[99mTc]Technetium labelled PnAo-azomycin glucuronides: a novel class of imaging markers of tissue hypoxia

Applied Radiation and Isotopes 57 (2002) 719–728

[99mTc]Technetium labelled PnAo-azomycin glucuronides: a novel class of imaging markers of tissue hypoxia Piyush Kumara, Leonard I. Wiebea,*, Rezaul H. Mannana,1, Zaihui Zhanga,2, Haiyan Xiaa, Alexander J.B. McEwanb a

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 b Cross Cancer Institute, Edmonton, Alberta, Canada T6G 2N8 Received 1 August 2001; received in revised form 8 October 2001; accepted 12 June 2002 This work is dedicated to the memory of our colleague and friend Dr. Richard M. Lambrecht

Abstract Azomycin glucuronate was coupled to a PnAO ligand to create azomycin-based ligands that would form watersoluble 99mTc-azomycin complexes for imaging hypoxic tissue. 1-b-D-(2-Nitroimidazolyl)glucuronic acid, 1, was synthesized by coupling 2-nitroimidazole with 1-a-bromo-2,3,4-tri-O-acetyl-6-methyl glucuronate, followed by deprotection. Reaction of 1 with 6-methyl-6-methylamino-HMPnAO (Pn-44) in the presence of BOP reagent in anhydrous dimethyl sulfoxide afforded the PnAO-glucuronides 5 and 6. Compound 5 was isolated in three rotomeric forms. Biological evaluation of 7 (99mTc-5) indicated selective binding to hypoxic EMT-6 cells, and cytotoxicity to fibroblasts and HeLa, sk24, sk23, and g361 cancer cell lines, at an IC20 o2.5 mg/ml. In vivo biodistribution of two formulations of 7 in Balb/c mice with EMT-6 tumor produced diverse results, with one formulation showing no tumor preference, and the other providing a tumor/blood ratio of 2.3 at 4 h post-injection. The latter formulation delineated tumor, large intestine and liver in scintigraphic images. r 2002 Elsevier Science Ltd. All rights reserved.

1. Introduction Decreased tissue oxygen tension (hypoxia) in tumor cells is thought to result in increased resistance of solid tumors to radiotherapy (Tomlinson and Gray, 1955). The design of hypoxic tumor tissue radiosensitizers (Adams et al., 1976; Brown and Workman, 1980) and the development of predictive imaging markers of hypoxia (Chapman et al, 1981), including radiolabelled misonidazole (Miso) and 123I-labeled iodoazomycin *Corresponding author. 3118 Dentistry/Pharmacy Center, University of Alberta, Edmonton, Alberta, Canada T6G 2N8.. E-mail address: [email protected] (L.I. Wiebe). 1 Present affiliation: Bureau of Biologics & Radiopharmaceuticals, Health Canada, Ottawa, Canada. 2 Present affiliation: Kinetek Pharmaceuticals, Inc., Vancouver, Canada.

arabinoside ([123I]IAZA) (Mannan et al., 1991), have focused primarily on the oxygen-sensitive, single electron reduction-activation of azomycins that form covalent adducts with tissue macromolecules in viable hypoxic cells (Biaglow et al., 1986). 1-(2-Hydroxy-3methoxy)propyl-2-nitroimidazole (Miso) was the first azomycin derivative used in clinical trials as a radiosensitizer for hypoxic cells (Urtasun et al., 1984) and when radiolabelled, as a predictive marker of hypoxia (Franko and Chapman, 1985; Urtasun et al, 1986a, b). 1-[a-D-(5-Iodo-arabinofuranosyl)]-2-nitroimidazole (IAZA), the first radioiodinated azomycin derivative for clinical SPECT imaging, has been used clinically to detect hypoxia in cancer (Parliament et al., 1991; Groshar et al., 1993; Urtasun et al., 1996), peripheral vascular disease of diabetes (Al-Arafaj et al., 1994), rheumatoid arthritis (McEwan et al., 1997) and blunt trauma of the brain (Vinjamuri et al., 1999). Despite the

0969-8043/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 4 3 ( 0 2 ) 0 0 1 8 8 - 4

P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 719–728

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demonstrated efficacy (Urtasun et al., 1996), clinical acceptance and commercial availability of 123I-radiopharmaceuticals remains low, in part because of expense and radiolabelling chemistry. N I NO 2 N

O HO

*

O NH HN

OH N

O

HO

NO 2

N N NO 2

MISO

N

IAZA

N N OH HO BMS-189781

A number of 99mTc-labelled, azomycin-based coordination complexes, including PNAO (Rumsey et al., 1994, 1995), BATO (Linder et al., 1993, 1995) and peptide derivatives (Zhang et al., 1999), have been prepared and tested as markers of tissue hypoxia. Of these, 99mTc-Propyleneamine oxa nitroimidazole (BRU59–21 [99mTc- BMS-189781]; Johnson et al., 2000), 99m Tc-propyleneamine nitroimidazole (BMS-181321; Linder et al., 1994) and 99mTc-HL-91 (Archer et al., 1995; Imahashi et al., 2000) have been among the most successful [99mTc]technetium agents developed and tested clinically. These compounds have not evolved as marketed radiopharmaceuticals, despite demonstrated preferential localization in hypoxic brain, myocardium and tumors, possibly because of their high lipophilicity, which contributes to their slow plasma clearance and high hepatic uptake. The synthesis, 99mTc radiolabelling, and preliminary biological evaluation of a novel class of moderately hydrophilic hexamethylpropyleneamine oxime coupled 2-nitroimidazole glucuronides (PnAO azomycin glucuronides 5 and 6) are now reported.

ppm downfield with respect to tetramethylsilane as an internal standard. Substitution patterns at C atoms were confirmed by 13C Jmod spin echo technique in which methyl and methine carbons are recorded as positive peaks and methylene and tertiary carbons as negative peaks. Cos.ser spectra, which present C–H shift correlation data in three dimensions (the 1H spectrum on the y-axis, the 13C spectrum on x-axis and C–H correlations as cross-peaks), were also recorded. The protons and carbons of the sugar moiety, ligand and nitroimidazole are represented by a single prime (0 ), a double prime (00 ) and no prime, respectively. When necessary, Fast atom bombardment (FAB) mass spectra were acquired, in lieu of elemental analysis, using a sodium probe on an AEIMS-12 mass spectrometer. Sodium-D-line (589 nm) optical rotations were determined at on a Perkin Elmer 241 polarimeter. Infrared spectra were recorded in KBr (pellet) on a Nicolet 1180/1280 FTIR instrument. UV spectra were recorded on a Philips PU 8700 series spectrophotometer. A gamma scintillation well counter (Beckman 8100) was used for radiometry of tissue and chromatographic samples. 99m Tc-Labelled complexes were analyzed on a Waters HPLC system (Waters 10 mm Radial-Pakt C-18 reverse phase cartridge), using a gradient eluting system with a flow rate of 2.0 ml/min: 0–17 min, 100% sodium acetate buffer (pH 5.6) to 100% THF; 17–20 min, 100% THF; 20–25 min, 100% THF to 100% buffer. Radioactivity of the eluent was measured on-line using an in-house design NaI (Tl) scintillation detector. Reaction mixtures were also analyzed chromatographically by ITLC-SG/ methyl ethyl ketone, ITLC-SG/saline, and Whatman No.1 paper/1:1 H2O:CH3CN, to determine the percentage of the labelled compound, pertechnetate, and reduced and hydrolyzed technetium species (RHT).

3. Chemistry 2. Materials and methods All chemicals used were of reagent grade. The solvents were dried over appropriate drying agents and freshly distilled before use. Dimethyl sulfoxide was purified by distillation under high vacuum and stored over molecular sieves. The progress of synthetic reactions was monitored by thin layer chromatography (TLC) using 250 mm Whatman MK6F silica gel micro TLC plates. Column chromatography was performed on Merck silica gel 60 (particle size 70–200 and 230–400 mesh ASTM). Melting points were determined on a Buchi capillary melting point apparatus and are uncorrected. 1 H and 13C NMR spectra were recorded on a Bruker AM-300 spectrometer in deuterated chloroform (CDCl3) and deuterium oxide (D2O), depending on the solubility of the product. Chemical shifts are reported in

The synthesis of 1-b-D-(2-nitroimidazolyl)-2,3,4-triO-acetyl-6-methyl glucuronate (3) proceeded by C-1 a-bromination of the protected methyl glucuronate 1 with HBr/AcOH (30% solution) at 221C (Scheme 1). Formation of the 1-a-bromoglucuronate was monitored by TLC. Excess HBr and acetic acid were removed after the reaction reached completion and the crude residue was dissolved in dichloromethane (150 ml) and washed with water (25 ml  2). The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated. 1-aBromo-2,3,4-tri-O-acetyl-6-methyl glucuronate 1a, so obtained, was redissolved in anhydrous acetonitrile (100 ml) and added to a mixture of mercuric cyanide (2.69 g, 10.6 mmol) and 1-trimethylsilyl-2-nitroimidazole 2a (14.5 mmol), which in turn was prepared by refluxing 2-nitroimidazole 2 (1.64 g, 14.5 mmol) with hexamethyldisilazane (HMDS) in the presence of 5% ammonium

P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 719–728 MeOOC

721

NO 2 O

OAc HN

OAc

N

Ac O

2 1

OAc

ii

i

NO 2

MeOOC O

Me3 Si N

OAc

N

iii Br Ac O

2a

NO 2 1a

MeOOC

OAc

N

O

N

OAc

Ac O 3

OAc iv NO 2

H OOC O

N

N

OH

HO OH 4

Scheme 1. Synthesis of 3 and 4. Reaction conditions: (i) HBr/AcOH; (ii) HMDS, reflux, 2 h; (iii) Hg(CN)2, 601C; 5 h (iv) 0.1 M. NaOH, 16 h, DOWEX 50 W  8 resin.

sulfate. The mixture was stirred under exclusion of moisture at 601C for 5 h, when 1a could no longer be detected by TLC in the reaction mixture. The solvent was removed under vacuum, and the crude residue taken in dichloromethane (125 ml) and filtered. The filtrate was washed with 30% potassium iodide (30 ml  2), dried over anhydrous sodium sulfate, filtered and evaporated to a viscous residue. The residue was subjected to silica gel column using gradients of toluene and ethyl acetate to yield 0.96 g (16%) of pure 3; Rf 0.27 (toluene:EtOAc, 6:4, v/v); mp 171–1731C; 1H NMR (CDCl3) d 1.94, 2.07 and 2.10 (three s, each for 3H of COCH3), 3.79 (s, 3H, COOCH3), 4.36 (d,J40 ;50 ¼ 9:0 Hz, 1H, H-50 ), 5.32–5.42 (merged dd, 2H, H-30 and H-40 ), 5.48 (d,J10 ;20 ¼ 9:0 Hz, 1H, H-20 ), 6.52 (d,J20 ;10 ¼ 9:0 Hz, 1H, H-10 ), 7.21 (d, J5;4 ¼ 1:5 Hz, 1H, H-4) and 7.45 (d, J4;5 ¼ 1:5 Hz, 1H, H-5) ppm; 13C NMR (CDCl3) d 20:10 and 20.40 (three COCH3), 53.16 (COOCH3), 68.94

(C-30 ), 70.68 (C-40 ), 72.09 (C-20 ), 74.91 (C-50 ), 82.75 (C10 ), 121.90 (C-4), 129.21 (C-5), 144.40 (C-2), 166.04 (COOCH3) and 168.89, 169.26, 169.59 (three COCH3) ppm; Anal. (C16H19N3O11) C, H, N. 1-b-D-(2-Nitroimidazolyl)glucuronic acid (4) was prepared by adding a cold aqueous solution of 0.1 M sodium hydroxide (6.02 mmol) to a pre-cooled solution of 3 (0.37 g, 0.86 mmol). This mixture was stirred at 51C (Scheme 1) with periodic monitoring by TLC. After 16 h, when no precursor was evident by TLC, the solution was de-ionized with DOWEX 50 W  8 resin to pH 5.0. The suspension was filtered and the filtrate was evaporated in vacuo to afford impure 4. Purification on a silica gel column, using gradients of chloroform/ methanol/water (60:40:5, v/v/v), afforded 0.247 g (99%) of pure 4; mp 2201C (dec.); 1H NMR (D2O) d 3.56 (d, J50 ;40 ¼ 9:0 Hz, of d; J30 ;40 ¼ 9:0 Hz, 1H, H-40 ), 3.72–3.60 (m, 2H, H-20 and H-30 ), 3.93 (d, J40 ;50 ¼ 9:0 Hz, 1H,

722

P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 719–728

H-50 ), 6.22 (d, J20 ;10 ¼ 8:5 Hz, 1H, H-10 ), 7.16 (d, J4;5 ¼ 1:0 Hz, 1H, H-5) and 7.72 (d, J5;4 ¼ 1:0 Hz, 1H, H-4) ppm; 13C NMR (D2O) d 72:24 (C-30 ), 74.13 (C-40 ), 76.40 (C-20 ), 79.31 (C-50 ), 85.46 (C-10 ), 124.17 (C-4), 129.12 (C-5), 146.13 (C-2) and 175.65 (COOH) ppm; Anal. (C9H11N3O8), C, H, N. The PnAO-azomycin glucuronides (5 and 6) were obtained by adding BOP reagent (0.89 g, 1.98 mmol) to a stirred solution of 4 (0.20 g, 0.79 mmol), Pn-44 (0.37 g, 1.18 mmol) and triethylamine (0.24 g, 2.29 mmol) in anhydrous dimethyl sulfoxide (2.5 ml) under complete exclusion of moisture (Scheme 2). TLC examination after 30 min showed no evidence of 4. The reaction was quenched by adding ice-water (15 ml). The cold mixture was filtered and the filtrate was lyophilized to yield a viscous mass that was purified on a flash silica gel column, using chloroform/ methanol/ water (80:20:1, v/v/v), to yield 0.26 g (59.3%) of 5 (a tertiary amide) and 0.18 g (40.7%) of 6 (a secondary amide). Compound 5 was isolated by HPLC in three rotameric forms (5a, 5b

and 5c) that differed in the chemical shifts for protons at methylene and CH3 located at C-600 (Scheme 2, specific carbon atoms are marked) and in the splitting pattern of protons at C-500 and C-700 . Small variations in the respective carbon chemical shifts for 5a–c were observed. PnAO-azomycin glucuronide (5a). Rf 0.12 (CHCl3:MeOH:H2O, 6:4:0.7 v/v); mp 159–1631C; 22 [a]D+8.371 (c ¼ 0:0083/ml); IR (KBr) 3446–3345 (N–H), 1664 (C ¼ O), 1546 cm1 (N ¼ O); UV (max) 323 nm; 1H NMR (D2O) d 0:85 (s,CH3 at C-600 ), 1.13–1.29 (four s; merged, four CH3 at C-300 and C-900 ), 1.69 (s, CH3 at C-200 ), 1.73 (s, CH3 at C-1000 ), 2.54 (m, CH2 at C-500 and C-700 ), 3.17 (d, Jgem ¼ 14:5 Hz, 1H, CH2 at C-600 ), 3.27 (d, Jgem ¼ 14:5 Hz, 1H, CH2 at C-600 ), 3.59–3.73 (m, 2H, H-20 and H-30 ), 3.79 (d, J30 ;40 ¼ 8:5 Hz, of d; J50 ;40 ¼ 9:0 Hz, 1H, H-40 ), 4.14 (d, J40 ;50 ¼ 9:0 Hz, 1H, H-50 ), 6.23 (d, J20 ;10 ¼ 8:5 Hz, 1H, H-10 ), 7.17 (d, J4;5 ¼ 0:9 Hz, 1H, H-5) and 7.69 (d, J5;4 ¼ 0:9 Hz, 1H, H-4) ppm; 13C NMR, (D2O) d 10:36 (CH3 at C-200 and

O

NO 2

NH 6 O NH 8

N

N

OH

NH 4 HO

N 11

1N

OH

HO

OH

6

NH 2 6 NH 8

NH 4 4 V

N 11

1N

OH

HO

OH 1

O

O

Pn-44

NO 2

N N

N

OH

4N HO 6

OH NH 8

H 2N

N 11 OH 5a-c

Scheme 2. Coupling products of Pn-44 and 4 to yield the secondary amide 6 and the rotationally-restricted tertiary amides 5a–c. Where, v=BOP reagent; Et3N/DMSO, 221C.

P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 719–728

C-1000 ), 20.42 (CH3 at C-600 ), 23.32, 23.43 and 23.78 (CH3 at C-300 and C-900 ), 38.03 (C-600 ), 44.38 (CH2 at C600 ), 49.27 (C-500 ), 49.47 (C-700 ), 60.69 (C-300 and C-900 ), 71.68 (C-30 ), 73.32 (C-40 ), 76.34 (C-20 ), 85.54 (C-10 ), 124.25 (C-4), 129.22 (C-5), 161.62 (C-200 and C-1000 ) and 171.45 (C ¼ O) ppm; +ve FAB (C24H42N8O9), M+ 37.9% (relative abundance). PnAO-azomycin glucuronide (5b). Rf 0.04 (CHCl3:MeOH:H2O, 6:4:0.7 v/v); mp 181–1851C; 22 [a]D +9.941 (c=0.0056/ml); IR (KBr) 3440–3350 (N-H), 1653 (C ¼ O) and 1545 (N ¼ O) cm1; UV (max) 323 nm; 1H NMR (D2O) d 0:94 (s, 3H, CH3 at C-600 ), 1.16–1.44 (merged s; 12H, four CH3 at C-300 and C-900 ), 1.74 (s, 3H, CH3 at C-200 ), 1.78 (s, 3H, CH3 at C1000 ), 2.65 (m, 4H, CH2 at C-500 and C-700 ), 3.25 (d, Jgem ¼ 15:0 Hz, 1H, CH2 at C-600 ), 3.31 (d, Jgem ¼ 15:0 Hz, 1H, CH2 at C-600 ), 3.63–3.78 (m, 2H, H-20 and H-30 ), 3.83 (d, J30 ;40 ¼ J50 ;40 ¼ 9:0 Hz, 1H, H-40 ), 4.20 (d, J40 ;50 ¼ 9:0 Hz, 1H, H-50 ), 6.25 (d, J20 ;10 ¼ 9:0 Hz, 1H, H-10 ), 7.20 (d, J4;5 ¼ 0:9 Hz, 1H, H-4) and 7.75 (d, J5;4 ¼ 0:9 Hz, 1H, H-5) ppm; 13C NMR (D2O) d 10:35 (CH3 at C-200 and C-1000 ), 20.34 (CH3 at C-600 ), 23.32, 23.43 and 23.78 (CH3 at C-300 and C-900 ), 38.03 (C-600 ), 44.30 (CH2 at C-600 ), 49.24 (C-500 ), 49.37 (C-700 ), 60.72 (C-300 ), 60.85 (C-900 ), 71.68 (C-30 ), 73.18 (C-40 ), 76.33 (C20 ), 78.24 (C-50 ), 85.53 (C-10 ), 124.26 (C-4), 129.19 (C-5), 145.20 (C-2), 161.32 (C-200 and C-1000 ) and 171.31 (C ¼ O) ppm. PnAO-azomycin glucuronide (5c). Rf 0.02 (CHCl3:MeOH:H2O, 8:2:0.1  3, v/v); viscous solid; IR (KBr) 3445–3341 (N–H), 1665 (C ¼ O) and 1546 (N ¼ O) cm1; 1H NMR (D2O) d 0:95 (s, 3H, CH3 at C-600 ), 1.24 and 1.29 (two s; 12H of four CH3 at C-300 and C-900 ), 1.72 (s, 3H, CH3 at C-200 ), 1.75 (s, 3H, CH3 at C1000 ), 2.71 (m, 4H, CH2 at C-500 and C-700 ), 3.23 (d, Jgem ¼ 14:5 Hz, 1H, CH2 at C-600 ), 3.31 (d, Jgem ¼ 14:5 Hz, 1H, CH2 at C-600 ), 3.61–3.73 (m, 2H, H-20 and H-30 ), 3.81 (d, J30 ;40 ¼ 9:0 Hz, of d, J50 ;40 ¼ 9:0 Hz, 1H, H-40 ), 4.17 (d, J40 ;50 ¼ 9:0 Hz, 1H, H-50 ), 6.22 (d, J20 ;10 ¼ 8:5 Hz, 1H, H-10 ), 7.16 (s, 1H, H5) and 7.69 (s, 1H, H-4) ppm; 13C NMR (D2O) d 10:37 (CH3 at C-200 and C-1000 ), 20.58 (CH3 at C-600 ), 23.39, 23.51, 23.82 and 23.90 (four s; CH3 at C-300 and C-900 ), 37.94 (C-600 ), 44.42 (CH2 at C-600 ), 49.36 (C-500 ), 49.43 (C-700 ), 60.36 (C-300 ) 60.52 (C-900 ), 71.70 (C-30 ), 73.35 (C400 ), 76.34 (C-200 ), 78.27 (C-50 ), 85.56 (C-10 ), 124.28 (C-4), 129.17 (C-5), 161.40 (C-200 ), 161.40 (C-1000 ) and 171.33 (C ¼ O) ppm. PnAO-azomycin glucuronide (6). Rf 0.12 (CHCl3:MeOH:H2O, 8:2:0.1  3, v/v); semisolid; IR (KBr) 3306 (sec. N–H), 1653 (sec. amidic C ¼ O) and 1548 (N ¼ O) cm1; UV (max) 323 nm; 1H NMR (CDCl3) d 0:96 (s, 3H, CH3 at C-600 ), 1.23 (s, 3H, CH3 at C-300 ), 1.26 (s, 3H, CH3 at C-900 ), 1.66 (s, 3H, CH3 at C-200 ), 1.68 (s, 3H, CH3 at C-1000 ), 2.68 (broad d; 4H, CH2 at C-500 and C-700 ), 3.22 (d, Jgem ¼ 15:0 Hz, 1H, CH2

723

at C-600 ), 3.29 (d, Jgem ¼ 15:0 Hz, 1H, CH2 at C-600 ), 3.58–3.72 (m, 2H, H-20 and H-30 ), 3.75 (d, J30 ;40 ¼ 8:5 Hz, of d; J50 ;40 ¼ 9:0 Hz, 1H, H-40 ), 4.16 (d, J40 ;50 ¼ 9:0 Hz, 1H, H-50 ), 6.19 (d, J20 ;10 ¼ 8:5 Hz, 1H, H-10 ), 7.10 (d, J4;5 ¼ 0:9 Hz, 1H, H-5), 7.62 (d, J5;4 ¼ 0:9 Hz, 1H, H-4) ppm; 13C NMR (D2O) d 10:02 (CH3 at C-200 and C-1000 ), 18.80 (CH3 at C-600 ), 22.67, 22.93, 23.10 and 23.30 (four CH3 at C-300 and C-900 ), 38.32 (C-600 ), 42.86 (CH2 at C600 ), 48.26 (C-500 and C-700 ), 62.29 (C-300 and C-900 ), 71.62 (C-30 ), 73.18 (C-40 ), 76.24 (C-20 ), 77.60 (C-50 ), 85.31 (C-10 ), 111.94 (C-4), 118.04 (C-5), 145.04 (C-2), 159.00 (C-200 and C-1000 ), 172.31 (C ¼ O) ppm. 3.1. Radiolabelling PnAO-azomycin glucuronide (5) was labelled with Tc by the following procedure: The pH of a solution of 5 (1.0 mg in 4 ml of normal saline), in a sterile, nitrogen-purged vial, was adjusted to either 7.6 (formulation 1; product 7a) or 8.6 (formulation 2; product 7b) with 0.1 N NaOH. This solution was reconstituted with Na99mTcO4 (nominally 0.15 GBq, 0.1 ml), followed by the addition of 0.5 ml of saturated stannous tartrate solution to reduce the pertechnetate. The resulting solution was then incubated at 651C for 15 min prior to use. Radiochemical purity was determined by HPLC, ITLC and paper chromatography. The radiolabelling yield for 99mTc-7a was 95% (formulation 1; pH 7.6) and 40% for 99mTc-7b (formulation 2; pH 8.6). The retention times by HPLC for 7a and 7b were identical (10.7 min). 99m

4. Biological assays All experimental animal protocols were approved by the University of Alberta Health Services Laboratory Animal Service Research Ethics Committee, in compliance with the regulations of the Canadian Council on Animal Care. In vitro oxygen-dependent binding was determined using a literature procedure (Jette et al., 1986). Briefly, EMT-6 cells were incubated under air (oxic), 1% oxygen (moderately hypoxic) or nitrogen (hypoxic) at 371C for up to 150 min, in the presence of 99mTc-PnAO-azomycin glucuronide (7a; 55–58 mCi; 0.2 ml). Following incubation, cells were disrupted and lysed, and the TCAinsoluble fraction was recovered, washed and counted for radioactivity. Data (Fig. 1) were corrected for cell survival, as determined by MTT assay, at the end of incubation. In vitro cytotoxicity of 5 and 6 against fibroblasts, and sk23, sk24, HeLa and g361 tumor cell lines (Fig. 2) was determined using a modified MTT-microculture tetrazolium assay (Chapman et al., 1983). Briefly, exponentially growing cells were harvested, counted and plated in 96-well microtitre plates containing 100 ml of

P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 719–728

724

RPMI-FBS medium per well. After 24 h, the compound to be tested, pre-dissolved (100 ml; 1.09 mM/ml) in RPMI-1640 (pH 7.5–7.8), was added to each well. After 2 days of incubation, MTT (250 mg; 50 ml of a 5 mg/ml solution) was added to each plate and the plates were incubated for an additional 4 h at 371C. The supernatant was aspirated and DMSO (100 ml) was added to each well, and after shaking to dissolve the formazan (Orbit Shaker, Labline, 200 rpm), the plates were read at 540 nm on a DynaTech model MR 600 microplate reader. The absorbance was determined in the standard and growth curves of each cell line used in the assay. In all experiments, untreated cells were in exponential growth at the completion of 4-day incubation period. In vivo biodistribution studies were performed in Balb/c mice bearing EMT-6 tumors. Mice (20–25 g.) were inoculated subcutaneously in the left flank with a suspension of murine EMT-6 cells (1  106 cells/ml). After 10–12 days, when the tumors reached 8–10 mm in diameter, each mouse received an intravenous (i.v.) dose of the test compound (9–11 kBq; 25–30 mCi in 0.1 ml) via tail vein. Mice (3 in each group) were sacrificed at 2 min, 30 min, 2, 4 and 8 h after injection. A blood sample 1.4E+06

Nitrogen Air 1%Oxygen

1.0E+06 8.0E+05 6.0E+05 4.0E+05 2.0E+05 0.0E+00 0

30

60 Time (min)

90

120

Fig. 1.

5. Results and discussion 5.1. Chemistry The overall strategy of the project was to design a hydrophilic azomycin-99mTc-PnAO ligand complex. The synthesis proceeded via preparation of the azomycin glucuronide 4 and its subsequent coupling to Pn-44. 1,2,3,4-Tetra-O-acetyl-6-methyl glucuronate, 1, was brominated with 30% HBr in glacial acetic acid to give 1-a-bromo-2,3,4-tri-O-acetyl-6-methyl glucuronate (1a) (Chernyak et al., 1991). At the same time, 2-nitro-1-Ntrimethylsilyl imidazole (2a) was prepared by heating 2nitroimidazole under reflux in hexamethyldisilazane, with complete exclusion of moisture. Coupling 2a to 1a afforded 1-b-D-(2-nitroimidazolyl)-2,3,4-tri-O-acetyl6-methyl glucuronate (3) in 16% yield (Scheme 1). The

5

6

1000 310 300 IC20 (ug/mL; Log plot)

DPM/106cells

1.2E+06

(approx. 1 ml) was collected by cardiac puncture upon euthanization (asphyxiation in dry CO2). Tissues including lung, heart, liver, muscle, kidney, spleen, stomach with contents, small intestine with contents were removed by dissection, weighed wet and counted, together with the remaining carcass, for 99mTc radioactivity. Data are presented in Table 1. Whole-body scintigraphic imaging of Balb/c mice bearing EMT-6 tumors, at predetermined intervals after injection, was performed with a Searle Gamma camera (Pho/Gamma III) and ADAC (CAM III) computer using a pinhole collimator. Three mice were dosed (37–44 MBq; 1–1.2 mCi) by i.v. injection of 7a. The mice, under Ketamine/Xylazine anaesthesia, were imaged in the supine, position (100–900 kilo counts per image) every 5 min for 30 min, and then at 1, 2, 3, 4, 5, 6 and 24 h after injection.

90

74.9

100

36.4 11.3

11 10

7.3 2.5

2.5

1 Fb

Sk23

Sk24

Hela

g361

Cell Lines

Fig. 2. In vitro cytotoxicity determination of 5 and 6 using the MTT assay.

P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 719–728

725

Table 1 Biodistribution of 7a in Balb/c mice bearing EMT-6 tumors (Data are %ID/g7S.D.; n ¼ 3) Tissue

Blood Heart Lungs Liver Spleen Stomacha Small intestineb Large intestinec Kidney Muscled Skind Tumor Brain Bladdere Carcassf

Time after injection 0.03 h

0.5 h

2h

4h

8h

4.1070.90 1.5070.40 3.0070.80 28.473.40 2.4070.90 1.3070.90 7.8074.10 0.9070.40 9.7074.60 0.8070.20 1.6070.60 1.5070.50 0.2070.06 3.0071.30 1.4070.20

0.5070.10 0.2070.07 0.5070.10 13.474.80 1.3070.40 4.1072.20 49.777.20 0.4070.04 1.0070.20 0.1070.04 0.3070.08 0.4070.10 0.01 2.0071.40 0.4070.20

0.2070.06 0.0570.02 0.4070.10 8.1071.20 1.9070.20 1.9071.20 5.5071.90 75.5715.5 0.5070.05 0.3070.10 0.2070.10 0.5070.10 0.01 5.3074.20 0.2070.08

0.2070.06 0.0570.01 0.2070.04 10.373.90 2.0070.40 1.6070.70 2.6071.80 68.6717.9 0.8070.60 0.1070.01 0.1070.05 0.3070.10 o0.01 3.8070.60 0.1070.06

0.0770.02 0.004 0.0870.02 6.4071.70 1.6070.40 0.3070.10 0.3070.10 25.077.40 0.4070.08 0.02 o0.01 0.2070.10 o0.01 0.10 0.1070.08

a

Stomach with contents. Small intestine with contents. c Large intestine with contents. d A section of tissue. e Bladder with contents. f Carcass includes remaining skin, muscle and tail (injection site). b

1

H NMR spectrum of this compound displayed a doublet (J ¼ 9:0 Hz) at d 6:52 (H-10 ), confirming the b conformation at the C-10 of the coupled product 3. The 13 C NMR spectrum of 3 showed characteristic signals for the C-60 (carbonyl) carbon of the ester glucuronide, and the C-2 of the nitroimidazole group at d 166:04 and 144.40 ppm, respectively. Further structural confirmation for 3 came from the infrared absorption bands at 1745 and 1450 cm1, which are specific for the stretching vibration bands of the ester carbonyl function and the asymmetric N ¼ O stretching frequency of the nitro group, respectively. Alkaline hydrolysis of 3, followed by neutralization, afforded 1-b-D-(2-nitroimidazolyl)glucuronic acid (4), in 99% yield. A shift in the infrared absorption band, from 1745 in 3 to 1715 cm1 in 4, confirmed hydrolysis of the ester. In addition, the resonances appeared significantly downfield for H-50 (d 0:44 ppm), and C ¼ O (d 9:61 ppm) and C-50 (d 4:40 ppm) in the respective 1H and 13C NMR spectra of 4, in comparison to 3, due to the strong de-shielding nature of the C-50 carboxyl group. The absence of the protective groups’ carbon resonances in 4 also confirmed deprotection of hydroxyl groups. Attempts to couple Pn-44 to the ester 3 (or activated esters prepared by replacing the methyl group of 3 with an active group like p-nitrophenol), or coupling 4 and Pn-44, using dicyclohexylcarbodiimide (DCC) as a coupling reagent, were futile. Coupling reactions between Pn-44 and amino acids have been observed to

proceed poorly in dimethylformamide (DMF), where the addition of benzotriazolyl-N-oxy-tris(dimethylamino)-phosphoniumhexafluoro phosphate (BOP reagent; Castro et al., 1975) and base led to the formation of a more lipophilic moiety and Pn-44 was not consumed at all (Archer, personal communication). In the current experiment the free acid, 4, was successfully coupled to Pn-44 in DMSO using BOP reagent in the presence of triethylamine under absolute anhydrous conditions (Scheme 2). The reaction was complete in 30 min. BOP-catalyzed coupling also led to the formation of secondary amide at the C-600 aminomethyl site and a tertiary amide at N-400 /N-800 . The coupling of 4 at a secondary amino site (N-400 /N-800 ) led to restricted rotation of the resulting glucuronide 5 around the tertiary amidic nitrogen. Three rotamers (5a, 5b and 5c) of this tertiary amide were isolated. Reaction of 4 with the primary amino group at C-600 of Pn-44, to form a secondary amidic nitrogen, did not introduce steric hindrance around this coupling site, and afforded a single secondary amide 6. The combined yield for all rotameric forms of 5 (5a, 5b and 5c) was 60%, while 6 was obtained in 40% yield. 1 H and 13C assignments of these coupled products are based on the Cos.ser experiments that showed a spatial correlation between the C-600 and the proton(s) hybridized to it. The formation of amides changes both 1H and 13C chemical shifts of the glucuronide moiety (H-50 , C-50 , C ¼ O) and the methylene carbons (C-300 , C-500 ,

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P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 719–728

C-700 , C-900 ) of Pn-44, as anticipated. It was found that H-50 , in all rotameric forms of the tertiary amide (5a, 5b and 5c), moved upfield by 0.60–0.66 ppm, C-50 by more than 1.0 ppm, and C ¼ O by nearly 4.3 ppm (in comparison to 4). The 13C chemical shift for the carbonyl group in secondary amides appears downfield in comparison to the tertiary amides (Silverstein et al., 1981). C-200 , C-300 , C-500 , C-700 , C-90 and C-10000 resonances were observed at downfield chemical shifts in tertiary amides 5a–c, in comparison to the secondary amide 6. Similar trends were observed in case of 6 where C ¼ O and C-50 appeared at d 172:31 and 77.60 ppm, respectively compared to d 171:33 and 78.27, respectively in 5a. In 6 the CH2NH2 carbon is shielded when 4 coupled with this amine at C-600 ; this methylene carbon appeared at a chemical shift of d 42:86 ppm (up by 3.87 ppm in comparison to Pn-44). In the case of 5a–c, where 4 formed an amidic bond at N-400 /N-800 , this methylene carbon was de-shielded in comparison to 6 and appeared at 44.30–44.42 ppm. In addition, the methyl group at C-600 appeared at d 20:32220:48 in 5a–c which is downfield (0.23–0.49 ppm) in comparison to Pn-44, while the same CH3 in 6 appeared upfield (at lower chemical shift, d 18:80) by 3.2 ppm in comparison to Pn-44. The infrared absorption band for the secondary amide, 6, appeared at a lower frequency (1653 cm1) in comparison to the stretching vibrations for the tertiary amides 5a–c (1664 cm1), reaffirming the tertiary nature of this amide. Also, sharp primary NH2 stretching vibration bands in 5a–c were observed at n ¼ 344623345 cm1. 1 H NMR study of 5a–c at elevated temperature (3481K) confirmed that these compounds are rotamers, since their proton spectra were virtually superimposible at this temperature. The protons for the C-500 , C-700 methylene groups, and the CH3 proton resonances at C300 , C-900 , became more compact at the higher temperature. A similar study with 6 did not change its proton spectrum pattern except that the aminomethyl proton resonances at C-600 changed from a broad doublet to a somewhat sharper doublet. The elevated-temperature 1 H spectrum of 6 was not superimposible on the spectra of 5a, 5b or 5c. The PnAO-azomycin glucuronide molecule (5a–c; Scheme 2) has three tetrahedral C-600 pendant coordinating arms, each of which can be involved in coordination with 99mTc. Indeed, radiolabelling studies with 99mTc confirmed the added complexity of the labelling products. One of the 99mTc-5 complexes (7a) demonstrated hypoxia-dependent binding to cellular macromolecules in vitro (Fig. 1). Hypoxic binding (8031 DPM/106 cells/ min under high purity N2) was substantially greater than binding under air (essentially zero), and was oxygen concentration sensitive, binding at 1296 DPM/106 cells/ min in 1% O2. The binding of 7a increased linearly with time under N2, while very little or no binding occurred

under oxic and 1% O2 conditions. The hypoxic binding was linear over the 0–150 min period of incubation. The cytotoxicity data presented in Fig. 2 indicate that 5 is cytotoxic to sk23 and g361 cancer cell lines (IC20 o2.5 mg/ml), but much less toxic to HeLa and sk24 cells and fibroblasts. In each case 6 was less cytotoxic than 5. The in vivo biodistribution of 7a in Balb/c mice bearing EMT-6 tumors was characterized by initial widespread biodistribution of radioactivity, consistent with the distribution expected from a small, highly diffusible, moderately lipophilic molecule (Table 1). These data depict slow clearance of radioactivity from every organ except from liver, spleen and large intestine (with contents), consistent with hepatobiliary clearance and fecal excretion. High concentrations of 7a in liver at early time periods (2 min) are suggestive of first pass clearance. At 2 h post-injection, tumor:blood (T/B) radioactivity ratios were similar to the ratios reported for IAZA and IAZP (Mannan, 1991), whereas the reported T/B ratios for [3H]MISO and [3H]FMISO are somewhat lower (1.9 at 2 h and 2.4 at 4 h, respectively; Rasey et al., 1987). At 8 h post injection, the T/B ratio wasD3, with approximately 0.2% of the injected dose remaining in the tumor. Although total uptake by tumor increased with time in the cases of IAZA and IAZP, 7a did not show any significant increase in uptake beyond 2 h. Comparative T/B data are summarized in Table 2. Two imaging studies were performed. One study, using 7a (with 95% radiochemical labelling), showed liver, spleen and large intestine (with contents) at 7 h, but the tumor was not visualized scintigraphically despite a T/B ratio of 2.3 (Table 2). In the other study

Table 2 A comparison of tumor/blood ratios of 7a with several wellcharacterized hypoxic markers Compound

Time (h)

Tumor/ blood ratio

Reference No.

99m

4 8 24 2 4 8 2 4 2 4 2 4 2 4 8

0.6 0.8 1.3 2.8 4.6 8.7 1.9 2.17 1.2 1.1 2.6 4.0 2.1 2.0 2.3

Zhang et al. (1995)

HNBAHP

125

IAZA

3

H-MISO H-FMISO 99m Tc-HL-91 3

99m

Tc-BMS-181321

7a (99mTc-5)

Mannan et al. (1991)

Chapman et al. (1981) Rasey et al. (1987) Imahashi et al. (2000) Johnson et al. (2000) This study

P. Kumar et al. / Applied Radiation and Isotopes 57 (2002) 719–728

using 7b, the tumor was visible at 3, 5 and 6 h post injection along with thyroid, liver, kidney, bladder, gastrointestinal tract and stomach. Product 7b contained two labelled components and about 40% free pertechnetate. Additional studies with free pertechnetate confirmed that this tumor uptake was not due to free 99mTcO 4 . Upon inspection of the structure of 7 it is clear that there are three coordinating arms, which creates the potential to form more than one radioactive coordination product. The biodistribution data of 7a and 7b, formed at different pH’s, show that the coupling structures obtained are, at least in part, pH directed. It is also conceivable that coordination of one or more of these complex structures could involve the imidazole ring, thereby altering the reduction potential of nitro group and changing the oxygen-dependency of hypoxic binding.

6. Conclusion A new class of azomycin nucleoside-PnAO ligands has been synthesized and characterized. In vitro studies demonstrate that they undergo hypoxia-dependent binding in vitro (formulation 7a), but this formulation is less selective in a tumor model. The desired hydrophilicity of these 99mTc complexes is offset by their chemical complexity (Scheme 2), which potentially gives rise to more than one 99mTc-radiolabelled species. Importantly, the nature of these 99mTc complexes is affected by the pH at which they are formulated, further complicating their suitability for clinical use. Additional radiolabelling and formulation studies, together with isolation and characterization of the radiolabelled products, will be required to determine their utility in hypoxia imaging.

Acknowledgements The authors gratefully acknowledge Alberta Cancer Board, (grant # RI-14) and Amersham International Inc., UK, for financial support. We thank Dr. C. Archer and Dr. A. King (Amersham International, UK) for helpful discussions and a generous supply of Pn-44, Dr. V. Somayaji (University of Alberta) for help with NMR data interpretations, and Aihua Zhou (University of Alberta) for assistance with the in vitro studies.

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