Synthesis of Oxytocin HYNIC Derivatives as Potential Diagnostic

Bioconjugate Chem. 2007, 18, 1560−1567

1560

Synthesis of Oxytocin HYNIC Derivatives as Potential Diagnostic Agents for Breast Cancer Alma D. Miranda-Olvera,† Guillermina Ferro-Flores,‡ Martha Pedraza-Lo´pez,§ Consuelo Arteaga de Murphy,§ and Luis M. De Leoń-Rodrı´guez*,† Instituto de Investigaciones Cientı´ficas, Universidad de Guanajuato, Guanajuato, Gto. Mexico, Gerencia de Aplicaciones Nucleares en la Salud, Instituto Nacional de Investigaciones Nucleares, Ocoyoacac, Edo. Mex., Mexico, and Instituto Nacional de Ciencias Me´dicas y Nutricioń Salvador Zubirań, Mexico City, Mexico. Received February 12, 2007; Revised Manuscript Received April 11, 2007

Two synthetic procedures for HYNIC oxytocin labeling were developed: one based on an orthogonal protection approach and the other with prelabeled (Boc)HYNIC-(Fmoc) amino acids. Both procedures were compared and applied to the preparation of several HYNIC-oxytocin derivatives where ligand position and amino acid (lysine and phenylalanine) were varied. Additionally, an oxytocin derivative labeled with HYNIC in the R-amino group of the Cys1 residue was also prepared. 99mTc-ethylendiaminediacetic acid (EDDA) labeling efficiencies were examined for all the derivatives, resulting in two candidates which showed affinity for the oxytocin receptor. Further biochemical experiments demonstrated that 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 could be used as a potential radiopharmaceutical for breast cancer diagnosis.

INTRODUCTION Recently, the development of target-specific diagnostic pharmaceuticals (TSP) has heightened the interest in studying the different chemical aspects involved in the production of these agents. In general, diagnostic TSP consist of a specific vector, a linker, a ligand, and a metal ion. The vector is the moiety acting as a carrier to the specific target. The usage of vectors such as organic compounds, antibodies, proteins, and peptides has been widely reported in the literature (1). However, peptides are of particular interest, given that sequence target specificity can be easily monitored by techniques such as phage display (2), and peptide synthesis and modification can be chemically accomplished by solid-phase peptide synthesis (SPPS). The linker is the intermediate group connecting the ligand with the vector and selection depends on either the metabolic characteristics desired for the TSP or the maintenance of the ligand as far as possible from the vector to minimize activity modification of the vector due to the proximity of the ligand. The ligand is the molecule in charge of forming a thermodynamically and kinetically stable complex with the metal ion of interest. The selection of the metal ion depends on its nuclear, physical, and magnetic properties, given a particular application. For instance, 99mTc is used in radiodiagnostics, and Gd3+ is used in contrast agents for magnetic resonance imaging (MRI). In particular, 99mTc is used in nearly 80% of all single-photon emission computed tomography (SPECT) diagnostic radiopharmaceuticals currently used in nuclear medicine (3), given its optimal halflife time (6 h), wide availability, and low-energy particle emission (4). Therefore, peptides labeled with ligands that form stable metal complexes with 99mTc are thoroughly studied as diagnostic tools (5). Among the chelators, HYNIC (2-hydrazinnicotinic acid) (6) (Figure 1a) is of particular interest, because * Corresponding author. Instituto de Investigaciones Cientı´ficas. Cerro de la Venada s/n. Guanajuato, Gto., C.P. 36040. Me´xico. Tel., Fax: +52-4737326252; E-mail: [email protected]. † Universidad de Guanajuato. ‡ Instituto Nacional de Investigaciones Nucleares. § Instituto Nacional de Ciencias Me´dicas y Nutricioń Salvador Zubirań.

Figure 1. (a) HYNIC and (b) Boc-HYNIC.

it shows a high labeling efficiency (rapid and high yield radiolabeling) and its usage with various co-ligands (e.g., ethylendiaminediacetic acid, tricine, and glucoheptonate) allows for easy modification of the hydrophilicity and pharmacokinetics of the 99mTc -labeled small peptides (7). Given the implications of chelate-labeled peptides, the study of the influence of ligand position within a peptide sequence on targeting specificity and properties of the peptide is of great interest. The most common way to attach a ligand to a peptide has been through the N-terminus or the -amino group of lysine by means of protected derivatives of the ligand for SPPS (Figure 1b). However, this limits the possibilities of creating peptide libraries for screening purposes. Additionally, several lysine-protected derivatives have been prepared (8, 9) and are commercially available (e.g., Bachem Bioscience Inc. USA, Advanced ChemTech Inc. USA, Merck KGaA, Germany) to specifically attach ligands to peptides through lysine residues by orthogonal protection procedures (10). Recently, a chemical procedure for introducing DOTA-lysine (DOTA-Lys) or DOTA-phenylalanine (DOTAPhe) within a peptide sequence using standard SPPS was reported, and thus, a small peptide library was created and monitored for peptide activity (11). Similarly, a HYNIC-Lys derivative (Scheme 1, compound 6) was reported for site-specific labeling of a calcitonin salmon peptide (12). In that work, the synthesis was accomplished by reacting Boc-HYNIC succinimide with NR-Fmoc lysine. However, the desired product was obtained in moderate yields after purification by HPLC, given the formation of a Boc-HYNIC-(NR-Fmoc Lys)2 subproduct. Thus, although the procedure is simple and partially works with amino acids and its derivatives containing a free aliphatic amino group (lysine), it has not been proven to work for amino aromatic amino acid derivatives, given its low nucleophilicity. Additionally, the moderate yields of the HYNIC-Lys and

10.1021/bc070047a CCC: $37.00 © 2007 American Chemical Society Published on Web 08/01/2007

Synthesis of Oxytocin HYNIC Derivatives

Bioconjugate Chem., Vol. 18, No. 5, 2007 1561

Scheme 1. Synthesis of HYNIC-Lys Derivativea

a (i) (Boc)2O, water-dioxane, TEA; (ii) BnBr, DIPEA, ACN; (iii) CH2Cl2, TFA; (iv) Boc-HYNIC, HBTU, DIPEA; (v) EtOH, H2, Pd/C; (vi) Fmoc-Succinimide, DMSO.

Scheme 2. Synthesis of HYNIC-Phe Derivativea

a (i) BnOCOCl, water-dioxane, Na2CO3; (ii) BnBr, DIPEA, ACN; (iii) Zn, EtOH, AcOH; (iv) Boc-HYNIC, HBTU, DIPEA, MW 100W; (v) EtOH, H2, Pd/C; (vi) Fmoc-Succinimide, DMSO.

subproduct formation are fully attributed to the free carboxylic acid of the NR-Fmoc amino acid. In spite of the existence of different chemical procedures for generating ligand-peptide libraries based on solid-phase peptide synthesis, there have been few reports related to the generation of these libraries for screening purposes of diagnostic or therapeutic agents. Therefore, in this work, we present a generalized procedure for the synthesis of HYNIC Lys (6) and Phe (12) (Schemes 1 and 2, respectively) derivatives based on functional group protection. To demonstrate the utility of these derivatives, a small library of the bioactive peptide oxytocin (OT) labeled with HYNIC was prepared. Additionally, the procedure was compared with an orthogonal protection approach for the controlled positioning of HYNIC in OT. Oxytocin is an important hypothalamic cyclic neurononapeptide (NH2-Cys1Tyr2Ile3Gln4Asn5Cys6Pro7Leu8Gly9-CONH2) that plays an important role in many reproductive and behavioral functions. OT structure-activity relationships have been widely studied, and the following characteristics may be noted: (a) The cyclic part of the peptide (Cys1-Cys6) is more important in conferring binding selectivity for the OT receptor (OTR) compared to the linear tripeptidic part (Pro7Leu8Gly9) (13); (b) the replacement of the CONH2 terminal group for COOH leads to less active OT derivatives; (c) the tripeptide tail of OT is important for binding, and Pro7Leu8 can be replaced by a variety of residues (moreover, OT derivatives with reduced potency are obtained when Pro7 is substituted by a residue that induces conformational constraints) (14); and (d) the deaminoCys1oxytocine derivative is more active than OT (15) and has higher stability to oxytocinase degradation (16). However, structureactivity relationships for OT-ligand conjugates are unknown. Recently, novel sites for OT receptor expression have been detected, including breast cancer cells, bone cells, myoblasts, cardiomyocytes, endothelial cells, and in ovarian carcinoma (17,

18). Although the mechanism is not completely understood, in tumors, OT acts as a growth regulator, and it has been demonstrated that in some cases it has an growth-inhibiting effect (mammarian carcinomas, neoplastic epithelial, nervous or bone cells, and ovarian carcinoma cells), while in others, it promotes cell proliferation (trophoblast and endothelium neoplastic cells) (19). Additionally, it has been recently recognized that the placental leucine aminopeptidase (P-LAP) acts as an OT-degrading aminopeptidase (oxytocinase) in endometrial adenocarcinoma tissues and ovarian cancer, and it has been suggested that the system OT-OTR-P-LAP might be involved in cellular growth for this type of cancers (18). Thus, we hypothesized that the presence of P-LAP might counterattack the growth-inhibiting effect of the OT degrading it. If such a mechanism exists, then it is important to consider that OTradiolabeled derivatives stable to P-LAP might serve as diagnostic or therapeutic agents for different cancer cells. As for OT-ligand conjugates, there are scarce reports (20, 21), and no OT libraries with different ligand position and nature have been reported specifically for HYNIC derivatives.

EXPERIMENTAL PROCEDURES General Procedures. 1H and 13C nuclear magnetic resonance spectra were taken using a JEOL Eclipse 200 MHz spectrometer and referenced to a tetramethylsilane standard. Chemical shifts are reported as parts per million (δ), with splitting patterns designated as singlet (s), doublet (d), triplet (t), multiplet (m), or double of doublets (dd). Coupling constants are reported in hertz. Melting points were taken in a Fisher Johns melting point apparatus. HPLC peptide analysis and purification were performed on a HP1050 instrument equipped with a 1050 series diode array UV-vis detector, while quantitative analysis was carried out on a C18 Jupiter column 5 µm, 250 × 4.6 mm 300

1562 Bioconjugate Chem., Vol. 18, No. 5, 2007

Å, and purification was done on a C18 Jupiter column 10 µm, 250 × 10 mm 300 Å, both from Phenomenex. Products were eluted at a flow rate of 1 mL/min (4.8 mL/min for semipreparative HPLC) using a linear gradient [(100:0) to (0:100) in 35 min] of H2O/TFA (0.1%) and acetonitrile/H2O/TFA (90/10/0.1). MS-ESI analysis was performed in an Agilent 1100 series instrument LC-MSD trap. Peptides were lyophilized in a FreeZone 2.5 Labconco lyophilizer. Microwave-assisted solid-phase peptide synthesis was carried out following Fmoc standard protocols. Microwave-assisted reactions were carried out in a Microwave Accelerated Reaction System, Mars X, version 047928, CEM corporation. Reaction conditions were as follows: swelling, 1 pulse of 120 s at 15 W; coupling, 4 pulses of 15 s at 100 W; capping, 3 pulses of 30 s at 75 W; deprotection, 3 pulses of 30 s at 75 W. To avoid racemization, cooling was applied after each pulse to keep reaction temperature below 60 °C. Peptides were synthesized on a 0.1 mmol scale using single-step couplings of 2 equiv Fmoc-amino acids, 2 equiv coupling agent and HOBt, and 6 equiv DIPEA in DMF and capping of free, unreacted amino groups with a 4.75 % v/v acetic anhydride solution in NMP. Coupling and capping completion was monitored with the ninhydrin test. Following linear peptide assembly, cleavage from the resin and final deprotection were carried out with TFA/ thioanisole/1,2-ethanedithiol/anisole (9:0.5:0.3:0.2) at 25 °C for 2 h. The resin was filtered off and the filtrate dried with a gentle nitrogen flow, and peptides were precipitated with cold diethyl ether (-20 °C). Precipitate was filtered, washed with cold ether, and dried. Into the resulting residue, a 10 mM solution of 2,2′bispyridyl disulfide in methanol was added. The resultant solution was stirred at room temperature, and peptide oxidation completion was monitored by HPLC. The solvent was removed, and the residue was redisolved in 10% acetic acid. Peptides were purified by reverse-phase HPLC, lyophilized, and characterized by MS-ESI. 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N-Hydroxybenzotriazole (HOBT), FmocL-amino acids, and resins (Wang resin for -COOH peptides and Rink amide for -CONH2 peptides) were obtained from Novabiochem (San Diego, CA). All other chemicals and solvents were purchased from commercial sources and were used without further purification. Synthesis. Compounds 2, 3, 4, 7, 8, 9, and 10 were prepared following procedures reported in the literature, and the characterization data (1H and 13C NMR, MS-ESI, elemental analysis) were in agreement with the proposed structure and what is reported (11). NR-(Carbobenzyloxy)-N[6-(tert-butoxycarbonylhydrazinpyridyn)-3-carboxyl]-L-Lysine Benzyl Ester (5). 6-Boc-hydrazinpyridyn-3-carboxylic acid (248 mg, 0.98mmol), HBTU (371 mg, 0.98 mmol), and DIPEA (0.88 g, 6.8 mmol) were dissolved, followed by the addition of compound 4 (362 mg, 0.98 mmol). The resultant solution was stirred at room temperature for 1 day, and reaction completion was monitored by HPLC (Gemini polymeric column, 5 µm, 250 × 4.6 mm, Phenomenex, linear gradient from 85% DI-H2O/15% MeCN to 10% DI-H2O/90% MeCN in 20 min, at a flow of 1.0 mL/min and spectrophotometric detection at 254 and 275 nm). After reaction completion, the solvent was removed by rotary evaporation, and the product was purified by chromatography column in silica gel 60 (70230 mesh), using ether as eluent. The fractions containing the product were collected, and the solvent was removed, yielding 0.53 g (90%) of colorless oil. Rf ) 0.36 (SiO2, ethyl acetate). 1H NMR (200 MHz, CDCl , 25 °C, TMS) δ 8.4(s, 1H), 7.8 (d, 3 J ) 8.8 Hz, 1H), 7.3 (s, 10H), 7.1 (s, 1H), 6.8 (s, 1H), 6.5 (d, J ) 8.8 Hz, 1H), 5.8 (s, 1H), 5.0 (m, 4H), 4.3 (m, 1H), 3.2 (m, 2H), 1.9-1.4 (m, 6H), 1.5 (s, 9H). 13C NMR (50 MHz, CDCl3,

Miranda-Olvera et al.

25 °C, TMS) δ 171, 165, 160, 155, 154, 146, 136, 134, 127, 121, 104, 81, 66, 65, 52, 38, 30, 28, 27, 21. MS-ESI: m/z 605 (calcd. 605) [M]+; Anal. (C32H39N5O7) C, H, N. N[6-(tert-butoxycarbonylhydrazinpyridyn)-3-carboxyl]-NR(fluorenilmethoxycarbonyl)-L-lysine (6). Compound 5 (300 mg, 0.49 mmol) was dissolved in ethanol (15 mL), and 10% Pd/C catalyst (40 mg) was added. The reaction mixture was set in a Parr hydrogenation apparatus and was purged with H2 (10×), and pressure was set to 40 psi for 2 days. At the end of the reaction, the solution was filtered to remove the catalyst, and the solvent was removed by rotary evaporation. The residue was vacuum-dried, yielding 180 mg (97%) of a white solid. Rf ) 0.9 (SiO2, MeOH) corresponding to the nonprotected amino acid derivative. mp ) 220 °C. 1H NMR (200 MHz, 25 °C, DMSO-d6) δ 8.9 (s, 1H), 8.6 (s, 1H), 8.5 (d, J ) 2.4 Hz, 1H), 7.9 (dd, J ) 2.4 Hz, 8.8 Hz, 1H), 7.3 (s, 1H), 6.5 (d, J ) 8.8 Hz, 1H), 3.5 (s, 2H), 3.2 (m, 2H), 1.8-1.2 (m, 6H), 1.4 (s, 9H). 13C NMR (50 MHz, 25 °C, DMSO-d6) δ 171, 166, 156, 148, 136, 121, 104, 80, 46, 44, 30, 28, 27, 23. MS-ESI: m/z 381 (calcd. 381) [M]+, 404 (calcd. 404) [M + Na]+; Anal. (C17H27N5O5) C, H, N. The obtained intermediate (50 mg, 0.13 mmol) was dissolved in DMSO (2 mL) and was set under inert atmosphere and 0 °C in an ice bath. Fmoc-succinimide (40 mg, 0.13 mmol) in DMSO (2 mL) was slowly added under inert atmosphere. The reaction mixture was allowed to stand at RT overnight, and the reaction completion was monitored by HPLC and MS-ESI. Reaction conversion was quantitative, and therefore the reaction mixture can be used without purification for SPPS. The solvent was removed by rotary evaporation, and the product was purified by column chromatography in silica gel 60 (70-230 mesh) using as eluents CHCl3, CHCl3/MeOH (50/50), and CHCl3/ MeOH (15/85) successively. The fractions containing the product were collected, and the solvent was removed, yielding 77 mg (98%) of off-white solid. Rf ) 0.74 (SiO2, CHCl3/MeOH 15/85). 1H NMR (200 MHz, 25 °C, DMSO-d6) δ 8.8 (s, 1H, -CH6-, arom. pyridine), 8.6 (s, 1H, Boc-NH-NH-), 8.5 (d, J ) 2.4 Hz, 1H, Boc-NH-NH-), 8.3 (s, 1H, -CH2-NHCO-pyridine), 7.9 (d, J ) 8.8 Hz, 1H, -CH4-, arom. pyridine), 7.8 (m, 2H, arom. Fmoc), 7.7 (d, J ) 7.2 Hz, 2H, arom. Fmoc), 7.5 (m, 2H, arom. Fmoc), 7.3 (m, 2H, arom. Fmoc), 6.5 (d, J ) 8.8 Hz, 1H, -CH3-, arom. pyridine), 4.3-4.2 (m, 3H, -CH-CH2OCO-, Fmoc), 3.9 (m, 1H, -CH-COOH), 3.2 (m, 2H, -CH2NHCO-pyridine), 1.8-1.5 (m, 4H, -(CH2)2-), 1.4 (s, 11H, -C(CH3)3, -CH2-). 13C NMR (50 MHz, 25 °C, DMSO-d6) δ 173 (-COOH), 165 (-NHCO-pyridine), 156 (-NHCOO-Fmoc and Boc), 148 (-C6H-, arom. pyridine), 143 (arom. Fmoc), 141 (arom. Fmoc), 139 (-C4H-, arom. pyridine), 129 (arom. Fmoc), 128 (arom. Fmoc), 127 (arom. Fmoc), 124 (-C5H-, arom. pyridine), 110 (-C3H-, arom. pyridine), 79 (-C(CH3)3), 66 (-CH2-, Fmoc), 54 (-CH-COOH), 47 (-CH-, Fmoc), 31 (-CH2), 28 (-CH2-), 25 (-C(CH3)3), 22 (-CH2-). MS-ESI: m/z 627 (calcd. 627) [M + Na]+; Anal. (C32H37N5O7) C, H, N. NR-(carbobenzyloxy)-N[6-(tert-butoxycarbonylhydrazinpyridyn)-3-carboxyl]-L-Phenylalanine Benzyl Ester (11). 6-Bochydrazinpyridyn-3-carboxylic acid (0.177 g, 0.7 mmol), HBTU (0.7 mmol, 0.266 g), and DIPEA (0.636 g, 4.9 mmol) were dissolved in acetonitrile (3 mL), followed by compound 10 (0.28 g, 0.7 mmol). The resultant mixture was irradiated by MW 100 W for 20 min. Solvent was removed by rotary evaporation, and the product was purified by chromatography in silica using ethyl ether as eluent. Fractions containing the product were collected, the solvent was removed by rotary evaporation, and the residue was dried under reduced pressure, yielding 0.35 g (80%) of a yellow oil. Rf ) 0.66 (SiO2, Et2O). 1H NMR (200 MHz, CDCl3, 25 °C, TMS) δ 8.6 (s, 1H), 7.9 (d, J ) 8.8 Hz, 1H), 7.5 (d, J ) 7.8 Hz, 2H), 7.2 (s, 10H), 6.9 (d, J ) 7.8 Hz, 2H), 6.6 (d, J


) 8.8 Hz, 1H), 5.2 (m, 4H), 4.6 (m, 1H), 3.0 (s, 2H), 1.5 (s, 9H). 13C NMR (50 MHz, CDCl3, 25 °C, TMS) δ 170, 164, 160, 155, 154, 146, 135, 133, 130, 128, 127, 121, 120, 104, 81, 66, 65, 54, 36, 28. MS-ESI: m/z 639 (calcd.) 639 [M]+; Anal. (C35H37N5O7) C, H, N. NR-(fluorenylmethoxycarbonyl)-N[6-(tert-butoxycarbonylhydrazinpyridyn)-3-carboxyl]-L-Phenylalanine (12). Compound 11 (0.2 g, 0.31 mmol) was dissolved in ethanol (15 mL), and 10% Pd/C catalyst was added (20 mg). The reaction mixture was set in a Parr hydrogenation apparatus and purged with H2 (10×), and the pressure was set to 40 psi for 2 days. At the end of the reaction, the solution was filtered to remove the catalyst, and the solvent was removed by rotary evaporation. The residue was vacuum-dried, yielding 125 mg (98%) of a yellow solid. Rf ) 0.9 (SiO2, MeOH), corresponding to the nonprotected amino acid derivative. mp ) 250 °C. 1H NMR (200 MHz, 25 °C, DMSO-d6) δ 11.0 (s, 1H), 8.9 (s, 1H), 8.8 (s, 1H), 8.7 (d, J ) 2.4 Hz, 1H), 8.2 (dd, J ) 2.4 Hz, 8.8 Hz, 1H), 7.7 (d, J ) 8.6 Hz, 2H), 7.6 (s, 1H), 7.2 (d, J ) 8.6 Hz, 2H), 6.5 (d, J ) 8.8 Hz, 1H), 3.9 (m, 1H), 3.1 (m, 2H), 2.1 (s, 2H), 1.4 (s, 9H). 13C NMR (50 MHz, 25 °C, DMSO-d ) δ 170, 164, 161, 156, 6 148, 139, 138, 130, 129, 128, 120, 105, 79, 54, 28. MS-ESI: m/z 415 (calcd. 415) [M]+; Anal. (C20H25N5O5) C, H, N. The obtained intermediate (50 mg, 0.13 mmol) was dissolved in DMSO (2 mL) and was set under inert atmosphere and 0 °C in an ice bath. Fmoc-succinimide (40 mg, 0.13 mmol) in DMSO (2 mL) was slowly added under inert atmosphere. The reaction mixture was allowed to stand at RT overnight, and reaction completion was monitored by HPLC and MS-ESI. Reaction conversion was quantitative, and therefore the reaction mixture can be used without purification for SPPS. The solvent was removed by rotary evaporation, and the product was purified by column chromatography in silica gel 60 (70-230 mesh) using as eluents CHCl3, CHCl3/MeOH (50/50), and CHCl3/ MeOH (15/85) successively. The fractions containing the product were collected, and the solvent was removed, yielding 74 mg (97%) of light yellow solid. Rf ) 0.58 (SiO2, CHCl3/ MeOH 15/85). 1H NMR (200 MHz, 25 °C, DMSO-d6) δ 9.0 (s, 1H, -CH6-, arom. pyridine), 8.8 (s, 1H, Boc-NH-NH-), 8.7 (d, J ) 2.4 Hz, 1H, Boc-NH-NH-), 8.0 (d, J ) 8.6 Hz, 1H, -CH4-, arom. pyridine), 7.8 (m, 2H, arom. Fmoc), 7.7-7.6 (m, 4H, arom. Fmoc), 7.5 (m, 2H, arom. Fmoc), -CH-, arom.), 7.3 (m, 2H, arom. Fmoc), 7.2 (d, J ) 8.0 Hz, 2H), 6.5 (d, J ) 8.6 Hz, 1H, -CH3-, arom. pyridine), 4.3-4.0 (m, 4H, -CH-COOH, -CH-CH2-OCO-, Fmoc), 3.0 (m, 2H, -CH2-CH-COOH), 1.4 (s, 9H, -C(CH3)3). 13C NMR (50 MHz, 25 °C, DMSO-d6) δ 174 (-COOH), 165 (-NHCO-pyridine), 156 (-NHCOO-Fmoc and Boc), 145 (-C6H-, arom. pyridine), 143 (arom. Fmoc), 141 (arom. Fmoc), 139 (-C4H- arom. pyridine), 136 (-CH- arom.), 131 (-CH- arom.), 129 (arom. Fmoc), 128 (arom. Fmoc and arom.), 127 (arom. Fmoc), 125 (-C5H- arom. pyridine), 120 (arom.), 110 (-C3H- arom. pyridine), 80 (-C(CH3)3), 49 (-CH-, Fmoc), 37(-CH2-), 26 (-C(CH3)3). MS-ESI: m/z 638 (calcd. 638) [M + H]+; Anal. (C35H35N5O7) C, H, N. Peptide Characterization. NH2-CYIQNCPLG-CONH2, MSESI: m/z 1007 (calcd. 1007) [M + H]+. NH2-CYIQNCPLG-COOH, MS-ESI: m/z 1008 (calcd. 1006) [M + H]+. HYNIC-NH-CYIQNCPLG-CONH2, MS-ESI: m/z 1142 (calcd. 1142) [M + H]+. HYNIC-NH-CYIQNCPLG-COOH, MS-ESI: m/z 1143 (calcd. 1141) [M + H]+. NH2-CYIQNCP(K-HYNIC)G-CONH2, MS-ESI: m/z 1157 (calcd. 1157) [M + H]+. NH2-CYIQNCP(K-HYNIC)G-COOH, MS-ESI: m/z 1158 (calcd. 1156) [M + H]+.


NH2-CYIQNCP(F-HYNIC)G-CONH2, MS-ESI: m/z 1191 (calcd. 1192) [M + H]+. NH2-CYIQNCP(F-HYNIC)G-COOH, MS-ESI: m/z 1192 (calcd. 1190) [M + H]+. Peptide Conjugates Technetium-99m Labeling. Radiolabeling was carried out in a glass vial by adding 10 µL (10 µg) of each HYNIC-OT derivative, 0.5 mL of tricine/ethylendiaminediacetic acid (EDDA) solution (40 mg of tricine plus 20 mg of EDDA in 1 mL of 0.2 M phosphate buffer), 25 µL of SnCl2 solution (1 mg/mL of stannous chloride in 10 mM HCl), and 740-1110 MBq (0.5 mL) of 99mTc-pertechnetate (99Mo/ 99mTc generator system, GETEC-Mexico), followed by incubation at 90 °C for 30 min in a dry block heater. Evaluation of Radiochemical Purity. Radiochemical purity analyses were performed by instant thin-layer chromatography on silica gel (ITLC-SG, Gelman Sciences), solid-phase extraction (Sep-Pak C-18 cartridges), and reverse-phase highperformance liquid chromatography (HPLC). ITLC-SG analysis was accomplished using three different mobile phases: 2-butanone to determine the amount of free 99mTcO - (R ) 1), 0.1 M sodium citrate pH 5 to determine 4 f 99mTc-coligand and 99mTcO - (R ) 1), and methanol/1 M 4 f ammonium acetate (1:1 v/v) for 99mTc-colloid (Rf ) 0). Rf values of the radiolabeled peptide in each system were 0.0, 0.0, and 0.7-1.0, respectively. The Sep-Pak cartridges were preconditioned with 5 mL of ethanol, followed by 5 mL of 1 mM HCl and 5 mL of air. An aliquot of 0.1 mL of the labeled peptide was loaded on the preconditioned Sep-Pak cartridge, followed by 5 mL of 1 mM HCl to elute free 99mTcO4-. The radiolabeled peptide was eluted with 3 mL of ethanol/saline (1:1), and the hydrolyzed-reduced 99mTc or 99mTc-colloid remained in the cartridge. HPLC analyses were carried out with a Waters instrument running Millenium software with both radioactivity and UVphotodiode array in-line detectors, and YMC ODS-AQ S5 column (5 µm, 4.6 × 250 mm) at a flow rate of 1 mL/min using the gradient system described above. In this system, retention times for free 99mTcO4-, 99mTc-coligand, and 99mTcEDDA/HYNIC-OT were 3 min, 3.5-4.5 min, and 12 min, respectively. Plasma Stability. A volume of 10 µL of the labeled peptide solution (0.8 µg/10 µL) was incubated at 37 °C with 1 mL of fresh normal female human plasma, and also with 1 mL of phosphate buffer as a control. Radiochemical stability was determined by taking samples of 50 µL at different times from 30 min to 24 h for analysis. The plasma protein in 50 µL of the sample was precipitated with acetonitrile and centrifuged at 1000 g for 5 min; the supernatant was removed, diluted 1:7 with saline solution, and analyzed by reverse-phase HPLC. Competition Assay. In order to evaluate the biological integrity of the labeled peptides, a radioimmunoassay (RIA) was carried out comparing the competition of the labeled peptides and the unlabeled peptide binding to a limited quantity of specific antibody (Oxytocin radioimmunoassay kit, Peninsula Laboratories Inc., Bachem). 125I-OT was used as the “gold” standard (IC50 ) 0.05 nM), and the unlabeled peptide (oxytocin) was used in the range 1-128 pg/100 µL (0.01-1.28 nM). 99mTcEDDA/HYNIC-Lys8-OT-CONH2 and 99mTc-EDDA/HYNICCys1-OT-CONH2 (both obtained with radiochemical purities higher than 96%) were purified by reverse-phase HPLC in order to eliminate the cold peptide and to obtain a radioligand concentration less than 1 pg/100 µL useful for the competition assay. A plot of % B (binding)/Bo (total binding less nonspecific binding) versus the log of the unlabeled OT concentrations was generated for each labeled peptide: 125I-OT, 99mTc-EDDA/ HYNIC-Lys8-OT-CONH2, and 99mTc-EDDA/HYNIC-Cys1-OT-


CONH2. The IC50 was calculated as the concentration of competitor (unlabeled OT) inhibiting 50% of the radioligand binding. Cell Lines. Human breast carcinoma cell MCF7 line was originally obtained from ATCC (USA). The cells were routinely grown at 37 °C, with 5% CO2 atmosphere and 100% humidity in RPMI medium supplemented with 10% newborn calf serum and antibiotics (100 µg/mL streptomycin). Internalization Assay and Nonspecific Binding. MCF7 cells (1 × 106) supplied with fresh medium were incubated in 6-well plates with about 200 000 cpm of 99mTc-EDDA/HYNIC-Lys8OT-CONH2 or 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 (0.2 nmol total peptide) for 2 h at 4 °C. Afterward, the preincubation cells were washed three times with ice-cold culture medium. Warmed culture medium was added to the plates, which were incubated in triplicate at 37 °C for 2, 4, 6, and 24 h in order to allow internalization. Cell-surface bound radioligand was removed in two steps of 5 min acid wash (50 mM glycine-HCl/ 100 mM NaCl, pH 2.8) at room temperature. Cells were solubilized by incubation with 1 N NaOH at 37 °C to determine internalized radioligand. Results were expressed as the percentage of total activity internalized. The nonspecific binding was determined in parallel but in the presence of 10 µM cold OT (Bachem-USA) to block OT receptor cells. Differences in the obtained cell binding data were evaluated with the Student t test. Imaging. Tumor uptake studies in mice were carried out according to the rules and regulations of the Official Mexican Norm 062-ZOO-1999. Athymic male mice (20-22 g) were kept in sterile cages with sterile wood-shavings bed; constant temperature, humidity, and noise; and 12:12 light periods. Water and feed (standard PMI 5001 feed) were given ad libitum. Tumors were induced by subcutaneous injection of MCF7 cells (1 × 106) resuspended in 0.2 mL of phosphate-buffered saline, into the backside of 6-7 week old nude mice. The sites of injection were observed at regular intervals for the appearance of tumor formation and progression. Athymic mice with induced tumors were used for imaging studies. 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 or 99mTc-EDDA/ HYNIC-Lys8-OT-CONH2 (∼1.11 MBq) in 0.04 mL was injected in the tail vein. The nude mice (n ) 4) with the implanted tumors were scanned with a gamma camera with a pinhole collimator 2 h after the radiopharmaceutical was injected in the tail vein.

RESULTS An orthogonal protection strategy was followed to prepare HYNIC-Lys and HYNIC-Phe as shown in Schemes 1 and 2. Thus, intermediates 4 and 10 were obtained in excellent yields. The later derivatives have the R-amino and carboxylic acid groups protected with Cbz and Bn, respectively. Intermediates 4 and 10 were reacted with Boc-HYNIC (Figure 1b) using HBTU as activating agent, to generate intermediates 5 and 11. While this stage was smooth for the lysine derivative, ligand conjugation with 10 was not completed even after several days of reaction. In a first attempt to drive to completion the ligand coupling step with 10, a succinimide active ester of Boc-HYNIC was used, giving similar results to those obtained with HBTU. Further attempts varying reaction conditions were also unsuccessful. As an alternative, microwave-assisted coupling was carried out, and thus, product 11 was obtained in high yield after 20 min irradiation at 100 W. The next steps for both amino acids proceeded without further complications, obtaining compounds 6 and 12 in good overall yields (76% and 61%, respectively) and enantiopurity.

Miranda-Olvera et al. Scheme 3

a

a (I) Oxytocin, (II) HYNIC-Lys8-oxytocin, (III) HYNIC-Phe8oxytocin, and (IV) HYNIC-Cys1-oxytocin. R ) -OH or -NH2.

Scheme 4. Synthesis of Oxytocin-HYNIC Peptides by SPPS Following an Orthogonal Protection Approacha

a (i) Dde-Lys or Dde-Phe (aa) coupling followed by Fmoc removal; (ii) succesive amino acid coupling; (iii) Dde removal with 2% hydrazine; (iv) Boc-HYNIC coupling; (v) protecting groups and peptide removal from solid support, cysteine oxidation, purification, and characterization.

The nanopeptide neurohypophysial hormone oxytocin (Scheme 3, I, R ) -NH2) was chosen as a model to demonstrate the utility of usage of derivatives 6 and 12 for the controlled HYNIC positioning of peptides during SPPS and henceforth for generating HYNIC-peptide libraries to screen specific activity. The linear part of OT was chosen as one of the targets to place HYNIC within the peptide based on previous knowledge. For this work, the Leu8 residue was selected as the point to be substituted by the HYNIC-Lys (Phe) residues (Scheme 3, II and III). Additionally, for comparison, the synthesis of II and III was also carried out following an orthogonal protection approach (Scheme 4) using a N-2-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethylamine (Dde)-protected lysine derivative (Figure 2a) commercially available or a p-aminophenylalanine derivative protected with Dde in the p-amino group recently prepared by our group (unpublished results) (Figure 2b). A HYNIC-R-amide-Cys1 derivative was also synthesized despite the fact that the ligand stays inside the cyclic part of the peptide (Scheme 3, IV). In all cases, peptides were prepared with both carboxylic and amide groups by using different solid supports. Synthesis of these derivatives and the OT native peptide was



Figure 2. (a) N-Dde-NR-Fmoc-lysine and (b) pN-Dde-NR-Fmoc-phenylalanine.

Figure 4. Uptake of 99mTc-EDDA/HYNIC-Lys8-OT-CONH2 in tumor MCF7 cells in athymic mouse (whole mouse).

Figure 3. Time-dependent internalization of (a) 99mTc-EDDA/HYNICLys8-OT-CONH2 and (b) 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 in unblocked and blocked MCF7 cells expressed as % of total activity (mean ( sd, n ) 3). Blocked cells were incubated with an additional receptor blocking dose of unlabeled OT in order to determine the nonspecific binding of radioactivity.

carried out using commercially available reagents following standard Fmoc SPPS protocols, and step reactions were assisted by microwave radiation in order to minimize reaction times and to demonstrate the suitability of using this procedure for the quick generation of peptide-ligand libraries. The microwaveassisted synthesis of the full peptides was accomplished very rapidly (ca. 40 min per peptide, not considering purification), which compared to classical SPPS protocols (ca. 30 h, not considering purification) is very attractive for generating peptide conjugate libraries. Following cleavage from the resin and total deprotection, cysteine oxidation for the crude peptides was obtained quantitatively in 5 min using 2,2′-bispyridyl disulfide. Crude cyclic peptides were purified by semipreparative HPLC yielding peptides I-IV. The global yield for peptides II and III prepared via the HYNIC prelabeled amino acids 6 and 12 was approximately 20% in both cases and 40% when the same peptides were prepared by an orthogonal protection approach, all carried out under the same nonoptimized reaction conditions

(reaction time, reactant equivalents, temperature, etc.). On the other hand, yields were not dependent on the type of resin used for the synthesis. Furthermore, higher global yields can be obtained when reaction conditions are independently optimized for each peptide for a given procedure. Peptide labeling efficiencies of these peptides with 99mTc and EDDA were tested, and the results showed low labeling yields for I and III (10% and 40%, respectively), while for II and IV, yields above 96% were obtained. In addition, a 40% 99mTcEDDA labeling efficiency for a Boc,Fmoc-deprotected HYNICPhe derivative of 12 was observed. Biological integrity of the labeled OT peptides 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 and 99mTc-EDDA/HYNIC-Lys8-OT-CONH2 was determined by a radioimmunocompetition binding assay. Thus, it was found that a 125I-OT peptide has an IC50 value of 0.05 nM, while peptides 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 and 99mTcEDDA/HYNIC-Lys8-OT-CONH2 have IC50 values of 0.2 and 1 nM, respectively. Therefore, IC50 values less than 10 nM indicate that both peptides maintain an important biological recognition. After 24 h in human plasma, the radiochemical purity of 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 remained higher than 90%, while the radiochemical purity of 99mTcEDDA/HYNIC-Lys8-OT-CONH2 decreased to 15%. Results of the in vitro assays showed a rapid internalization and a specific receptor binding of 99mTc-EDDA/HYNIC-Lys8OT-CONH2 and 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 to MCF7 cells, since there were significant differences in the percentage of uptake between blocked receptor and unblocked receptor cells at different times (p < 0.05) (Figure 3). In vivo images of 99mTc-EDDA/HYNIC-Lys8-OT-CONH2 and 99mTcEDDA/HYNIC-Cys1-OT-CONH2 showed that the highest nonspecific uptake was found in the kidneys and in lower amounts in the liver (Figures 4 and 5). However a clear tumor uptake


Miranda-Olvera et al.

Figure 5. Uptake of 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 in tumor MCF7 cells in athymic mouse: (a) whole mouse; (b) mouse with dissection of internal viscera to highlight tumor uptake.

The lower peptide yields obtained for the prelabeled amino acid approach when compared to the orthogonal protection procedure can be attributed to an increase in peptide aggregation in the resin, given the higher probability of hydrogen bonding formation due to the HYNIC unit being readily attached to the solid support. The HYNIC-OT radiolabeling results show that 99mTc is complexed to the peptide through HYNIC and also that labeling efficiency depends on the nature of the amino acid where HYNIC is attached. The poor 99mTc labeling efficiency for III can be attributed to a lower electron-donation ability of the hydrazine group of HYNIC toward the metal, provoked by the conjugation of the ligand to the p-amino aromatic amino acid and not by the conjugate-peptide structure. As previously mentioned, it has been shown that OT receptors have weak selectivity to peptides sharing the same cyclic part (from Cys1 to Cys6). This indicates that the cyclic part of OT is more important in conferring binding affinity for the OT receptor compared with the linear tripeptidic part of the hormone (from Pro7 to Gly9) (13). However, our results showed that peptideligand conjugation in the tripeptide part diminishes binding affinity to a higher extent than when the peptide is labeled in the Cys1 residue. This is opposite to what has been hypothesized previously on the basis of what is known of the nonconjugated peptide (18, 19). 99mTc-EDDA/HYNIC-Lys8-OT-CONH degradation in plasma 2 and its poor in vivo tumor uptake might be caused by the effect of the oxytocinase enzyme, which acts on the N-terminal Cys1 residue (16). Furthermore, the plasma stability and in vivo tumor uptake of 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 could support the hypothesis that the introduction of HYNIC-Cys1 produces a protective steric effect on the amide bond susceptible to hydrolysis by P-LAP, which acts on the N-terminal Cys1 residue. These results justify further in vitro and in vivo evaluations of 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 as a potential radiopharmaceutical for diagnosis of OT receptor overexpressing tumors and might be useful for further understanding of the OT cancer regulation mechanisms, particularly where OT has shown to have an inhibitory growth effect. Finally, we can conclude that, when designing peptide-based ligand conjugates for diagnostic or therapeutic applications and determining the best ligand position, the use of conventional wisdom (such as known structure-activity relationships for the nonconjugated peptide) as prescribed by numerous studies may be a time-saving, and on occasion convenient, approach. However, as this work has demonstrated, challenging conventional wisdomswhile time-consuming and seemingly foolhardyscan also lead to exciting possibilities. This research has provided and demonstrated the utility of different chemical tools to generate ligand-peptide libraries when searching for that metal-peptide conjugate that can have the desired characteristics.

was only observed for 99mTc-EDDA/HYNIC-Cys1-OT-CONH2 (Figure 5).

ACKNOWLEDGMENT

DISCUSSION

This work was supported by CONCYTEG Mexico grants no. GTO-2002-C01-6029 and GTO-2003-C02-11517.

In this work, unlike the report indicated above for the synthesis of the lysine derivative 6 (12), blocking the carboxylic acid group was done to minimize side reactions such as mixed ligand-amino acid anhydride formation, and thus, 6 was prepared in a good overall yield. Moreover, as for the phenylalanine derivative, the observed lack of reactivity of 10 can be explained by the weaker nucleophilicity of the p-amino group in Phe when compared to the -amino aliphatic group of the lysine derivative. Furthermore, the MW-assisted ligand coupling with 10 gives an alternative for the future efficient synthesis of Phe-ligand peptide libraries when either an orthogonal protection or a prelabeled amino acid approach is followed.

APPENDIX. ELEMENTAL ANALYSES Compound 5: Anal. (C32H39N5O7) C, H, N calcd. 63.46, 6.49, 11.56; found 63.53, 6.51, 11.60. Intermediate before compound 6: Anal. (C17H27N5O5) C, H, N calcd. 53.53, 7.13, 18.36; found 53.61, 7.10, 18.39. Compound 6: Anal. (C32H37N5O7) C, H, N calcd. 63.67, 6.18, 11.60; found 63.79, 6.20, 11.58. Compound 11: Anal. (C35H37N5O7) C, H, N calcd. 65.71, 5.83, 10.95; found 65.43, 5.84, 10.96. MS-ESI: m/z 639, (calcd.) 639 [M]+.



Intermediate before compound 12: Anal. (C20H25N5O5) C, H, N calcd. 57.82, 6.07, 16.86; found 57.88, 6.05, 16.84. Compound 12: Anal. (C35H35N5O7) C, H, N calcd. 65.92, 5.53, 10.98; found 65.69, 5.50, 10.95.

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(12) Greenland, W. E., Howland, K., Hardy, J., Fogelman, I., and Blower, P. J. (2003) Solid-phase synthesis of peptide radiopharmaceuticals using Fmoc-N-epsilon-(hynic-Boc)-lysine, a technetiumbinding amino acid: application to Tc-99m-labeled salmon calcitonin. J. Med. Chem. 46, 1751-1757. (13) Gimpl, G., and Fahrenholz, F. (2001) The oxytocin receptor system: structure, function, and regulation. Physiol. ReV. 81, 629683. (14) Hruby, V. J., Chow, M. S., and Smith, D. D. (1990) Conformational and structural considerations in oxytocin-receptor binding and biological activity. Annu. ReV. Pharmacol. Toxicol. 30, 501-534. (15) Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., et al. (1986) Crystal structure analysis of deaminooxytocin: conformational flexibility and receptor binding. Science 232, 633-636. (16) Luciani, N., Marie-Claire, C., Ruffet, E., Beaumont, A., Roques, B. P., and Fournie-Zaluski, M. C. (1998) Characterization of Glu350 as a critical residue involved in the N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2): insights into its mechanism of action. Biochemistry 37, 686-692. (17) Zingg, H. H., and Laporte, S. A. (2003) The oxytocin receptor. Trends Endocrinol. Metab. 14, 222-227. (18) Morita, T., Shibata, K., Kikkawa, F., Kajiyama, H., Ino, K., and Mizutani, S. (2004) Oxytocin inhibits the progression of human ovarian carcinoma cells in vitro and in vivo. Int. J. Cancer 109, 525-532. (19) Cassoni, P., Sapino, A., Marrocco, T., Chini, B., and Bussolati, G. (2004) Oxytocin and oxytocin receptors in cancer cells and proliferation. J. Neuroendocrinol. 16, 362-364. (20) Bussolati, G., Chinol, M., Chini, B., Nacca, A., Cassoni, P., and Paganelli, G. (2001) 111In-labeled 1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid-Lys8-vasotocin: a new powerful radioligand for oxytocin receptor-expressing tumors. Cancer Res. 61, 4393-4397. (21) Chini, B., Chinol, M., Cassoni, P., Papi, S., Reversi, A., Areces, L., Marrocco, T., Paganelli, G., Manning, M., and Bussolati, G. (2003) Improved radiotracing of oxytocin receptor-expressing tumors using the new [111In]-DOTA-Lys8-deamino-vasotocin analogue. Br. J. Cancer 89, 930-936. BC070047A

Synthesis of Oxytocin HYNIC Derivatives as Potential Diagnostic

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