IN VIVO STABLE HG-197(M) COMPOUNDS, METHOD FOR THE PRODUCTION THEREOF AND USE THEREOF IN NUCLEAR MEDICAL DIAGNOSTICS AND ENDORADIONUCLIDE THERAPY (THERANOSTICS)
20210228748 · 2021-07-29
Assignee
Inventors
- Hans-Jürgen PIETZSCH (Heidenau, DE)
- Martin WALTHER (Ottendorf-Okrilla, DE)
- Thomas WÜNSCHE (Neusalza-Spremberg, DE)
Cpc classification
A61K51/0472
HUMAN NECESSITIES
A61K47/6849
HUMAN NECESSITIES
C07B2200/05
CHEMISTRY; METALLURGY
C07F17/02
CHEMISTRY; METALLURGY
International classification
A61K47/68
HUMAN NECESSITIES
C07B59/00
CHEMISTRY; METALLURGY
C07F17/02
CHEMISTRY; METALLURGY
Abstract
The present invention relates to in vivo stable .sup.197(m)Hg compounds according to formula (E) for use in nuclear medical diagnostics and endoradionuclide therapy (theranostics), particularly the treatment of cancer, a method for the production of the .sup.197(m)Hg compounds comprising the step of radiolabeling of organic precursor compounds with NCA .sup.197(m)Hg by electrophilic substitution; and the use of the .sup.197(m)Hg compounds for nuclear medical diagnostics and endoradionuclide therapy (theranostics), particularly the treatment of cancer.
Claims
1. A .sup.197(m)Hg compound according to the following formula ##STR00044## wherein: both .sup.197(m)Hg-substituents Ar and Y are linked by at least one aliphatic and/or aromatic spacer molecule; the curved line is the aliphatic and/or aromatic spacer molecule; Ar is an unsubstituted or substituted -aryl or -heteroaryl group; and Y is an unsubstituted or substituted -aryl or -heteroaryl group.
2. The .sup.197(m)Hg compound according to claim 1, wherein the aliphatic and/or aromatic spacer comprises 4-40 Atoms, the atoms comprising 2-5 heteroatoms selected from N, O, S and P.
3. The .sup.197(m)Hg compound according to claim 1 wherein both .sup.197(m)Hg-substituents Ar and Y are the same, according to formulas (I*.sub.bridge), (Ia*.sub.bridge) or (Ib*.sub.bridge) ##STR00045##
4. The .sup.197(m)Hg compound according to claim 3 wherein the aliphatic and/or aromatic spacer is connected to both .sup.197(m)Hg-substituents in ortho position to the bond to .sup.197(m)Hg.
5. The .sup.197(m)Hg compound according to claim 1 having a specific activity of at least 100 GBq/μmol based on the amount of mercury.
6. The .sup.197(m)Hg compound according to claim 1, wherein Ar and/or Y comprise at least one amino acid, peptide, protein, antibody, oligonucleotide, alkaloid residue and/or aliphatic spacer.
7. The .sup.197(m)Hg compound according to claim 1, wherein —Y is selected from unsubstituted or substituted phenyl groups as shown in formula (IV) ##STR00046## wherein R.sup.7 is selected from H, unsubstituted or substituted alkyl groups, alkoxy groups with formula —OR.sup.8, amide groups with formula —CON(R.sup.8).sub.2, carboxy groups with formula —COOR.sup.8, aryl or heteroaryl groups, wherein R.sup.8 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -aryl or -heteroaryl groups.
8. The .sup.197(m)Hg compound according to claim 3, wherein n is 1, according to formula (VI) ##STR00047## wherein X is selected from H, unsubstituted or substituted alkyl groups, alkoxy groups with formula —OR.sup.1, amide groups with formula —CON(R.sup.1).sub.2, carboxy groups with formula —COOR.sup.1, aryl or heteroaryl groups, wherein R.sup.1 is selected from H, unsubstituted or substituted C1 to C15-alkyl, succinimidyl, -aryl or -heteroaryl groups.
9. A method for nuclear medical diagnostics and endoradionuclide therapy of cancer comprising the step of administering to a subject in need thereof a pharmaceutical composition containing a therapeutically effective amount of the .sup.197(m)Hg compound according to claim 1.
10. A method for the production of .sup.197(m)Hg compounds according to claim 1 comprising: a) providing an organic precursor compound, b) synthesizing no carrier added (NCA).sup.197(m)Hg, and c) radiolabeling of the organic precursor compound with the no carrier added (NCA) .sup.197(m)Hg by electrophilic substitution.
11. The method according to claim 10, wherein the organic precursor compound is an organotin precursor compound, a boron precursor compound or a silicon precursor compound.
12. The method according to claim 11, wherein the organic precursor compound is a trialkyl-tin precursor compound.
13. The method of claim 10, wherein the synthesis of NCA .sup.197(m)Hg according to step b) is carried out by irradiation of gold (Au) with a cyclotron.
14. The method according to claim 10, wherein the radiolabeling of the organic precursor compound according to step c) is carried out at a pH value between pH 1.0 and 5.0 to form asymmetric .sup.197(m)Hg compounds.
15. The method according to claim 10, wherein the radiolabeling of the organic precursor compound according to step c) is followed by reaction of activated ester groups by ester hydrolysis, reaction with amino groups or reaction with hydroxyl groups of an amino acid, peptide, protein, antibody, oligonucleotide, alkaloid residue and/or aliphatic spacer.
16. The method according to claim 11 wherein in step a) an organic precursor compound according to formula (E.sub.bridge-prec) is provided: ##STR00048## wherein both Ar and Y are linked by at least one aliphatic and/or aromatic spacer molecule, wherein Ar is an unsubstituted or substituted -aryl or -heteroaryl group, and Y is an unsubstituted or substituted -aryl or -heteroaryl group; M is Sn, B or Si; R.sup.10 is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups, and i is 2 or 3.
17. An organic precursor compound according to the formula (E.sub.bridge-prec) ##STR00049## wherein both Ar and Y are linked by at least one aliphatic and/or aromatic spacer molecule, wherein Ar is an unsubstituted or substituted -aryl or -heteroaryl group, and Y is an unsubstituted or substituted -aryl or -heteroaryl group; M is Sn, B or Si; R.sup.10 is selected from H, unsubstituted or substituted C1 to C15-alkyl, -aryl or -heteroaryl groups, and i is 2 or 3.
Description
FIGURES AND EXAMPLES
[0211] The present invention will now be further explained by the following non-limiting figures and examples.
[0212]
[0213]
[0214]
[0215]
[0216]
GENERAL SYNTHETIC TECHNIQUES
[0217] All Chemicals were used without further purification and in the highest degree of purity.
[0218] Sodium hydroxide in suprapur quality was purchased from Merck (Darmstadt, Germany). Methyl isobutyl ketone (MIBK) was purchased from Sigma-Aldrich (St. Louis, USA). The routine activity measurement was performed with an Isomed 2000 from MED (Nuklear-Medizintechnik Dresden GmbH, Dresden, Germany) calibrated by γ-ray spectroscopy measurements after decaying .sup.197(m)Hg. ICP-MS measurements were carried out on an ELAN 9000 (PerkinElmer SCIEX, Waltham, USA).
[0219] Gamma-Ray Spectroscopy
[0220] For γ-ray spectroscopy measurements a reverse electrode HPGe detector (CANBERRA GR2018, 19.6% rel. efficiency) in a low-background Pb shielding was used with the sample at 10 cm distance from the detector end cap. It was operated with the software InterWinner version 7.1. The system was calibrated using a mixed standard solution (57Co, 85Sr, 88Y, 60Co, 109Cd, 113Sn, 137Cs, 139Ce, 203Hg, 241Am) with a volume of 0.38 mL in the tip of a 1.5 mL Eppendorf vial. The energy depending detector efficiency was calculated from these calibration points using the algorithms of the spectroscopy software. The samples were measured in similar geometry, but smaller volume of 1-10 μl in the tip of a 1.5 mL Eppendorf vial thus, no further corrections were necessary with except of decay correction. Pile-up effects were observed, especially at higher activities. Nevertheless, no corrections are made, because the effects are less than the simple standard deviation and thus negligible. For the determination of Hg-activities only the γ-ray lines >100 keV have been used, in particular for the isomer 197mHg only the lines ˜134 keV and ˜165 keV of the isomeric transition and for the isomer 197Hg only the lines ˜191 keV and ˜269 keV are discussed in the activity calculation.
[0221] NMR and IR Spectroscopy
[0222] .sup.1H and .sup.13C NMR spectra were recorded with a Varian Inova-400 spectrometer. The chemical shifts were reported relative to the standard tetramethylsilane (TMS). IR spectra were measured with a Fisher Scientific Nicolet iS5 FTIR spectrometer.
[0223] Thin Layer Chromatography (TLC)
[0224] Thin layer chromatography was performed using RP18 plates (Merck), developed in a 1:1 mixture H.sub.2O with 0.1% trifluoroacetic acid (TFA) (A) and CH.sub.3CN with 0.1% TFA (B) and analyzed with a Raytest Linearanalyser RITA.
[0225] Radio-TLC is the detection of radioactive species separated by TLC with radiation detector to determine the radiochemical purity or to quantify the radioactive species.
[0226] The radiochemical yield is the yield of the radionuclide and was calculated by the specific activity of the .sup.197(m)Hg compound divided by the specific activity of the no carrier added (NCA).sup.197(m)Hg.
[0227] High-Performance Liquid Chromatography (HPLC) Measurements
[0228] Radiochemical purity was determined by radio-HPLC. All HPLC runs are performed under the same conditions with the same HPLC-equipment. Column: Zorbax C18 column with inner diameter of 8 mm. Mobile phase: H.sub.2O with 0.1% TFA (A) and CH.sub.3CN with 0.1% TFA (B). Flow rate: 3 mL/min. HPLC gradient of B phase: in 0 to 20 min from 45% to 80%, in 20 to 25 min from 80% to 100%.
[0229] Mass Spectrometry (Electrospray Ionization (ESI)-MS, Matrix-Assisted Laser Desorption/Ionization (MALDI)-MS)
[0230] For mass spectrometry a QuadroLC by Micromass with electrospray ionisation (ESI) mode and a Bruker MALDI-TOF MS instrument (MALDI) were used.
1. Synthesis of an Organic Precursor Compound
N.SUP.1.,N.SUP.3.-bis(3-iodobenzyl)isophthalamide
[0231] ##STR00023##
[0232] 3-iodobenzylamine hydrochloride salt (4 g, 14.84 mmol) was dissolved in chloroform (100 ml) in a 250 ml round-bottomed flask. To this was added triethylamine (10.3 ml, 0.074 mol) followed by isophthaloyl chloride (1.51 g, 7.42 mmol). The flask was fitted with a CaCl.sub.2) drying tube and the colourless solution was left to stir at room temperature overnight. The reaction was monitored by TLC using 19:1 dichloromethane (DCM)/methanol (MeOH). The reaction mixture was washed with 3:1 water/saturated NaHCO.sub.3(aq.) (3×50 ml), then with 0.1 M HCl.sub.(aq.) (3×50 ml), then with deionized water (2×30 ml). The product is mostly insoluble in chloroform and precipitates during the aqueous washes, thus further dilution with chloroform helps separation. The product was purified by simple recrystallization of cooling the chloroform. Impurities dissolved in the solvent were decanted. This process was repeated to increase yield. The product was washed lightly with cold chloroform and after drying left a white powder (1.02 g, 92% yield).
[0233] .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm): 8.23 (s, 1H), 7.92 (dd, J=7.8, 1.6 Hz, 2H), 7.64 (s, 2H), 7.59 (d, J=7.9 Hz, 2H), 7.48 (t, J=7.8 Hz, 1H), 7.28 (s, 1H), 7.04 (d, J=7.8 Hz, 2H), 6.84 (d, J=5.3 Hz, 2H), 4.52 (d, J=5.8 Hz, 4H),
[0234] .sup.13C NMR (101 MHz, CDCl.sub.3) δ (ppm): 166.57, 140.37, 136.93, 134.52, 130.64, 130.40, 129.27, 127.32, 125.67, 94.78, 43.61.
N.SUP.1.,N.SUP.3.-bis(3-(trimethylstannyl)benzyl)isophthalamide
[0235] ##STR00024##
[0236] N.sup.1,N.sup.3-bis(3-iodobenzyl)isophthalamide (0.97 g, 1.63 mmol) was dissolved in 1,4-dioxane (20 ml) in a 50 ml 3-necked round-bottomed flask. A glass bubbler allowed argon to bubble through the solution with a coiled water condenser attached to the top along with a bubble counter to monitor argon flow. A catalytic amount of tetrakis(triphenylphosphine)palladium(0) (20.4 mg, 16.3 μmol) orange crystals were added forming a clear pale yellow solution. This was followed by an excess of hexamethylditin (3.16 ml, 15.26 mmol). Rinsing of sample phials and addition funnel brought the total solvent volume to 30 ml. The reaction mixture was heated by an oil bath (125° C.) and stirred for 8 h. The reaction was monitored by TLC using 1:1 ethanol (EtOH)/n-hexane. The reaction mixture turned a dark orange with a cloudy precipitate. This was filtered to remove most of the brown precipitate. The solvent was removed by evaporation and the product purified by flash column chromatography using EtOH/n-hexane. Drying yielded a white powder (0.164 g, 15% yield).
[0237] .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ (ppm): 9.11 (broad t, 2H), 8.38 (s, 1H), 8.01 (dd, J=7.8, 1.6 Hz, 2H), 7.58 (t, J=7.8 Hz, 1H), 7.53-7.22 (m, 8H), 4.47 (d, J=5.9 Hz, 4H), 0.25 (s, 18H),
[0238] .sup.13C NMR (101 MHz, CDCl.sub.3) δ (ppm): 166.41, 143.39, 137.31, 135.72, 135.46, 134.92, 130.12, 129.18, 128.61, 128.22, 125.51, 44.68, −9.35.
2. Production of No-Carrier-Added .SUP.197(m).Hg
[0239] The irradiations were performed at a Cyclone 18/9 cyclotron (IBA, Louvain la Neuve, Belgium, 18 MeV protons) located at Dresden-Rossendorf. A 1.0 mm aluminum foil (high purity aluminum, 99.999%) from Goodfellow (Huntingdon, England) was used as vacuum window. As target material massive high purity gold disks (23 mm diameter, 2 mm thickness, N5 purity 99.999%) were purchased from ESPI (Ashland, USA). Alternative gold targets consisted of a gold foil (12.5×12.5 mm, 0.25 mm thickness, 99.99+%) or a small gold disk (10 mm diameter, 0.125 mm thickness, 99.99+%, Pt content: 45±5 ppm quantified per ICP-MS) between an aluminum disk (22 mm diameter, 1 mm thickness, 99.0%, hard) and an aluminum lid (23 mm diameter, 99.0%, hard) purchased from Goodfellow (Huntingdon, England). Hydrochloric acid (30%) and nitric acid (65%) were purchased from Roth (Karlsruhe, Germany) in Rotipuran® Ultra quality. Deionized water with >18 MΩcm resistivity was prepared by a Milli-Q® system (Millipore, Molsheim, France). LN resin was purchased from Triskem International (Bruz, France). The gold target was irradiated for 120 min with a 25 μA current of 10 MeV protons resulting in 200 MBq of .sup.197(m)Hg. The irradiated gold foil was dissolved in 700 μl of aqua regia (freshly prepared 1 h before EOB from 525 μl 30% HCl+175 μl 65% HNO.sub.3) at room temperature. The gold disk was completely dissolved after 50 to 60 min. The column preparation was carried out directly before use by loading 3.6 g LN resin slurried with 10 ml of 6 M HCl onto the column and rinsing with additional 30 ml of 6 M HCl. After dilution of the 700 μl product solution with 300 μl 6 M HCl, this mixture was loaded onto the column and eluted with 6 M HCl in 1 ml aliquots.
[0240]
3. Radiolabeling of the Organic Precursor Compound with the No Carrier Added (NCA) .SUP.197(m).Hg by Electrophilic Substitution
General Synthetic Procedure for Synthesis of Diphenyl.SUP.nat.Mercury Compounds (Reference) Based on Sn-Precursors:
[0241] A solution of one equivalent mercury (II)-chloride was added to a solution of two equivalents tin-precursor in acetonitrile. The immediately starting precipitation of the product was completed by addition of ice cooled diethyl ether after 2 h mixing at room temperature. Centrifugation followed by washing the residue with cold diethyl ether results in a colorless microcrystalline product.
Bis(4-(N-succinimidyl)benzoate)mercury (II) (Reference)
[0242] ##STR00025##
[0243] A solution of one equivalent mercury (II)-chloride (5.5 mg, 20 μmol) in 1.5 ml acetonitrile was added to a solution of two equivalents tin-precursor N-succinimidyl-4-(tri-n-butylstannyl)benzoate (21 mg, 41 μmol) in 1.5 ml acetonitrile. The immediately starting precipitation of the product was completed by addition of ice cooled diethyl ether after 2 h mixing at room temperature. Centrifugation followed by washing the residue with cold diethyl ether results in a colorless microcrystalline product.
C.sub.22H.sub.16HgN.sub.2O.sub.8, Chemical Formula:
[0244] Molecular Weight: 636.97 g/mol,
[0245] .sup.1H-NMR (400 MHz, DMSO-D.sub.6) δ (ppm): 2.89 (s, 8H); 7.77 (d, 4H); 7.99 (d, 4H),
[0246] .sup.13C-NMR (100 MHz, DMSO-D.sub.6) δ (ppm): 25.5 (CH.sub.2); 123.5 (C); 128.8 (CH); 137.8 (CH); 161.9 (C); 162.0 (C); 170.3 (C), yield: 7 mg (15.4 μmol; 77%),
[0247] ESI.sup.+ m/z: 637 [M].sup.+; 539 [M-NHS].sup.+.
General Synthetic Procedure for Synthesis of Radiolabeled Diphenyl-Mercury Species—Based on Sn-Precursors
[0248] The .sup.197(m)Hg chloride stock solution in 0.2 M HCl is adjusted to pH 6 by adding 100 μl 0.2 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer and 5-10 μl 1 M NaOH. A solution of 1-10 μg trialkyltin precursor in 50-100 μl dimethyl sulfoxide (DMSO) is added to this buffered .sup.197(m)Hg chloride solution and mixed at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (acetonitrile (ACN)/H.sub.2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).
[.SUP.197(m).Hg] Bis(4-(N-succinimidyl)benzoate)mercury(II)
[0249] ##STR00026##
[0250] The .sup.197(m)Hg chloride solution in 0.2 M HCl is adjusted to pH 6 by adding 100 μl 0.2 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer and 5-10 μl 1 M NaOH. A solution of 10 μg (20 nmol) N-succinimidyl-4-(tri-n-butylstannyl)benzoate in 100 μl DMSO is added to 110 μl of this buffered .sup.197(m)Hg chloride solution (45 MBq [.sup.197(m)Hg] mercury) and mixed at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (ACN/H.sub.2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).
[0251] Radiochemical yield (TLC): ≥95%,
[0252] Radiochemical purity (TLC): ≥95%
[0253] Radio-TLC: R.sub.f=0.45 (ACN/H.sub.2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
General Synthetic Procedure for Synthesis of Diaryl/Heteroaryl.SUP.nat.Mercury Compounds (HPLC Reference)-Based on B-Precursors
[0254] (See Ref. Partyka et al., J. Organometallic Chemistry):
[0255] A mixture of one equivalent mercury (II)-acetate (5 μmol), ten equivalents boronic acid (50 μmol) and ten equivalents cesium carbonate (50 μmol) in 1 ml propane-2-ol was tempered at 50° C. for 20 h. After cooling and drying the mixture by rotary evaporation the product was extracted from the residue with toluene or THF purified by HPLC and identified by mass spectrometry.
Di(thiophen-2-yl)mercury
[0256] ##STR00027##
[0257] A solution of one equivalent mercury (II)-acetate (1.6 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents 2-thienylboronic acid (6.4 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.
C.sub.8H.sub.6HgS.sub.2, Chemical Formula:
[0258] Molecular Weight: 366.85 g/mol,
[0259] ESI.sup.+ m/z: 369 [M].sup.+.
Bis(5-carboxythiophen-2-yl)mercury
[0260] ##STR00028##
[0261] A solution of one equivalent mercury (II)-acetate (1.6 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents 5-(Dihydroxyboryl)-2-thiophenecarboxylic acid (8.5 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.
C.sub.10H.sub.6HgO.sub.4S.sub.2, Chemical Formula:
[0262] Molecular Weight: 454.86 g/mol,
[0263] ESI.sup.+ m/z: 457 [M].sup.+.
Di(ferrocenyl)mercury
[0264] ##STR00029##
[0265] A solution of one equivalent mercury (II)-acetate (1.6 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents ferroceneboronic acid (11.5 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.
C.sub.20H.sub.18Fe.sub.2Hg, Chemical Formula:
[0266] Molecular Weight: 570.64 g/mol,
[0267] ESI.sup.+ m/z: 573 [M].sup.+.
Bis(5-carboxypyridin-3-yl)mercury
[0268] ##STR00030##
[0269] A solution of one equivalent mercury (II)-acetate (1.6 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents 5-(dihydroxyboryl)-3-pyridinecarboxylic acid (8.3 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.
C.sub.12H.sub.8HgN.sub.2O.sub.4, Chemical Formula:
[0270] Molecular Weight: 444.02 g/mol,
[0271] ESI.sup.+ m/z: 447 [M].sup.+.
(5-Carboxythiophen-2-yl)(phenyl)mercury
[0272] ##STR00031##
[0273] A solution of one equivalent phenylmercury acetate (1.7 mg, 5 μmol) in 0.5 ml propan-2-ol was added to a solution of ten equivalents 5-(dihydroxyboryl)-2-thiophenecarboxylic acid (8.5 mg, 50 μmol) and cesium carbonate (16 mg, 50 μmol) in 1.0 ml propan-2-ol and mixed at 50° C. for 20 h.
C.sub.11H.sub.8HgO.sub.2S, Chemical Formula:
[0274] Molecular Weight: 404.83 g/mol,
[0275] ESI.sup.+ m/z: 407 [M].sup.+.
General Synthetic Procedure for Synthesis of Radiolabeled Diaryl/Heteroaryl.SUP.−.Mercury Species
[0276] Based on B-Precursors
[0277] A solution of 10-100 μg aryl boronic acid precursor in 50-100 μl ethanol is added to the intended amount .sup.197(m)Hg acetate solution in 0.2 M sodium acetate. The pH of the mixture is then adjusted to pH 8 by adding 100 μl 0.2 M 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer and shaken at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (acetonitrile (ACN)/H.sub.2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).
Di(thiophen-2-yl)mercury
[0278] ##STR00032##
[0279] Radiochemical yield (TLC): ≥95%,
[0280] Radio-TLC: R.sub.f=0.2 (ACN/H.sub.2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
Bis(5-carboxythiophen-2-yl)mercury
[0281] ##STR00033##
[0282] Radiochemical yield (TLC): ≥95%,
[0283] Radio-TLC: R.sub.f=0.9 (ACN/H.sub.2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
Di(ferrocenyl)mercury
[0284] ##STR00034##
[0285] Radiochemical yield (TLC): ≥95%,
[0286] Radio-TLC: R.sub.f=0.1 (ACN/H.sub.2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
Bis(5-carboxypyridin-3-yl)mercury
[0287] ##STR00035##
[0288] Radiochemical yield (TLC): ≥95%,
[0289] Radio-TLC: R.sub.f=0.9 (ACN/H.sub.2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
(5-carboxythiophen-2-yl)(phenyl)mercury
[0290] ##STR00036##
[0291] This heteroleptic diaryl mercury compound is accessible in a two-step procedure (analogous to the asymmetric phenylmercury dithiocarbamate derivatives (see next section):
Step 1: Synthesis of .SUP.197(m).Hg Phenylmercury Chloride
[0292] The .sup.197(m)Hg chloride stock solution in 0.2 M HCl is diluted by adding 100 μl water and 100 μl ethanol to improve the solubility of the tin precursor and the lipophilic intermediate. A solution of 10 μg trimethylstannyl benzene precursor in 50 μl dimethyl sulfoxide (DMSO) is added to this acidic .sup.197(m)Hg chloride solution and mixed at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (acetonitrile (ACN)/H.sub.2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).
Step 2: Reaction of the .SUP.197(m).Hg Phenylmercury Chloride with the Aryl Boronic Acid
[0293] A solution of 50 μg 5-carboxy-2-thienylboronic acid in 50 μl ethanol is added together with 100 μl 0.2 M sodium acetate to the .sup.197(m)Hg phenylmercury chloride. The pH of the mixture is then adjusted to pH 8 by adding 100 μl 0.2 M 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer and shaken at 50° C. for 1 h. The completion of the reaction is confirmed by TLC control (acetonitrile (ACN)/H.sub.2O 90:10 (v/v) with 0.1 vol-% trifluoroacetic acid (TFA), instant thin layer chromatography medium (iTLC)-silica gel (SG) and RP18 material).
[0294] Radiochemical yield (TLC): ≥60%,
[0295] Radio-TLC: R.sub.f=0.45 (ACN/H.sub.2O 90:10 (v/v) with 1 vol-% trifluoroacetic acid (TFA, RP-18).
Synthesis of Asymmetric Radiolabeled Aryl-Mercury-Dithiocarbamate Derivatives
[.SUP.197(m).Hg] (Diethylcarbamothioyl)thio)(4-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)benzoyl-amido)-mercury (II)
Step 1: Phenyl-.SUP.197(m).Hg—Cl Derivatives
[0296] ##STR00037##
[0297] 2 μg of the tin precursor N-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)-4-(tributylstannyl)benzamide (K08-15) dissolved in 20 μl DMSO was added into 50 μl 0.1 M HCl solution containing 45.5 MBq [.sup.197(m)Hg]HgCl.sub.2. The reaction mixture was shaken overnight at 25° C. (>12 h). Acidic environment is needed to avoid the formation of symmetric diphenyl mercury species. Excess of organotin precursors were decomposed slowly in acid environment.
Step 2: pH-.SUP.197(m).Hg-Dithiocarbamate Derivatives
[0298] ##STR00038##
[0299] The pH of the phenyl mercury chloride derivatives (step 1) was adjusted to pH 6, adding about 200 μl 0.2 M MES buffer (pH 6.0 to 6.2) and about 10 μl 0.2 M NaOH, before the dithiocarbamate ligand is added. Then 20 μg dithiocarbamate (cw04) containing 50 μl 0.2 M MES buffer (pH 6.0 to 6.2) were added into mixture quickly. Then, the reaction mixture was shaken at 50° C. for 60 min.
[0300] Radiochemical purity was determined by radio-HPLC (see
4. Ester Hydrolysis
Bis(4-carboxylphenyl)mercury(II) (Reference)
[0301] ##STR00039##
[0302] To a solution of 23 mg (36 μmol) Bis(4-(N-succinimidyl)benzoate)mercury(II) in 2 ml dimethylformamide (DMF) 2.88 μl 2.5 N NaOH (72 μmol) and 1 ml water were added. After mixing 2 h at 50° C. the completion of the reaction was confirmed by TLC control (DCM/MeOH 50:1 (v/v), DC silica gel 60 F.sub.254). The pH was adjusted to pH 3 by addition of acetic acid then the solvent was removed by rotary evaporation and residue redissolved in 2 ml DMF. The product was precipitated by addition of 20 ml cold diethyl ether, filtrated and dried under vacuum, resulting in a white solid.
C.sub.14H.sub.10HgO.sub.4, Chemical Formula:
[0303] Molecular Weight: 442.82 g/mol,
[0304] .sup.1H-NMR (400 MHz, DMSO-D.sub.6, AcOH-D.sub.4) δ (ppm): 7.52 (d, 4H); 7.83 (d, 4H),
[0305] .sup.13C-NMR (100 MHz, DMSO-D.sub.6, AcOH-D.sub.4) δ (ppm): 129.6 (CH); 131.0 (C); 137.7 (CH); 161.6 (C); 168.4 (C), yield: 15.3 mg (34 μmol; 94%),
[0306] ESI.sup.+ m/z: 443 [Hg-M].sup.+.
[.SUP.197(m).Hg] Bis(4-carboxyphenyl)mercury (II)
[0307] ##STR00040##
[0308] The solution of [.sup.197(m)Hg] Bis(4-(N-succinimidyl)benzoate)mercury (II) is adjusted to pH 9 by adding 10 μl 1 M NaOH and mixed for 1 h at 50° C. The completion of the reaction is confirmed by TLC control (ACN/H.sub.2O 90:10 (v/v) with 0.1 vol-% TFA, ITLC-SG and RP18 material). Finally, the pH is adjusted to pH 6-7 by addition of 10 μl 1 M HCl.
[0309] Radiochemical yield (TLC): ≥95%,
[0310] Radiochemical purity (TLC): ≥95%
[0311] Radio-TLC: R.sub.f=0.6 (ACN/H.sub.2O 90:10 (v/v) with 0.1 vol-% TFA, RP-18).
5. Synthesis of the [.SUP.197(m).Hg] Bis(4-carboxyphenyl)mercury (II)-mAb Cetuximab (C225) Conjugate by Prelabeling with the Labeled Active Ester
[0312] ##STR00041##
[0313] The solution of [.sup.197(m)Hg] Bis(4-(N-succinimidyl)benzoate)mercury (II) is added to a solution of 1 mg size-exclusion chromatography (SEC) purified C225 antibody in HEPES buffer at pH 8. After mixing the pH is adjusted to pH 8.5. After 1 h at 37° C. the progress of the reaction is confirmed by TLC control. (ACN/H.sub.2O 90:10 (v/v) with 0.1 vol-% TFA, ITLC-SG and RP18 material). Unreacted active ester residues were quenched by adding 10 μl 1 M tris(hydroxymethyl)aminomethane (TRIS) solution and separated using a PD10 desalting column.
[0314] Radiochemical yield (TLC): ≥50-70%,
[0315] Radiochemical purity (TLC): ≥95%,
[0316] Radio-TLC: R.sub.f=0 (ACN/H.sub.2O 90:10 (v/v) with 0.1 vol-% TFA, RP-18).
6. Synthesis and Stability of Compound (3*)
[0317] ##STR00042##
[0318] The synthesis is schematically shown in the following scheme:
##STR00043##
[0319] Compound (3*) was characterized by UV (
[0320] In vivo stability of (3*) was tested (
[0321] The results (
[0322] To test the actual in vivo stability of 3*, a biodistribution was performed on healthy rats and the results (
Preparation of 3,7-bis(2-bromobenzyl)-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one (1)
[0323] A 250 ml round-bottomed flask, with magnetic flea, was charged with 1,3-diphenylpropan-2-one (8.03 g, 38.2 mmol), followed by THF (100 ml) and stirred until a clear, pale yellow solution formed. To this was added 2-bromobenzylamine (14.20 g, 76.3 mmol, Alfa Aesar), a 37 wt % aqueous solution of formaldehyde (11.4 ml, 152.6 mmol) and a catalytic amount of ethanoic acid (a few drops). The reaction mixture was refluxed at 65° C. overnight for 19 h forming a dark yellow solution. TLC confirmed the complete conversion of the ketone starting material (Rf≈0.8, 1:1 EtOAc:hexane, KMnO.sub.4 stain). The ethanoic acid was neutralized by adding saturated Na—HCO.sub.3(aq) until the reaction mixture was slightly alkaline. The THF was removed by evaporation, the reaction mixture dissolved in DCM (50 ml) and washed with water (3×20 ml). The aqueous layers were combined and extracted with DCM (5 ml). The organic layers were combined, washed with brine (10 ml), dried with anhydrous Na.sub.2SO.sub.4 and filtered. The DCM was evaporated, leaving a brown solid, which was recrystallized by dissolving in boiling EtOH and slowly cooling to room temperature, affording 1 as white crystals (17.95 g, 28.5 mmol, 75%). TLC Rf≈0.8 (1:1 EtOAc:hexane, KMnO.sub.4 stain). >99% HPLC purity. Anal. ESI-MS: calculated [M+H]+ 631.0783, found 631.0781. 1H NMR (400 MHz, CDCl3): δ 7.64 (dd, J=8.0, 1.2 Hz, 2H, Ar), 7.53 (dd, J=7.6, 1.7 Hz, 2H, Ar), 7.34 (td, J=7.5, 1.3 Hz, 2H, Ar), 7.31-7.27 (m, 3H, Ar) 7.24-7.16 (m, 9H, Ar), 3.85 (s, 4H, NCH.sub.2Ar), 3.42 (dd, J=186.0, 10.7 Hz, 8H, CCH2N). 13C NMR (101 MHz, CDCl3): δ 210.89 (C═O), 142.75, 137.37, 133.31, 131.71, 129.21, 127.98, 127.41, 127.03, 126.72, 125.21, 64.87 (CCH.sub.2N), 61.25 (NCH.sub.2Ar), 54.64 (PhCCO).
Preparation of 9-butyl-1,5-diphenyl-3,7-bis(2-(trimethylstannyl)benzyl)-3,7-diazabicyclo[3.3.1]nonan-9-ol (2)
[0324] 1 (1.70 g, 2.7 mmol) was charged into an oven-dried 250 ml round-bottomed Schlenk flask with a magnetic flea, on a Schlenk line, under argon and sealed with a rubber septum. Anhydrous THF (50 ml) was syringed into the flask to form a suspension. Carefully, dropwise addition of nBuLi (2.5 M in hexane, 5.4 ml, 13.5 mmol), keeping the temperature below the boiling point, then reacted with the suspension to form a clear yellow solution. Me.sub.3SnCl (1 M in THF, 13.5 ml, 13.5 mmol) was syringed drop-wise 30 min later, eventually causing the reaction mixture to turn colorless. After leaving to stir throughout the night (19 h), the reaction mixture was carefully quenched with EtOH and then water. Organic solvents were removed by evaporation. More water was then added, into a final 40 ml solution, then NaHCO.sub.3 to form an alkaline phase pH≈8 that was extracted with DCM (3×40 ml). Afterwards, all organic phases were combined and washed with brine solution (40 ml), dried with anhydrous Na.sub.2SO.sub.4, solids filtered off and the remaining solution evaporated leaving a crude brown oily residue. Recrystallization with Et.sub.2O formed a brown ppt. that was filtered off. Evaporation of the remaining Et.sub.2O left 2 g of a crude brown oily residue. Column chromatography purification (dry-loaded onto Alox (basic 90) as the desired product proved unstable on silica) with a slow gradient of EtOAc (0% to 5%) in hexane yielded 2 as a white solid (200 mg, 0.23 mmol, 9%). TLC: Rf≈0.7 (Alox plate, 15% EtOAc:hexane, 12 stain). >90% HPLC purity. Anal. ESI-MS: calculated [M+H]+ 857.2666, found 857.2677. 1H NMR (400 MHz, CDCl3) δ 7.80 (t, J=7.0 Hz, 2H, SnAr-o), 7.49-7.11 (m, 16H, Ar), 3.79 (s, 2H, NCH2Ar), 3.77 (s, 2H, NCH2Ar), 3.37 (dd, J=42.7, 11.3 Hz, 4H, CCH2N), 2.88 (dd, J=51.6, 10.6 Hz, 4H, CCH2N), 2.27 (s, 1H, OH), 1.26 (m, 2H, Bu-C1), 0.64 (td, J=14.2, 6.7 Hz, 2H, Bu-C3), 0.44 (s, 9H, SnMe3), 0.38 (s, 9H, SnMe3), 0.36 (t, J=7.3 Hz, 3H, Bu-C4), 0.02 (m, 2H, Bu-C2). 13C NMR (101 MHz, CDCl3): δ 145.29, 145.25, 144.47, 136.22, 136.08, 129.26, 129.02, 128.93, 128.61, 127.85, 127.35, 127.16, 126.84, 126.18, 125.67, 65.61, 64.38, 64.29, 60.30, 47.32, 30.48, 30.24, 29.58, 26.12, 23.34, 13.71, −7.44 (SnMe3), −7.47 (SnMe3).
Preparation of 9-butyl-8,10-diphenyl-6,10:8,12-dimethanodibenzo[c,f][1,9]diaza[5]mercuracyclotetradecan-9-ol (3)
[0325] A 1.5 ml Eppendorf LoBind hinge-top tube was charged with 2 (26 mg, 0.03 mmol) and HgCl.sub.2 (8.1 mg, 0.03 mmol) in THF (1.5 ml) and mixed at 50° C. for 5 h. Evaporation of THF left a yellow oily residue. HPLC analysis showed no remaining starting material. Purification by HPLC yielded a white solid (7.2 mg, 5.25 mmol, 33%). ˜90% HPLC purity. Anal. ESI-MS: calculated [M+H].sup.+ 731.2925, found 731.2926. 1H NMR (600 MHz, CDCl3): δ 7.54 (d, J=7.8 Hz, 4H), 7.48 (dd, J=6.9, 1.4 Hz, 1H), 7.45 (dd, J=7.1, 1.4 Hz, 1H), 7.43-7.12 (m, 11H), 7.09 (td, J=7.4, 1.5 Hz, 1H), 3.62 (s, 2H, NCH2Ar), 3.51 (s, 2H, NCH.sub.2Ar), 3.33 (dd, J=198.0, 11.5 Hz, 4H, CCH.sub.2N), 2.99 (dd, J=305.4, 11.4 Hz, 4H, CCH.sub.2N), 1.73 (s, 1H, OH), 1.44 (t, J=8.5 Hz, 2H, Bu-C1), 0.74 (h, J=7.3 Hz, 2H, Bu-C3), 0.40 (t, J=7.3 Hz, 3H, Bu-C4), −0.04 (p, J=7.9 Hz, 2H, Bu-C2). 13C NMR (101 MHz, CDCl3): δ 171.33 (HgC1), 170.92 (HgC1), 146.84 (HgC2), 146.44 (HgC2), 141.78 (Ph-i), 139.08 (HgC6), 138.84 (HgC6), 128.96 (Ph-m), 128.95 (Ph-p), 127.98 (HgC3), 127.83 (HgC3), 127.23 (HgC4), 127.16 (HgC4), 126.72 (HgC5), 126.67 (HgC5), 126.41 (Ph-o), 75.09 (COH), 67.71 (NCH.sub.2Ar), 66.99 (NCH.sub.2Ar), 60.17 (CCH.sub.2N), 60.09 (CCH.sub.2N), 46.25 (CPh), 31.39 (Bu-C1), 25.76 (Bu-C2), 25.99 (Bu-C3), 13.62 (Bu-C4). 199Hg NMR (108 MHz, CDCl3): δ −684.5.
Preparation of .SUP.197m,g.Hg
[0326] The radionuclide was prepared by the bombardment of high purity 197Au target (99.99+%, 10 mm diameter, 0.125 mm thickness, Safina, Czech Republic) with a deuteron beam of the cyclotron U-120M in the Nuclear Physics Institute of the CAS, Czech Republic. The irradiations were per-formed using 15.8 MeV deuterons at the beam current of 10 μA for 4 h. It resulted in ˜0.58 GBq of 197gHg and ˜1.14 GBq of 197mHg at EOB, respectively. After arrival at HZDR, Germany, the irradiated targets were dissolved in aqua regia (700 μl), prepared from 30% HCl(aq) (525 μl) and 65% HNO3(aq) (175 μl) (purity Trace-Select, Sigma-Aldrich), and diluted with 6M HCl(aq) (300 μl). The resulting solution had a total activity of ˜0.9 GBq. 0.5 μl (˜1.57 MBq) was removed as a reference substance and the rest of the solution was carefully loaded onto a prepared column filled with 3.6 g of LN resin (LN-B100-A, 100-150 μm, TRISKEM, France) that had been soaked for 15 min in 6M HCl(aq), rinsed slowly with 6M HCl(aq) (30 ml), capped with a frit, overlayered with ca. 1 cm of sand and finally rinsed with 6M HCl(aq) (80 ml). After loading the target solution, the column was slowly washed with 6M HCl(aq) (6×1 ml) fractions, minor activity being detected from the 5th fraction, the fraction volume was reduced (6×0.5 ml). Most of the activity was eluted in the 9th-11th fractions.
[0327] Radiolabeling Procedure for Stability Tests:
[0328] After pH-adjustment of the hydrochloric acid solution of [197(m)Hg]HgCl2(aq) (˜55 MBq, 20 μl, pH 1), by addition of 0.5 M HEPES buffer (pH 8, 200 μl), EtOH (200 μl), 6 M NaOH(aq) (11 μl), and 1 M NaOH(aq) (2 μl), a 1 mg/ml acetonitrile solution of 2 (12 μl, 14 nmol) was added. This solution (pH 6, 445 μl) was mixed at 50° C. for 1 h. The radiochemical yield of 3* was determined by radio-TLC (iTLC ACN+0.1% TFA, RP-18 TLC 9:1 ACN: H2O+0.1% TFA) as >95%. Purification and solvent change were carried out with a C8 cartridge (500 mg). After washing with water the major product was eluted with 7:3 EtOH:H2O from the cartridge. The last 2 fractions contained ˜16 MBq and ˜10 MBq respectively. The ˜16 MBq fraction had 3×200 μl extracted (˜4 MBq each). These fractions then had 1 competitor added each (1 mg/ml, 10 μl): tris(2-mercaptoethyl)ammonium oxalate, glutathione and Na.sub.2S. The mixtures were left at rt and checked by radio-TLC after 5 min, 1 h and 2 d, the only degradation observed was ˜4% after 2 d in the Na.sub.2S mixture. The ˜10 MBq fraction was divided into 2×500 μl. The first lot was used to test the stability of 3* in the highly aqueous solvent system necessary for biodistribution studies, this was diluted from 70% EtOH(aq) to ˜10% EtOH(aq) with brine (3.5 ml). Transferal to a fresh vial showed negligible loss in activity and radio-TLC of the solution showed good stability. The other 500 μl lot was used to test the volatility of 3*: firstly the sample was diluted with 70% EtOH(aq) (500 μl) and the vial heated to 50° C. whilst a stream of dry nitrogen was blown onto the 1 ml solution for 1 h until the solution volume had been reduced to ˜350 μl. Transferal to a fresh vial showed negligible loss in activity and measurement of the remaining solution showed no observable loss by evaporation.
[0329] Determination of Distribution Coefficient of (3*):
[0330] Shake flask method: Into a 10 ml glass vial was added n-octanol (500 μl), 0.05 M HEPES buffer solution (pH 7.4, 475 μl) and a 1:1 EtOH:H2O solution of 3* (25 μl). The vial was shaken for 30 s, then 400 μl extracted from each phase and centrifuged separately. 2×100 μl was taken from each phase and the intensity of radioactivity was measured by a gamma counter and averaged.
[0331] Human Serum Stability Assay:
[0332] Human serum “off the clot” (5 ml) stored at −20° C. was slowly thawed on ice and filtered using syringe filters with a pore size of 0.2 μm. Two aliquots of filtered serum (2×220 μl) were mixed with 1 M HEPES/NaOH buffer (pH 7.4, 2×45 μl). Separately, 2×200 μl solution (1:1 EtOH:H2O, pH 6) of 3* (˜4 MBq) and 197(m)HgCl2/EDTA (˜5 MBq, 10 μg EDTA) had 1 M HEPES/NaOH buffer (pH 8.0, 2×20 μl) added to increase solution pH to 7.4. Then 135 μl of each 197(m)Hg-radiolabeled sample was added to one of the previously prepared serum/buffer solutions (265 μl) and incubated for 1 h at 37° C. 50 μl aliquots were then taken and mixed with 50 μl of 2× Laemmli sample buffer (Bio-Rad Laboratories), N.B. no reducing agent was added and the samples were not heated. The mixtures were then analyzed by non-reducing SDS-PAGE with acrylamide concentrations of 5% in the stacking gel and 20% in the resolving gel. 2 μl of each sample were loaded into each gel well. The SDS-PAGE was run at r.t. and 80 V until the dye front reached the resolving gel and then increased to 140-160 V. After electrophoresis, the gel was washed for 1 min with H.sub.2O and then exposed to a high-resolution phosphor imaging plate (GE Healthcare) for 10 min and the exposed plate scanned (Amersham Typhoon 5 Scanner, GE Healthcare) to measure an autoradiograph. The gel was then stained with PageBlue protein staining solution (Thermo Fisher Scientific, Coomassie G-250).
CITED NON-PATENT LITERATURE
[0333] R. L. Greif, W. J. Sullivan, G. S. Jacobs, R. F Pitts (1956) Distribution of radiomercury administered as labelled chlormerodrin (neohydrin) in the kidneys of rats and dogs. J. Clin. Investig. 35, 38-43. [0334] D. B. Sodee (1964) Letters to the Editor. Hg-197 as a Scanning nuclide. J. Nuc. I. Med. 5, 1964, 74-75. [0335] B. Matricali (1969) Brain scanning by means of .sup.197Hg-labelled neohydrin. Psychiatr. Neurol. Neurochir. 72, 89-95. [0336] M. Walther, S. Preusche, S. Bartel, G. Wunderlich, R. Freudenberg, J. Steinbach, H.-J. Pietzsch (2015) Theranostic mercury: .sup.197(m)Hg with high specific activity for imaging and therapy. Applied Radiation and Isotopes 97, 177-181. [0337] M. Walther, O. Lebeda, S. Preusche, H.-J. Pietzsch, J. Steinbach (2016) Theranostic mercury Part 1: A New Hg/Au separation by a resin based method. Abstract 16.sup.th International Workshop on Targetry and Target Chemistry. [0338] G. N. George, R. C. Prince, J. Gailer, G. A. Buttigieg, M. B. Denton, H. H. Harris, I. J. Pickering (2004) Mercury Binding to the Chelation Therapy Agents DMSA and DMPS and the Rational Design of Custom Chelators for Mercury. Chem. Res. Toxicol. 17, 999-1006. [0339] F. S. Mishkin (1966) Clinical brain scanning with .sup.203Hg Neohydrin. J. Indiana State Med. Assoc. 59 (12), 1435-1438. [0340] T. W. Clarkson, L. Magos (2006) The Toxicology of Mercury and Its Chemical Compounds. Critical Reviews in Toxicology. 36, 609-662. [0341] Remington's Pharmaceutical Sciences (1975) 15th Edition. Editor: A. Osol and J. E. Hoover. Mack Publishing Co., Easton, Pa. 18042. [0342] D. V. Partyka, T. G. Gray (2009) Facile syntheses of homoleptic diarylmercurials via arylboronic acids. J. Organometallic Chem. 694, 213-218.