Syntheses of Functionalized Indium Porphyrins for Monoclonal

NOVEMBER/DECEMBER 1996 Volume 7, Number 6 © Copyright 1996 by the American Chemical Society

ARTICLES Syntheses of Functionalized Indium Porphyrins for Monoclonal Antibody Labeling Catherine H. Bedel-Cloutour,*,† Laurent Mauclaire,‡ Annie Saux,† and Michel Pereyre† Laboratoire de Chimie Organique et Organome´tallique, URA 35, Universite´ Bordeaux I, 351 cours de la Libe´ration, F-33405 Talence Cedex, France, and CIS Biointernational, BP 32, 91192 Gif sur Yvette Cedex, France. Received July 27, 1995X

The syntheses and characterizations of five differently hydrosoluble monosubstituted aryl porphyrins are reported. Their metallation with indium-111 was achieved and provided tracers with strong specific activities. The covalent coupling between indium-111 porphyrins and BSA served as a model reaction for the definition of the best experimental coupling conditions; the transposition to the labeling of anti-CEA monoclonal antibody was realized. The conjugates exhibited an in vitro good mAb-labeling efficiency, as well as a good preservation of immunoreactivity.

INTRODUCTION

The attachment of radioactive metal ions to monoclonal antibodies for cancer diagnosis and therapy has gained considerable interest in recent years (1-5). Radionuclides of interest for radioimmunoimaging should emit γ-ray energy of greater than 120 keV (which can be detected by conventional scintillation cameras), have a short half-time (24-72 h), and have a rapid clearance rate in order to obtain the maximum tumor-to-background ratio (6). Indium-111 strictly a γ-emitter, is wellsuited for these purposes, and tumor uptake of antibodies labeled with it is generally higher than that with iodinelabeled antibodies (7). Chelators that can hold radiometals with high stability under physiological conditions * To whom correspondence should be addressed. Catherine H. Bedel-Cloutour, Laboratoire de Technologie Enzymatique, URA 1442, Universite´ de Technologie de Compie`gne, BP 529, 60205 Compiegne Cedex, France. Telephone: (33) 44 23 44 08. Fax: (33) 44 20 39 10. † Universite ´ Bordeaux I. ‡ CIS Biointernational. X Abstract published in Advance ACS Abstracts, August 15, 1996.

S1043-1802(96)00045-6 CCC: $12.00

are essential to avoid excessive radiation damage to nontarget cells. This led to the development of bifunctional chelating agents possessing functional groups capable of binding a metal ion and allowing covalent linkage to proteins. In many of the numerous approaches, poly(aminocarboxylic acids) (EDTA, DTPA, and their derivatives) have been covalently bound to mAbs and complexed with indium-111 (8-10). It is essential for effective imaging that the radioactive metal ion remains complexed by the mAb-chelator conjugate. The loss of In-111 from its chelate in vivo depends on the rate at which In ligands exchange with other ligands; these are dependent on the metal involved, but they are strongly influenced by the structure of the chelator (11). Chelators containing highly basic groups [e.g. poly(aminocarboxylates)] will bind H+ vigorously at physiological pH, and this can reduce the effective value of the stability constant by a large factor. Many of the radiometal chelates formed from these chelating agents are not stable enough to prevent transcomplexation to serum proteins such as transferrin or conalbumin (12). Efforts to enhance the stability of these complexes under physiological conditions have afforded new analogues of © 1996 American Chemical Society

618 Bioconjugate Chem., Vol. 7, No. 6, 1996

Bedel-Cloutour et al.

characterization of these chelates with indium(III) and finally describe the syntheses of the conjugates of the indium porphyrin derivatives and bovin serum albumin (BSA) and a whole monoclonal antibody, the anticarcinoembryonic monoclonal antibody (anti-CEA). MATERIALS AND METHODS

Figure 1. Structures of porphyrins synthesized in this study.

DTPA. The addition of branching groups to the ethylenediamine backbone of DTPA has been achieved and led to more stable chelates (13-15). Bifunctional derivatives of DTPA have been developed in which the carbon backbone of the chelate has a linking group inserted for attachment to the antibody such that all eight coordination sites on DTPA are preserved (16). In the same way, there has also been considerable interest in the development of macrocyclic poly(aminocarboxylate) chelators (17, 18). Particularly, derivatives of DOTA form extremely stable complexes with a variety of radiometals (19). This type of macrocyclic agent offers promise for antibody labeling. As an alternative to the many methods reported in the literature, we have developed bifunctional chelating porphyrinic agents to label antibodies with indium-111. We selected porphyrins as 111In chelating agents to be conjugated to antibodies for a main reason. Tetrapyrrolic macrocycles of indium belong to the class III type (20) and are stable in vivo against demetallation (21). We have previously performed model-coupling reactions between symmetrical and unsymmetrical mesosubstituted (carboxyphenyl)indium porphyrins and amino acid ester derivatives and defined the best experimental conditions yielding the amide derivatives (22). Also, several monoclonal IgG and F(ab′)2 fragments were labeled in vitro with the indium-111 derivative of meso-5-(p-carboxyphenyl)-10,15,20-tritolylporphyrin (TTCPP111InCl) using the ester-activated method [N-hydroxysuccinimide/1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] (23). Up to 10 molecules of tracer were bonded to mAb and 1.5 to its F(ab′)2 fragment. The absence of any significant differences in the results of ELISA tests carried out with the metallated, unmetallated, and unmodified antibody suggested that immunoreactivity was not altered by covalent linkage between indium porphyrin and mAbs. In this report, we describe the syntheses of hydrosoluble and monofunctionalized meso-tripyridinylarylporphyrins (Figure 1), more compatible with the biological medium and possessing various function susceptible to covalent coupling, not only to amino group side chains but also to carboxylic, thiol, alcohol, and phenol groups as well as to aldehyde functions generated by oxidation of carbohydrate moieties. We report the preparation and

Reagents (analytical grade) were used without further purification. General Methods. 1H NMR spectra were recorded at 25 °C on Hitachi Perkin-Elmer R-24B (60 MHz) and Brucker WM 250 spectrometers operating at 200 MHz FT. Unless otherwise specified, the solvent was deuteriochloroform with TMS as the internal standard. The chemical shifts, δ, are given in parts per million, and coupling constants are in hertz. Abbreviations used were as follows: br, broad; d, doublet; dd, doublet of doublet; m, multiplet; o, octet; q, quadruplet; s, singlet; and t, triplet. Optical spectra in the Soret and visible region were measured on a Beckman-25 spectrophotometer, and infrared spectra were recorded on a Perkin-Elmer 683 spectrometer; the samples were prepared as a 0.5% dispersion in KBr pellets. Mass spectrometry was carried out using a Micromass 16F instrument for the aldehydes and a Nermag R1010B instrument for desorption ionization (DI-MS). FABMS studies were carried out in the Laboratoire de Spectrome´trie de Masse, CNRS, BP 22, F-69399 Vernaison, France; the FAB-MS matrices used were thioglycerol and 3-nitrobenzyl alcohol (NBA). HPLC analysis and preparations were carried out on a Varian 5000 Liquid Chromatograph coupled with a Varian 2550 multiwavelength detector set at 420 nm or a Waters 590 Programmable HPLC pump. An analytical silica column, Micro Pak Si 10 (4.6 × 250 mm, 20 µm particles, 60 Å pore size, column 1), or a preparative silica column (40 × 200 mm, 20 µm particles, 60 Å pore size, column 2) was used for un-ionized free base porphyrins and their indium derivatives; for the quaternized macrocycles, free bases, and indium derivatives, a Nucleosil C4 column was used (4.6 × 250 mm, 10 µm particles, 300 Å pore size, column 3). TLC microplates of silica gel were used. Liquid chromatography was performed on a silica gel column (25 × 200 mm, 230-400 mesh), and anion exchange resin Dowex 1 × 8 (200-400 mesh, chloride form) was used for quaternized free bases and indium derivatives. Porphyrin Syntheses. meso-10-(3-Nitro-4-fluorophenyl)-5,15,20-tri(4′-N-pyridinyl)porphyrin (1). 3-Nitro-4fluorobenzaldehyde. Following a published procedure (24), a mixture of concentrated sulfuric acid (94 mL) and nitrite-free nitric acid (12.5 mL) was cooled to -5 °C and under argon was treated dropwise with 4-fluorobenzaldehyde (23.8 g, 191.5 mmol) while the temperature was kept below 5 °C. Then the reaction mixture was allowed to stand at room temperature for 3 h and then poured on ice (500 g). The nitro derivative precipitated and was filtered off through a sintered glass frit. The solid was washed with water and dried. Recrystallization from diethyl ether gave the pure aldehyde (70%, 16.6 g, 134 mmol). Mp: 46 °C [lit. (24) 46.5 °C]. NMR (60 MHz, CDCl3): 7.5 (q, 1H), 8.4-8.06 (m, 1H), 8.56 (dd, 1H), 10.03 (s, 1H). MS: m/e (relative intensity) 170 (MH+, 8.5), 168 (M+ - 1, 100). Porphyrin 1. 4-Pyridinecarboxaldehyde (7.13 g, 66.6 mmol) and 3-nitro-4-fluorobenzaldehyde (2.25 g, 13.3 mmol) were added to propionic acid (200 mL), and the mixture was brought to reflux. Pyrrole (5.37 g, 80 mmol)

Bioconjugate Chem., Vol. 7, No. 6, 1996 619

Radioactive Indium Porphyrins and Antibody Labeling

Table 1. 250 MHz 1H NMR Chemical Shifts of the Protons in Free Base Porphyrinsa

compound 1

2

6

3

7

a

Σ 3′-NO2, 4′-F

4′-F

4′-O(CH2)4NC8H4O2

4′-O(CH2)4NH2

4′-OCH2COOEt

solvent CDCl3

CDCl3

CDCl3

CDCl3

CDCl3

4

4′-OCH2COOH

CD3COOD

5

4′-OCH2COOPhSMe

CDCl3

pyrrole

pyridyl

8.89 (d, 2H), J ) 4.9 Hz 8.87 (s, 4H: 2,3,17,18) 8.82 (d, 2H), J ) 4.9 Hz

9.07 (d, 6H: 3′,5′), J ) 5.5 Hz 8.18 (d, 6H: 2′,6′), J ) 5.3 Hz

8.90 (d, 2H, J ) 4.8 Hz 8.85 (s, 4H: 2,3,17,18) 8.82 (d, 2H), J ) 4.8 Hz 8.96 (d, 2H), J ) 4.9 Hz 8.85 (s, 4H: 2,3,17,18) 8.81 (d, 2H), J ) 4.9 Hz 8.96 (d, 2H), J ) 4.8 Hz 8.85 (s, 4H: 2,3,17,18) 8.81 (d, 2H), J ) 4.8 Hz 8.95 (d, 2H), J ) 4.9 Hz 8.85 (s, 4H: 2,3,17,18) 8.82 (d, 2H) J ) 4.9 Hz 9.00 (m, 8H) 8.96 (d, 2H), J ) 4.8 Hz 8.87 (s, 4H: 2,3,17,18) 8.83 (d, 2H), J ) 4.8 Hz

9.06 (d, 6H: 3′,5′), J ) 5.9 Hz 8.18 (d, 6H: 2′,6′), J ) 5.9 Hz 9.04 (d, 6H: 3′,5′), J ) 5.7 Hz 8.16 (d, 6H: 2′,6′), J ) 5.7 Hz

phenyl 8.92 (m, H2′)

NH

Σ

-2.92 (s, 2H)

8.47 (o, H6′), JHHo ) 8.5 Hz JHHm ) 2.3 Hz JHFm ) 4.2 Hz 7.74 (dd, H5′), JHHo ) 8.5 Hz JHFo ) 10.4 Hz 8.15 (m, 2H: 2′,6′)

-2.91 (s, 2H)

7.49 (dd, 2H: 3′,5′), J ) 8.8 Hz 8.08 (d, 2H: 2′,6′), J ) 8.6 Hz 7.27 (d, 2H: 3′,5′), J ) 8.6 Hz

-2.88 (s, 2H)

phtha: 7.86 (m, 2H), 7.7 (m, 2H) OCH2: 4.28 (m, 2H) NCH2: 3.87 (m, 2H)

9.03 (d, 6H: 3′,5′), J ) 5.8 Hz 8.15 (d, 6H: 2′,6′), J ) 5.8 Hz

8.08 (d, 2H: 2′,6′), J ) 8.6 Hz 7.27 (d, 2H: 3′,5′), J ) 8.6 Hz

-2.86 (s, 2H)

[CH2]2: 2.04 (m, 4H) OCH2: 4.27 (t, 2H) H2NCH2: 2.90 (t, 2H) [CH2]2: 2.02 (m, 4H)

9.05 (d, 6H: 3′,5′), J ) 5.9 Hz 8.16 (d, 6H: 2′,6′), J ) 5.9 Hz

8.13 (d, 2H: 2′,6′), J ) 8.7 Hz 7.32 (d, 2H: 3′,5′), J ) 8.7 Hz

9.29 (m, 6H: 3′,5′) 8.64 (m, 6H: 2′,6′) 9.08 (d, 6H: 3′,5′), J ) 5.9 Hz 8.2 (d, 6H: 2′,6′), J ) 5.9 Hz

8.16 (m, 2H: 2′,6′) 7.36 (m, 2H: 3′,5′) 8.15 (d, 2H: 2′,6′), J ) 8.6 Hz 7.35 (d, 2H: 3′,5′), J ) 8.6 Hz

-2.87 (s, 2H)

OCH2CO: 4.93 (s, 2H)

-

CH2Me: 4.42 (q, 2H), J ) 7.1 Hz CH2CH3: 1.42 (t, 3H), J ) 7.1 Hz OCH2: 4.99 (s, 2H)

-2.89 (s, 2H)

PhSMe: 7.71 (m, 2H), 7.54 (m, 2H) OCH2: 5.06 (s, 2H) SCH3: 2.5 (s, 3H)

Residual absorptions from TMS for CHCl3 (7.25 ppm) and CH3COOH (2.06 ppm).

was then added, and the mixture was refluxed for a further 1 h and concentrated to dryness. The solid obtained was dissolved in DMF and the resulting solution allowed to cool overnight at 0 °C. The purple slurry of mixed porphyrins (1.46 g) containing the six possible macrocycles was then filtered and fractionated by preparative silica gel liquid chromatography (bed volume, 390 mL) with a gradient elution (CHCl3 containing 0 to 4% methanol). Porphyrin 1 was eluted with CHCl3/CH3OH (97/3) in a 1.9% yield (1.72 mg, 0.25 mmol). NMR (CDCl3): Table 1. Electronic absorption spectrum (CHCl3): λmax (/512) 417, 512.5 (1), 546 (0.36), 587 (0.39), 643 nm (0.12). FAB-MS: m/e (relative intensity) 681 (MH+, 65), 391 (100), 307 (61). IR (KBr): 540 (NO2), 1130 cm-1 (CF). meso-10-(4-Fluorophenyl)-5,15,20-tri(4′-N-pyridinyl)porphyrin (2). We followed the same protocol as for porphyrin 1, using 4-fluorobenzaldehyde (1.65 g, 13.3 mmol). In this case, the total crude material was dissolved in the minimum amount of DMF and precipitated by the addition of methanol. Porphyrin 2 was isolated from the six macrocycles (2.5 g) by column chromatography on silica gel (bed volume, 390 mL) with CHCl3/CH3OH (97/3) as eluent (2.6%, 222.5 mg, 0.35 mmol). NMR (CDCl3): Table 1. Electronic absorption spectrum (CHCl3): λmax (/511) 415.5, 511.5 (1), 545.5

(0.32), 586.5 (0.31), 643 nm (0.12). FAB-MS: m/e (relative intensity) 636 (MH+, 39), 391 (68), 149 (100). IR (KBr): 1085 cm-1 (CF). meso-10-[4-(4-Aminobutoxy)phenyl]-5,15,20-tri(4′-N-pyridinyl)porphyrin (3). 4-(Phthalimidobutoxy)benzaldehyde. 4-Hydroxybenzaldehyde (2.44 g, 0.02 mol), N-(4bromobutyl)phthalimide (5.64 g, 0.02 mol), and anhydrous K2CO3 (13.82 g, 0.1 mol) were mixed in anhydrous DMF (100 mL). The mixture was stirred at room temperature for 5 h. After filtration and washings with water, the solution was extracted with chloroform. The organic layer was shaken with 3 M NaOH and dried over NaHCO3 and the solvent evaporated to give the pure aldehyde (70%, 4.53 g, 14 mmol). Mp: 114-115 °C. NMR (60 MHz, CDCl3): 1.87 (m, 4H), 3.72 (m, 2H), 4.05 (m, 2H), 6.91 (d, 2H), 7.70 (m, 6H), 9.82 (s, 1H). MS: m/e (relative intensity) 323 (M+, 3), 202 [(CH2)4NC8H4O2+, 44.6], 160 (CH2NC8H4O2+, 100). meso-10-[4-(4-Phthalimidobutoxy)phenyl]-5,15,20-tri(4′N-pyridinyl)porphyrin (6). The same protocol as for porphyrin 1 was followed, using 4-(4-phthalimidobutoxy)benzaldehyde as the functionalized aldehyde (4.3 g, 13.3 mmol). The six macrocycles (1.4 g) were separated by preparative HPLC (column 2; flow rate, 12 mL/min; binary linear gradient of 0 to 3% CH3OH with chloroform). Combined fractions corresponding to porphyrin


6 were vacuum rotary evaporated to leave the pure porphyrin (2.2%, 239.62 mg, 0.29 mmol). NMR (CDCl3): Table 1. Electronic absorption spectrum (CHCl3): λmax (/516) 419.5, 516 (1), 552 (0.51), 590 (0.35), 647 nm (0.23). FAB-MS: m/e (relative intensity) 835 (22), 391 (100), 307 (72). Porphyrin (3). Porphyrin 6 (48.9 mg, 59.7 µmol) was dissolved in THF (50 mL); anhydrous hydrazine (5 mL) was added, and the mixture was brought to reflux for 2 h and then filtered. The solution was extracted with chloroform; the organic layer was washed with KHCO3 (pH 9) and a saturated ammonium chloride solution and then dried with NaHCO3 and concentrated to dryness under vacuum. The purity of porphyrin 3 was verified by TLC (silica gel plate; eluent, 97/3 CHCl3/MeOH) (95%, 39.9 mg, 56.7 µmol). NMR (CDCl3): Table 1. Electronic absorption spectrum (CHCl3): λmax (/516) 420, 516.5 (1), 551.5 (0.55), 591 (0.34), 650.5 nm (0.58). IR (KBr): 2960 (CH2), 1245 cm-1 (ether). meso-10-[4-(Carboxymethoxy)phenyl]-5,15,20-tri(4′-Npyridinyl)porphyrin (4). 4-Formylphenoxyacetic Acid. Adapting a published procedure (25), 4-hydroxybenzaldehyde (48.85 g, 0.4 mol) and NaOH (16 g, 0.4 mol) were mixed in water (500 mL), and potassium chloroacetate (57.01 g, 0.43 mol) was added. The solution was brought to reflux for 1 h and then left until room temperature was reached. pH was adjusted to 8-9 by addition of sodium hydrogenocarbonate; phenol precipitated and was eliminated by filtration. A subsequent adjustment to pH 1 by concentrated HCl addition precipitated 4-formylphenoxyacetic acid. The pure aldehyde was filtrated, washed with ethanol and ether, and dried under vacuum (21%, 15.13 g, 84 mmol). Mp: 194 °C [lit. (25) 192-195 °C]. NMR (60 MHz, DMSO-d6): 4.8 (s, 2H), 7.05 (d, 1H), 7.82 (d, 2H), 9.85 (s, 2H). MS: m/e (relative intensity) 180 (M+, 19.4), 179 (M+ - 1, 16.8), 121 (p-CHOPhO+, 100), 93 (PhO+, 43.1), 65 (C5H5+, 40.7). Ethyl (4-Formylphenoxy)acetate. N,N′-Carbonyldiimidazole (6.81 g, 42 mmol) was dissolved in anhydrous THF (200 mL), and 4-formylphenoxyacetic acid (7.21 g, 40 mmol) was added. The mixture was brought to reflux for 30 min, and then absolute ethanol (1.53 mL, 42 mmol) was added. The resulting solution was further brought to reflux for an additional 1 h. Solvent was evaporated and the resulting solid dissolved in chloroform. The organic solution was washed with dilute HCl and water and dried. Once chloroform evaporated, the crude product was purified by liquid chromatography on silica gel (25 × 200 mm; eluent, CHCl3). The aldehyde was then recrystallized in hot ether (80%, 6.66 g, 32 mmol). Mp: 154 °C [lit. (26) 155 °C]. NMR (60 MHz, CDCl3): 1.27 (t, 3H), 4.25 (q, 2H), 4.70 (s, 2H), 6.96 (d, 2H), 7.80 (d, 2H), 9.90 (s, 1H). MS: m/e (relative intensity) 208 (M+, 100), 135 (M+ - COOEt, 91.6), 105 (PhOCH2+, 56.9), 77 (C6H5+, 36.9). meso-10-[4-(Carboethoxymethoxy)phenyl]-5,15,20-tri(4′N-pyridinyl)porphyrin (7). The same protocol as for porphyrin 2 was followed to synthesize porphyrin 7 using the aldehyde described above (2.77 g, 13.3 mmol). The six possible macrocycles (2 g) were separated by preparative HPLC (column 2; flow rate, 12 mL/min; gradient elution, CHCl3 containing 0 to 3% methanol). Porphyrin 7 was isolated after elimination of the solvent under vacuum (5.6%, 535 mg, 0.74 mmol). NMR (CDCl3): Table 1. Electronic absorption spectrum (CHCl3): λmax (/515) 418.5, 515 (1), 550.5 (0.44), 589 (0.31), 646 nm (0.19). FAB-MS: m/e (relative intensity) 720 (MH+, 100), 391 (67), 307 (65). Analytical HPLC (column 1; flow rate, 2 mL/min; gradient elution, CHCl3 (relative intensity) 0 to 5% MeOH within 2 min): tR ) 6 min 50 s.


Porphyrin 4 (27). Porphyrin 7 (51.97 mg, 72.2 µmol) was dissolved in THF (50 mL). Ester hydrolysis was carried out by addition of methanolic sodium hydroxide (1 mL) [NaOH (4 g, 0.1 mol), H2O (5 mL), EtOH (50 mL)]. The reaction mixture was then refluxed for 1 h; solvent was flashed off and residue dissolved in chloroform. The resulting solution was washed with 0.1 M HCl; precipitate was filtered and dissolved in methanol. Insoluble materials were eliminated, and solvent was evaporated under vacuum. Porphyrin 4 was obtained in a 95% yield (47.45 mg, 68.6 µmol). NMR (CD3COOD): Table 1. Electronic absorption spectrum (CHCl3): λmax (/511) 413, 511 (1), 546.5 (0.45), 586.5 (0.35), 643 nm (0.22). FABMS: m/e (relative intensity) 692 (MH+, 25), 391 (90), 307 (100). IR (KBr): 1725 (COOH), 1225 cm-1 (ether). 4-(Methylthio)phenyl Ester of meso-10-[4-(Carboxymethoxy)phenyl]-5,15,20-tri(4′-N-pyridinyl)porphyrin (5) (28). To porphyrin 4 (17.29 mg, 25 µmol) and in solution in THF (30 mL) was added DCC (51.58 mg, 0.25 mmol) in DMF (5 mL); the solution was cooled to 0 °C, and 4-(methylthio)phenol (70.1 mg, 0.5 mmol) was added. The mixture was maintained under stirring at room temperature for 24 h. The solvent was evaporated to dryness, and the residue was chromatographied on a silica gel column (bed volume, 390 mL; eluent, 97/3 chloroform/ methanol) (78%, 15.87 mg, 19.5 µmol) NMR (CDCl3): Table 1. Electronic absorption spectrum (CHCl3): λmax (/513) 417, 513 (1), 548 (0.44), 588 (0.32), 644.5 nm (0.22). Analytical HPLC (column 1; flow rate, 2 mL/min; gradient elution, CHCl3 containing 3 to 5% MeOH within 1 min): tR ) 1 min 50 s. Quaternization of Porphyrins 1, 2, and 4. The following general procedure was followed. The free base porphyrin (0.13 mmol) was dissolved in DMF (10 mL), and an excess of iodomethane (20 mL) was added. The mixture was stirred and heated to 40 °C for 2 h and then cooled to room temperature; porphyrin iodide salt was precipitated by addition of diethyl ether, recovered by filtration, and dried at 130 °C in a vacuum oven. The cationic porphyrin was dissolved in methanol and converted to the chloride form with an anion exchange resin (eluent, methanol). The cationic free base porphyrins were characterized by their UV-visible (in MeOH) and 1 H NMR (in CD3OD) spectra which were in good agreement with the proposed structures. meso-10-(3-Nitro-4-fluorophenyl)-5,15,20-tris-(4′-Nmethylpyridinyl)porphyrin Trichloride Salt (Me-1). Yield: 88% (95.2 mg, 114.4 µmol). Electronic absorption spectrum: λmax (/513) 417, 513 (1), 549.5 (0.45), 589 (0.37), 647.5 nm (0.17). NMR: Table 2. IR (KBr): 1505, 1220 cm-1. meso-10-(4-Fluorophenyl)-5,15,20-tris(4′-N-methylpyridinyl)porphyrin Trichloride Salt (Me-2). Yield: 87% (89.03 mg, 113.1 µmol). Electronic absorption spectrum: λmax (/514) 422, 514.5 (1), 551 (0.45), 589.5 (0.36), 646.5 nm (0.14). NMR: Table 2. DI-MS: m/e (relative intensity) 715 (M+ - 2Cl-, 12), 680 (M+ - 3Cl-, 100). IR (KBr): 1110 cm-1. meso-10-[4-(Carboxymethoxy)phenyl]-5,15,20-tris(4′N-methylpyridinyl)porphyrin Trichloride Salt(Me-4). Yield: 70% (76.73 mg, 91 µmol). Electronic absorption spectrum: λmax (/517) 427.5, 517.5 (1), 556 (0.58), 592 (0.42), 627.5 nm (0.22). NMR: Table 2. FAB-MS: m/e (relative intensity) 842 (MH+, 5), 391 (55), 214 (100). IR (KBr): 1760, 1715 (COOH), 1210 cm-1 (ether). Radioactive Indium Derivatives of Cationic Porphyrins Me-1, Me-2, and Me-4 and Free Bases 3 and 5. A solution of 37 MBq 111InCl3 in 0.05 M HCl (370 MBq/ mL; specific activity, 17.02 TBq/mg) was placed in a tube and evaporated to dryness under a stream of nitrogen.



Table 2. 250 MHz 1H NMR Chemical Shifts of the Protons in Quaternized Free Base Porphyrins in CD3ODa

compound

Σ

pyrrole

Me-1

3′-NO2, 4′-F

9.13 (br s, 8H)

Me-2

4′-F

9.06 (br s, 8H)

Me-4

4′-OCH2COOH

9.08 (br s, 8H)

a

pyridyl 9.39 (m, 6H: 8.94 (m, 6H: 9.40 (m, 6H: 8.94 (m, 6H: 9.40 (m, 6H: 8.96 (m, 6H:

3′,5′) 2′,6′) 3′,5′), J ) 6 Hz 2′,6′), J ) 6 Hz 3′,5′) 2′,6′)

phenyl

Σ

9.07 (m, 2H: 2′,6′) 7.68 (m, H5′) 8.18 (m, 2H: 2′,6′) 7.54 (dd, 2H: 3′,5′), J ) 8.8 Hz 8.14 (m, 2H: 2′,6′) 7.39 (m, 2H: 3′,5′)

N+Me: 4.86 (s, 9H) N+Me: 4.85 (s, 9H) N+Me: 4.84 (s, 9H) OCH2: 5.04 (s, 2H)

Residual absorption from TMS (3.32 ppm).

Then indium-115 trichloride (1.35 × 10-7 mol) and porphyrin (1.4 × 10-7 mol) in solution in acetic acid containing 3.3% trifluoroacetic acid (250 µL) were added, and the mixture was heated to 140 °C for 3 h. Porphyrins Me-1, Me-2, and Me-4. Once the reaction was completed, the pH was adjusted to 4-5 by 1 M NaOH. Radiolabeled indium porphyrins were purified on anion exchange resin as described for the quaternization (eluent, distilled water). Fractions (1 mL) were collected, and 111In radioactivity was measured by γ counting. Radioactive indium porphyrin fractions were pooled, and the solvent was evaporated under vacuum. meso-10-(3-Nitro-4-fluorophenyl)-5,15,20-tris(4′-Nmethylpyridinyl)porphyrin Indium(III) Tetrachloride(Me1-In). Yield: 75%. Electronic absorption spectrum (MeOH): λmax (/561) 428.5, 521.5 (0.2), 561 (1), 604.5 nm (0.34). Specific activity A (MBq/mg): 547.6. meso-10-(4-Fluorophenyl)-5,15,20-tris(4′-N-methylpyridinyl)porphyrin Indium(III) Tetrachloride (Me-2-In). Yield: 85%. Electronic absorption spectrum (MeOH): λmax (/562) 429.5, 523 (0.2), 562 (1), 606.5 nm (0.39). Specific activity A (MBq/mg): 573.5. meso-10-[4-(Carboxymethoxy)phenyl]-5,15,20-tris(4′-Nmethylpyridinyl)porphyrin Indium(III) Tetrachloride (Me4-In). Yield: 87%. UV-visible (MeOH): λmax (/564) 433.5, 525 (0.23), 564.5 (1), 609.5 nm (0.48). Specific activity A (MBq/mg): 540.2. Porphyrins 3 and 5. When the reaction was completed, the pH was adjusted to 6-7 with 1 M NaOH. In order to completely remove unreacted carrier-free 111In/115In, the solution was extracted with chloroform (2 × 10 mL); chloroformic solutions were combined and washed several times with distilled water to neutrality. The chloroform was then evaporated under vacuum. meso-10-[4-(4-Aminobutoxy)phenyl]-5,15,20-tris(4′-Nmethylpyridinyl)porphyrin Indium(III) Tetrachloride(3-In). Yield: 76%. Electronic absorption spectrum (CHCl3): λmax (/559) 426.5, 520.5 (0.23), 559.5 (1), 600 nm (0.4). Specific activity A (MBq/mg): 629. meso-10-[4-(Carboxymethoxy)phenyl]-5,15,20-tris(4′-Npyridinyl)porphyrin Indium Chloride 4′-Methylthio)phenyl Ester (5-In). Yield: 53%. Electronic absorption spectrum (CHCl3): λmax (/559) 424, 522 (0.16), 559 (1), 598.5 nm (0.29). Specific activity A (MBq/mg): 555. Quaternization of Porphyrin 5-In. The indium porphyrin 5-In (67.35 mg, 7 × 10-2 mmol) was dissolved in methyl iodide (5 mL), and the mixture was maintained under stirring at 40 °C for 2 h. Then, an excess of methyl iodide was eliminated under vacuum, and the residue was dissolved in methanol (2 mL). The cationic indium porphyrin was converted to the chloride form with an anion exchange resin in the Cl- form (eluent, methanol). Fractions (1 mL) were collected, and the radioactivity was

measured. Radioactive fractions were pooled, and the solvent was evaporated to dryness. meso-10-[4-(Carboxymethoxy)phenyl]-5,15,20-tris(4′-Nmethylpyridinyl)porphyrin Indium Pentachloride 4′(Dimethylthio)phenyl Ester (Me-5-In). Yield: 74% (60.3 mg, 5.18 × 10-2 mmol). Specific activity A (MBq/mg): 481. BSA Labeling. BSA was obtained from Sigma Chimie and was used without further purification. [111In/115In]Porphyrins Me-1-In, Me-2-In, and Me-5In. To 6.9 × 10-2 µmol (37 MBq) of indium porphyrin derivatives in water (200 µL) was added 15 µL of a BSA solution in water (4.6 × 10-4 M). The volume was adjusted to 1 mL by addition of water. The mixture was maintained under stirring at room temperature for 1.5 h and then chromatographied on a Sephadex G75 column (10 mL). Fractions (1 mL) were collected; the conjugate porphyrin-BSA was eluted within the third to eighth fractions. The yields of coupling determined by γ counting of 111indium radioactivity for Me-1-In and Me-2-In were 95 and 97%, respectively, with specific activities A of 66.6 MBq/mg. In the case of the activated ester Me-5-In, the same protocol was used, leading to a porphyrin-BSA conjugate in a 67% yield (A ) 48.1 MBq/mg). [111In/115In]Porphyrins 3-In and Me-4-In: Carbodiimide Activation. The indium derivative (6.9 × 10-2 µmol, 37 MBq) was dissolved in water (200 µL). To this solution were added 30 µL of NHS solution (2.29 × 10-3 M) and 46 µL of EDAC solution (1.5 × 10-3 M) both in 12.5 mM sodium phosphate (pH 6). The mixture was stirred in the dark at room temperature for 1 h before BSA in water was added (6.9 × 10-3 µmol, [porphyrin]/ BSA] ) 10). The stirred reaction mixture was kept at room temperature in the dark for an additional 1 h. The same protocol, as described above, was used to purify the labeled BSA. Using these conditions, the coupling efficiency was 89% for 3-In (A ) 66.6 MBq/mg) and 38% for Me-4-In (A ) 29.6 MBq/mg). Preparation of Anti-CEA mAb-Radioactive Porphyrin Conjugates. A commercially available antibody directed against carcinoembryonic antigen (anti-CEA mAb; CIS-Bioindustries Co., France) was used in the entire mAb form. Experiments were run at this company. [111In/115In]Porphyrins Me-1-In, Me-2-In, and Me-5In. All conjugations were carried out in water at pH 6.5-7 and room temperature for 12 h (Me-1-In and Me2-In) and 2 h (Me-5-In). Radioactive indium porphyrin derivative (19.5 × 10-2 µmol, 103.6 MBq) was dissolved in water (1 mL), and 2.6 × 10-2 µmol of mAb was added (stoichiometry of chelate-antibody ) 7.5). After incubation, the porphyrin-anti-CEA mAb conjugates were further isolated from the uncoupled porphyrins by cen-


trifugation in an Amicon Centriflo CF 50 membrane cone (molecular weight cutoff of 50 000). The labeling yields were 60% for Me-1-In, 75% for Me-2-In, and 35% for Me5-In, with specific activities of 14.8, 18.5, and 7.4 MBq/ mg, respectively. [111In/115In]Porphyrins 3-In and Me-4-In. For these two macrocycles, the same protocol as described previously for BSA (carbodiimide activation) was used. After incubation, the porphyrin-anti-CEA mAb conjugates were purified by centrifugation in a Centricon membrane cone and were counted for radioactivity. Labeling efficiencies were ∼100% for 3-In (A ) 25.9 MBq/mg) and 91% for Me-4-In (A ) 22.2 MBq/mg). HPLC Analysis of Radioactive Porphyrin-mAb Conjugates. The labeled conjugates were analyzed by HPLC using a Nucleosil C4 column (column 3; flow rate, 1.2 mL/ min) and detection at 420 nm. The elution medium was 0.1% trifluoroacetic acid (solvent A) and acetonitrile/ solvent A (60/40 by volume) (solvent B). A linear gradient of solvent B in solvent A was developed over 10 min. Whatever the conjugate was, retention times were the same for coupled and uncoupled mAbs (10 min 20 s), the nonbonded porphyrins being eluted after the mAb fractions. Labeling of Anti-ACE mAb without Isotopic Dilution: Derivatives Me-1-In, Me-2-In, and 3-In. For the insertion of 370 MBq indium-111 in these macrocycles (26 × 10-2 µmol), we followed the same experimental conditions as described before, without addition of 115InCl3, and obtained for each case a mixture of free base and indium derivative free from any carrier-free 111In. The yields of metallation determined by γ counting of 111In radioactivity in the extract were 75% for Me-1-In (A ) 1.28 GBq/ mg), 85% for Me-2-In (A ) 1.54 GBq/mg), and 76% for 3-In (A ) 1.54 GBq/mg). Conjugations with anti-CEA mAb were carried out in sodium hydrogen carbonate (0.5 M, pH 8.5) at room temperature for 12 h, using an input stoichiometry of chelate/antibody of 10/1 and an antibody concentration of 26 µM. We followed the same protocol as for isotopic dilution labeling. Taking into account the high radioactivity present, after incubation, the porphyrin (111In and free base derivative)-anti-CEA mAb conjugates were further isolated from the unreacted porphyrin derivatives on a 10 mL Sephadex G100 column presaturated with HSA. Antibody fractions, eluting in the void volume, were pooled and counted for radioactivity. Coupling efficiencies were ∼95% with Me-1-In (A ) 66.6 MBq/mg) and ∼100% with Me-2-In (A ) 81.4 MBq/mg) and 3-In (A ) 74 MBq/mg). Immunoreactivity. The immunoreactivity of the radiolabeled conjugates Me-2-In-mAb and 3-In-mAb was determined by passage over a CEA immunoadsorbent in the Laboratoire de Controˆle et Qualite´ of CISBioindustries Co. The percentage of activity in the absorbed fraction was related to the total activity recovered. Recoveries from the affinity columns were between 55 and 60% for the 3-In-anti-CEA mAb conjugate and between 40 and 45% for the Me-2-In-anti-CEA mAb conjugate. RESULTS AND DISCUSSION

Syntheses of Substituted Free Base Porphyrins. Taking into account the numerous functions present in a protein, various reactions can be used to covalently attach compounds. In particular, this attachment can be achieved with the amino acid residues of the lateral chains of proteins, e.g. amino or carboxy groups (lysine and arginine, glutamic and aspartic acid), the sulfhydryl group of cysteine, the hydroxy group of serine and


tyrosine, the imidazole group of histidine, and the various functional groups of the aromatic amino acid residues. The purpose of this investigation was to synthesize a series of differently monosubstituted arylporphyrins possessing at their periphery three pyridinyl groups, whose quaternization gives hydrosoluble molecules, and only one functional phenyl group, to avoid possible ulterior reticulation during the course of coupling reactions (Figure 1). By analogy with Sanger’s reagent (29), which allows the quantitative arylation of N-terminal amines as well as -amine groups of lysine, imidazole, histidine, alcohol, and thiol functions of serine, tyrosine, and cysteine residues, fluorine was introduced in the para position of the phenyl group (Figure 1, 1 and 2). Micheel et al. (24) have shown that the mesomeric effect of the nitro group, in 3-nitro-4-fluorobenzaldehyde, is sufficient to initiate fluoride lability; we synthesized these porphyrins to compare the influence of the porphyrinic macrocycle on the C-F bond with and without the presence of the NO2 substituent. To react with the carbohydrate moieties, located outside the antigen binding domain of the antibody molecule and carboxy groups of the Fc region, the free base 3 was synthesized. To maintain a sufficient nucleophilic character of the peripheral nitrogen, four sp3 carbons were introduced. Since the coupling of a carboxylate to an amine site on a protein is a common strategy, a carboxylic substituent was introduced. A spacer was used to move it away from the macrocycle (porphyrin 4) since, when it is directly bonded to the phenyl ring, Bedel-Cloutour et al. (23) have shown that reactivity of COOH is decreased and synthesis of the corresponding acid chloride is incomplete. Finally, porphyrin 5, derived from the latter, was synthesized; 4-(methylthio)phenol ester presents a great interest (28). In neutral medium, it acts as a nonactivated ester; methylation of the thioether activates the ester, leading to a good leaving benzoate group. The reaction scheme to produce new cationic periphery porphyrins was based on literature precedents (30-33). The free bases corresponding to the general formula meso-(pyridinyl)3(Σ-phenyl)PH2 were prepared by condensation of pyrrole with a mixture of 4-pyridinecarboxaldehyde and the corresponding substituted benzaldehyde by a modification of the mixed-aldehyde method described by Little et al. (30). Porphyrins 1 and 2 were obtained by direct synthesis with their corresponding aldehyde: either the commercially available 4-fluorobenzaldehyde or its synthetic nitrated derivative (24) (Figure 2). The synthetic routes used to obtain free bases 3-5 are shown in Figure 3 for their precursors and in Figure 4. The initial step, conversion of the commercially available 4-hydroxybenzaldehyde to the corresponding N-(4-butyl)phthalimido derivative (34, 35), provides a precursor for condensation with pyrrole and 4-pyridinecarboxaldehyde to produce after isolation and purification meso-10-[4-(4-phthalimidobutoxy)phenyl]-5,15,20tri(4′-N-pyridinyl)porphyrin (6). The phthalimido group was removed by hydrolysis following the typical method using hydrazine (36), leading to the aminoporphyrin 3. In the case of porphyrin 4, 4-hydroxybenzaldehyde was converted into its acetoxyl derivative, which was subsequently esterified with ethanol in the presence of N,N′-carbonyldiimidazole. The condensation of ethyl (4-formylphenoxy)acetate along with 4-pyridinecarboxaldehyde and pyrrole gave, after purification, the



Figure 2. Chemistry for the synthesis of macrocycles 1 and 2.

Figure 3. Chemistry for the synthesis of macrocycles 6 and 7.

esterified porphyrin 7. Hydrolysis of the ester with alcoholic sodium hydroxide gave the acid 4 (27). Finally, the synthesis of the activated ester 5 was carried out by esterification of porphyrin 4 with 4(methylthio)phenol in the presence of dicyclohexylcarbodiimide (28). All free base porphyrins were characterized by UVvisible spectroscopy, mass spectrometry, and 1H NMR spectroscopy. Porphyrins 1-7 gave UV-visible spectra typical of meso-tetraaryl-substituted porphyrins (37), i.e. a strong

B band (around 418 nm) and four Q bands (510-650 nm), the intensities of which decreased with increasing wavelengths. The spectra reported in Materials and Methods all fit this general pattern. Mass spectra of compounds 1, 2, 4, 6, and 7 were measured by positive FAB techniques where M + 1 peaks appeared as parent ions. The 1H NMR spectra of free bases 1-7 were straightforward and were readily assigned on the basis of chemical shift correlations and integration measurements. Proton chemical shifts are reported in Table 1. Our results were in agreement with literature data



Figure 4. Chemistry for the synthesis of macrocycles 3-5.

reported previously for meso-5-phenyl-10,15,20-tri(4′-Npyridinyl)porphyrin (38). The introduction of asymmetry into the molecule leads to an inequivalence of pyrrolic protons; except with compound 4, a splitting pattern is observed: one singlet corresponding to the four protons H2, H3, H17, and H18 and two asymmetrical doublets, each corresponding to two protons, H7 and H8 being equivalent pairwise to H12 and H13, respectively. They have their proton resonances split into an AB quartet which is reduced almost to first order at 250 MHz (J ∼ 5 Hz). Pyridinyl ring signals were found to be independent of the substituent present on the molecule; they appear at ∼9.1 and ∼8.2 ppm (protons H3′ and H5′ and H2′ and H6′, respectively). For the compounds monosubstituted on the phenyl ring, the meta and ortho protons appear as doublets at ∼8.1 and 7.3 ppm. In compound 1, the meta proton adjacent to the fluorine appears as a doublet of doublets (JHH ) 8.5 Hz, JHF ) 10.4 Hz), whereas the ortho proton H6′ appears as an octet at 8.5 ppm and the ortho proton H2′ as a multiplet at ∼8.9 ppm. All the NH resonances appear in the range of -2.9 ppm, except for compound 4 since the NH signal was not visible in CD3COOD. For compounds 3-7, integrations of the aromatic regions relative to the phenyl substituent resonances are diagnostic of the purity of these derivatives. Quaternization and Metallation of Substituted

Free Base Porphyrins. The corresponding triiodine salts of cationic porphyrins 1, 2, and 4, e.g. Me-1, Me-2, and Me-4, were prepared using a variation of the method reported in the literature (Figure 5) (39). The trichloride salts were synthesized by passing the cationic porphyrins through an ion exchange column. They were characterized by UV-visible spectroscopy, mass spectrometry, and infrared spectroscopy (see Materials and Methods); proton chemical shifts of cationic porphyrins at ambient temperature are listed in Table 2. The porphyrin ring protons appear at ca. 9.1 ppm as a broad singlet; the monomer-dimer equilibrium could explain the broadening of this NMR signal. Chemical shifts of pyridyl protons were in accordance with data obtained previously for unsubstituted meso-5-phenyl-10,15,20-tris(4′-N-methylpyridinyl)porphyrin (33). Quaternization of pyridyl rings does not modify chemical shifts of phenyl protons. Porphyrin 3 was not quaternized since it is impossible to convert it into its trichloride salt without quaternizing the primary amine; the basicities of these various amine functions differ. The pKa values of the primary amine vary from 10 to 12, whereas it is equal to 5.2 in the case of pyridine (40). In the case of porphyrin 5, the quaternization was conducted after metallation of the molecule, since the thioether function is also methylated to give the activated ester which must be used immediately for further coupling. For metallation with indium, we applied the method



Figure 5. General scheme for the quaternization of free base porphyrins.

Figure 6. Metallation of the quaternized free base porphyrins.

we have already used for meso-substituted phenyl porphyrins (22), by refluxing a slight excess of metal salt, a mixture of 111InCl3 and 115InCl3, with the porphyrin base in glacial acetic acid (Figure 6). To improve the solubilization of the free base, 3.3% trifluoroacetic acid was added. Hydrosoluble indium derivatives of macrocycles Me1, Me-2, and Me-4, Me-1-In, Me-2-In, and Me-4-In, respectively, were purified on anion exchange resin; the corresponding indium complexes of porphyrins 3 and 5, e.g. 3-In and 5-In, were purified by chloroformic extraction, a method we perfected earlier (21), suitable for further in vivo use. The yields of metallation, determined by γ counting of 111In radioactivity of the different pure derivatives, were between 75 and 90% except for that for compound 5-In (53%); this lower yield confirms the instability of the ester in acidic medium. These radioactive derivatives were obtained with a specific activity of ∼555 MBq/mg, smaller than the one observed with 5-(p-carboxyphenyl)-10,15,20-tritolylporphyrin 111indium(III) chloride (23) since, in this case, we used an isotopic dilution of indium-115 and -111. The electronic spectra of metalloporphyrins consist of an intense band near 420 nm (Soret band) and two bands between 500 and 600 nm, R and β bands (37); on metallation of our macrocycles, the four-banded etio-type visible spectra lead to the normal visible spectra for a metalloporphyrin (see Materials and Methods). The absorption bands observed appear at about the same wavelengths irrespective of the macrocycle. Indium Porphyrin-Bovine Serum Albumin Conjugates. Coupling with BSA served as a model for the optimization of the experimental conditions and was conducted with macrocycles Me-1-In, Me-2-In, 3-In, Me4-In, and Me-5-In. Just prior to BSA coupling, 5-In was quaternized with CH3I and purified by passing it through an anion exchange column in the Cl- form; Me-5-In was obtained in a 74% yield with a specific activity of 481 MBq/mg.

A radioactive porphyrin/BSA molar ratio of 10 was used, and all reactions were conducted in the dark to prevent porphyrin photooxidation. Labeling of BSA with Me-1-In, Me-2-In, and Me-5In derivatives was carried out in water, within 1.5 h, and conjugates were further purified by Sephadex G75 size exclusion chromatography, to give preparations in which 95 and 97% of the radiolabel, Me-1-In and Me-2-In, respectively, was bound to BSA. In the case of the activated ester Me-5-In, the coupling yield was lower (67%). For porphyrin Me-4-In, we followed the method described by Lavallee et al. (41) involving formation of the activated carboxylate by reaction with N-hydroxysuccinimide (NHS) in the presence of 1-ethyl-3-[3′-(dimethylamino)propyl]carbodiimide hydrochloride (EDAC), followed by conjugation with BSA ([porphyrin]/[BSA] ) 10). The conjugate Me-4-In-BSA was purified, as previously described, by chromatography of the conjugation mixture on a Sephadex G75 column; 38% of the conjugate was recovered with a specific activity of 29.6 MBq/mg. An identical protocol was followed to synthesize the conjugate 3-In-BSA in an 89% yield. Indium Porphyrin-mAb Conjugates. One area of oncology where radiolabeled monoclonal antibodies are most likely to play an important clinical role is in the evaluation of patients with colorectal carcinomas (4). While carcinoembryonic antigen (CEA) is not specific to tumors, high concentrations of this antigen occur in a variety of tumors, particularly in colorectal cancer (42), and for this reason, it has been used as a target in conjunction with a labelled anti-CEA mAb (43-46). Taking into account the previous results, we transposed these reactions to label the anticarcinoembryonic antigen monoclonal antibody (anti-CEA mAb). For all derivatives, we followed an experimental protocol identical to that for BSA, using an input stoichiometry of chelate/antibody of 7.5/1; the first assays were performed with the derivatives metallated with an


isotopic dilution of indium trichloride. Under these conditions, excellent labeling yields were obtained for derivatives 3-In and Me-4-In (∼100 and 91%, respectively). In particular, the amino derivative 3-In, which gave only 38% of the conjugate after 2 h of contact with BSA, required a longer contact time to react; labeling was nearly quantitative. In contrast, with fluoro derivatives Me-1-In and Me-2-In, a lower labeling efficiency was obtained (60%, 75%). The presence of a nitro substituent did not improve the coupling yield; on the contrary, the influence of its steric hindrance was predominant. With the activated ester Me-5-In, as for BSA, labeling efficiency was low. Specific activities were between 7.4 and 25.9 MBq. Covalent coupling was verified by analytical reversedphase HPLC. Under the conditions used (see Materials and Methods), no uncoupled radioactive porphyrin was detected whatever the conjugate. It is noteworthy that porphyrin-mAb conjugates in aqueous solution, kept at room temperature and daylight, were stable; after 48 h, the integrity of the chelates was maintained. In order to improve the specific activities of the conjugates, we performed the metallation of macrocycles Me-1, Me-2, and Me-3 in the presence of a higher radioactivity (370 MBq), without isotopic dilution; after coupling, conjugates were purified on a Sephadex G100 column to eliminate unreacted indium-111-porphyrins. Using these conditions, the coupling efficiency was between 95 and 100%. The number of porphyrin residues per mAb molecule was given by the product of the fraction of counts associated with mAb (mAb-bound radioactivity) (0.95-1) with the total porphyrin/mAb ratio used in the conjugation reaction (10/1); we obtained radioimmunoconjugates with an average number of indium porphyrin groups attached to each mAb molecule of 9.5-10 and an average specific activity of 73 MBq/ mg. Despite the great number of indium porphyrins covalently coupled to the monoclonal antibody, these conjugates displayed a relatively good retention of antibody immunoreactivity. This preliminary result needs more investigations which will be carried out in the near future. CONCLUSION

The syntheses and characterizations of new functionalized radioactive indium porphyrins, which hold metal with extreme stability, under physiological conditions, are an important step in the field of labeling. The experiments described above demonstrate the feasibility of coupling these radioactive derivatives, with high yield, to a monoclonal antibody in view of tumor detection. Indium porphyrin-labeled anti-CEA mAbs scanning could significantly impact patient management as a complementary tool to preoperative evaluation of a patient with colorectal cancer. In vivo delivery studies of these conjugates are currently under way. ACKNOWLEDGMENT

The financial support of CIS-Bioindustries and the Etablissement Public Re´gional d’Aquitaine (Poˆle Ge´nie Biologique et Me´dical) is gratefully acknowledged. We thank Paul O’Brart, who accepted the task of improving the English of the text. LITERATURE CITED (1) Meares, C. F. (1986) Chelating Agents for the Binding of Metal Ions to Antibodies. Nucl. Med. Biol. 13, 311-318.

Bedel-Cloutour et al. (2) Hnatowich, D. J. (1990) Antibody Radiolabelling, Problems and Promises. Nucl. Med. Biol. 17, 49-55. (3) Liu, Y., and Wu, C. (1991) Radiolabelling of Monoclonal Antibodies with Metal Chelates. Pure Appl. Chem. 63, 427463. (4) Kramer, E. L., and Larson, S. M. (1991) Tumor Targeting with Radiolabeled Antibody for Diagnosis and Therapy. Immunol. Allergy Clin. North Am. 11, 301-339. (5) Baranowska-Kortylewicz, J., Dalrympe, G. V., Quadri, S. M., and Harrison, K. A. (1995) Radiolabeling of Antibodies for Therapy and Diagnosis. In Methods in Molecular Biology, 51: Antibody Engineering Protocols (S. Paul, Ed.) pp 423439, Humana Press Inc., Totowa, NJ. (6) Kozak, R. W., Waldmann, T. A., Atcher, R. W., and Gansow, O. A. (1986) Radionuclide-conjugated Monoclonal Antibodies: a Synthesis of Immunology Inorganic Chemistry and Nuclear Science. Trends Biotechnol. 4, 259-264. (7) Fairweather, D. S., Bradwell, A. R., Dykes, P. W., Vaughan, A. T., Watson-James, S. F., and Chandler, S. (1983) Improved Tumor Localisation Using Indium-111 Labelled Antibodies. Br. Med. J. 287, 167-170. (8) Meares, C. F., and Goodwin, D. A. (1984) Linking Radiometals to Proteins with Bifunctional Chelating Agents. J. Protein Chem. 3, 215-228. (9) Wang, T. S. T., Srivastava, S. C., Fawwaz, R. A., Giacomini, P., Ferrone, S., Richards, P., Hardy, M. A., and Alderson, P. O. (1984) A Comparison of the Cyclic Anhydride and Mixed Anhydride Methods for 111In-DTPA Chelation to Monoclonal Antibodies. NuklearMedizin 4, 193-195. (10) Fawwaz, R. A., Wang, T. S. T., Estabrook, A., Rosen, J. M., Hardy, M., Alderson, P. O., Srivastava, S. C., Richards, P., and Ferrone, S. (1985) Immunoreactivity and Biodistribution of Indium-111-Labeled Monoclonal Antibody to a Human High Molecular Weight-Melanoma Associated Antigen. J. Nucl. Med. 26, 488-492. (11) Margerum, D. W., Cayley, G. R., Weatherburn, D. C., and Pagenkopf, G. K. (1978) Kinetics and Mechanisms of Complex Formation and Ligand Exchange. ACS Monogr. 174, 1-220. (12) Cole, W. C., De Nardo, S. J., Meares, C. F., McCall, M. J., De Nardo, G. L., Epstein, A. L., O’Brien, H. A., and Moi, M. K. (1987) Comparative Serum Stability of Radiochelates for Antibody Radiopharmaceuticals. J. Nucl. Med. 28, 83-90. (13) Brechbiel, M. W., and Gansow, O. A. (1991) BackboneSubstituted DTPA Ligands for 90Y Radioimmunotherapy. Bioconjugate Chem. 2, 187-194. (14) Kozak, R. W., Raubitschek, A., Mirzadeh, S., Brechbiel, M. W., Junghaus, R., Gansow, O. A., and Waldmann, T. A. (1989) Nature of the Bifunctional Chelating Agent Used for Radioimmunotherapy with Yttrium-90 Monoclonal Antibodies: Critical Factors in Determining in vivo Survival and Organ. Cancer Res. 49, 2639-2644. (15) Cummins, C. H., Rutter, E. W., Jr., and Fordyce, W. A. (1991) A Convenient Synthesis of Bifunctional Chelating Agents Based on Diethylenetriaminepentaacetic Acid and their Coordination Chemistry with Yttrium(III). Bioconjugate Chem. 2, 180-186. (16) Brechbiel, M. W., Gansow, O. A., Atcher, R. W., Schlom, J., Esteban, J., Simpson, D., and Colcher, D. (1986) Synthesis of 1-(p-Isothiocyanatobenzyl) Derivatives of DTPA and EDTA. Antibody Labeling and Tumor Imaging Studies. Inorg. Chem. 25, 2772-2781. (17) Moi, M. K., Meares, C. F., and De Nardo, S. J. (1988) The Peptide Way of Macrocyclic Bifunctional Chelating Agents. Synthesis of 2-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic Acid and Study of Its Yttrium(III) Complex. J. Am. Chem. Soc. 110, 6266-6267. (18) Cox, J. P. L., Graig, A. S., Helps, L. M., Jandowski, K. J., Parker, D., Eaton, M. A. W., Millican, A. T., Millar, K., Beeley, N. R. A., and Boyce, B. A. (1990) Synthesis of C- and N-Functionalised Derivatives of 1,4,7-Triazacyclononane1,4,7-triylacetic Acid (NOTA), 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayltetraacetic Acid (DOTA), and Diethylenetriaminepentaacetic Acid (DTPA): Bifunctional Complexing Agents for the Derivatisation of Antibodies. J. Chem. Soc., Perkin Trans. 1, 2567-2576. (19) Meares, C. F., Moi, M. K., Diril, H., Kubis, D. L., McCall, M. J., Deshpande, S. V., De Nardo, S. J., Snook, D., and


Radioactive Indium Porphyrins and Antibody Labeling Epenetos, A. A. (1990) Macrocyclic Chelates of Radiometals for Diagnosis and Therapy. Br. J. Cancer, Suppl. 10, 2126. (20) Buchler, J. W. (1975) Static Coordination Chemistry of Metalloporphyrins. In Porphyrins and Metalloporphyrins (K. M. Smith, Ed.) pp 157-231, Elsevier Scientific, Amsterdam. (21) Cloutour, C., Ducassou, D., Pommier, J. C., and Vuillemin, L. (1982) Chloro Indium-111 Tetraphenylporphyrin and its Possible Clinical Application. Int. J. Radiat. Isot. 33, 13111318. (22) Bedel-Cloutour, C. H., and Gacherieu, C. (1987) Functionalization of Tetrapyrrolic Macrocycles: Free Bases and Indium Derivatives, Models for Protein Labeling. Main Group Met. Chem. 10, 109-128. (23) Bedel-Cloutour, C. H., Maneta-Peyret, L., Pereyre, M., and Bezian, J. H. (1991) Synthesis of a Monoclonal AntibodyIndium-111-Porphyrin Conjugate. J. Immunol. Methods 144, 35-41. (24) Micheel, F., and Noffz, D. (1957) Reactions of 3-Nitro-4fluorobenzaldehyde with Amino Acids. Chem. Ber. 90, 15861589. (25) Cavallini, G., and Ravenna, F. (1951) Thiosemicarbazones. Farmaco (1946-1952) 6, 145-149. (26) Elkan, T. (1886) On the Isomers of Aldehydophenoxyacetic Acid. Beritche XIX, 3041-3054. (27) Molinaro, F. S., Little, R. G., and Ibers, J. A. (1977) Oxygen Binding to a Model for the Active Site in Cobalt-Substituted Hemoglobin. J. Am. Chem. Soc. 99, 5628-5632. (28) Kouge, K., Koizumi, T., Okaı¨, H., and Kato, T. (1987) Peptide Synthesis in Aqueous Solution. I. Application of p-Dialkyl-sulfoniophenols as a Water-Soluble Coupling Reagent. Bull. Chem. Soc. Jpn. 60, 2409-2418. (29) Sanger, F. (1945) Free Amino Groups of Insulin. Biochem. J. 39, 507-515. (30) Little, R. G., Anton, J. A., Loach, P. A., and Ibers, J. A. (1975) The Synthesis of Some Substituted Tetraarylporphyrins. J. Heterocycl. Chem. 12, 343-349. (31) Little, R. G. (1981) The Mixed-Aldehyde Synthesis of Difunctional Tetraarylporphyrins. J. Heterocycl. Chem. 18, 129-133. (32) Williams, G. N., Williams, R. F. X., Lewis, A., and Hambright, P. (1979) Synthesis, Characterization and Copper Incorporation into 5-(4-pyridyl)-10,15,20-triphenylporphyrin. J. Inorg. Nucl. Chem. 41, 41-44. (33) Sari, M. A., Battioni, J. P., Dupre, D., Mansuy, D., and Le Pecq, J. B. (1990) Interaction of Cationic Porphyrins with DNA: Importance of the Number and Position of the Charges and Minimum Structural Requirements for Intercalation. Biochemistry 29, 4205-4215. (34) Kojima, S., Onishi, A., Tanaka, H., and Itakura, J. (1977) p-(Alkoxyalkoxy)-benzylidene-p′-cyanoanilines. Jpn. Kokai Tokkyo Koho 7787, 139.

(35) Ito, H., Nitta, A., Tanaka, T., Kamio, H., and Tsuboi, K. (1985) Halo-substituted Aromatic, Alicyclic, and Heterocyclic Ethers. Jpn. Kokai Tokkyo Koho 60161, 942. (36) March, J. (1992) in Advanced Organic Chemistry. Reactions, Mechanisms, and Structure, 4th ed., pp 293-500, Wiley-Interscience, New York. (37) Gouterman, M. (1978) Optical Spectra and Electronic Structure. In The Porphyrins (D. Dolphin, Ed.) Vol. III, pp 1-165, Academic Press, New York. (38) Shamin, A., Worthington, P., and Hambright, P. (1981) Synthesis and Characterization of Phenyl/4-pyridyl meso Substituted Porphyrins. J. Chem. Soc. Pak. 3, 1-3. (39) Hambright, P., and Fleischer, E. B. (1970) The Acid-Base Equilibria, Kinetics of Cu-Ion Incorporation and AcidCatalyzed Zinc-Ion Displacement from the Water-Soluble Porphyrin R,β,γ,δ-Tetra(4-N-methylpyridyl)porphine. Inorg. Chem. 9, 1757-1761. (40) Gordon, A. J., and Ford, R. A. (1972) in The Chemist’s Companion. A Handbook of Practical Data, Techniques, and References, pp 1-80, Wiley-Interscience, New York. (41) Roberts, J. C., Figard, S. D., Mercer-Smith, J. A., Svitra, Z. V., Anderson, W. L., and Lavallee, D. K. (1987) Preparation and Characterization of Copper-67 Porphyrin-antibody Conjugates. J. Immunol. Methods 105, 153-164. (42) Gold, P., and Freedman, S. O. (1965) Specific Carcinoembryonic Antigens of the Human Digestive System. J. Exp. Med. 122, 461-485. (43) Goldenberg, D. M., Wlodkowski, T. J., Sharkey, R. M., Silberstein, E. B., Serafini, A. N., Garty, I. I., Vanheertum, R. L., Higginbothamford, E. A., Kotler, J. A., Balasubramanian, N., Swayne, L. C., Hansen, H. J., and Pinsky, C. M. (1993) Colorectal Cancer Imaging With Iodine-123-Labelled CEA Monoclonal Antibody Fragments. J. Nucl. Med. 34, 6170. (44) Webster, W. B., Harwood, S. J., Carroll, R. G., and Morrissey, M. A. (1992) Pharmacokinetics of Indium-111Labelled B72.3 Monoclonal Antibody in Colorectal Cancer Patients. J. Nucl. Med. 233, 498-504. (45) Halpern, S. E., Dillman, R. O., Amox, D., Hagan, P. L., Burks, R., Dillman, J., Perdikakis, B., Merchant, B., Frincke, J., and Schweighardt, S. (1992) Detection of Occult Tumor Using Indium-111-Labeled Anticarcinoembryonic Antigen Antibodies. Arch. Surg. (Chicago) 127, 1094-1100. (46) Curtet, C., Vuillez, J. P., Daniel, G., Aillet, G., Chetanneau, A., Visset, J., Kremer, M., Thedrez, P., and Chatal, J. F. (1990) Feasibility Study of Radioimmunoguided Surgery of Colorectal Carcinomas Using Indium-111-CEA-Specific Monoclonal Antibody. Eur. J. Nucl. Med. 17, 299-304.

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