Bloconjugate Chem. 1993, 4, 326-333
328
Synthesis, Characterization, and in Vitro Evaluation of Nitroimidazole-BAT0 Complexes: New Technetium Compounds Designed for Imaging Hypoxic Tissue K. E. Linder, Y. W. Chan, J. E. Cyr, D. P. Nowotnik,' W. C. Eckelman, and A. D. Nunn Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543-4000. Received March 9, 1993
Several technetium-99 BATO (boronicacid adduct of technetium dioximes)complexesTcX(dioxime)aBR (X = C1) that contain a boron cap R which bears a 2- or 4-nitroimidazole moiety have been prepared from either TcCl(dioxime)a or from Tc(dioxime)3(pOH)SnCla [dioxime = dimethyl glyoxime (DMG) or cyclohexanedione dioxime (CDO)]. Two hydroxy analogs (X = OH) were isolated by treatment of the corresponding chloro complexes with aqueous NaOH. The complexes have been characterized by elemental analysis, mass spectrometry, NMR, UV/vis spectroscopy, and high-performance liquid chromatography. These complexes have the potential for selective retention in hypoxic tissue, by a mechanism believed to be the result of nitro reduction. The electrochemistry and enzymatic reduction of these complexes was studied to assess the potential for reduction in uiuo. The nitroreductase enzyme xanthine oxidase was shown to reduce the nitroimidazole group on the complexes99TcOH(DMG)3BBN0~ and 99TcOH(DMG)3BprenNO~ under anaerobic conditions in the presence of hypoxanthine. However, the results indicated that the rate of reduction might be slow in uiuo, limiting the suitability of these compounds for imaging of regions of hypoxia.
INTRODUCTION Recently, there has been considerable interest in the concept of imaging hypoxia (areas of low oxygen tension) with radiolabeled derivatives of nitroimidazoles. Other groups have reported the binding of 14C-( 1 - 4 ) ,3H- ( 5 , 6 ) , szBr- (7, 8), and lSF-labeled (9-12) 2-nitroimidazole derivatives to hypoxic cells or tissues in cerebral ischemia (13),myocardial infarction (6, 10, 11, 14), and malignant tumors or tumor spheroids ( 1 -5,12,15). Reports of iodinelabeled nitroimidazole derivatives (14, 16-19) have also been published recently. I t was apparent to us that the development of a technetium-based hypoxia-localizing nitroimidazole would be a useful contribution to the field of nuclear medicine, as it would make hypoxia imaging available to the majority of nuclear medicine centers. One class of technetium compounds that we have studied are nitroimidazole-BAT0 complexes TcX(dioxime)sBR (X = C1, OH; R = a nitroimidazole derivative, BATO = boronic acid adduct of technetium dioxime (20,21);see Figure 1). Technetium BATO compounds have already proved to be useful for perfusion imaging of the heart and brain (22), and BATOs derivatized with an isothiocyanato functionality have been used to radiolabel antibodies (23, 24). It seemed reasonable to incorporate a nitroimidazole functionality into the boron cap of one of these molecules, as one approach to the design of a technetium-based hypoxia imaging agent. The syntheses of the nitroimidazole boronic acids appear in a separate report (25).The structures of these compounds are shown in Figure 2. In this report we describe the syntheses, characterization, and electrochemistry of the BATO complexesderived from nitroimidazole boronic acids, and their initial evaluation in uitro using an assay which evaluates their ability to be reduced by the nitroreductase enzyme xanthine oxidase. As the uptake of l8F-labeledmisonidazolein vivo was found to be dependent on the degree of hypoxia, whereas the uptake of labeled metronidazole was less selective (26), the reduction properties of the nitroimidazole boronic acids
Figure 1. The general structure of BATOs [TcX(dioxime)sBR (dioxime = DMG, CDO)]. The R' groups represent methyl, in
DMG. Alternatively, two R' groups combined represent CH2CH2CH2CH2, in CDO. X = C1 or OH. R is the substituent containing the nitroimidazole moiety. and nitroimidazole-BATOs were compared to those of misonidazole and metronidazole.
EXPERIMENTAL PROCEDURES Materials and Reagents. Dimethylglyoxime (DMG, Eastern Chemicals) and cyclohexanedione dioxime (CDO, Fluka) were used as received. The syntheses of the nitroimidazole boronic acids are described elsewhere (25). Misonidazole was obtained as a gift from Prof. K.Krohn, University of Washington. Metronidazole (Aldrich), 2 4 troimidazole (Aldrich), tris(hydroxymethy1)aminoethane (Fisher Scientific),Ru(acac)s (Strem),andCdS04 (Johnson Mathey Puratronic) were used as received. BuNBF4 (Aldrich),a supporting electrolyte, was recrystallized twice from MeOH/HzO or acetone/ether, dried, and stored under vacuum. The complexes Tc(DMG)3(p-OH)SnC13'3HzO, Tc(CD0)3(pOH)SnC13, TcCl(DMG)3, and TcCl(CD0)s were prepared as described previously (21). All solvents were reagent grade or better and used as received. Water was purified using a Millipore MilliQ system. HPLC measurements were made on either a 15-cm Hamilton PRP-1 10-wm column (AlltechAssociates),using a mobile phase of acetonitrile/O.l M NH40Ac (pH 4.6) at
l043-l802/93/ 2904-0326$O4.OO/O 0 1993 American Chemical Society
Bioconlugete Chem., Vol. 4, No. 5, 1993 327
Nitroimidazole-BAT0 Complexes
'"I YN
(HO)ZBCBZCH~CH~N
NO2 (HO)ZBProPNOZ
(HO)ZBCE-CECHZN
? YN
NO2 (A0)2BprenN02
Figure 2. The structures of the nitroimidazole boronic acids ((H0)zBR).
a flow rate of 2.0 mL/min, or on a 4.6 X 300 mm Nucleosil C-8 column (Alltech Associates) using a mobile phase of acetonitrile/O.l M citric acid (pH 2.3) at a flow rate of 1.5 mL/min. Infrared measurements were recorded as KBr pellets on a Sirius 100 FT-IR instrument. UV/visible spectra were recorded using a Hewlett-Packard H P 8451A photodiode-array spectrophotometer. Air-sensitive reduced solutions were analyzed under nitrogen by carefully transferring them via syringe into a spectroscopy cell sealed with a rubber septum (flushed with N2). Fast atom bombardment mass spectra were obtained on a VG-ZAB2F spectrometer. lH NMR spectra were recorded on a JEOL GX-270 instrument. Elemental analyses were performed in-house by the Microanalytical Department. Xanthine oxidase (XOD, xanthine:oxygen Oxidoreductase, EC 1.1.3.22; isolated from cow milk) was obtained from Boeringer Mannheim as a suspension in ammonium sulfate solution (3.2 mol/L in (NH4hS04, pH -8, [EDTA] = 10 mmol/L). The solution had a specific activity of about 1 unit/mg of protein and contained 20 units/mL. The enzyme was stored a t 4 OC until use. Xanthine (20 mg/L) and hypoxanthine (0.01M) solutions were prepared by stirring the purines (Sigma)in water or phosphate buffer a t near boil until dissolved. All pH 7.4 phosphate buffer (0.025 or 0.1 M) contained 20 mg/L of Na2EDTA.2H20. Solutions were freshly prepared for each assay. Synthesis and Characterization of Technetium Complexes. Synthesis of 99TcCZ(CDO)3BBN02. To TC(CDO)~(~-OH)S~C~~*~H~O (90.3 mg, 0.11 mmol) in 10 mL of warm acetonitrile was added (OH)2BBN02 (31 mg, 0.129 mmol), followed by 1.5 mL of 3 N HC1. The solution was heated gently for 30 min., 10 mL of 1 M HC1 was added, and the solution was cooled to room temperature. The resulting orange solid was isolated by suction filtration, washed with 5 mL of 1N HC1 and 5 mL of H20, and dried in uucuo to give 61 mg (78%) of crude product. The product was dissolved in 10 mL of warm acetonitrile and filtered, and an equal volume of 1 N HC1 was added dropwise. Analytically pure microcrystals precipitated on cooling. Anal. Calcd for C2sH34NgBClOsTc: C, 43.68; H, 4.45; N, 16.37. Found: C, 43.59; H, 4.43; N, 16.20. Synthesis of 99TcCl(DMG)3BBN02.To Tc(DMG)&OH)SnCl3'3H20 (55 mg, 0.074 mmol) in 10 mL of warm acetonitrile was added (OHhBBN02 (25 mg, 0.10 mmol), followed by 1 mL of 3 N HC1. The solution was heated gently for 30 min, 10 mL of 1N HC1 was added, and the solution was cooled to room temperature. After a slight
delay, red-orange crystals precipitated. These were isolated by suction filtration, washed with 2 X 5 mL of 1N HC1, and dried in uucuo. A second crop was isolated after 6 h. The overall crude yield was 41 mg (80.5%). The product was partially purified by recrystallization from 10 mL of boiling acetonitrile, to which was added 2 mL of 1N HC1 and 15 mL of water. Further recrystallization from 2 mL of boiling acetonitrile, 1mL of CH2C12, and 1 drop of 1N HC1yielded pure product on slow evaporation. Anal. Calcd for TcCl(DMG)3BBNO2'0.5HzO (C22H29NgBClOs.5Tc): C, 37.71, H, 4.17; N, 17.99. Found: C, 37.62; H, 4.33; N, 17.63. S y n t h e s i s of 99TcOH(DMG)3BBN02. T o TcC1(DMGhBBN02 (45 mg, 0.065 mmol) in 2 mL of warm acetonitrile were added 3 drops of 2 N NaOH and 2 mL of H2O. The resulting suspension was stirred with gentle heating for 15min. During this time, all starting material dissolved and was converted to desired product, as determined by HPLC (Nucleosil C-8, 50/50 CH&N/O.l M NH~OAC, 1.5mL/min; t R of TcCl(DMG)3BBN02= 5.83, t R of TcOH(DMG)sBBN02 = 4.88 min). Water (2 mL) was added, and the solution was heated until precipitation of product began. Orange microcrystals (30.5 mg) were isolated after the solution was cooled to room temperature. A second crop (6 mg, 80% yield overall) was isolated after 4 h. The product was washed with water and dried in uucuo for 1 h. Synthesis of 99Tc Cl (DMG)3BPhEtN02. To Tc(DMG)3(pOH)SnC13'3H20 (77.1 mg, 0.104 mmol) in 5 mL of acetonitrile was added (OH)2BPhEtN02 (28.8 mg, 0.11 mmol), followed by 2 drops of concentrated HC1. The solution was heated at a boil for 30 min, during which time an orange solid precipitated (68 mg, 93 % yield). Product was washed well with acetonitrile and dried in uucuo. This crude solid (identity unknown, not TcCl(DMG)3BPhEtN02) was dissolved in 1 mL of DMF, 2 mL of acetonitrile, and 1mL of 1M HC1 and boiled for 30 min. HPLC analysis (PRP-1,70/30 CHsCN/O.l M NHdOAc, 2 mL/min) demonstrated that starting material ( t R = 1.17 min) was slowly converted to desired product ( t R = 2.35 min) over this time. Crystals (31 mg, 40% overall yield) formed upon cooling overnight. Anal. Calcd for TcC1(DMG)3BPhEtN02*0.5DMF(C24.5H33Ng.5BC108.5T~): C, 39.64;H,4.81;N, 17.92. Found: C,39.80,H,4.60;N, 17.55. Synthesis o f g g ~ c C ~ ( ~ ~ ~ To ) ~Tc(DMG)3p r o p ~ ~ ~ . (p-OH)SnC13.3H20 (143 mg, 0.193 mmol) in 10 mL of acetonitrile was added (0H)zBpropNOz (41.8 mg, 0.21 mmol), followed by 3 mL of 2 N HC1. The reaction mixture was heated gently for 30 min, 50mL of 1N HC1was added, and the solution was cooled to room temperature. The resultant flocculant orange solid was isolated by suction filtration, washed with 1 M HC1 and H20, and dried in uucuo to yield 97 mg (78%) of crude product. This was dissolved in 1mL of CHCl3 and chromatographed on a 1 x 10 cm silica gel column that was conditioned and eluted with CHC13. The major orange band was evaporated to dryness, redissolved in 3 mL of CH2C12, and treated with 30 mL of hexane. Analytically pure solid was isolated in 57% yield overall. Anal. Calcd for ClsH2sNgBClOsTc:C, 33.58;H,4.38;N, 19.58. Found: C,33.66;H,4.20;N, 19.28. Synthesis of 99TcCZ(DMG)3BprenN02.To Tc(DMG)&OH)SnC1$3H20 (135.5 mg, 0.183 mmol) in 10 mL of acetonitrile was added (0H)zBprenNOz (39.4 mg, 0.20 mmol), followed by 3 mL of 2 N HC1. The reaction mixture was heated gently with stirring for 30 min, 15 mL of 1N HC1 was added, and the solution was cooled to room temperature. An orange oil formed, which was stirred with scratching until the product solidified. The crude
328 Bioconjugate Chem., Vol. 4, No. 5, 1993
solid (85 mg, 73%) was isolated by suction filtration, washed with H20, and dried in vacuo. It was then dissolved in a minimal volume of CHCl3 and chromatographed on a 2.5 X 8 cm silica gel column that was conditioned and eluted with 10% ethyl acetate/90% CHCl3. The major orange band was collected as four fractions, the first of which was discarded after analysis by HPLC demonstrated the presence of a lipophilic impurity. The other fractions were pooled and rotary evaporated to an oil, and the oil was redissolved in 5 mL of acetonitrile. Treatment with 15 mL of 0.5 N HC1 caused precipitation of an orange microcrystalline solid. Synthesis of 99TcOH(DMG)aprenN02. To TcC1(DMG)sBprenNOz (100 mg, 0.156 mmol) in 2 mL of warm acetonitrile were added 3 drops of 2 N NaOH and 2 mL of HzO. The solution was heated with stirring for 1h, the volume was reduced using an N2 stream, 2 mL of H2O was added, and the product was extracted into 3 X 4 mL of CHzC12. The organic layer was washed with water (3 X 4 mL), and 5 mL of acetonitrile was added to the organic phase. The solvent was removed by rotary evaporation to yield an orange oil. Addition of water (2 mL) caused precipitation of an oily solid, which was stirred, with scratching, until completely solid. The product was isolated by suction filtration, washed with water, and airdried to yield 99 mg of orange brown product. Synthesis of 99TcC1(DMG)3BB4N02. A mixture of TcCl(DMG13 (32.7 mg, 0.068 mmol) and (OH)zBB4N02 (17.7 mg, 0.073 mmol) in 5 mL of acetonitrile and 0.5 mL of 2 N HC1 was heated with stirring for 15 min. Solvents were then removed by evaporation. The product was purified on a silica gel column eluted with 40/60 ACNI CH2Clz. The major orange band was evaporated to dryness and recrystallized from ether (61% yield). Anal. Calcd .~C~H~~O for T C C ~ ( D M G ) ~ B B ~ N O ~ * O (Cz2H8N908BClTc.O.2C4HloO): C, 38.75; H, 4.28; N, 17.84. Found: C, 38.39; H, 4.21; N, 17.44. Synthesis of 99TcCl(CD0)d3B4N02. A mixture of TcCl(CD0)3 (25.07 mg, 0.046 mmol) and (OH)2BB4N02 (11.6 mg, 0.048 mmol) was dissolved in 5 mL of ACN and 0.5 mL of 3 M HC1 and heated with stirring for 30 min. An orange solid was precipitated by adding 5 mL of 1M HCl, collected by filtration (crude yield, 83%), and recrystallized from ether. Anal. Calcd for C28H34N~O&LBTC-O.~CJI~~O: C,44.65; H, 4.87; N, 15.62. Found: C, 44.81; H, 4.95; N, 16.05. Synthesis of 99mTcComplexes. 99mTc-BAT0nitroimidazoles were prepared by methods similar to those reported previously (23)involving addition of the boronic acid derivative to freeze-dried tris-dioxime kits. The radiochemical purities (RCPs) of these complexes were determined using reversed-phase high-performance liquid chromatography (using the PRP-1 and Nucleosil C8 systems, described above). RCPs were typically >go%. Electrochemical Studies. Instrumentation and experimental conditions for electrochemical studies in DMF were as described elsewhere (27). The CV of Ru(acac)3 was determined on a daily basis to compensate for variations in the reference potential. All measured potentials were corrected to an absolute peak reduction potential for Ru(acac)s of -1.210 V vs Ag/AgNOs (acetonitrile) at Hg (-0.790 V vs SCE at Pt). Determination of Rate of Reduction by Xanthine Oxidase Catalysis. Standardization of Enzyme Solutions. The specific activity (units/mL) of the xanthine oxidase solutions was assayed by measuring the rate of XOL-catalyzed formation of uric acid from xanthine,
Linder et el.
following the procedure of Bray (28). Based upon the assay results, 0.5 units of enzyme was added per study. Effect of 5%DMF on the activity of XOD. A xanthine oxidase suspension (10 pL) was diluted to 1mL with 990 pL of pH 7.4 phosphate/EDTA buffer. To a quartz cuvette was added 1.0 mL of xanthine solution (20 mg/L of water) followed by 1.75 mL of phosphate buffer and 150 pL of DMF. The reaction was initiated by adding 100 pL of diluted enzyme solution. Absorbance at 292 nm was monitored for 10min, and data were converted to "change in absorbance units/min" relative to a comparable reaction in the absence of DMF (Table VI). Similar studies were performed with 0 and 10% DMF. Appropriate UV/vis blanks contained water in place of xanthine solution. XOD-Catalyzed Reduction of Nitroimidazole Boronic Acids and Nitro Compounds. To a 3-mL quartz cuvette was added solutions of hypoxanthine (10 pmol) and nitro compound (0.25 pmol) dissolved in sufficient pH 7.6 phosphosaline (PBS; 0.025 M phosphate in saline) or pH 7.4 phosphate buffer (0.025 M) to bring the total volume to 2 mL. The cuvette was sealed with a rubber septum and purged of oxygen by passage of a stream of ultra high purity argon through the solution. Meanwhile, to a 5-mL siliconized vial was added 1.05 mL of PBS or phosphate buffer followed by about 150 pL of XOD suspension. The vial was crimp sealed and deoxygenated by passing a flow of argon over the surface of the enzyme solution for exactly 15 min. To initiate the reaction, 500 pL of the deoxygenated enzyme solution (0.5 units) was added to the degassed nitro solution via a gas-tight noncoring syringe. The final assay solution (2.5 mL) was 0.2 units/mL in XOD, 100pM in nitro compound, and 4 mM in hypoxanthine. The cuvette was inverted to mix and then monitored by UVvis (vs PBS buffer blank) to follow the disappearance of the nitro group absorbance. Both the spectrum of the solution from 280 to 450 nm and absorbance a t 326 nm were recorded every 5 min. Data were automatically stored to disk for later retrieval and analysis. The absorbance a t 326 nm was converted to concentration, and plots of log[concentrationl vs time were prepared using Excel software. All of the 2-nitroimidazole boronic acids, misonidazole, and metronidazole were tested as described above. Data for the 4-nitroimidazole compound (OH)2BB4N02 was obtained in the same fashion, except that the absorbance was monitored at 310 nm instead of 326 nm. XOD-Catalyzed Reduction of 99TcOH(DMG)a(R = BBNO2, BprenN02). To a 3-mL quartz cuvette was added 0.2 pmol of the *TcOH(DMG)3R complex in 125 pL of DMF, 1.0 mL of 0.01 M hypoxanthine solution in 0.1 M phosphate/EDTA buffer (pH 7.41, and 0.875 mL of 0.1 M sodium phosphate buffer. The cuvette was sealed with a rubber septum and was purged of oxygen by passage of a stream of ultra high purity argon through the solution. Meanwhile, to a 5-mL siliconized vial was added 1.05 mL of pH 7.4 phosphate buffer (0.1 M) and approximately 150 pL of XOD suspension. The vial was crimp sealed and deoxygenated by passing a flow of argon over the surface of the enzyme solution for exactly 15 minutes. To initiate the reaction, 500 p L of enzyme solution (0.5 units) was added to the degassed nitro solution via a gastight noncoring syringe. The final assay solution (2.5 mL) was 0.2 units/mL in XOD, 80-100 pM in nitro compound, 4 mM in hypoxanthine, and 5 % in DMF. The spectrum of the resulting solution was recorded from 280 to 600 nm every 15 min for 10 h. Data were stored to disk for later retrieval and analysis as above. Similar assays were
Bloconjugate Chem., Vol. 4, No. 5, i993
Nltrolmidarole-BAT0 Complexes
conducted with (OHhBBN02 and (0H)zBprenNOz to allow direct comparison of boronic acid and Tc complex data in 5% DMF. Control reactions (in the absence of enzyme or in the absence of hypoxanthine) were also carried out, substituting phosphate buffer for reagents not added. In addition, a control reaction substituting the non-nitro compound 99TcC1(DMG)3BPhwas carried out.
Reversible in Normoxic Tissue
R
n 4
w
-
[ N Further reduction in hypoxic tissue
\>NO2.-
Reductases
R
329
N \>NHoH N R
?
1
v Binding to cellular components
Figure 3. Proposed mechanism for the preferential entrapment
RESULTS AND DISCUSSION
of nitroimidazoles in hypoxic tissue.
Synthesesand Characterizationof NitroimidazoleBATOs. Synthesis of the wTc-BATO nitroimidazoles from either Tc(dioxime)3(~-OH)SnC13 or TcCl(dioxime)s (dioxime = DMG or CDO) usually proved to be straightforward, following procedures described previously (21) for the synthesis of non-nitroimidazole BATOs (the exception was TcCl(DMG)sBPhEtNOz, which gave an unidentified polar product, which converted to the required BATO on recrystallization from HC1-acidified DMF). Isolated yields of 7 0 4 0 % could be obtained after heating the Tc starting material with a nitroimidazole boronic acid in HC1-acidifiedacetonitrile. The structures of the nitroimidazole boronic acids used are shown in Figure 2. Two BATO-nitroimidazole compounds, in which the axial X ligand (Figure 1)was hydroxyl, were also prepared. On treatment of the chloro compounds TcCl(DMG)3BBNO2 and TcCl(DMG)sBprenN02 with NaOH, chloro to hydroxy exchange to form TcOH(DMG13BBN02 and TcOH(DMG)sBprenNOzwas observed, as has been noted for other BATO compounds (29). These hydroxy compounds must not be vacuum-dried after isolation, or the material will turn deep brown, as observed for other BATOs with an axial hydroxyl ligand (30). A sample of TcOH(DMG)@prenNOzthat was vacuum-dried overnight yielded a molecular ion corresponding to the mass of the dimer: RB(DMG),Tc-O-Tc(DMG),BR After recrystallization, satisfactory analyses could be obtained on all chloro nitroimidazole BATO compounds, as determined by NMR, mass spectrometry, IR, and elemental analysis.l The infrared spectra of the isolated products all showed two strong nitro bands (v, and vSp) (31,321in the range 1535-1546 and 1360-1400 cm-1, respectively, indicating that the nitroimidazole moiety had survived the reaction conditions required for the synthesis of the BATOs. All compounds showed similar UV/visible spectra. All of the absorption bands found in a normal BATO are also present in the nitroimidazole BATOs. However, the peak a t 320 also contains a significant contribution from a A r* transition (33)from the nitroimidazole cap. In the lH NMR spectra of all nitro BATO complexes, the DMG protons are found as a multiplet (18H) at 6 2.352.45 ppm, and the CDO protons are found at 6 1.8 (12H) and 3.0 (12H) ppm. These chemical shifts are not significantly different from those observed for other BATOs (20,21).The dioxime protons, if observed, fall at about 6 14.8 ppm. In CDC13, these protons appear as a sharp singlet, but in CD2Cl2, this peak is quite broad. The phenyl protons of both the BBNOz and BB4N02 complexes appear as a pair of doublets a t 6 7.2 and 7.8 ppm, consistent with para substitution. The benzyl protons (s,2H) fall at 6 5.2 ppm for both the CDO and DMG BB4N02 complexes and a t 6 5.6 for the corresponding BBNO2 complexes.
-
There was insufficent material to obtain an elemental analysis on gsTcCl(DMG)sBprenNOz.
The imidazole protons of both the CDO and DMG BB4N02 compounds appear as a pair of singlets at 6 7.47.5and 7.7 ppm (1H each). In the TcCl(DMG)aBpropNOz and TcCl(DMG)sPhEtNOncomplexes, where the protons of the 2-nitroimidazole group are adjacent to one another, the protons also appear as two singlets (1H each) (6 7.1, 7.2, and 6.9, 7.05 ppm respectively). In the spectrum of both TcCl(DMG)sBBN02 and TcCl(CD0)3BBN02, the imidazole protons appear to be coincidentally equivalent and fall as a singlet at 6 7.15 (2H) in both compounds. Fast atom bombardment mass spectral (FAB-MS) fragmentation patterns for the nitroimidazole BATOs were similar to those observed for other BATOs (34). A molecular ion and protonated molecular ion were seen for all of the chloro compounds in the positive mode. Loss of the axial ligand (as HC1) was also observed. All of the benzyl compounds (CDO and DMG complexes with BBN02 and BB4N02 caps) also showed loss of the 2-nitroimidazole group under positive FAB conditions. In the negative mode, ions corresponding to a mass of [M HI- and [M - HC11- were noted for all of the chloro compounds. FAB-MS of the hydroxyl complexTcOH(DMG)3BBN02 showed only a very weak molecular ion, as has been observed previously for other BATO hydroxy complexes (21). The base peak in the spectrum of this complex corresponded to [M + H - HzOl+ or [M - H201- under FAB positive and negative conditions, respectively. Like the chlorobenzyl complexes, loss of the 2-nitroimidazole group was noted in the positive mode, for the BBNOz cap only. A molecular ion was noted in the spectrum of the propene analog TcOH(DMG)@prenNOz, as well as fragment ions due to loss of water, as noted above for the BBNOz complex. Electrochemical Studies. In vivo reduction of the nitro group to a reactive hydroxylamine species, followed by binding to cellular components (Figure 3), has been proposed as the mechanism by which nitroimidazoles are selectivelyretained in hypoxic tissue (35).As the reduction potential of nitroaromatic compounds appears to be an important determinant of a compound’s ability to progress along this pathway, we decided to study the electrochemistry of the nitroimidazole BATOs and their boronic acid precusors. Ideally, the electrochemical behavior of these complexes should be studied under conditions which mimic those in vivo;i.e. under aqueous conditions at pH 7.4. However, the majority of the nitroimidazole-BATOs are not sufficently soluble in water to achieve a concentration level suitable for normal electrochemical studies (ca. 0.2 mM) (27). As a result, the nitroimidazole-BATOs were examined in DMF. Although the observed electrochemical reductive processes for nitroimidazoles are different in DMF and water (in DMF, a one-electron reduction of the nitro group has been reported (361, while in aqueous media, a single irreversible four-electron process was observed (37)),Breccia et al. (36)demonstrated good correlation
330
Llnder et el.
Bioconjugate Chem., Vol. 4, No. 5, 1993
Table I. Cyclic Voltammetry Data for Nitroimidazole Compounds in DMFs nitrocentered reduction
compound ~
BATOcentered reduction
~~
BATO-Nitroimidazoles 99T~(C1)(DMG)3BBN02 -1.58 99Tc(Cl)(DMG)3BB4NOz -1.84 Vc(Cl)(DMG)3BPhEtN02 -1.56 Vc(Cl)(DMG)sBpropN02 -1.64 ?I'c(Cl)(DMG)sBprenNOz -1.60 99Tc(Cl)(CD0)3BBN02 -1.56 ?I'c(Cl)(CD0)3BB4NOz -1.84 %Tc(OH)(DMG)sBBNOz -1.56 ?I'c(OH)(DMG)sBprenNOz -1.58
-1.31 -1.30 -1.29 -1.34 -1.32 -1.22 -1.23 -1.83 -1.83
"Simple"BATOsb 99Tc(Cl)(DMG)3Ph 99T~(C1)(DMG)3B~ 99TC(Cl)(CDO)sPh ?I'c(OH)(DMG)~Bu
-1.30 -1.35 -1.24 -1.84, -2.56
0 Scan rate = 100 mV/s. All solutions were 0.2-0.7 mM in sample and 0.1 M in tetrabutylammonium tetrafluoroborate. Hanging mercury drop working electrode vs AgiAgNOa reference. From ref 27.
*
between reduction potentials of nitroimidazoles determined in DMF and water. Cyclic voltammetry at Hg in DMF of the N-substituted nitroimidazoles showed a reduction/oxidation couple which can be ascribed to reduction of the nitro substituent (37). Peak potential values for nitro and BATO core reduction are tabulated in Table I. Note that, in DMF, the reduction potentials for the BATO "core" occur at a less negative value than those of the nitro group, indicating that, in DMF, the BATO "core" may be easier to reduce than the nitro group. The reduction potentials for the BATO "core" in DMF are similar to those observed for "simple" BATOs (27). The nitro reduction potentials of the 2-nitroimidazole-BATOs (and the boronic acids) fall between those of misonidazole and metronidazole. On the basis of these data we cannot predict whether the BATO-nitroimidazoles will be reduced in hypoxic tissue. The order of ease of reduction of DMGBATO complexes in DMF is BBN02 >> BPhEtNO2 > BpropNO2 >> BprenNOz, with BBNO2 being closest to misonidazole. More detail on the electrochemistry of BATO nitroimidazole derivatives will appear in a separate report. Reduction of Nitroimidazole-BATOs by Xanthine Oxidase. As the electrochemistry studies demonstrated that the reduction potentials of the a-nitroimidazoleBATOs were similar to those of misonidazole (providing the first indication of potential for imaging of hypoxic tissue), it was necessary to demonstrate that these nitroimidazole-BAT0 complexes could be reduced by nitroreductase enzymes. The rates of reduction relative to misonidazole (38) would provide some additional indication of the potential of these new technetium complexes for imaging hypoxia in vivo. Xanthine oxidase (XOD) is a convenient model nitroreductase to use with these Tc-nitroimidazole-BAT0 compounds, as it is a readily available enzyme that has already been shown to reduce a variety of nitro heterocyclic compounds (39).In vivo, XOD catalyzes the oxidation of hypoxanthine to xanthine and of xanthine to uric acid: xanthine
+ 0, + H 2 0
-
urate + Hz02
XOD shows very broad specificity for both oxidant and reductant. Reducing substrates include purines, such as xanthine and hypoxanthine, aldehydes, and NADH (28). Oxidizingsubstrates include 0 2 , pyrimidines (40),and nitro
-06
I
~
0
10
40
ea
ea
IW
110
44a
Tima (minutes)
Figure 4. Reduction of nitroimidazole boronic acids by xanthine oxidase. compounds such as misonidazole (41, 42) and fluoromisonidazole (43). Therefore, even in the absence of oxygen, a substrate will be reduced, provided some other suitable source of oxidant is added. The XOD assay described here is based on methods previously reported (for example, see ref 43). It provides a measure of the effectiveness of a nitroimidazole derivative (as the oxidizing agent) in the XOD-catalyzed reduction of hypoxanthine in the absence of oxygen. Reduction of Nitroimidazole Boronic Acids. In a typical reaction of the boronic acid (0H)zBBNOz with xanthine oxidase and hypoxanthine under anaerobic conditions, hypoxanthine is first oxidized to xanthine and then to uric acid, as determined by HPLC (data not shown). At the same time, the UV absorbance peak at 326 nm (which is attributable to the nitro group) disappears. Figure 4 shows a comparison of the rate of decrease of the nitro absorbances of (OHIzBBNOz, (OH)ZPhEtNOz, (0H)zBB4N02, (OH)zBprenNOn, misonidazole, and metronidazole under identical assay conditions. Under the conditions tested, all of the 2-nitroimidazole boronic acids reacted with XOD at a rate that was equal to or faster than that observed for the 2-nitroimidazole compound misonidazole. The nitro group of the 4-nitroimidazole boronic acid (OH)zBB4NOz was consumed a t a much slower rate, and metronidazole (a 5-nitroimidazole) was found to be almost unreactive in this assay. XOD-Catalyzed Reduction of 99Tc-BATO-Nitroimidazoles. As DMF was added to help solubilize the Tc complexes, the effect of DMF on xanthine oxidase was tested. Enzyme activity in the standard assay drops as the concentration of DMF is increased. At 5% DMF, activity was 76 % of that observed in the absence of DMF. This was deemed significant but tolerable. Unfortunately, the 99Tc-chloro-BATO-nitroimidazole compounds were not sufficiently water-soluble, even if 5% DMF was added to help solubilize the Tc complex. Therefore, enzyme studies on Tc-BATO-nitroimidazoles were conducted with the more soluble hydroxy analogs "TCOH(DMG)~BR(29). A p l o t showing t h e reaction between TcOH(DMGIsBprenNO:! and hypoxanthine in the presence of XOD under anaerobic conditions is shown in Figure 5. A slow but significant drop in absorbance at 326 nm is seen, consistent with loss of the nitro group. Onlyminor changes are noted in other portions of the BATO spectrum. After the reaction is complete, the final spectrum of the reaction mixture is very similar to that of non-nitro imidazoleBATO compounds (21). In control experiments performed in the absence of XOD or the absence of hypoxanthine, no reaction was seen over 16 h. When the non-nitro containing BATO TcCl(DMG)3BPhwas used, no reaction
Bloconjugete Chem., Vol. 4, No. 5, 1993 331
Nkroimidazole-BAT0 Complexes
Table 11. Comparison of Enzyme Assay Data and Redox Potentials for Nitroimidazole Boronic Acids and Standards
T1p for nitro group loss (n) 13.0 2.8 min (6) 21.6 f 1.7 min (5) 39.0f 1.4 min (4) 120 min (2) 36.5 1.7 min (4) >20 h (2)
*
(OH)2BBN02 (0H)zBprenNOz (OH)zBPhEtN02 (OH)zBB4N02 misonidazole metronidazole In DMF at mercury working electrode (0.1M in TBA BF,) vs Ag/AgNO3 (ACN) reference. Scan rate = 100 mV/s. In DMF this is a quasi-reversible one-electron process.
-
I I
0 0
200
100
m
m
lax,
1200
1100
1 m
nme elapaed (minutes)
Figure 5. Comparison of rate of reduction of 99Tc(OH)(DMG)aprenNOz and its boronic acid precursor.
occurred. These results suggest that only the nitroimidazole group is reduced in the presence of XOD, without dramatically affecting the rest of the BATO core. This is of interest because, in electrochemical reduction of BATO nitro compounds at mercury electrodes in DMF, BATO core reduction preceeds nitro reduction. These results demonstrate the importance of enzyme specificity in this reaction. Similar spectral results were obtained in the reaction of XOD with TcOH(DMG13BBN02. In the presence of enzyme, significant changes occurred in the nitro absorption region, with little change in the other peaks in the UV/vis spectrum. In the no enzyme control, only a 1% decrease in absorbance was noted at all wavelengths (indicative of minor precipitation) over 8 h. Comparison to Boronic Acids. To allow a direct comparison of the rate of reduction of a nitroimidazole boronic acid to its Tc complex, the enzymatic reduction of (H0)2BprenNOz was also studied in 5% DMF. Figure 5 displays data from that study, comparing the rate of disappearance of the nitro group in TcOH(DMG)3BprenNOz to its boronic acid precusor. These results show that the unchelated boronic acid was reduced by XOD at a much faster rate than was the corresponding BATO compound. A similar result was obtained when TcOH(DMG)3BBN02was compared to (H0)2BBN02 (data not shown). This may be due in part to changes in the reduction potential of the nitro group. On coordination to technetium, the boron changes from three- to fourcoordinate, which appears to decrease the reduction potential of the nitro group. It may also be that high lipophilicity of BATOs (44) relative to misonidazole (45) and/or steric hindrance from the rather bulky Tc complex hinder(s) binding of the nitro group at the active site of the enzyme. Comparison with Results from the Literature. The rates that we observed for the XOD-catalyzed reduction of misonidazole (initial rate of 6.2 nmol/min/unit of XOD) were comparable to initial rates reported by Josephy et al. (42) (6 nmol/min/unit), Clarke et al. (46) (8 f 2 nmol/ min/unit) and Prekeges (5.7 + 0.7 nmol/min/unit) (43). Clarke et al. (46) have also reported that XOD reduced metronidazole very slowly; this is consistent with our observed half-life of >>20hours. As expected from previous studies that show 4- and 5-substituted nitroimidazoles to be harder to reduce (47) than are 2-nitro compounds, both the 4-nitro-substituted boronic acid and the Tc-BB4N02 compound were reduced at a significantly slower rate than any of the 2-nitro compounds. A study by Clarke et al. (46) indicated that the redox potential (E1p) and the rate of XOD-catalyzed nitroreduction show good correlation in a series of 15 nitroimidazoles, using xanthine as the electron donator. Based
Epc(volta) for NO2 reduction’ -1.52 -1.56 -1.53 -1.80 -1.49 -1.62
*
on Clarke’s results, the predicted order of rate of enzyme reduction in our nitroimidazole boronic acids should be miso > (OH)2BBN02 = (OH12BPhEtN02 > (OH)2BprenNOz > metronidazole >> (OH)2BB4N02. However, the observed order (Table 11)for these compounds was, in fact, (OH)2BBN02 > (0H)zBprenNOn > miso = (OH).$PhEtN02 > (OH)2BB4N02 >> metronidazole. There was no correlation between the rate of enzymatic reduction and redox potential (using data on the 2 4 troimidazole derivatives shown in Table 11; r2 = 0.134, n = 4). The reaction of (OH)2BPhEtN02 with XOD was considerably slower TI/^ = 40 min) than that of the methylene analog (OHhBBN02, (T112 = 13 min) despite the fact that the one- electron reduction potential of the BPhEt ligand indicates that it should be slightly easier to reduce. These data suggest that some factor other than redox potential is affecting the rate of reduction in these boronic acids (a finding that is not unexpected for an enzyme-catalyzed reaction). A similar result was reported recently by O’Connor et al. (481, who found, in a series of nitroacridines, that xanthine oxidase exhibited significant substrate specificity beyond that imposed by redox potential. CONCLUSIONS
We have synthesized and characterized a series of BATO-nitroimidazole complexes and conducted a preliminary assessment of their potential as radiopharmaceuticals for imaging of hypoxic tissue by studying their electrochemical and enzymic reduction. In DMF the nitro reduction potentials of the 2-nitroimidazole-BATOs (and the boronic acids) fall between those of misonidazole and metronidazole, indicating that these compounds may possess slightly suboptimal reduction potentials. Overall, these data would indicate that the 2-nitroimidazole-substitutedBATOs have reduction potentials in the range that might be anticipated as promising for compounds which are expected to bind in hypoxic tissue. The 4-substituted nitroimidazoles are more difficult to reduce than metronidazole, suggesting that these compounds will probab’y not be as useful as the 2-nitroimidazoles for imaging hypoxia. Under anaerobic conditions, xanthine oxidase rapidly reduces nitroimidazole boronic acids at a rate that is, unexpectedly, faster than that observed for misonidazole. The enzyme also reduces the nitro group of BATOnitroimidazoles, although the initial rate is slower than that of the corresponding boronic acid by at least a factor of 10. This may, in part, be due to a negative shift in redox potential upon coordination to Tc, or it may be due to the relatively high steric bulk and/or lipophilicity of the technetium BATO complexes examined in this study. These results suggest that the BATO-nitroimidazoles have some of the properties required for a hypoxia imaging
332 Bioconjete Chem., Vol. 4, No. 5, 1993
agent, but that reduction in vivo could be slower than desired. However, BATO-nitroimidazoles and their boronic acid precursors are clearly recognized as substrates for xanthine oxidase. This is of note because technetium is not a naturdy occuring element, and literature examples where technetium complexes are recognized by enzymes are rare. A technetium-labeled ribonucleoside was shown to be an inhibitor of ribonuclease U2 (49),and technetiumlabeled progestin derivatives displayed in vitro binding to the progesterone receptor (50). Previous reported examples of technetium complexes which are believed to be metabolized by enzyme catalysis are TcO(ECD1 (511, an ester derivative of Tc-(~-M~O-SAL~TAME)+ (52),and Tc(53). All of these complexes are (CNC(CH3)2(COOCH3)s+ de-esterified in serum or tissue homogenate. While in vivo reduction has been proposed to explain the behavior of certain technetium cationic complexes (541,we believe that the BATO-nitroimidazoles are the first example of a series of technetium complexes in which enzymatic reduction has been demonstrated. These promising results lead us to examine a nitroimidazole derivative of another technetium complex (PnAO)(55),which has demonstrated promise as an imaging agent for hypoxia in an in vivo model (56). ACKNOWLEDGMENT
We thank K. Krohn for providing misonidazole, K. Ramalingam and N. Raju for the syntheses of the nitroimidazole boronic acids, and A1 Bauer for writing the Basic program for the automatic data collection from the UV/vis spectrometer. Supplementary Material Available: Details of the 270MHz 'H-NMR spectra, FAB mass spectra (positive and negative ion modes), and IR spectra (4 pages). Ordering information is given on any current masthead page.
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