Bioconjugate Chem. 2000, 11, 175−181
175
Single Isomer Technetium-99m Tamoxifen Conjugates Duncan H. Hunter* and Leonard G. Luyt Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5B7. Received August 20, 1999; Revised Manuscript Received December 3, 1999
To produce an imaging agent for breast cancer using a technetium-99m-labeled agent specific for estrogen receptors, an N2S2 bifunctional chelator was conjugated to Z- and E-aminotamoxifens through an amide linker. These bioconjugates have been chelated with both technetium-99m and rhenium. For the Z-isomer, chelation with rhenium in the presence of sodium acetate yields a mixture of two isomers, anti and syn, in a 1:1 ratio and in the presence of hydroxide results in only the anti isomer. Both the Z- and E-tamoxifen conjugates have been chelated with technetium-99m at the tracer level yielding a single isomer product, which is assigned as anti based on chromatographic comparison to the rhenium complexes. Radiochemical yields were consistently greater than 80%, with Sep-Pak column purification yielding a final product with >99% radiochemical purity and no residual starting material. Both in vitro and in vivo biological evaluation of the tamoxifen chelates indicated very limited estrogen receptor binding.
INTRODUCTION
Breast cancer is the leading form of cancer in females, with one out of every eight women in North America developing the disease in their lifetime (1). The proper diagnosis and therapy of breast cancer is continuing to evolve with an ever increasing number of diagnostic procedures and therapeutic agents including nuclear medicine techniques (2). Estrogen receptor (ER) status of the tumor is used as a guide for the selection of appropriate tumor therapy, with the drug tamoxifen (1) playing a leading role in the therapy of ER positive carcinomas (3). Since over 60% of breast cancer tumors contains estrogen receptors, the discovery of an estrogen receptor specific radiopharmaceutical is desirable. Not surprisingly, tamoxifen has been the target of several radiolabeling studies including 3H (4, 5), 18F (6), 111In (7), 123I (8), 125I (9), and 131I (10), but labeling with 99mTc has not been reported. Given the widespread use of technetium-99m for radiopharmaceuticals, our previous experience with a radiolabeled tamoxifen, and our recent preparation of an N2S2 bifunctional chelator, the attachment of 99mTc via a linker from tamoxifen to an N2S2 bifunctional chelator was attempted. In determining the location for attachment of a chelator to tamoxifen, the potential metabolism of the radiopharmaceutical was considered. The metabolism of tamoxifen in vivo has been extensively studied (11, 12), with the primary active metabolites in humans being 4-hydroxytamoxifen with the OH on ring-2 and N-demethylated compounds. No metabolites have been found involving modification of phenyl ring-3. Placing a metal chelator on phenyl ring-3 may be advantageous, as the resultant metal complex will be able to undergo in vivo metabolism congruous to what occurs for tamoxifen. The large size of a technetium chelator makes it important to attach the chelator at a region of tamoxifen that can manage steric bulk without sacrificing receptor binding affinity. Table 1 presents the relative binding * To whom correspondence should be directed. Phone: (519) 661-3122. Fax: (519) 661-3022. Email:
[email protected].
Table 1. Relative Binding Affinities of Tamoxifen Analogues Modified at Ring 3, for Rat Uterine Estrogen Receptors (10)
relative binding affinity (estradiol ) 100) R
Z isomer
E isomer
NH2 (2) I (3) H (tamoxifen, 1)
1 3.3 0.2
0.9 0.1 ∼0.02
affinity (estradiol ) 100) for tamoxifen derivatives modified at the 4-position of ring-3 (10). Compared to tamoxifen, the relative binding affinities of the 4-amino (2) and 4-iodo (3) tamoxifens show that ring-3 accommodates additional steric bulk with some improvement in estrogen receptor binding. However, a recently published X-ray crystal structure of tamoxifen bound in the estrogen receptor binding site suggests that only limited steric bulk can be accommodated at this position (13). On the basis of the metabolism studies and on the relative binding affinity of tamoxifen derivatives, it was decided that the preferred location for a technetium chelator would be ring-3 of tamoxifen. The bifunctional chelator chosen for conjugation to tamoxifen is the symmetrical N2S2 ligand 4 (14). This chelator forms conjugates with amine containing compounds, which upon chelation can produce a single isomer metal complex without the need for HPLC separation. This will greatly ease the purification and characterization of both technetium-99m and rhenium bioconjugates. Using this
10.1021/bc990110z CCC: $19.00 © 2000 American Chemical Society Published on Web 02/19/2000
176 Bioconjugate Chem., Vol. 11, No. 2, 2000
Figure 1. Bifunctional chelator and the proposed [99mTc]tamoxifen analogues.
N2S2 bifunctional chelator, the [99mTc]tamoxifen analogues Z-5 and E-5 shown in Figure 1 are proposed as potential estrogen receptor imaging agents. EXPERIMENTAL SECTION
Chemicals were purchased from commercial sources (Sigma-Aldrich Canada Ltd., Mississauga, Ontario, and Lancaster, Georgetown, Ontario) as reagent grade and were used as received with the following exceptions: 3,3bis(triphenylmethylthio-acetamidomethyl)propanoic acid 4 (14), trans-bis(ethylenediamine)dioxorhenium(V) chloride (15), and the aminotamoxifens Z-2 and E-2 (10) were all prepared according to literature procedures and had physical and spectroscopic properties consistent with those reported previously. 1H, 13C, and two-dimensional NMR spectra were collected on a Varian Gemini-300 or Varian Gemini-200 spectrometer with TMS as an internal reference. Infrared spectra were recorded in the range 4000-600 cm-1 on a Bruker IFS 5500 FT-IR with a Spectra-Tech diffuse reflectance accessory (DRIFT); UV spectra were recorded on a Hewlett-Packard 8451A Diode Array spectrophotometer. Routine mass spectra (MS) and negative ion fast-atom bombardment mass spectra (FABMS) were measured on a Finnigan MAT 8200 spectrometer. HPLC was performed using a Dionex DX 300 HPLC with a Waters µBondpak C18-RP column under an eluent gradient (44% acetonitrile/56% 10 mM KH2PO4 ramped to 53% acetonitrile/47% 10 mM KH2PO4 over 20 min). The UV detector was set at 249 nm, and the γ counter window was at 140 ( 30 keV. Elemental analyses were performed by Guelph Chemical Laboratories Ltd. (Guelph, Ontario). Na[99mTc]TcO4- was obtained from a commercial 99Mo/99mTc generator as supplied by Dupont. The receptor binding affinity was performed in the laboratories of J. A. Katzenellenbogen, Department of Chemistry, University of Illinois, Champagne-Urbana, IL. Mice were used with approval of the University of Western Ontario University Council on Animal Care and were cared for in accordance with the Canadian Council on Animal Care and the Animals for Research Act of the Province of Ontario. 2-(4-(3,3-Bis(triphenylmethylthio-acetamidomethylene)propanamido)phenyl)-1-(4-[2-(N,N-dimethylamino)ethoxy]phenyl)-1-phenyl-1(Z)-butene (Z-6). The amine Z-2 (112 mg, 0.291 mmol), 4 (210 mg, 0.275 mmol), dicyclohexylcarbodiimide (63.5 mg, 0.308 mmol), 1-hydroxybenzotriazole (49.6 mg, 0.324 mmol), and 40 µL of collidine (0.36 mmol) were added to 20 mL of CH2Cl2. The mixture was stirred under argon for 1 day at
Hunter and Luyt
which time the white precipitate (dicyclohexylurea, 56 mg) was filtered. The filtrate, with additional CH2Cl2, was transferred to a separatory funnel and washed with 1 M HCl (three times), 0.5 M Na2CO3 (two times), and saturated NaCl. The organic layer was dried over MgSO4 and filtered, and the solvent was removed by rotary evaporation. The crude product was then dried under vacuum (0.1 mmHg, RT) yielding 334 mg. A 1H NMR spectrum of the crude product indicated a mixture of Z-6, dicyclohexylurea, and a small quantity of starting material. Purification was carried out by flash chromatography (silica, column 23 × 80 mm), with ethyl acetate as the first eluent and 20% methanol/80% ethyl acetate to elute the product. This second eluent was placed in a flask and the solvent was removed by rotary evaporation, followed by drying under vacuum (0.05 mmHg, RT) to give a pale yellow solid. This was recrystallized from chloroform/hexanes yielding 203 mg (65% yield) of Z-6 as fine buff white crystals: mp 187-189 °C; 1H NMR spectrum (δ, CDCl3, RT) 0.89 ppm (t, 3H, CH3, J ) 7.4 Hz), 1.78 (m, 1H, CH), 1.90 (d, 2H, CH2), 2.29 (s, 6H, NCH3 × 2), 2.42 (q, 2H, CH2NMe2, J ) 7.4 Hz), 2.52 (m, 2H, CH2N), 2.65 (t, 2H, CH2N, J ) 5.8 Hz), 3.09 (s, 4H, CH2-S × 2), 3.18 (m, 2 H, CH2N), 3.92 (t, 2H, CH2O, J ) 5.8 Hz), 6.55 (ar, 2H), 6.77 (ar, 2H), 6.83 (m, 2H, NH × 2), 7.03 (ar, 2H), 7.20-7.40 (ar, 37 H), 9.76 (s, 1H, NH); 13C NMR spectrum (δ, CDCl , RT) 13.61 ppm (CH ), 28.80 3 3 (CH2 of ethyl), 36.03 (SCH2 × 2), 37.90 (CH2), 38.34 (CH), 40.01 (NCH2 × 2), 45.73 (NCH3 × 2), 58.14 (CH2NMe2), 65.30 (OCH2), 67.79 (CPh3), 113.35 (CdC), 118.98 (Cd C), 126.35 (ar), 126.97 (ar), 127.08 (ar), 127.98 (ar), 128.10 (ar), 129.27 (ar), 129.41 (ar), 130.07 (ar), 131.79 (ar), 135.57 (ar), 136.48 (ar), 137.70 (ar), 137.97 (ar), 140.74 (ar), 143.84 (ar), 156.57 (ar), 169.50 (CdO), 170.06 (Cd O); UV (95% ethanol) 296 nm (14 300 M-1 cm-1), 251 nm (25 000 M-1 cm-1), 202 nm (151 400 M-1 cm-1); Anal. calcd for C73H72N4O4S2: C, 77.35; H, 6.40; N, 4.94; O, 5.65; S, 5.66. Found: C, 77.80; H, 6.53; N, 5.22; O, 6.09; S, 5.30. 2-(4-(3,3-Bis(triphenylmethylthio-acetamidomethylene)propanamido)phenyl)-1-(4-[2-(N,N-dimethylamino)ethoxy)phenyl)-1-phenyl-1(E)-butene (E-6). The preparation of E-6 followed the procedure for Z-6 using 69 mg of E-2 (0.178 mmol) and 136 mg 4 (0.178 mmol). After column chromatography, E-6 was recrystallized from chloroform/hexanes yielding 139 mg (0.123 mmol, 69% yield) of fine buff white crystals: mp 200201.5 °C; 1H NMR spectrum (δ, CDCl3, RT) 0.92 ppm (t, 3H, CH3), 1.78 (m, 1H, CH), 1.89 (d, 2H, CH2), 2.38 (s, 6H, NCH3 x2), 2.48 (m, 4H, CH-N × 2 and CH2), 2.79 (t, 2H, CH2NMe2, J ) 5.7 Hz), 3.08 (s, 4H, CH2-S × 2), 3.16 (m, 2 H, CH-N × 2), 4.09 (t, 2H, CH2O, J ) 5.7 Hz), 6.85 (m, 2H, NH × 2), 6.86-7.43 (ar, 43 H), 9.75 (s, 1H, NH); 13C NMR spectrum (δ, CDCl3, RT) 13.60 ppm (CH3), 28.81 (CH2 of ethyl), 36.04 (SCH2 × 2), 37.90 (CH2), 38.29 (CH), 39.98 (NCH2 × 2), 45.56 (NCH3 × 2), 58.01 (CH2NMe2), 65.46 (OCH2), 67.74 (CPh3), 113.95 (CdC), 118.90 (CdC), 125.55 (ar), 126.94 (ar), 127.29 (ar), 128.07 (ar), 129.39 (ar), 130.04 (ar), 130.47 (ar), 130.74 (ar), 136.25 (ar), 136.48 (ar), 137.64 (ar), 138.06 (ar), 141.29 (ar), 143.29 (ar), 143.83 (ar), 157.25 (ar), 169.51 (CdO), 170.02 (CdO); Anal. calcd for C73H72N4O4S2: C, 77.35; H, 6.40; N, 4.94; O, 5.65; S, 5.66. Found: C, 77.60; H, 6.08; N, 4.94; O, 6.00; S, 5.32. 2-(4-(3,3-Bis(thioacetamidomethylene)propanamido)phenyl)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1-phenyl-1(Z)-butene (Z-7). Z-6 (149 mg, 0.131 mmol) was dissolved in 4 mL of methylene chloride and was cooled to 0 °C on an ice bath. To the clear and
Single Isomer Tc-99m Tamoxifen Conjugates
colorless stirred solution was added 4 mL of cold (0 °C) trifluoroacetic acid resulting in an immediate yellow color. Triethylsilane (50.2 µL, 0.30 mmol) was then added with the mixture slowly turning to a clear colorless solution. After 30 min the reaction was warmed to RT and was transferred to a separatory funnel with an additional 20 mL of methylene chloride. The mixture was washed with H2O and 1 M Na2CO3. The organic layer was placed in a flask and the solvent was removed by rotary evaporation. The resulting solid was repeatedly triturated with hexane to remove the triphenylmethane, leaving behind Z-7 as a pale yellow solid: HPLC, tr ) 25.0 min, area ) 99.1%. 2-(4-(3,3-Bis(thioacetamidomethylene)propanamido)phenyl)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1-phenyl-1(E)-butene (E-7). The preparation of E-7 followed the identical procedure as used for Z-7. The dithiol E-7 was recovered as a white solid: HPLC E-7 (21.1 min, 90.6%), Z-7 (24.5 min, 9.4%). anti- and syn-2-(4-(3,3-Bis(thioacetamidomethylene)propanamido)phenyl)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1-phenyl-1(Z)-butene Oxorhenium (V) [anti- and syn-(Z)-8]. A flask was charged with Z-7 (85 mg, 0.131 mmol), ReOCl3(PPh3)2 (111 mg, 0.133 mmol), and 5 mL of 1 M sodium acetate in methanol (degassed by bubbling argon through the solvent). The reaction was heated at reflux under an argon blanket for 5 h. After cooling to room temperature, the solvent was removed by a rotary evaporator and dried under 0.3 mmHg overnight. The resulting pale orange powder was washed well with water leaving 72 mg as a mixture of anti-(Z)-8 and syn-(Z)-8 (63% crude yield): HPLC (UV at 249 nm) 16.00 min (46.1% area), 16.75 min (42.0%); HRMS FAB (negative ion, thioglycine and oxalic acid matrix) m/z calcd for C35H40N4O5S2187Re 847.1998; found 847.1998 (100%); calcd for C35H40N4O5S2185Re 845.1970; found 845.1970 (59%). anti-2-(4-(3,3-Bis(thioacetamidomethylene)propanamido)phenyl)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1-phenyl-1(Z)-butene Oxorhenium (V) [anti-(Z)-8]. Z-6 (222 mg, 0.196 mmol) was deprotected as previously described. The crude Z-7 was dissolved in 25 mL of methanol, to which was added trans-bis(ethylenediamine)dioxorhenium (V) chloride (84 mg, 0.208 mmol) and 8 mL of 10% tetraethylammonium hydroxide aqueous solution. The reaction mixture was heated at reflux for 1 h and cooled to RT, and 10 mL of water was added. The methanol volume was reduced by a rotary evaporator, resulting in precipitation of anti(Z)-8 as a pale orange powder (108 mg, 56% yield): mp 129-132 °C; 1H NMR spectrum (δ, CDCl3, RT) 0.87 ppm (t, 3H, J ) 7.4 Hz, CH3), 1.20 (t, 12H, J ) 7.1 Hz, CH3 of NEt4), 2.25 (s, 6H, NCH3 × 2), 2.39 (q, 2H, J ) 7.4 Hz, CH2 of ethyl), 2.50 (m, 1H, CH), 2.62 (m, 4H, CH2NMe2 and CH2CO), 2.89 (m, 2H, CHN × 2 axial), 3.17 (q, 8H, J ) 7.1 Hz, CH2 of NEt4), 3.80/3.88 (AB, 4H, CH2S × 2, J ) 17.1 Hz), 3.92 (t, 2H, OCH2), 4.01 (m, 2H, CHN × 2 equatorial), 6.54 (ar, 2H), 6.75 (ar, 2H), 7.01 (ar, 2H), 7.07-7.33 (ar, 5H), 7.40 (ar, 2H), 9.10 (s, 1H, NH); 13C NMR spectrum (δ, CDCl3, RT) 7.53 (CH3 of NEt4), 13.67 ppm (CH3), 28.85 (CH2 of ethyl), 38.79 (SCH2 × 2), 40.33 (CH2CO), 41.19 (CH), 45.80 (NCH3 × 2), 52.36 (CH2 of NEt4), 55.35 (NCH2 × 2), 58.21 (CH2NMe2), 65.63 (OCH2), 113.49 (CdC), 119.13 (CdC), 126.45 (ar), 128.04 (ar), 128.22 (ar), 129.35 (ar), 129.97 (ar), 131.88 (ar), 135.65 (ar), 136.75 (ar), 137.67 (ar), 140.60 (ar), 143.78 (ar), 156.61 (ar), 170.03 (NHCdO), 196.04 (NCdO); IR 969 cm-1 (RedO); HPLC anti-(Z)-8 16.4 min; HRMS FAB
Bioconjugate Chem., Vol. 11, No. 2, 2000 177
(negative ion, thioglycine and oxalic acid matrix) m/z calcd for C35H40N4O5S2187Re 847.1998, found 847.2005 (100%). 2-(4-(3,3-Bis(thioacetamidomethylene)propanamido)phenyl)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1-phenyl-1(Z)-butene Oxotechnetium99m (V) (Z-5). A 3 mL conical vial was charged with 28.1 mCi freshly eluted [99mTc]TcO4- in 500 µL of normal saline, 400 µL of 0.5 mM Z-7 (50% ethanol/water solution), 40 µL of 1 M NaOH and 80 µg of SnCl2 in 40 µL of 0.1 M HCl. The vial was sealed with a Teflon-lined cap and heated at 90 °C for 30 min. After cooling to room temperature, a 29.7 µCi aliquot (2.5 µL) was checked by HPLC and found to contain 16.6% [99mTc]TcO4- and 83.4% product. The remaining 26.1 mCi was placed onto a Sep-Pak (Waters C18 1 cm3) and was eluted with 3 mL of distilled water removing 3.22 mCi. No additional radioactivity was being eluted with additional water. The Sep-Pak was then flushed with 1 mL of ethanol removing 18.9 mCi. An additional ethanol flush removed only 0.06 mCi and was not used. An aliquot of the ethanol eluent was checked by HPLC, with one γ peak appearing at a retention time of 16.6 min (area ) 100.00%). The ethanol eluent was placed into a small test tube in a warm water bath, and air was bubbled through it until the volume was reduced to less than 0.1 mL. To this was added 9.0 mL of saline solution (2% ethanol, 0.1% Tween), and the solution was filtered through a sterile 0.22 µm Millex GS filter yielding 15.2 mCi of dissolved product. Purity of the radiopharmaceutical was checked by HPLC analysis of a 53 µCi aliquot (30 µL) resulting in a single peak on the γ trace, eluting at 16.6 min with 100.00% purity. The UV chromatogram shows very little UV absorbable impurities, indicating that the dithiol starting material has been removed. Radiochemical yield corrected for decay is 71%. 2-(4-(3,3-Bis(thioacetamidomethylene)propanamido)phenyl)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1-phenyl-1(E)-butene Oxotechnetium99m (V) (E-5). A 3 mL conical vial was charged with 517 µCi freshly eluted [99mTc]TcO4- in saline, 160 µL of 0.5 mM E-7 (50% ethanol/water solution filtered through a 0.22 µm syringe tip filter), 20 µL of 1 M NaOH and 0.04 mg SnCl2 in 20 µL of 0.1 M HCl. The vial was sealed with a Teflon lined cap and heated at 90 °C for 30 min. After cooling to room temperature an 75.0 µCi aliquot (40 µL) was checked by HPLC and found to contain 35.8% [99mTc]TcO4- (RT ) 3.0 min.), 58.4% anti-(E)-5 (RT ) 11.9 min) and 5.8% anti-(Z)-5 (RT ) 16.1 min). The remaining 349 µCi was placed onto a Sep-Pak (Waters C18, 1 cm3) and was flushed with 2 mL of distilled water removing 127 µCi. No additional radioactivity was being eluted with additional water. The Sep-Pak was then eluted with 1 mL of ethanol removing 213 µCi. The Sep-Pak was found to contain only 7 µCi of residual radioactivity. An 31 µCi aliquot of the ethanol eluent was checked by HPLC and was found to contain 0.3% [99mTc]TcO4- (RT ) 2.5 min), 93.9% anti-(E)-5 (RT ) 10.8 min) and 5.8% anti-(Z)-5 (RT ) 16.0 min). The radiochemical yield is 61%, with a radiochemical purity of 99.7%, and an E:Z ratio of 16:1. Biodistribution of anti-(Z)-5. An in vivo biodistribution study was carried out using female C3H mice (18.5-22.0 g; 20-25 weeks of age). The [99mTc]-anti-(Z)tamoxifen [anti-(Z)-5] was dissolved in a solution of saline containing 2% ethanol and 0.1% Tween (pH 5-5.5). A total of 100 µL of this solution was injected into the tail vein of the mouse with the syringe being counted preand postinjection. The mice were sacrificed at 1, 3, 24,
178 Bioconjugate Chem., Vol. 11, No. 2, 2000
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Table 2. Selected 1H NMR (CDCl3, 300 MHz) Chemical Shifts (ppm) for Z- and E-6
protons
Z-6
E-6
OCH2 (of ethoxy) NCH2 (of ethoxy) ortho of Ar-1 meta of Ar-1
3.92 2.52 6.77 6.55
4.09 2.79 >6.86 >6.86
Table 3. Comparison of 1H NMR (300 MHz, δ ppm) Spectroscopy for Protons near the Rhenium Chelation Site (Z)-8 anti
9 anti
10 syn
anti
syn
NCH2 (axial) 2.96 2.63 3.31 2.87 (equatorial) 3.92 3.78 3.88 3.92 SCH2 3.65/3.76 3.57/3.64 3.48/3.79 3.62/3.74 3.62/3.76
3.42 3.86 3.76/3.84 4.04/4.09
Table 4. In Vivo Biodistribution of (anti)-Z-5 in Mice at Various Time Points (4 or 5 mice/time point) % injected dose/gram of tissue at four time points tissue
1h
3h
24 h
48 h
blood lung liver kidney uterus muscle
0.88 ( 0.20 0.54 ( 0.17 7.78 ( 2.39 1.43 ( 0.69 0.30 ( 0.10 0.15 ( 0.12
0.47 ( 0.18 0.28 ( 0.10 4.00 ( 0.75 0.27 ( 0.05 0.16 ( 0.07 0.10 ( 0.10
0.15 ( 0.12 0.49 ( 0.39 1.24 ( 0.51 0.19 ( 0.16 0.18 ( 0.16 0.26 ( 0.32
0.10 ( 0.05 0.28 ( 0.19 0.66 ( 0.26 0.23 ( 0.10 0.33 ( 0.23 0.31 ( 0.19
or 48 h postinjection with four or five mice at each time point. A blood sample was immediately removed followed by dissection of the lungs, liver, kidneys, uterus, muscle, and tail. The tissues were weighed and then stored in a freezer until all the dissections were complete. The tissues were then counted in a γ well counter along with standards with a total count time per sample of 10 min. The radioactivity uptake, as presented in Table 4, is expressed as the percentage of the injected dose per gram of tissue. Estrogen Receptor Binding Affinity. The relative binding affinity (RBA) value for anti-(Z)-5 was determined by a previously reported competitive radiometric binding assay (16). Lamb uterine cytosol, diluted to approximately 1.3 nM of receptor, was incubated with 10 nM [3H]estradiol for19 h at 0 °C. Free ligand was removed by absorption onto dextran-coated charcoal. The ligand concentration was altered from 10-4 to 10-9 M. Estradiol was run as a control with 50% inhibition occurring at 10 nM. The binding affinity of anti-(Z)-5 relative to estradiol ()100) was too low to measure, with a RBA of 95%, with the sole contaminant, as discovered by HPLC analysis, being (E)-8. In this reaction, the anionic product remained in solution, allowing for it to be precipitated as a tetraethylammonium salt by the removal of methanol. This single isomer product was characterized by 1H and 13C NMR spectroscopy, which allowed for its assignment as anti-(Z)-8. Table 3 presents a comparison of the chemical shifts of anti-(Z)-8 to two other anti-rhenium products (14). The chemical shifts and coupling constants for the protons of the chelate core are consistent with this isomer being the anti-epimer. This is especially apparent for the NCH2 axial protons, where the antiepimers invariably appear between 2.6 and 3.0 ppm, with two large coupling constants. The chromatographic behavior of this isomer is also consistent with it being the anti-isomer, in that it elutes as the first product as seen in Figure 3d. The product was further characterized by high-resolution FAB mass spectrometry with the parent signal for the 187Re complex at 847.2005 m/z and by FTIR spectroscopy with the RedO absorption occurring at 969 cm-1. Radiolabeling and Biological Evaluation. Radiolabeling of the dithiol tamoxifen analogue 7 was performed using Na[99mTc]TcO4 with tin(II) chloride as the reducing agent. Under mildly basic conditions (0.1 M sodium acetate) and D-gluconate, very little chelation occurred with low radiochemical yields. When 1 M NaOH
180 Bioconjugate Chem., Vol. 11, No. 2, 2000
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Figure 5. HPLC chromatograms (radiometric γ detector) of [99mTc]tamoxifen analogues: (a) crude reaction yielding anti-Z5; (b) anti-Z-5 after C18 Sep-Pak purification; (c) anti-E-5 after C18 Sep-Pak purification.
Figure 3. HPLC chromatograms (UV detector) of Z-8: (a) rhenium chelation yielding both anti-Z-8 and syn-Z-8; (b) after mixture is exposed to 1 M NaOH; (c) co-injection; (d) rhenium chelation under basic conditions yielding only anti-Z-8.
Figure 4. Previously reported anionic rhenium complexes (13, 20).
was added to the reaction medium, radiochemical yields of over 80% were achieved. The addition of D-gluconate for ligand exchange, did not improve the yield or product purity and was, therefore, not further employed. As seen in Figure 5a, the chelation for the Z-isomer is very clean, yielding essentially a single product with an HPLC retention time of 16.6 min and a small trailing peak. The crude chelation reaction mixture typically contained traces of residual [99mTc]TcO4- eluting just after the solvent front. For the E-isomer, the crude reaction mixture was also checked by HPLC analysis with the γ trace, indicating that pertechnetate, E-5, and Z-5 are all present with the desired E-isomer being obtained in 79% radiopurity. Purification was carried out for both isomers using a C18 Sep-Pak column. The aqueous solution was passed through the column, with the pertechnetate being washed out with water. Elution with ethanol yielded the [99mTc]tamoxifen product 5. HPLC analysis of these eluents (Figure 5b for Z-5 and Figure 5c for E-5) indicates the product is >99% radiochemically pure, although the E-isomer is contaminated with 5.8% of Z-5.
For biodistribution experiments, the ethanol eluent volume was reduced and a saline solution containing 2% ethanol and 0.1% Tween-80 was added. The solution was then filtered through a sterile 0.22 µm filter yielding the final pure product with a radiochemical yield (decay corrected) of 71%. HPLC analysis again indicated a radiochemical purity of >99%. It should be mentioned that Z-5 is not soluble in saline, but is soluble in saline with 10% ethanol. It was necessary to introduce the surfactant Tween-80 in order to keep the concentration of ethanol to an appropriate level (2%) for injection into mice. One requirement for a radiopharmaceutical to be suitable for a biodistribution study is that it be free of any nonradioactive tamoxifen dithiol (7), as this would potentially bind to the estrogen receptors, thereby competing with the radiopharmaceutical. HPLC analysis of the 99mTc reaction product indicated that there existed very little residual dithiol, as deduced by the lack of UV absorbance at 25 or 21 min (for Z-7 and E-7, respectively). Since the dithiol is being used in a very large excess as compared to the pertechnetate, it was presumed that the dithiol is being oxidized during the reaction. To validate this hypothesis, a study of the oxidation of Z-7 was carried out by HPLC monitoring. It was determined that oxidation is occurring when the dithiol is heated in the presence of sodium hydroxide solution, presumably with oxygen as the oxidant. Oxidation also occurs at room temperature, but at a much slower rate. All oxidation products elute with or shortly after the solvent front and are removed by the Sep-Pak purification. The biodistribution results of anti-(Z)-5 in C3H female mice are summarized in Table 4 as percentage of the injected dose per gram of tissue. C3H mice were chosen for this study to allow for comparison of the results to a previous study of radioiodinated tamoxifen derivatives (10). Four time points for sacrificing were used, including 24 and 48 h time points in order to allow for comparison to a previous study of [131I]tamoxifen, where the maximal uterus uptake occurred between 12 and 48 h (10). The overall radioactivity found in the tissues was very low,
Single Isomer Tc-99m Tamoxifen Conjugates
with an average 9.5% of the injected dose being recovered at the 1 h time point. This suggests very rapid clearance of the radiopharmaceutical. Since the uterus is a major target organ for estrogens, it was expected that significant uptake would be observed in that tissue. Selective uterine uptake was not observed in this biodistribution study, with the percent injected dose per gram of uterine tissue remaining below 0.4% throughout all of the time points. A blocking study was not performed due to this low uterine uptake. An estrogen receptor binding study of the rhenium analogue anti-(Z)-8 using lamb uterine tissue gave a relative binding affinity of
