applying the sample, the plates were run in ethyl acetate to remove any adsorbed impurities. Radio-TLC was initially analyzed using a System 200 Imaging Scanner (BioScan, Washington, DC); sheets were then cut into strips and counted using an automated gamma counter (LKB 1282, Wallac, Finland). Highpressure liquid chromatography was performed using a Beckman System Gold HPLC equipped with a model 126 programmable solvent module, a model 168 diode array detector, a model 170 radioisotope detector, and a model 406 analogue interface module. Reversed-phase chromatography utilized a Waters XTerra C18 column (4.6 × 250 mm, 5 µ) and a Waters XTerra C18 column (19 × 50 mm) for analytical and semipreparative runs, respectively. Proton NMR spectra were obtained on a Varian Mercury 300 spectrometer. Chemical shifts are reported in δ units; solvent peaks are referenced appropriately. Mass spectra were obtained on a JEOL SX-102 high-resolution mass spectrometer (FAB, EI, and GC-MS),

an Applied Biosystems Voyager DE Pro (MALDI), or an Agilent 1100 LC/MSD Trap SL (electrospray). High-resolution mass spectra were obtained using a JEOL SX-102 high-resolution mass spectrometer. Cells and Culture Conditions. SH-SY5Y (38), a thrice-cloned subline of the NET-expressing human neuroblastoma cells SK-NSH (39), was obtained from American type Culture Collection (Manassas, VA). Evidence for the presence of somatostatin receptors in the SH-SY5Y cell line has been reported (40–42). The incubation medium for these cells was made by mixing 500 mL of RPMI 1640 (GIBCO), 55 mL of fetal calf serum, and 2.7 mL of penicillin-G/streptomycin (10 000 U of penicillin and 10 000 µg of streptomycin in 1 mL of 0.85% saline). SK-N-SH cells stably transfected with SSTR2 (SK-N-SHsst2) were derived as described previously (5). The mRNA level in these cells, relative to that in parent SK-N-SH line, was 28.3 ( 5.8 as determined by quantitative RT-PCR (5). A medium consisting of 500 mL MEM, 15% FBS, 12 mL penicillin/streptomycin (200 U/mL penicillin and 200 µg/mL streptomycin), 6 mL nonessential amino acids, 6 mL L-glutamine, and 300 mg G418 was used for the culturing these cells. The cells were grown at 37 °C in a humidified incubator containing 5% CO2. Cell viability was evaluated prior to each experiment by trypan blue dye exclusion (43), and was 95–98% for all studies. 2-Iodo-4-methylbenzoic Acid (1a). A solution of sodium nitrite (747 mg; 10.8 mmol) in 6 mL of water was added to a suspension of 2-amino-3-methylbenzoic acid [(Ryan Scientific, Isle of Palms, SC, or Maybridge Building Blocks, Cornwall, UK) 1.51 g; 10 mmol] in cold dilute sulfuric acid (2 mL conc H2SO4, 12 g ice, and 18 mL water) at 2–3 °C over a period of 10 min. The mixture was stirred for 2 h at 2–3 °C, and a solution of KI (2.28 g; 10.8 mmol) in 5 mL of water was then added over a period of 15 min while maintaining the temperature at 2–3 °C. The mixture was subsequently heated in an oil bath maintained at 60 °C for 1 h. The reaction mixture was partitioned between ethyl acetate and water, and the aqueous layer was washed twice with ethyl acetate. The combined ethyl acetate layer was washed twice with saturated sodium thiosulfate solution and once with brine. The dried ethyl acetate layer was

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evaporated, and the crude product was chromatographed on silica gel using 70:30:0.1 ethyl acetate/hexanes/acetic acid to yield 1.5 g solid (57%). It was further purified by precipitation from an ethyl acetate solution by hexanes: mp 134–136 °C [lit. (44) mp 128–130 °C]. The melting point was similar to that of a commercial sample (Maybridge Building Blocks, Cornwall, UK)—individually and mixed. 2-(Trimethylsilyl)ethyl 2-iodo-4-methylbenzoate (2a). Polymer-bound DCC [(Argonaut Technologies, Inc., San Carlos, CA, now a division of Biotage, Uppsala, Sweden), 5.0 g; 5 mmol] was added to a stirred solution of 1a (930 mg, 3.6 mmol), DMAP (45 mg; 0.4 mmol), and trimethylsilylethanol (570 mg; 4.8 mmol) in 35 mL of dry ethyl acetate. The heterogeneous mixture was stirred at 20 °C under argon atmosphere for 3–4 h. The polymer was filtered and washed with ethyl acetate, which was evaporated from the combined filtrate using a rotary evaporator under aspirator vacuum. The residual oil was chromatographed using a gradient of 0–5% ethyl acetate/hexanes to yield 1.0 g (77%) of 2a as an oil: 1H NMR (CDCl3) 0.07 (s, 9H), 1.15 (m, 2H), 2.33 (s, 3H), 4.41 (m, 2H), 7.18 (ddd, 1H), 7.72 (dd, 1H), 7.83 (dd, 1H). MS (EI+), m/z: 362 (M+), 245. HRMS (EI+) Calcd for C13H19IO2Si (M+): 362.0199. Found: 362.0197 ( 0.0004 (n ) 2). 2-(Trimethylsilyl)ethyl 2-bromo-4-methylbenzoate (2b). The title compound was synthesized from 2-bromo-4-methylbenzoic acid similar to 2a above but using DCC (not polymersupported). Starting from 2.15 g (10 mmol) of 2-bromo-4methylbenzoic acid, 2.5 g (80%) of 2b was obtained as an oil: 1 H NMR (CDCl3) 0.07 (s, 9H), 1.15 (m, 2H), 2.36 (s, 3H), 4.42 (m, 2H), 7.15 (ddd, 1H), 7.49 (m, 1H), 7.71 (dd, 1H). MS (GCEI+), m/z: 313 and 315 (M+), 299 and 301 (M – CH3)+, 271 and 273 (100%), 227 and 229, 197 and 199 (M – OCH2CH2SiMe3). 2-(Trimethylsilyl)ethyl 4-Bromomethyl-2-iodobenzoate (3a). A mixture of 2a (1.02 g; 2.82 mmol), N-bromosuccinimide (473 mg; 2.66 mmol), 1,1′-azo-biscyclohexanenitrile, and 1,2dichloroethane (DCE; 25 mL) was heated at reflux under an incandescent lamp for 3 h. DCE was evaporated from the cooled mixture, and the residue was partitioned between ethyl acetate and water. The aqueous layer was extracted twice with ethyl acetate, and the pooled ethyl acetate solution was washed with sodium thiosulfate solution and dried. The ethyl acetate was evaporated, and the residual oil was chromatographed on silica gel using a gradient of 0–3% ethyl acetate in hexanes to yield 541 mg (44%) of an oil: 1H NMR (CDCl3) 0.08 (s, 9H), 1.15 (m, 2H), 4.39 (s, 2H), 4.43 (m, 2H), 7.42 (ddd, 1H), 7.76 (dd, 1H), 8.00 (ddd, 1H). MS (EI+), m/z: 440 and 442 (M+), 412 and 414, 397 and 399 (100%), 361, 334. HRMS (EI+) Calcd for C13H17BrIO2Si (M – H)+: 438.9226. Found: 438.9231. This compound was contaminated with less than 10% of the dibromo derivative (based on NMR data) and was used as such for the synthesis of 4a. 2-(Trimethylsilyl)ethyl 2-Bromo-4-bromomethylbenzoate (3b). This compound was synthesized from 2b following the procedure described for the preparation of 3a. Starting from 670 mg (2.13 mmol) of 2b, 526 mg (63%) of 3b was obtained as an oil: 1H NMR (CDCl3) 0.08 (s, 9H), 1.15 (m, 2H), 4.42 (s, 2H), 4.43 (m, 2H), 7.37 (ddd, 1H), 7.68 (ddd, 1H), 7.74 (dd, 1H). 2-(Trimethylsilyl)ethyl 4-[ N1,N2-Bis-(tert-butyloxycarbonyl)guanidinomethyl]-2-iodobenzoate (4a). N1,N2-bis-(tertbutyloxycarbonyl)guanidine (412 mg; 1.6 mmol) was added to a stirred suspension of sodium hydride (60% dispersion in mineral oil) (62.5 mg; 1.6 mmol) in 6 mL of dry DMF under argon and the mixture stirred at room temperature for 5 min, whereby a homogenous solution was obtained. A solution of 3a (504 mg; 1.14 mmol actual) in 2.3 mL of DMF was added

Vaidyanathan et al.

to the above solution of the guanidine derivative and the reaction mixture stirred at room temperature for 10–15 min The reaction mixture was partitioned between water and ethyl acetate and the aqueous layer extracted a couple of times with ethyl acetate. The pooled ethyl acetate solution was washed once with brine, dried, and evaporated. The crude mixture was chromatographed on silica gel with a gradient of 5–20% ethyl acetate in hexanes to yield 591 mg (83%) of a white solid: m.p. 103–105 °C. 1H NMR (CDCl3) 0.08 (s, 9H), 1.15 (m, 2H), 1.38 (s, 9H), 1.48 (s, 9H), 4.43 (m, 2H), 5.17 (s, 2H), 7.32 (dd, 1H), 7.75 (dd, 1H), 7.91 (dd, 1H). MS (FAB+), m/z: 620 (M + H)+, 564, 508, 436, and 392. HRMS (FAB+) Calcd for C24H39IN3O6Si (M + H)+: 620.1653. Found: 620.1654 ( 0.0016 (n ) 2). 2-(Trimethylsilyl)ethyl 2-Bromo-4-[ N1,N2-bis-(tert-butyloxycarbonyl)guanidinomethyl]benzoate (4b). This compound was prepared following a procedure similar to that used for the synthesis of 4a. Starting with 820 mg (2.1 mmol) of 3b, 551 mg (46%) of a white solid was obtained. 1H NMR (CDCl3) 0.06 (s, 9H), 1.12 (m, 2H), 1.35 (s, 9H), 1.47 (s, 9H), 4.41 (m, 2H), 5.13 (s, 2H), 7.24 (dd, 1H), 7.55 (d, 1H), 7.73 (dd, 1H). MS (FAB+), m/z: 572 and 574 (M + H)+, 516 and 518, 460 and 462, 388 and 390, and 344 and 346. HRMS (FAB+) Calcd for C24H3979BrN3O6Si (M + H)+: 572.1792. Found: 572.1786 ( 0.0021 (n ) 2). 4-[N1,N2-Bis-(tert-butyloxycarbonyl)guanidinomethyl]-2iodobenzoic Acid (5a). Tetrabutyl ammonium fluoride (0.18 mL of 1 M solution in THF; 0.18 mmol) was added dropwise to a solution of 4a (40 mg; 0.06 mmol), and the mixture was stirred at 20 °C for 3–4 h. THF was evaporated, and the residue was partitioned between ethyl acetate and 1 N HCl. The aqueous layer was extracted twice with ethyl acetate, and the pooled ethyl acetate solution was washed with brine and dried. Evaporation of ethyl acetate yielded 30 mg (89%) of an oil: 1H NMR (CDCl3) 1.35 (s, 9H), 1.49 (s, 9H), 5.15 (s, 2H), 7.29 (dd, 1H), 7.87 (d, 1H), 7.92 (d, 1H), 9.28 (br d; 2H). MS (FAB+), m/z: 520 (M + H)+, 408. HRMS (FAB+) Calcd for C19H27IN3O6 (M + H)+: 520.0945. Found: 520.0931 ( 0.0000 (n ) 2). This compound was carried to the next step without further purification. 4-[N1,N2-Bis-(tert-butyloxycarbonyl)guanidinomethyl]-2bromobenzoic Acid (5b). This compound was prepared from 4b (30 mg; 0.05 mmol) in a fashion similar to the preparation of 5b in an almost quantitative yield and was used for the next step without further purification. 1H NMR (CDCl3) 1.36 (s, 9H), 1.49 (s, 9H), 5.18 (s, 2H), 7.25 (dd, 1H), 7.58 (d, 1H), 7.88 (dd, 1H), 9.4 (br d; 2H). N-Succinimidyl 4-[N1,N2-Bis-(tert-butyloxycarbonyl)guanidinomethyl]-2-iodobenzoate (6a). Polymer-bound DCC (400 mg; 0.4 mmol) and N,N-dimethylaminopyridine (3 mg) in that order were added to a solution of 5a (147 mg; 0.28 mmol) in 3 mL of dry ethyl acetate. The mixture was stirred at 20 °C under a positive pressure of argon for a few minutes after which N-hydroxysuccinimide (72 mg; 0.6 mmol) was added and then further stirred for 1 h at 20 °C. The resin was filtered off, washed with ethyl acetate, and the filtrate concentrated. Silica gel chromatography using a gradient of 25–40% ethyl acetate in hexanes yielded 50.8 mg (41%) of a foamy solid: m.p. 79–80 °C. 1H NMR (CDCl3) 1.40 (s, 9H), 1.50 (s, 9H), 2.90 (s, 4H), 5.18 (s, 2H), 7.40 (dd, 1H), 8.02 (d, 1H), 8.08 (d, 1H), 9.36 (br d; 2H). MS (FAB+), m/z: 617 (M + H)+, 561, 505, 460. HRMS (FAB+) Calcd for C23H30IN4O8 (M + H)+: 617.1108. Found: 617.1104 ( 0.0010 (n ) 2). N-Succinimidyl 4-[N1,N2-Bis-(tert-butyloxycarbonyl)guanidinomethyl]-2-bromobenzoate (6b). This compound was prepared from 5b (30 mg) in 49% yield following the procedure for the preparation of 6a. 1H NMR ((CD3)2SO) 1.32 (s, 9H),

Radioiodinated MIBG–Octreotate Conjugate

1.39 (s, 9H), 2.90 (s, 4H), 5.10 (s, 2H), 7.45 (dd, 1H), 7.78 (dd, 1H), 8.05 (dd, 1H). 2-(Trimethylsilyl)ethyl 4-[N1,N2-Bis-(tert-butyloxycarbonyl)guanidinomethyl]-2-trimethylstannylbenzoate (7). The title compound was synthesized from either 3a or 3b using similar procedures (procedure using 3a is given below). A mixture of 3a (128 mg; 0.21 mmol), hexamethylditin (652 mg; 2 mmol), (Ph3)2PdCl2 (100 mg; 0.14 mmol) in 4 mL of dioxane was heated at reflux under argon for 1 h. The mixture was cooled to room temperature and passed over a bed of Celite, which was then washed with ethyl acetate. The solvents from the combined filtrate were evaporated, and the residual oil was loaded onto a silica bed. The nonpolar byproducts were washed out with a large excess of hexanes, and the product and polar byproducts were eluted with ethyl acetate. The ethyl acetate was evaporated from latter fractions and the residue chromatographed on silica gel using 5% ethyl acetate in hexanes to yield 107 mg (79%) of an oil: 1H NMR (CDCl3) 0.07 (s, 9H), 0.24 (s, 9H [119Sn-H, d]), 1.13 (m, 2H), 1.31 (s, 9H), 1.48 (s, 9H), 4.38 (m, 2H), 5.22 (s, 2H), 7.24 (dd, 1H), 7.56 (d, 1H), 8.03 (dd, 1H), 9.40 (br d; 2H). MS (FAB+), m/z: cluster peaks at 658 (M + H)+, 642, 602, 546, 514, 414, 356. HRMS (FAB+) Calcd for C27H48N3O6Si120Sn (M + H)+: 658.2334. Found: 658.2328 ( 0.0000 (n ) 2). N-Succinimidyl 4-[N1,N2-Bis-(tert-butyloxycarbonyl)guanidinomethyl]-2-(trimethylstannyl)benzoate (8). A solution of tetrabutylammonium fluoride (317 µL of 1 M in THF; 0.32 mmol) was added to a solution of 7 (76.1 mg; 0.12 mmol) in 1.73 mL of dry THF, and the mixture was stirred at 20 °C for 1–2 h. THF was evaporated and the residue partitioned between water and ethyl acetate. The aqueous layer was extracted twice with ethyl acetate, and the combined ethyl acetate fractions were washed with brine and dried. Ethyl acetate was evaporated and the crude product dried. Polymer-bound DCC 163 mg (0.16 mmol) and 1.4 mg of DMAP were added to a solution of the above acid in 1.3 mL of dry ethyl acetate. Five minutes later, N-hydroxysuccinimide (31 mg; 0.27 mmol) was added and the mixture stirred at 20 °C under argon for 2–3 h. The resin was filtered and washed with ethyl acetate. Ethyl acetate was evaporated from the filtrate, and the residue was purified by thick-layer chromatography using 25% ethyl acetate in hexanes to yield 36.2 (46%) mg of an oil: 1H NMR (CDCl3) 0.25 (s, 9H [119Sn-H, d]), 1.35 (s, 9H), 1.48 (s, 9H), 2.90 (br s, 4H), 5.22 (s, 2H), 7.30 (dd, 1H), 7.63 (d, 1H), 8.21 (dd, 1H), 9.40 (br d; 2H). MS (FAB+), m/z: cluster peaks at 655.2 (M + H)+, 543. HRMS (FAB+) Calcd for C26H39N4O8120Sn (M + H)+: 655.1784. Found: 655.1907 ( 0.0026 (n ) 4). N-(4-Guanidinomethyl-2-iodobenzoyl)-D-Phe1-octreotate (12a). Compound 6a (3.4 mg; 5.5 µmol) was added to a solution of 9 (6.2 mg; 5.2 µmol) in 0.1 mL of 0.1 M DIEA in NMP. The homogeneous mixture was heated at 45 °C for 30 h. The progression of the reaction was periodically checked by HPLC. For this, the reversed-phase analytical column was eluted with a gradient consisting of 0.1% HOAc (w/v) in each water (solvent A) and acetonitrile (solvent B) at a flow rate of 1 mL/min. The percentage of solvent B was increased from 30% to 60% over 15 min and to 100% in the next 15 min and then held at 100% for 5 min. Under these conditions, the retention times for peptide 9, compound 6a, and the product 10a were 11.3, 25.5, and 29.0 min, respectively. Several analytical HPLC runs of the reaction mixture were performed to yield 3 mg of 10a. MS m/z: Calcd for C78H100IN13O18S2 1698.7419. Found (LCMS-ESI) 1699.6 (MH+), 849.7 (MH2+). Ethanolamine (0.98 mL) was added to a solution of 10a in 0.98 mL of ethanol and left at 20 °C. The removal of the Dde group was complete in 30 min as judged by HPLC. The HPLC was conducted using the same solvents as above at a flow rate of 1 mL/min; the percentage of B was

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increased from 10% to 40% over a period of 30 min and to 100% in the next 10 min and then held at 100% for 5 min The retention times of 10a and 11a under these conditions were 45.5 and 38.6 min, respectively. Several analytical HPLC runs were performed to obtain 2.5 mg of 11a. MS m/z: Calcd for C68H88IN13O16S2: 1534.5408. Found (MALDI): 1356.4 (M + Na – 2Boc)+, 1334.8 (MH – 2Boc)+. A 95:2.5:2.5 (v/v/v) mixture of TFA/water/triisopropylsilane (0.2 mL) was added to the above peptide (11a; 2.5 mg), and the mixture left at 20 °C for 30 min. HPLC (conditions as above for the deprotection of 10a) indicated complete disappearance of 11a (tR ) 38.6 min) and appearance of a peak at 24.8 min. The solvents were evaporated with a stream of argon and the residue redissolved in 0.5 mL of ethanol, and the ethanol evaporated. The process of coevaporation with ethanol was repeated once, and finally the residue was triturated with ether to obtain 1.9 mg of 12a. MS m/z: Calcd for C58H72IN13O12S2: 1334.3092. Found (MALDI): 1356.26 (M + Na)+, 1334.27 (MH)+. N-(2-Bromo-4-(guanidinomethyl)benzoyl)-D-Phe1-octreotate (12b). A solution of 9 (3.2 mg; 2.7: mol) and 6b (4.8 mg; 8.4: mol) in 0.1 mL of 0.1 M DIEA in NMP was incubated at 20 °C for 20 h whereupon HPLC (same conditions as for the synthesis of 10a; tR ) 25.0 and 27.9 min for 6b and 10b, respectively) indicated the complete consumption of 9. Semipreparative HPLC (same conditions as for analytical with a flow rate of 10 mL/min) of the reaction mixture yielded 1–2 mg of 10b (tR ) 22.0 and 24.0 min for 6b and 10b, respectively). MS m/z: Calcd for C78H100BrN13O18S2 1651.7414. Found (FAB+) 1652.5 and 1650.5 (M)+, 1552.5 and 1550.5 (M – Boc)+, 1452.4 and 1450.4 (M – 2Boc)+. Ethanolamine (0.25 mL) was added to a solution of the above peptide (10b) in 0.25 mL ethanol, and the deprotection reaction was allowed to proceed at 20 °C. HPLC (same conditions as for the deprotection of 10a) at 60 min indicated the complete disappearance of 10b (tR ) 44.3 min) and the appearance of a peak at 38.5 min. Semipreparative HPLC of the reaction mixture (same conditions as analytical except for a flow rate of 10 mL/min; tR ) 35.3 min) yielded 1.2 mg of 11b. MS m/z: Calcd for C68H88BrN13O16S2 1487.5403. Found (FAB+) 1488.5 and 1486.5 (M)+, 1288.5 and 1286.5 (M – 2Boc)+. A 95:2.5:2.5 mixture of TFA/water/triisopropylsilane (0.2 mL) was added to the above peptide and the mixture incubated at 20 °C for 30 min. HPLC (same conditions as used for the conversion of 10b to 11b) indicated complete removal of Boc groups (tR for 12b ) 22 min). MS m/z: Calcd for C58H72BrN13O12S2 1287.3087. Found (MALDI) 1310.5 and 1308.5 (M + Na)+, 1288.5 and 1286.5 (MH)+. N-[4-(N′,N′′-Bis-tert-butyloxycarbonyl)guanidinomethyl2-(trimethylstannyl)benzoyl]-D-Phe1-octreotate (11c). A solution of 8 (13.8 mg; 21.1 µmol) and 9 (9.4 mg; 7.9 µmol) in 0.15 mL of 0.1 M DIEA in NMP was incubated at 20 °C for 7 d. The reaction progress was monitored by the same reversed-phase HPLC conditions that were used for the synthesis of 10a and 10b. The retention times of 8 and 10c were 29.0 and 32.1 min, respectively. The reaction mixture was subjected to semipreparative HPLC using the conditions used for the isolation of 10b to yield 5.5 mg of 10c. MS m/z: Calcd for C81H109N13O18S2Sn 1735.6510. Found (MALDI) cluster peaks around 1537.5 (M – 2Boc)+. Ethanolamine (0.25 mL) was added to a solution of the above peptide in ethanol (0.25 mL) and the mixture left at 20 °C. HPLC (same conditions as for the synthesis of 11a and 11b) indicated that the removal of the Dde group was complete in about an hour (tR ) 47.1 and 39.1 min for 10c and 11c, respectively). The reaction mixture was subjected to semipreparative HPLC (same conditions as for 11b; tR ) 36.5 min) to isolate 2.8 mg of 11c. MS m/z: Calcd for C71H97N13O16S2Sn 1571.4499. Found (MALDI) cluster peaks around 1375.2 (M – 2Boc)+; (FAB) cluster peaks around 1572.0 (MH)+, 1474.1, 1372, 1354.9.

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N-(4-Guanidinomethyl-2-[131I]iodobenzoyl)-D-Phe1-octreotate ([131I]12a). A mixture of 12b (50 µg), 131I (0.2–4.0 mCi) and 25 µL of glacial acetic acid in a closed ½-dram vial was heated at 100 °C for 20 min The entire reaction mixture was injected into the analytical reversed-phase HPLC column and eluted with the gradient conditions used for the synthesis of 12a. The HPLC fractions containing [131I]12a were isolated and the radioactivity concentrated using a C18 solid-phase cartridge as reported for the isomeric compound (36). Paired-Label Uptake of [125I]MIBG and [131I]12a by SH-SY5Y and SK-N-SH-sst2 cells as a Function of Time. Cells (5 × 105 per well in 2 mL) in 6-well plates in the respective medium was incubated for 24 h. The medium was removed, and the medium containing 100 nCi of each tracer (in 2 mL) was added to the wells. The cells were incubated at 37 °C for 15, 30, 60, 120, and 240 min, and then the medium was aspirated and the cells were washed with the medium and solubilized in 1 N NaOH. Cell-associated radioactivity was determined by counting for both 125I and 131I in an automated gamma counter. For each time point, the assay was performed in triplicate. To determine whether the uptake was NETmediated, a parallel assay was performed in the presence of 1.5 µM DMI. The results are expressed as the percentage of total activity that was associated with the cells. Time-Dependent Paired-Label uptake of [131I]12a and [125I]I-Gluc-TOCA by SK-N-SH-sst2 Cells. This assay was performed essentially like the above except that a different tracer pair was employed and the assay was done using only the transfected cell line. To determine the extent of SSTR-mediated uptake, a parallel assay was performed in the presence of 10 µM octreotide. Internalization of [131I]12a versus That of [125I]I-GlucTOCA and [125I]OIBG-Octreotate by SK-N-SH-sst2 Cells. These assays were performed in a paired-label format following a procedure reported for octreotate derivatives in another cell line (36). Briefly, about 200 000 CPM of each tracer was incubated with 5 × 105 cells at 37 °C for 30 min, 1, 2, 3, and 4 h in the presence or absence of 1 µM octreotide. The cell culture supernatants were removed, and the cells were incubated with an acidic (pH 5) buffer to remove surface-bound radioactivity. Cells were finally lysed with 1 N NaOH and counted for radioactivity. The percentage of input radioactivity that was present in the intracellular compartment was determined. Specific internalized radioactivity, defined as the difference in internalized radioactivity in the presence and absence of 1 µM octreotide, was plotted as a function of time. Paired-Label Washout of [125I]MIBG and [131I]12a from SK-N-SH-sst2 Cells. The cells were allowed to take up the tracers for 4 h as in the uptake experiment described above. The medium containing unbound tracers was removed, and the cells were supplemented with fresh medium. Subsequently, the cell-associated radioactivity was determined as above at 0, 4, 8, 16, 24, and 48 h. A paired-label Student’s t test was used to determine the statistical significance of the difference in the measured values for 125I and 131I.

RESULTS AND DISCUSSION Radioiodinated MIBG has been widely used for the diagnosis of a number of neuroendocrine tumors as well as imaging the pathophysiology of the heart. MIBG labeled with the β-emitter 131 I has been used in the targeted radiotherapy of the above neoplasms. With the hope that a conjugate of [*I]MIBG and an octreotate derivative may deliver a higher cumulative radiation dose to the tumor, we have developed such a fusion molecule and subjected it to preliminary in vitro evaluation of SSTR2- and NET-mediated accumulation in human cancer cells. It was envisaged that the easiest way to append an MIBG moiety to a peptide would be to introduce a carboxylic acid function on

Vaidyanathan et al. Scheme 1. Synthesis of N-Succinimidyl 2-Bromo-4-guanidinomethylbenzoate and N-Succinimidyl 4-guanidinomethyl-2-iodobenzoate

the MIBG molecule and conjugate it to the N-terminus of the peptide to form an amide bond. We chose to modify MIBG at its 4-position because we showed earlier that the introduction of functional groups at this position did not interfere appreciably with NET-related biological properties of MIBG (45). To avoid the potential interference of the carboxyl function present in the peptide in the direct coupling of MIBG derivative with a COOH group, we set forward to synthesize its active ester. Thus, our target was compound 6a (Scheme 1) wherein the guanidine function of the active ester of an MIBG derivative with an added carboxyl group was protected with Boc groups. Both 6a and the corresponding trimethylstannyl derivative 8 were prepared as shown in Schemes 1 and 2. The starting material for the iodo derivative, 2-iodo-4methyl benzoic acid (1a), was synthesized from 2-amino-4methylbenzoic acid. A standard of the final MIBG–octreotate conjugate (12a) and the tin precursor (11c) were synthesized following protocols reported earlier (36) for the isomeric peptide derivatives (Scheme 3). Several sets of different conditions were utilized for the radioiodination of 11c but less than 1% radiochemical yields of [*I]11a, which can be converted to the final target [*I]12a, were obtained. Although difficulty in radioiodinating compounds with a carbonyl moiety ortho to the trialkyltin group has been reported, utilizing very harsh conditions has resulted in decent radiochemical yields (46). Attempts to radioiodinate 7 to [*I]4a under a variety of conditions including those reported by the above authors for their target compound also were futile, and it was not possible to convert the free acid of 7 (R ) H) to unlabeled 5a. Modest radiochemical yields (15–20%) were obtained, however, for the iododestannylation of 8 under relatively mild conditions. It was possible to obtain radioiodinated 4-guanidinomethyl-2-iodobenzoic acid by the nucleophilic iododebromination of the corresponding bromo precursor in 85–90% radiochemical yields (experimental details of the above reactions are not provided). Therefore, a bromo derivative of the peptide conjugate (12b) was synthesized (Schemes 1 and 3). Heating a mixture of this peptide and radioiodine in acetic acid yielded the target compound [*I]12a (Scheme 4) albeit in modest radiochemical yields (3–36%; n ) 10). Higher yields might have been obtained with higher amounts of the starting bromo peptide; however, the difference in HPLC retention times between 12a and 12b was only 2–3 min, which precluded the use of larger amounts of 12b for this reaction.

Radioiodinated MIBG–Octreotate Conjugate Scheme 2. Synthesis of N-Succinimidyl 4-(N, N′-Bistert-butyloxycarbonyl)guanidinomethyl-2-trimethylstannyl Benzoate

The accumulation and retention properties of the MIBGoctreotate ([131I]12a) conjugate were evaluated in two cell lines that express both SSTR2 and NET. First, the uptake of [125I]MIBG and [131I]12a as a function of time was determined in a pairedlabel format using SH-SY5Y (38, 40) and SK-N-SH-sst2 (5) cell lines. Both cell lines demonstrated significant uptake of [125I]MIBG, which appeared to plateau at 4 h (Figure 1). As seen with the wildtype SK-N-SH cells (2, 47, 48), the uptake was NET-mediated as shown by its abrogation by DMI treatment. The level of [125I]MIBG uptake in these cell lines was similar to that seen typically in the parental line SK-N-SH (49). While specific uptake of radioiodinated MIBG in the SH-SY5Y line has been shown to be similar to the SK-N-SH cell line (50), this is the first time the specific uptake of MIBG has been demonstrated in the SSTR2-cloned line. Unlike the case with [125I]MIBG, the uptake of [131I]12a did not reach a plateau in either cell line. The total uptake of [131I]12a in SK-N-SHsst2 cells was similar to that for [125I]MIBG at all time points (34.9 ( 2.4% vs 43.8 ( 1.2% at 4 h; p < 0.05), while it was substantially lower in the SH-SY5Y line (5.4 ( 0.3% vs 35.9 ( 1.2%). However, DMI did not decrease the uptake of [131I]12a in either cell line to the same degree as was seen for [125I]MIBG. This suggests that, while there may be an NET-mediated component to its cell accumulation, the uptake of [131I]12a in these cell lines, especially in the SK-N-SH-sst2 cells, might be predominantly related to SSTR2 binding. Although SSTR2 expression in SHSY5Y cells has been demonstrated (41), to our knowledge, its expression level relative to that in SK-N-SH cells has not been reported. On the other hand, quantitative RT-PCR showed that the transfected cells express about a 28-fold higher amount of SSTR2 mRNA than that by the SK-N-SH line. To investigate the extent of SSTR2 involvement in the uptake of [131I]12a by SK-N-SH-sst2 cells, another paired-label assay was performed in which the time-dependent uptake [131I]12a was compared to that of [125I]I-Gluc-TOCA. Among various labeled octreotide derivatives, [125I]I-Gluc-TOCA and other sugarconjugated congeners have demonstrated high accumulation in

Bioconjugate Chem., Vol. 18, No. 6, 2007 2127 Scheme 3. Synthesis of Octreotate Conjugates 12a, 12b, 12c, 13a, and 13b

SSTR2-expressing tumor cells and xenografts (51, 52). Surprisingly, the uptake of [131I]12a was 8–10-fold higher (p < 0.05) than that for [125I]I-Gluc-TOCA at all time points (Figure 2). Importantly, the uptake was reduced to about 20% when the cells were coincubated with 10 µM octreotide, demonstrating again that the uptake of [131I]12a in this cell line is predominantly mediated by SSTR2. The absolute magnitude of total cellular uptake of [131I]12a was lower than that seen in the previous experiment (see Figure 1) and of the intracellularly trapped radioactivity from another experiment (see below; Figure 3). We speculate that this might be due to the use of different passages of cells in different experiments. However, the assays were performed in a pairedlabel format, and therefore, any changes in receptor and/or transporter expression affected the uptake of both tracers used to the same level, making the comparison meaningful. An exciting observation from these results is that [131I]12a exhibits considerably higher SSTR2-mediated cell uptake compared with [125I]I-Gluc-

2128 Bioconjugate Chem., Vol. 18, No. 6, 2007

Vaidyanathan et al.

Scheme 4. Synthesis of Radioiodinated MIBG–Octreotate (12a)

TOCA, which perhaps is one of the best agents for targeting SSTR2-positive tumors. One of the motivations for development of a hybrid molecule derived from MIBG and octreotate is the feasibility of augmenting the retention of radioactivity in the tumor caused by the possible translocation of the peptide–receptor conjugate to lysosomes after internalization. If the radioactivity were trapped in the lysosomes and, in this manner, NET-mediated egress from the cell was avoided, this could lead to the enhanced residence time in tumor cells, a feature of obvious advantage with such a long-lived radionuclide as 131I. We first determined the extent of internalization of [131I]12a by SK-N-SHsst2 cells and compared it to that of [125I]IGluc-TOCA and [125I]OIBG-octreotate (36)—a position isomer of [131I]12a—in two separate paired-label experiments. As depicted in Figure 3, the intracellularly trapped radioactivity from [131I]12a steadily increased with time. In concert with the results from the uptake assay, the specific (total minus that in presence of 1 µM octreotide) internalized radioactivity from [131I]12a was substantially higher (3–4-fold; p < 0.05) than that from [125I]I-Gluc-

Figure 2. Paired-label uptake of (A) [125I]I-Gluc-TOCA and (B) [131I]12a by SK-N-SH-sst2 cells as a function of time in the presence (circle) and absence (triangle) of 1 µM octreotide. The cells were incubated with both tracers for various periods of time in the presence or absence of unlabeled octreotide, and the cell-associated radioactivity was determined. Note that the Y-axis scales are not the same for the two plots.

Figure 3. Paired-label specific internalized radioactivity (total minus that in presence of 1 µM octreotide) over a period of 4 h in SK-NSH-sst2 cells: (A) [131I]12a (circle) vs [125I]OIBG-octreotate (triangle) and (B) [131I]12a (circle) vs [125I]I-Gluc-TOCA (square). See text for details.

Figure 1. Paired-label time-dependent uptake of [125I]MIBG (bottom panel) and [131I]12a (top panel) by SH-SY5Y cells (left panel) and SK-N-SH-sst2 (right panel) in the presence (triangle) and absence (circle) of DMI. Cells (5 × 105 per well) were allowed to take up both tracers in the presence or absence of DMI over various time points, and the cell-associated radioactivity was determined.

TOCA. Interestingly, the internalized radioactivity of [131I]12a was also higher than that for [125I]OIBG-octreotate. OIBG-octreotate and 12a differ only in the position of iodine on the aromatic ring (Chart 1). In their seminal work, Wieland and colleagues have shown that, among the three iodobenzylguanidine derivatives, 2-iodobenzylguanidine (OIBG) is the weakest norepinephrine analogue (53, 54). Thus, the above results tempt us to speculate that the enhanced residence time of [131I]12a in SK-N-SHsst2 cells is mediated partly by NET. Finally, an assay was performed to determine the extent of egress of [131I]12a, in comparison to that of [125I]MIBG, from SK-N-SHsst2 cells. The results from this paired-label study are shown in Figure 4. Up to 24 h, the radioactivity from [131I]12a was retained by these cells to a higher extent than from

Radioiodinated MIBG–Octreotate Conjugate

Figure 4. Paired-label washout of [125I]MIBG (square) and [131I]12a (circle) from SK-N-SH-sst2 cells in the presence (open) and absence (closed) of 1.5 µM DMI. Cells were allowed to take the two tracers for a period of 4 h; the free radioactivity-containing medium was removed and supplemented with fresh medium with or without DMI. The cell-associated radioactivity was determined periodically afterwards.

[125I]MIBG possibly due to the hypothesized lysosomal trapping. As observed before (45), DMI obliterated the retention of [125I]MIBG; on the other hand, it did not have any effect on the retention of [131I]12a. Because DMI blocks the NETmediated uptake, this again demonstrates that NET plays only a minimal role in the uptake of [131I]12a.

CONCLUSIONS In summary, we have developed a method for the synthesis of a conjugate of radioiodinated MIBG and octreotate. While it demonstrated considerable uptake in a tumor cell line expressing both NET and SSTR2, it appears that the predominant pathway for its uptake is via the somatostatin receptor. This might be due to the fact that 12a, compared to MIBG, lost its affinity to NET due to the presence of the large peptide moiety. MIBG with a COOH function at its 4-position had an uptake 8% that of MIBG in SK-N-SH cells (unpublished results). A conjugate with a long, flexible, and/or cleavable linker may have to be interposed between MIBG and octreotate moieties. On the other hand, the MIBG– octreotate conjugate exhibited excellent retention in these cells that express both NET and SSTR2.

ACKNOWLEDGMENT This work was supported by grants CA93371 and CA42324 from the National Institutes of Health. The authors want to thank Philip Welsh for his excellent technical assistance.

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