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BATO complexes derived from dimethoxy dioximes: Synthesis, characterization and biodistribution
Vol. 22,No. 5, pp. 625-634, 1995 Copyright 0 1995ElsevierScienceLtd Printedin Great Britain. All rights reserved 0969-8051195 $9.50+ 0.00
NW/. Med. Biol.
Pergamon
0969-8051(94)00143-X
BAT0 Complexes Derived from Dimethoxy Dioximes: Synthesis, Characterization and Biodistribution KONDAREDDIAR RAMALINGAM, SILVIA S. JURISSON, BRUCE L. KUCZYNSKI, RICHARD DI ROCCO, RAMA K. NARRA. DAVID P. NOWOTNIK* and ADRIAN D. NUNN The Bristol-Myers Squibb Pharmaceutical Research Institute, Route 206 & Provinceline Road, Princeton, NJ 08543, U.S.A. (Accepted 28 November 1994)
To prepare less lipophilic BAT0 complexes, two new methoxy-substituted dioximes were synthesized: cis-4,5-dimethoxycyclohexane-1,2-dione dioxime (DMCDO) and 1,4-dimethoxybutane-2,3-dione dioxime (DMDMG). 99mTcC1(DMCDO),BMe (BMe = methylboronic acid) was prepared and characterized. Reversed-phaseHPLC analyses of 9P”TcC1(DMCDO),BMe and 99mTcC1(DMCDO),-p-TBA (p-TBA = ptolylboronic acid) indicated that both of these complexes were mixtures of four enantiomeric pairs of diastereomers. Attempted preparation of a BAT0 complex from DMDMG gave a mixture of products. In rats, 99mTcC1(DMCDO),BMedisplayed more rapid liver and renal clearance than 99mTcC1(CDO),BMe, but WmTcCI(DMCDO),BMe and 99mTcC1(DMCDO),-p-TBA displayed low uptake in both heart and brain
Introduction
tracer, TcCl(DMG),B2MP, it is 3.8 (Feld and Nunn, 1989)]. Such high lipophilicity values are of concern in applications which might require tracers to cross lipid membranes in vtvo (Pirro et al., 1994) or where it is necessary to minimize non-specific binding. In other series of complexes, addition of methoxy substituents have been used with some success when a reduction of lipophilicity was required (Nowotnik and Nunn, 1992). In this paper, we describe the synthesis and preliminary evaluation of BATOs (Fig. 1) prepared from two novel dioximes which contain methoxy substituents: cis-4,5-dimethoxy-1,2cyclohexanedionedioxime (DMCDO) and dimethoxydimethylglyoxine (DMDMG). The chemistry of these two new classes of BATOs has been studied on both the macroscopic Tc-99 level and the radiopharmaceutical Tc-YYm level. Rat biodistributions were performed with two of the DMCDO BATOs, and compared to the previously reported data for TcCl(CDO),MeB.
The BAT0 (Treher et ul., 1989) class of compounds (Boronic acid Adducts of Technetium dioximes) have been studied for a variety of applications in nuclear medicine: for myocardial perfusion imaging (Narra et al., 1989) cerebral perfusion imaging (Narra et al., 1990). antibody labelling (Linder et al., 1991a, b), imaging of regions of hypoxia (Linder et al., 1993) and receptor imaging (Nunn et al., 1991). The BAT0 complexes have the general formula TcCl(dioxime),BR, where BR is a boronic acid adduct. These complexes are formed by a template synthesis, in which the technetium, dioxime. boronic acid and chloride ion come together in situ to form the resultant TcCl(dioxime)3BR complex (Linder et al., 1990). To date the dioxime has been limited primarily to 1,2-cyclohexanedionedioxime (CDO) and dimethylglyoxime (DMG), while the R group on the boronic acid has been varied from an OH group, to alkyl, aryl and molecules which might target specific sites in the body (Nunn et al., 1990). The BATOs are neutral complexes and the DMG and CD0 analogs are highly lipophilic [the estimated log P for the myocardial perfusion tracer, TcCl(CDO),MeB, is 4.6, and for the brain perfusion
Experimental
*Author for correspondence, at: Guilford Pharmaceuticals, 661I Tributary Street, Baltimore, MD 21224, U.S.A. 625
Physical methods HPLC separations were run using a system consisting of two Altex 1lOA pumps, a Rheodyne 4125 injector, a Kratos 747 UV-visible flow through detector (for Tc-99 complexes), a NaI well type detector
Kondareddiar
626
Ramalingam
controlled by a Tennelec system (for Tc-99m complexes) and a SpectraPhysics 4270 integrator connected to Labnet software. Analyses were run on either a Hamilton PRP-1 (15 cm) column with a mobile phase of 65/35 ACN/O. 1 M NH,OAc (pH 4.6) and a flow rate of 2 mL/min or a Nucleosil C, (15 cm) column with a mobile phase of SO/SO ACN/O.I M citric acid (pH 2.4) and a flow rate of 1 mL/min. FTIR specta (KBr pellets), NMR spectra (‘H and 13C, CD+&), mass specta and elemental analyses were run by the appropriate analytical departments at Bristol-Myers-Squibb in Lawrenceville. Log k’ and log P determinations The log k’ values were determined from an HPLC method described previously (Nowotnik et al., 1993). The PRP- I (15 cm) reverse phase column was used with a mobile phase composed of 65/35 ACN/O.l M NH,OAc (pH 4.6) and a flow rate of 2 mL/min. Nitrate was used to determine the void volume (Nowotnik and Narra, 1993). Diethyl(2S,3S)(-)2,3-dimethoxysuccinate Freshly prepared and finely powdered dry silver oxide (276.66 g, 1.19 mol) was added in small portions to an externally cooled, vigorously stirring solution of diethyl-(2S,3S)-( -)tartrate (81.5 g, 0.395 mol) in methyl iodide (5OOg, 3.5 mol). After stirring for I h at 10°C the reaction mixture was refluxed for 12 h and then filtered. The brown solid was washed with ether. The combined organic solution was evaporated to dryness on a rotary evaporator. The residue was distilled to give 83 g of diethyl(2S,3S)-( -)2,3-dimethoxysuccinate (89.5%). b.p. 1lS”Cj5 mm. Lit b.p. (Mori, 1974): 106108”C/ 4 mm. ‘HNMR (CDCI,): 3.41 ppm (s, 6H, CO&H,), 3.75 ppm (s, 6H, OCH,), 4.09 ppm (s, 2H, C2-H, and C3-H). (2S,3S)-( -)dimethoxybutane-
1,4-diol
To a stirred slurry of lithium aluminum hydride (35.2 g, 0.94 mol) in ether (600 mL) was added drop-
e/ ul.
wise a solution of diethyl-(2S,3S)-( - )2.3-dimethoxysuccinate (83 g. 0.35 mol) in dry ether (400 tnL). The reaction mixture was refluxed for 6 h and then allowed to stir overnight at room temperature. Excess lithium aluminum hydride was carefully destroyed by the successive addition of water (35.0 mL), 15% sodium hydroxide (35 mL) and water (150 mL). The reaction mixture was filtered, the filter cake was washed with acetone (6 x 200 mL), and the combined filtrate was evaporated on a rotary evaporator. The oil obtained was distilled under reduced pressure to give 36 g of (2S,3S)-( -)2,3-dimethoxybutane-I .4diol (75%). b.p. 92-93‘CO.l mm. Lit b.p. (Mori. 1974): 92297”C/O. 1 mm. ‘HNMR (CDCI,): 3.54 ppm (s, 8H, OCH, and OH), 3.78 ppm (m, 4H, CH2), 4.05 ppm (s, 2H, C2-H and C3-H). (2S,3S)-( -)2,3-dimethoxybutanetoluenesulphonate
1.4-dial
di-p-
To a cooled (OC) stirring solution of (2&3S)(-)dimethoxybutane-1,4-diol (36.0 g, 0.24 mol) in dry pyridine was added p-toluenesulfonyl chloride (112 g, 0.56 mol) and the resulting mixture was left at 5°C for 24 h. The reaction mixture was poured into ice-water and the oil that separated was extracted with ether (4 x 200 mL). The ether solution was washed with 5% HCI (2 x 200 mL) and dried with sodium sulfate. Evaporation of the ether afforded a viscous oil. Yield 70.0 g (64%). The compound was used for the next step without further purification. ‘HNMR (CDCI,): 2.42 ppm (s, 6H, -CH,), 3.27 ppm (s, 6H, OCH,), 3.5 ppm (t, 2H, C2-H and C3-H), 7.41 ppm (d, 4H, ArH). 7.64 ppm (d, 4H, ArH). (3S,4S)-(-)3,4-Dimethoxyhexane-l,&dinitrile To a solution of (2S,3S)-( -)dimethoxybutane-l,4diol di-p-toluenesulfonate (70 g. 0.153 mol) in dry DMSO (150 mL) at room temperature was added NaCN (18.5 g, 0.38 mol) in portions over a period of 3 days. The reaction mixture was stirred for a further 72 h at which time the clear brown solution was poured into water (2 L) and extracted with methylene
MeO
OMe
\f
I
MC?0
Cl
‘R’
TcC~(DMDMG)~BR Fig. 1. General structures of the BATOs derived from methoxy-substituted
dioximes
BAT0
complexes derived from dimethoxy dioximes
chloride (6 x 500 mL). The methylene chloride solution was washed with water (3 x 500 mL) and dried with sodium sulfate. Evaporation of the methylene chloride afforded a yellow solid. Yield 15.8 g (61.8%). m.p. 71-72°C. Lit m.p. (Cope and Mehta, 1964) 71.8-72.2”C. ‘HNMR (CDCI,): 2.66ppm (d, 4H, CH,), 3.5 ppm (s, 6H, OCH,), 3.72 ppm (2H, t, C3-H and C4-H). Dimethyl (3S,4S)-3,4-dimethoxyadipate A stirred solution of (2S,3S)-( -)3,4-dimethoxyhexane- 1.6-dinitrile (15.8 g, 0.94 mol) in dry methanol was saturated with dry HCl. The mixture was refluxed for 2 h and then the methanol was removed on a rotary evaporator. The remaining solution was diluted with water and the oil that formed was extracted with ether (3 x 300mL). Removal of the solvent afforded a brown oil, which was added to a solution of thionyl chloride in methanol (20 mL of thionyl chloride was added to 150 mL of methanol at - 78°C) and kept overnight in a refrigerator. The methanol was evaporated on a rotary evaporator and the residue was dissolved in ether (200mL). The ether solution was washed with sodium bicarbonate solution (2 x 200 mL) and water (2 x 300 mL) and dried with sodium sulfate. The solvent was removed and the residue was distilled to give 17.8 g of dimethyl(3S,4S)-(-)3,4-dimethoxy adipate (81.5%). b.p. 12&22C/0.5mm. Lit. b.p. (Cope and Mehta, 1964) 85’C/O.O6mm. ‘H-NMR (CDCl,): 2.53 ppm (m, 4H, CH,), 3.42 ppm (s, 6H, COCH,), 3.76 ppm (s, 6H, OCH,), 3.96 ppm (m, 2H, C‘3-H and C4-H). 1,2-Bis(trimethylsilylo.~y)-4,5-dimetho.~ycyclohexene A 500mL three necked flask was fitted with a stirrer, addition funnel and a reflux condenser and maintained under nitrogen atmosphere. The flask was charged with 200 mL of dry toluene and 1.84 g (0.08 mol) of sodium. The solvent was brought to gentle reflux and the stirrer was operated until the sodium was fully dispersed. A mixture of dimethoxy adipate (4.68 g, 0.02 mol) and trimethylsilylchloride (8.68 g, 10.1 mL, 0.08 mol) in 50 mL of dry toluene was added over 3 h. The reaction was exothermic and a dark purple precipitate formed. A gentle reflux was maintained throughout the addition. After 5 h of additional stirring, the contents of the flask were cooled and then filtered under a nitrogen atmosphere. The pale yellow filtrate was transferred to a distilling flask, the solvent was evaporated under reduced pressure and residue was distilled. b.p. 75-78’C/ 1 mm. Yield 2.8 g (47%).
1,4Cyclohexadiene (21.4 g, 0.27 mol), potassium acetate (0.066 mol) and iodine (0.134 mol) in acetic acid (400 mL) were stirred at RT for 12 h. Potassium acetate (30.0 g, 0.3 mol) was added and the mixture was refluxed for 7 h. After cooling, water (5 mL) was
627
added and the solvent was removed in vacua. The residue was taken up in ether (250 mL), and washed with aqueous sodium thiosulfate solution to remove the brown color. After drying, solvent was removed in vacua and cyclohex-4-ene-cis- 1,2-diol monoacetate was distilled. Yield 18.0 g, b.p. 94C/2mm. MS: (M + H)+ = 157. The cis-monoacetate (5.0 g) and sodium methoxide (1 .Og) in dry methanol (60 mL) were heated to reflux for 1 h, cooled, and neutralized with carbon-dioxide. The solvent was removed in vacua and the residue was extracted with ether. Removal of ether afforded cyclohex-4-ene-cis - 1,2diol. m.p. 8&82’C. Lit m.p. (Ali and Owen, 1958): 80-8 I (C. 2-lodo-4,5-dimethoxycyclohexanone Iodine (93.2 g, 0.0125 mol) and a solution of pyridine (0.4 g) in 5 mL of methylene chloride at 0 ‘C were added to a suspension of silver chromate (3.3 1 g, 0.01 mol) and 4A molecular sieves (5 g) in 25 mL of methylene chloride and stirred for 10 min. A solution of dimethoxy cyclohexene (1.46 g, 0.01 mol) in 15 mL of methylene chloride was added dropwise over 5 min to the ice-cooled suspension, and then stirred for 30min at 0°C. The cooling bath was removed and the reaction mixture was stirred for an additional hour at room temperature. The dark-brown mixture was filtered through a pad of celite, washed with aqueous sodium thiosulfate (5%) and dried with sodium sulfate. The crude product obtained after concentration was purified by column chromatography (silica gel, hexane-ethyl acetate 7: 3) to give the title compound as a light yellow oil. IR (neat) 17lOcm~‘. ‘H-NMR (CDCI,): 2.12-3.12 ppm (m. 4H, CH?), 3.42 and 3.48 ppm (s, 6H, OCH,), 3.82-3.98 ppm (m, 2H, CH), 4.75 ppm (m, IH, CHI). MS: (M + NH,)’ = 302. 4.5Dimethoxycycloheuanone-2-nitrate
ester
To a solution of 2-iodo-4,5-dimethoxycyclohexanone (1 g, 3.5 mmol) in acetonitrile (2 mL) was added a solution of silver nitrate (0.67 g, 4 mmol) in 2 mL of acetonitrile. After stirring for 24 h at RT the mixture was filtered, the silver bromide was washed with ether, and the combined filtrate and washings were evaporated at 30mm (3OC). The residue was taken up in ether, washed with water, and dried. Removal of the solvent afforded the nitrate ester. Yield 0.5 g (65%). ‘H-NMR (CDCI,): 2.28-2.84 ppm (m. H, CH,), 3.42 and 3.48 ppm (s, 6H, OCH,). 3.7553.85 ppm (m, 2H, CH), 4.05 ppm (m. 1H, CHONO,). MS: (M + H)+ = 237. cis-Dimethoxycyclohe.uane-1,2-dione
dioxime
To a mixture of hydroxylamine hydrochloride (1.05 g, 0.015 mol) and sodium acetate (1.64 g, 0.02 mol) in methanol (10 mL) was added a solution of the nitrate ester (1.14 g, 0.005 mol) in methanol (5 mL) and the mixture was stirred at RT for 12 h. Silica gel (5.0 g) was added to this reaction mixture
Kondareddiar Ramalingam et crl
62X
and the methanol was removed on a rotary evaporator to afford a free flowing powder. The free flowing powder was loaded on to a silica gel column (50 g) and eluted first with 9.5:0.5 methylene chloride-methanol followed by 9: 1 methylene chloride-methanol. Fractions containing the product were collected and evaporated to give a tan yellow solid. Yield 0.61 g (60%). The product was crystallized from acetone. m.p. 180-181 ‘C. ‘H-NMR (DMSOd6): 2.66 ppm (m, 4H, CH,), 3.29 ppm (s, 6H, OCH,), 3.64 ppm (t, J = 6.0 Hz, lH, CH), 11.23 ppm (s, 2H, OH). MS: (M + H)+ = 203; (M + NH,)+ = 220. Anal. Calcd. for: C,H,,N20,: C, 47.51, H, 6.98, N, 13.86. Found: C, 47.62, H, 7.13, N, 13.87. 1,4-Dimethoxy-2-butene Sodium hydride (24g, 60% in mineral oil) was washed with anhydrous ether (100 mL) and then suspended in dry THF (200 mL). 2-butene-1,4-diol (22 g, 0.25 mol) was added, the mixture was cooled to 0°C and then stirred at 0°C for 20 min. Methyl iodide (20 mL) was added and the reaction mixture was allowed to warm to RT while stirring. The reaction mixture became exothermic (+ 10 min) at which time it was cooled with an ice bath. After cooling for 20 min the reaction mixture was allowed to warm to room temperature and stirred at RT overnight. The THF was evaporated and the residue was extracted with ether (3 x 150 mL) and dried with sodium sulfate. Evaporation of the ether afforded an oil which was distilled under diminished pressure. Yield 14 g (48%). b.p. 85-87”C/25 mm. ‘H-NMR (CDCI,): 3.31 ppm (s, 6H, OCH,), 3.98 ppm (d, 4H, CH?). 5.82 ppm (t, 2H, CH = CH). 2-Iodo-4,Sdimethoxljbutanone 1,4-Dimethoxy-2-butene (5.8 g, 0.05 mol) was reacted with silver chromate (16.55 g, 0.05 mol) and iodine (16.51 g, 0.065 mol) and pyridine (2 g) in methylene chloride in the presence of 4 8, molecular sieves (25 g). The reaction mixture was filtered, the methylene chloride solution was washed with sodium thiosulfate and water, and then dried with sodium sulfate. Evaporation of the methylene chloride afforded a yellow oil. Yield 6.5 g. ‘H-NMR (CDCI,): 3.31 and 3.33ppm (s, 6H, OCH,), 3.64.4ppm (m, 4H, CH2). 4.8 ppm (m, lH, CHI). MS: (M+H)+ =261. 4,5-Dimethoxy-1,2-butanedione
product formed was purified by column chromatography (silica gel, methylene chloride--methanol~8:2). Yield 1.5 g. The product was crystallized from water, m.p. 165-66°C. ‘HNMR (CDCI,): 6 3.2 ppm (s, 6H, OCH,), 4.25ppm (s, 4H, CHJ. I I.8 ppm (s, 2H, NOH). MS: (M + H)+ = 177. Anal. Calcd. for: C,H,,N,O,: C, 40.90, H, 6.87, N, 15.9. Found: C. 40.80, H. 7.09, N, 15.81.
NH,99Tc0, (0.05142 g; 0.284 mmol) was dissolved of H,O. DMCDO (0.18113g; in ca 15mL 0.897 mmol) and methylboronic acid (0.02093 g; 0.340 mmol) dissolved in 5 mL of EtOH were added to the stirring NH,TcO, solution. The reaction mixture was heated gently; SnCI, (0.125 18 g; 0.659 mmol) dissolved in I mL of cone HCl and diluted with 1 mL of H,O was added dropwise over 5 min to the stirring reaction mixture. The reaction mixture became an intense reddish-brown in color. The reaction was stirred with heat for ca 1 h. An equal volume of CH,Cl, (ca 25 mL) was added and the reaction mixture was allowed to sit at room temperature overnight. The CH2Cl, fraction was collected and the aqueous fraction then exhaustively extracted with CHJl,. The combined CH,C& fractions were dried through anhydrous Na,SO, and concentrated to UI 2 mL. The CH,Cl, fraction was purified by silica gel chromatography (1 x 10 cm;. Four green and one orange band (unidentified) were eluted with CH2ClI. CHCI, eluted the orange product band. partially separating the diastereomers (Vide @a). The product was collected in five fractions, The five fractions were analyzed by HPLC [Nucleosil C, (15 cm), 50150 ACN/O. 1 M citric acid (pH 2.4). 1 mL/min, 400 nm], and showed that a partial separation of diastereomers had been achieved (fractions l-5 from the column had retention times in the range 6.9-7.6 min). Crystallization from CH$I,/MeOH/ HCI yielded solid from two of the fractions, The orange solids from fractions 1 and 2 were collected. washed with MeOH/HCl and vacuum dried. Mass Spectra (FAB): Fraction
1:
Fraction 2:
M +/(M - H) (M - Cl)+/(M - HCI) M+/(M-H)(M - Cl)+/(M - HCl)
= 762,:761 = 727/726 =762!‘761 = 727.‘726
dioxime
2-Iodo-4,5-dimethoxybutanone (6.5 g, 0.0256 mol) was reacted with a solution of silver nitrate (8.7 g, 0.044 mol) in acetonitrile (25 mL) at RT for 24 h. The silver iodide formed was filtered, and the solvent was removed under vacuum to afford the nitrate ester. Yield 2.5 g. ‘HNMR (CDCI,): fi 3.31 and 3.42 ppm (s, 6H, OCH,), 3.742 ppm (m, 4H, CH,), 5.7 ppm (m, lH, CHNO,). The crude nitrate ester obtained above (2.0 g) was reacted with hydroxylamine hydrochloride and sodium acetate in methanol and the
FTIR Specta (KBr pellets): Fraction
1: C=N B-O Fraction 2: C=N B-O
1633cm-‘, N-O 1048cm.‘, 1096 cm ’ 1633 cm ’ , N-O 1049 cm I, 1099 cm ’
The following were added to a 6 mL glass vial: methylboronic acid (2-3 mg, 33-50 mmol), DMCDO
BAT0
complexes derived from dimethoxy dioximes
(0.24.4 mg, IO-20 mmol), 0.2 mL of aqueous citric acid (100 mg/mL, 95.1 mmol), 0.1 mL of DTPA (20 mg/mL 0.5 M NaOH, 5.1 mmol), 0.2 mL of saturated NaCl, 5 p L of &Cl, solution (0.1 g/mL in cone HCl diluted to 4 ml, 2.6 mmol). The vial was sealed and 0.5 mL of 99mTc04m/saline (21.8 mCi generator eluent) was added. The vial was heated at 70°C for 15 min and cooled. The reaction mixture was purified by a solid-phase extraction procedure described previously (Jurisson et al., 1991). The lipophilic product was adsorbed onto a plug of PRP-1 resin. The resin was washed three times with 0.5 mL of 25% EtOH/saline to remove the non-radioactive constituents, then the product eluted by washing the resin with 0.5 mL of EtOH. The RCP of the purified complex was found to be 90.3%. The ethanolic solution was diluted with saline to obtain a sample (24 mCi/O.l mL) suitable for rat biodistribution studies. The product had the same retention time on HPLC analysis as “9TcCI(DMCDO),BMe.
Table I. Reaction conditions and HPLC results in the attempted preparation of 99mTcCl(DMDMG)1BBu Peak retention times (min)
RCP (%)
(a) pH 2, no non-essential ingredients
0.90 I .39
x7.3 12.7
(2) pH 4 with NH,OAc, no non-essential ingredients
0.X6 0.98 I .39
20. I 50.0 2Y.9
(3) Kit 2 conditions, heated at 7o’C for ISmin
0.99 1.39
62.3 37.7
(4) pH 4 with Na,citrate, no non-essential ingredients
0.76 I .42
66.9 33.1
(5) Kit 4 conditions heated at 7o’C for I5 min
0.75 I .40
52.2 47.8
Kit conditions
Purification of kit 4 (Table 1) on a plug of PRP-1 resin by adsorbing the complex of interest, washing once with 0.5 mL of 25% EtOH-saline and eluting with 0.5 mL of EtOH yielded the 1.44 min peak in 73% RCP. Biodistribution
This uncapped tris(dioxime) complex (Linder et al., 1990) was prepared following the procedure described above for 99mTcCl(DMCDO),BMe but omitting the methylboronic acid. HPLC analysis (same system as above; retention time of 3.03 min) gave an RCP of ca 80% for the desired complex. The complex was purified by solid-phase extraction; the complex was adsorbed onto the PRP-1 resin, washed three times with 25% EtOH-saline and eluted with 0.5 mL of EtOH. After purification, the RCP of the complex was 97%. To insure that this complex was the uncapped TcCl(DMCDO),, ca 2mg of (HO),BMe was added to the purified complex in EtOH, followed by two drops of 2 M HCl. This resultant solution was heated at 70’ C for 10 min. ‘““TcCI(DMCDO).,B-pTBA A complex was prepared following the procedure described above for 99mTcCl(DMCDO),BMe, substituting the equivalent molar quantity of p-tolueneboronic acid @-TBA) for MeB. EtOH (0.2 mL) was also added to the kit to help dissolve the pTBA. HPLC analysis (as above) showed four pairs of diastereomers (retention times of 12.4, 13.1, 13.6 and 14.3min) with a combined RCP of ca 83%. The complex was purified on a plug of PRP-1 resin; the compound was adsorbed onto the PRP-1 resin, washed twice with 0.5 mL of 25% EtOH-saline, once with 0.5 mL of 50% EtOH/saline and eluted with 0.8 mL of EtOH. This gave the desired complex with an RCP of cu 96%. The ethanolic solution was diluted with saline for a rat biodistribution study. Attempted synthesis (BBu = n-hutylhoronic
OJ’ yHnTcCI(DMDMG)lBBu, acid)
Several reaction conditions were examined for the preparation of this complex, as listed in Table 1.
629
studies
Biodistribution studies were conducted using male rats anesthetized with sodium Nembutal (50 mg/kg, i.p.). The agents were injected into the external jugular vein as a 0.1 mL bolus and the rats were sacrificed 5 and 60 min later. The organs and blood were collected and the radioactivity in each sample was counted to determine the injected dose per organ and per gram of organ. Five rats were used for each time point. Gamma cameru stud?) An anesthetized male rat was positioned (anterior view) for gamma camera acquisition and then injected i.v. with 2.1 mCi of 99mTcCl(DMCDO),BMe (prepared as above). Immediately following the injection, a 30 min (1 frame/min) dynamic acquisition was initiated using a pinhole collimator. For each frame of the dynamic acquisition, counts in the liver region of interest (ROI) were obtained using the gamma camera histogram function. The liver washout data (liver ROI counts) was extrapolated from percent injected dose at 30 min and compared to a typical Cardiotec histogram of liver washout (Narra et a/.. 1989).
Results and Discussion Syntheses of‘ the dimethoxy dioximes Syntheses of cis-4,5-dimethoxycyclohexanedione dioxime (DMCDO) and 1,4-dimethoxy-2,3-butanedione dioxime (DMDMG), were undertaken for the purpose of producing more hydrophilic BAT0 complexes. There are a few examples of the synthesis of substituted cyclohexanedione dioximes in the literature, so some preliminary studies had to be undertaken to develop a successful method of synthesis cis-4,5-dimethoxycyclohexanedione dioxime (DMCDO).
Kondareddiar Ramalingam CI trl
MICAH,CH,I
CO,C,Hs
CH,OH
UAIH, b diethyl ether
TsCl
CH,OTs
b
pyridine
NaCN / DMSO
m30, b
C&OH / HCI
I,
CH,OH
CH,CO,CH,
WCY), QCW,
CH,OTs
WP
JI
1
CY%c=H,
0
Na / xylene
HOAc
W,),Si~
0
OSi(CH,),
Scheme 1. Attempted synthesis of 4,5-dimethoxycyclohexane- I ,2-dione via the reduction of diethyl 3,4-dimethoxyadipate. Based on Ruhlmann’s report (Ruhlmann, 1971) on the successful synthesis of 1,2-cyclopentanedione using an acyloin condensation, the route shown in Scheme 1 was devised. Dimethyl (3S,3S)-3,4dimethoxyadipate (1) was prepared according to the published report (Mori, 1974) from diethyl-L-tartrate. Acyloin condensation of this diester in the presence of trimethylsilyl chloride produced a complex product mixture. Purification of this mixture by column chromatography afforded 15% of the desired 4,5-dimethoxy-1,2-bis(trimethylsilyloxy)cyclohexene (2). However, oxidation of this compound with acetic acid and cupric acetate did not yield the desired diketone (3). Therefore, this route was abandoned due to the difficulty in isolating the silyl ether and oxidizing the ether to diketone. Instead a five step route (Scheme 2) was investigated. The first step of
the procedure was that of Krow et al. (1977), whereby cyclohexene- 1,4-diene was converted to the diol. The diol was smoothly converted to the methyl ether with methyl iodide and sodium hydride. The next step involved the oxidation of the olefin to a a-iodocyclohexanone. The method was based on a literature report (Cardillo and Shimizu, 1977) of the synthesis of a-iodocyclohexanone from cyclohexene. Oxidation of 4,5-dimethoxycyclohexene with silver chromate and iodine in methylene chloride afforded the iodoketone in 46% yield. The iodoketone was readily converted to the nitrate ester by reaction with silver nitrate in acetonitrile. Conversion of the nitrate ester to the 1,2-diketone proceeded smoothly. However, isolation of the diketone product from the reaction mixture proved difficult. One reason for this is the fact that the 1,2-diketone may exist as an enol
CH30
HO NELH/CH,I CH3CN
+ CH30
HO
12/CH2C12
I
Ag2Cr04
NH20H. cn3c0pa
c-I30
5LizYic-t30 5
HCl
CH30
CH30
AgN03/CH3CN CH30
CH30 4
Scheme 2. Synthesis of 4,5-dimethoxycyclohexane- I ,2-dione dioxime.
3
BAT0
Ho
Ho
NaH, CH,I
MeO
THF
MeO
l3
4W, CH,CN
complexes derived from dimethoxy
*
MeO
2
631
AS,CrO, 1 I,
ME!0 32
cw4
3
N&OH. HCI
I
Me0
I-
04
Mt30
dioximes
MeO M&)
b
NaOAc, -OH 0
0 NOH
NOH
Scheme 3. The synthesis of DMDMG.
tautomer. The highly polar enolic product partitions into the aqueous layer rather than the organic layer which may make isolation of the product in the diketo form difficult. Since the diketone is ultimately needed as its dioxime derivative, the nitrate ester was reacted with hydroxylamine hydrochloride and sodium acetate in ethanol to afford the dioxime. DMDMG was prepared from butene-1,4-diol by a procedure similar to that described above for DMCDO (Scheme 3).
developing a linear plot of log k’ and log P (octanol/water) for a set of standard organic compounds. The mathematical equation relating to k’ and log P was found to be log P = (log k’/0.36) + 0.23. The log P values estimated for 99”TcCI(DMCDO),BMe and 99mTcC1(CDO),BMe using this system are shown in Table 2. As expected the lipophilicity of 99mTcCI(DMCDO),BMe is much less than that of 99”TcC1(CDO),BMe. While DMCDO BAT0 complexes appear as a broad peak on the PRP-1 HPLC system, each comFormation of technetium complexes plex can be resolved into four peaks on a Nucleosil BAT0 complexes were formed from DMCDO and C8 reversed-phase column. As shown in the schematic representation in Fig. 2, the DMCDO BATOs two boronic acids, methylboronic acid (MeB), and p-tolylboronic acid (p-TBA), and a number of reacare a mixture of four enantiomeric pairs of diastereomers, which are formed as a result of the cis tion conditions were examined for the formation of a complex derived from DMDMG and n-butylconfiguration of the methoxy groups on the cyclohexane ring and the presence of the seventh ligand about boronic acid (BBu). For the DMCDO complexes, the 99Tc and 99mTc the Tc. For 99mTcCI(DMCDO),BMe, the retention times of this quartet are 6.5, 6.9, 7.1 and 7.5 min, BAT0 complexes were formed under conditions while for 99mTcCl(DMCDO),-p-TBA the retention similar to that described previously for CD0 complexes (Linder et al., 1990; Jurisson et al., 1991). times are 12.4, 13.1, 13.6 and 14.2min. Purification of 99TcCl(DMCDO),BMe by silica The initial radiochemical purities of the DMCDO gel chromatography partially separated two of complexes were lower than had been observed for the four diastereomers. These were isolated and their CD0 analogs, but RCPs could be improved each analyzed by FTIR and mass spectroscopy. by using a solid-phase extraction procedure. 99mTcC1(DMCDO)JBMe can also be formed in a The mass specta were consistent with the assignment of structure (Fig. 1) while FTIR spectrum of two-step process involving the isolation of the 99TcCl(DMCDO),BMe was similar to that reported intermediate uncapped complex 99mT~Cl(DMCDO)1 previously for 99TcCl(CDO),BMe (Treher et a/., followed by capping with methylboronic acid, which 1989). One of the two isolated diastereomers was also has been reported for other BAT0 complexes (Linder characterized by ‘H and ‘)C-NMR spectroscopy. The et al., 1990). The 99mTcDMCDO BAT0 complexes appeared as analytical data are consistent with the structural assignment shown in Fig. 1, indicating that the two a broad single peak on the Hamilton PRP-1 HPLC system. This system has been used previously for the isolated complexes are diastereomers. Attempts to prepare 99mT~Cl(DMDMG)3BB~ reestimation of log P. The system is calibrated by sulted in mixtures of products. Reaction conditions which were investigated are listed in Table 1. Based *The expected retention time of DMDMG-BBu was calcuon the retention times of 99mTcCI(DMCDO)3BMe, lated based on the observed difference in log k’ values for CDO-MeB and DMCDO-MeB of 0.97 (1.52 and 0.55, ““‘TcCl(CDO),BMe, and 99”TcCI(DMG),BBu on respectively). If we assume that the log k’ difference for DMG-BBu and DMDMG-BBu is comparable (0.97), we can calculate an approximate retention time for DMDMG-BBu on PRP-I (65/35 ACN/O.l M NH,OAc, 2 mL/min). The log k’ for DMG-BBu is 1.20. If the log k’ difference between DMG-BBu is ca 0.97, then log k’ for DMDMG-BBu is ca 0.2. A log k’ value of 0.2 indicates a retention time of ca 1.4 min when the void volume is 0.55 min.
Table 2. Log P values of BAT0 complexes estimated from HPLC lee k’ data log P Compound
Compound TcCI(CDO),MeB TcCI(DMCDO),MeB *(Estimated
4.6 I .7
TcCI(DMG),BBu TcCI(DMDMG),BBu*
as described in the text).
log P 3.x 1.4
Kondareddiar Ramalingam el al.
bMe
OMe
Fig. 2. Schematic representation of the four diastereomers of TcCl(cis-DMCDO),BR.
the PRP-1 system, the retention time of 99mTcCl(DMDMG),BBu was estimated to be 1.4 min*. A product with this retention time was observed, but could not be isolated from the other reaction products. The 0.75 min peak observed in kits 4 and 5 is believed to be a Tccitrate complex. Based on our prior experience with the BAT0 complexes (Treher et al., 1989; Linder et al., 1990; Jurisson et al., 1991) it is clear that complex yields with DMCDO and DMDMG are inferior to their corresponding CD0 and DMG complexes, In the case of DMCDO, it is possible that the two ether oxygen atoms can compete with the oximes when binding to technetium, so lowering the yield(s) of the BAT0 reaction precursors. In the caseof DMDMG, while the separation and flexible link between the ether groups make it less likely that these groups can compete with the oximes compared to DMCDO, it is possible that the formation of a six-membered ring resulting from intramolecular hydrogen-bond formation between an ether group and its adjacent oxime group reduces the chelating ability of this dioxime. Biodistribution
studies
The results of biodistribution studies of 99mTcCl(DMCDO),BMe and 99mTcCl(DMCDO),-pTBA in rats are shown in Tables 3 and 4. Part of the motivation for developing these less lipophilic agents was to overcome our concern that the BATOs might be so lipophilic that they would readily penetrate into a phospholipid bilayer but remain trapped in the hydrophobic membrane interior. We reasoned that an agent that was somewhat less lipophilic, might retain enough water solubility that it would be able to move back out of the membrane and into the
aqueous cytoplasmic compartment. The biodistribution data show, however, that the brain uptake of these agents is quite low relative to the DMG and CD0 based BATOs [reported previously (Narra et al., 1989)]. Whereas reduced lipophilicity of the methoxy BATOs should increase the ability of an agent to leave the hydrophobic membrane interior once therein, it would also make it more difficult to get in since there would be less free energy available to penetrate the initial membrane resistancepresented by the rigid spacing between phospholipid polar head groups (Pirro et al., 1994). In addition, the increase in size of these agents would increase this resistance. These factors may be related to the reduction of brain “uptake” seen in the biodistribution data compared to their unsubstituted analogs (Narra et al., 1989). As shown in Fig. 4, the liver washout profile of 99mTcCl(DMCDO),BMe is considerably more rapid than that of 99mTcCl(CDO),BMe. In addition, the renal clearance of 99mTcCl(DMCDO),BMe is more rapid than that for 99mTcCl(CDO),BMe, resulting in lower ‘background’ levels for the methoxy BAT0 compared to the unsubstituted compound. However, heart uptake of 99”TcCl(DMCDO),BMe is less than adequate at 0.32% ID at 5 min for imaging.
Conclusions Two routes to 4,5-dimethoxycyclohexane- 1,2dione dioxime (DMCDO) were investigated. This compound was prepared in good overall yield via an iodo nitrate ester intermediate. A similar route was used to prepare 1,4-dimethoxybutane-2,3-dione dioxime (DMDMG). The 99Tccomplex, TcCl(DMCDO),BMe was prepared and characterized. HPLC analyses of the ““TcCl(cis-DMCDO),BR complexes Table 4. Biological
Table 3. Biological
distribution
of 99mTcCI(DMCDO),MeB
distribution
in rats
% i.d./organ 5 min
of ‘gmTcC1(DMCDO)~B-p-TBA rats % i.d./orean
60 min
5 min
Tissue
Mean
SD
Mean
SD
Brain Blood Heart Lungs Kidneys Liver Muscle Bone Stomach Upper intestine Lower intestine
0.03 1.22 0.32 0.49 3.39 Il.96 IS.48 5.36 2.92 10.98 3.09
0.004 0.816 0.016 0.066 1.503 1.142 2.728 0.699 3.61 6.991 2.037
0.02 2.24 0.12 0.19 0.85 I .73 7.77 2.13 0.55 35.47 1.51
0.009 0.153 0.019 0.029 0.169 0.510 I.619 0.305 0.713 3.639 0.313
Tissue
Mean
SD
Mean
SD
Brain Blood Heart Lungs Kidneys Liver Muscle Bone Stomach Upper intestine Lower intestine
0.05 10.49 0.55 0.89 4.69 17.4s 15.80 8.01 2.33 12.93 2.17
0.013 1.274 0.052 0.047 0.835 2.302 2.413 0.853 1.180 2.860 0.345
0.02 3.24 0.31 0.67 I .89 5.14 10.78 3.94 0.44 40.14 1.15
0.002 0.359 0.045 0.163 0.3 I3 0.434 1.573 0.460 0.118 2.845 0.101
in
BAT0
complexes derived from dimethoxy
h3?
The DMCDO dioxime ligand does produce BAT0 complexes which are more hydrophilic than unsubstituted analogs, and the preliminary data reported here indicates that methoxy substitution increases liver and renal clearance. However, despite a reduction in lipophilicity, there is no evidence for a significant improvement in membrane permeability. Therefore, BATOs based on “hydrophilic” dioximes merit further study because of their potential for faster hepatic clearance, although applications for these BATOs may be limited to targetting sites which do not require physical diffusion across lipid membranes.
BAT0 complexes
I Hydrophilic impurities
References
J I
I
I
I
0
5
10
15
Rt (min) Fig. 3. Reversed-phase HPLC chromatogram of TcCl(DMCDO),-p-TBA. HPLC analysis (Nucleosil C, (15 cm), 50.‘50 ACN/O.I M citric acid (pH 2.4) Flow-l mlimin).
indicated that the product is a mixture of four diastereomers. Had trans-DMCDO been used instead. a similar mixture of complexes would have resulted. However, trans-DMCDO is itself a mixture of D- and L-enantiomers, and use of a single enantiomer should result in the formation of a single stereoisomeric BAT0 complex. The initial radiochemical yields of methoxy-substituted BAT0 complexes was lower than previously obtained with their unsubstituted analogs. Lowering of radiochemical yields is attributed to interaction of the ether group with either the metal and/or the oxime groups. In the case of the dioxime DMDMG, a product with the HPLC retention time expected for the BAT0 complex was observed, but this product could only be obtained in low yield.
0
dioximes
5
IO
15
20
25
30
35
40
Time (min) Fig. 4. Comparison TcCl(CDO),MeB
of the liver uptake and washout of and TcCl(DMCDO),MeB in rats.
Ali E. and Owen L. (1958) Alicyclic Glycols. Part XIV. The tetralin-2: 3-diols and cyclohex-4-ene-1 : 2-diols. J. Cken~. Sot. 86, 106661073. Cardillo G. and Shimizu M. (1977) Oxidation of olefins with silver chromate-iodine. A new and facile synthesis of a-iodo ketones. J. Org. Gem. 42, 426811270. Cope A. C. and Mehta A. S. (1964) Molecular asymmetry of olefins. II. The absolute configuration of traris-cyclooctene. J. Am. Chem. SW. 86, 5626-5630. Feld T. and Nunn A. D. (1989) A chromatographic method for the measurement of the lipophilicity of lipophilic technetium complexes, J. Label. Compound Radiopharm. 26, 274-276. Jurisson S., Hirth W.. Linder K.. Di Rocco R. J., Narrd R. K., Nowotnik D. P. and Nunn A. D. (1991) Chloro - hydroxy substitution on technetium BAT0 (TcCl(dioxime),BR) complexes, Nucl. Med. Rio/. 18, 7355744. Krow G. R., Carmosin R. and Mancuso A. (1977) A simple synthesis of 4-cyclohexene-1,2-ci-diol and its USC as a precursor of a functionalized synthon. Org. Prep. Proc. hr. 9, 285-288. Linder K. E., Malley M. F., Gougoutas J. Z., Unger S. E. and Nunn A. D. (1990) Neutral, seven-coordinate dioxime complexes of technetium(II1): synthesis and characterization. Inorg. C/rem. 29, 242882434. Linder K. E., Wen M. D., Nowotnik D. P., Malley M. F., Gougoutas J. Z., Nunn A. D. and Eckelman W. C. (199la) Technetium-labeling of monoclonal antibodies with functionalized BATOs: 1. TcCl(DMG),PITC (PITC = phenylisothiocyanate). BioconjtrRate Chem 2, 160~170.’ Linder K. E., Wen M. D.. Nowotnik D. P., Ramalingam K.. Sharkev R. M.. Yost F., Narra R. K., Nunn A. D. and Eckelman W. C. (199lb) Technetium-labeling of monoclonal antibodies with functionalized BATOs: 2. TcCl(DMG),CPITC (CPITC = carboxyphenylisothiocyanate) labeling of B72.3 and NP-4 whole antibodies and NP-4 F(ab’)?. Bioconjugnle Chem. 2, 407-414. Linder K., Chan Y.-W., Cyr J. E., Nowotnik D. P.. Eckelman W. C. and Nunn A. D. (1993) Synthesis, characterization, and in vitro evaluation of nitroimidazole-BAT0 complexes: new technetium compounds designed for imaging hypoxic tissue. Bioconjugcrte Chcm. 4, 326-333. Mori K. (1974) Synthesis of cxo-brevicomin. The pheromone of western pine beetle, to obtain optically active forms of know absolute configuration. Tetruhedron 30, 4223 4227. Narra R. K., Nunn A. D.. Kuczynski B. L., Feld T.. Wedeking P. and Eckelman W. C. (1989) A neutral technetium-99m complex for myocardial imaging. J. Nucl. Med. 30, 1830 -1837. Narra R. K., Nunn A. D., Kuczynski B. L.. Di Rocco R.
634
Kondareddiar
J., Feld T., Silva D. A. and Eckelman W. C. (1990) A neutral lipophilic technetium-99m complex for regional cerebral blood flow imaging. J. Nucl. Med. 31, 1370--l 377. Nowotnik D. P. and Narra R. K. (1993) A comparison of methods for the determination of dead time in a reversedphase high-performance liquid chromatography system used for the measurement of lipophilicity. J. Liy. Chromutogr. 16, 3919-3932. Nowotnik D. P. and Nunn A. D. (1992) Technetium compounds for imaging myocardial and cerebral perfusion. Drug News and Perspectiws 5, 174-183. Nowotnik D. P., Feld T. and Nunn A. D. (1993) An examination of some reversed-phase HPLC systems for the determination of lipophilicity. J. Chromatogr. 630, 105-l 15. Nunn A. D., Feld T. and Narra R. K. (1990) Quantitative structure distribution relationships (QSDRs) of BATOs. In Technetium and Rhenium in Chemistry and Nuclear Medicine, Vol. 3, pp. 399-404.
Ramalingam
PI (II
Nunn A. D., Jurisson S., Linder K. II. and Eckeiman W. C. (1991) Boronic acid adducts of rhenium dioximc and technetium-99m dloxime complexes containing a biochemically active group. Eur. EP 441. 491 Pirro J. P., Di Rocco R. J.. Narra R K. and Nunn A. D. (1994) The relationship between iit ri/ro memhr-ant permeability and in riro single-pass brain cxtractlon bf several -SPECT imaging aienis. .I. lVvlc<,(.,kl<,t/. 35, 1514~1519. Ruhlmann K. (1971) Die Umsetzung von Carbonsaureestern mit Natrium in Gegenwart von Trimethylchlorsilan. Synthesis 236&252. Treher E. N., Francesconi L. C.. Gougoutas J. %.. Malley M. F. and Nunn A. D. (1989) Monocapped rris(dioxime) complexes of technetium(II1): synthesis and structural characterization of TcX(dioxime),B-R (X = C1,Br; dioxime = dimethylglyoxime, cyclohexanedione dioxime; R = CH,, C,H,). Inorg. Chem 28, 341 l-3416.
NW/. Med. Biol.
Pergamon
0969-8051(94)00143-X
BAT0 Complexes Derived from Dimethoxy Dioximes: Synthesis, Characterization and Biodistribution KONDAREDDIAR RAMALINGAM, SILVIA S. JURISSON, BRUCE L. KUCZYNSKI, RICHARD DI ROCCO, RAMA K. NARRA. DAVID P. NOWOTNIK* and ADRIAN D. NUNN The Bristol-Myers Squibb Pharmaceutical Research Institute, Route 206 & Provinceline Road, Princeton, NJ 08543, U.S.A. (Accepted 28 November 1994)
To prepare less lipophilic BAT0 complexes, two new methoxy-substituted dioximes were synthesized: cis-4,5-dimethoxycyclohexane-1,2-dione dioxime (DMCDO) and 1,4-dimethoxybutane-2,3-dione dioxime (DMDMG). 99mTcC1(DMCDO),BMe (BMe = methylboronic acid) was prepared and characterized. Reversed-phaseHPLC analyses of 9P”TcC1(DMCDO),BMe and 99mTcC1(DMCDO),-p-TBA (p-TBA = ptolylboronic acid) indicated that both of these complexes were mixtures of four enantiomeric pairs of diastereomers. Attempted preparation of a BAT0 complex from DMDMG gave a mixture of products. In rats, 99mTcC1(DMCDO),BMedisplayed more rapid liver and renal clearance than 99mTcC1(CDO),BMe, but WmTcCI(DMCDO),BMe and 99mTcC1(DMCDO),-p-TBA displayed low uptake in both heart and brain
Introduction
tracer, TcCl(DMG),B2MP, it is 3.8 (Feld and Nunn, 1989)]. Such high lipophilicity values are of concern in applications which might require tracers to cross lipid membranes in vtvo (Pirro et al., 1994) or where it is necessary to minimize non-specific binding. In other series of complexes, addition of methoxy substituents have been used with some success when a reduction of lipophilicity was required (Nowotnik and Nunn, 1992). In this paper, we describe the synthesis and preliminary evaluation of BATOs (Fig. 1) prepared from two novel dioximes which contain methoxy substituents: cis-4,5-dimethoxy-1,2cyclohexanedionedioxime (DMCDO) and dimethoxydimethylglyoxine (DMDMG). The chemistry of these two new classes of BATOs has been studied on both the macroscopic Tc-99 level and the radiopharmaceutical Tc-YYm level. Rat biodistributions were performed with two of the DMCDO BATOs, and compared to the previously reported data for TcCl(CDO),MeB.
The BAT0 (Treher et ul., 1989) class of compounds (Boronic acid Adducts of Technetium dioximes) have been studied for a variety of applications in nuclear medicine: for myocardial perfusion imaging (Narra et al., 1989) cerebral perfusion imaging (Narra et al., 1990). antibody labelling (Linder et al., 1991a, b), imaging of regions of hypoxia (Linder et al., 1993) and receptor imaging (Nunn et al., 1991). The BAT0 complexes have the general formula TcCl(dioxime),BR, where BR is a boronic acid adduct. These complexes are formed by a template synthesis, in which the technetium, dioxime. boronic acid and chloride ion come together in situ to form the resultant TcCl(dioxime)3BR complex (Linder et al., 1990). To date the dioxime has been limited primarily to 1,2-cyclohexanedionedioxime (CDO) and dimethylglyoxime (DMG), while the R group on the boronic acid has been varied from an OH group, to alkyl, aryl and molecules which might target specific sites in the body (Nunn et al., 1990). The BATOs are neutral complexes and the DMG and CD0 analogs are highly lipophilic [the estimated log P for the myocardial perfusion tracer, TcCl(CDO),MeB, is 4.6, and for the brain perfusion
Experimental
*Author for correspondence, at: Guilford Pharmaceuticals, 661I Tributary Street, Baltimore, MD 21224, U.S.A. 625
Physical methods HPLC separations were run using a system consisting of two Altex 1lOA pumps, a Rheodyne 4125 injector, a Kratos 747 UV-visible flow through detector (for Tc-99 complexes), a NaI well type detector
Kondareddiar
626
Ramalingam
controlled by a Tennelec system (for Tc-99m complexes) and a SpectraPhysics 4270 integrator connected to Labnet software. Analyses were run on either a Hamilton PRP-1 (15 cm) column with a mobile phase of 65/35 ACN/O. 1 M NH,OAc (pH 4.6) and a flow rate of 2 mL/min or a Nucleosil C, (15 cm) column with a mobile phase of SO/SO ACN/O.I M citric acid (pH 2.4) and a flow rate of 1 mL/min. FTIR specta (KBr pellets), NMR spectra (‘H and 13C, CD+&), mass specta and elemental analyses were run by the appropriate analytical departments at Bristol-Myers-Squibb in Lawrenceville. Log k’ and log P determinations The log k’ values were determined from an HPLC method described previously (Nowotnik et al., 1993). The PRP- I (15 cm) reverse phase column was used with a mobile phase composed of 65/35 ACN/O.l M NH,OAc (pH 4.6) and a flow rate of 2 mL/min. Nitrate was used to determine the void volume (Nowotnik and Narra, 1993). Diethyl(2S,3S)(-)2,3-dimethoxysuccinate Freshly prepared and finely powdered dry silver oxide (276.66 g, 1.19 mol) was added in small portions to an externally cooled, vigorously stirring solution of diethyl-(2S,3S)-( -)tartrate (81.5 g, 0.395 mol) in methyl iodide (5OOg, 3.5 mol). After stirring for I h at 10°C the reaction mixture was refluxed for 12 h and then filtered. The brown solid was washed with ether. The combined organic solution was evaporated to dryness on a rotary evaporator. The residue was distilled to give 83 g of diethyl(2S,3S)-( -)2,3-dimethoxysuccinate (89.5%). b.p. 1lS”Cj5 mm. Lit b.p. (Mori, 1974): 106108”C/ 4 mm. ‘HNMR (CDCI,): 3.41 ppm (s, 6H, CO&H,), 3.75 ppm (s, 6H, OCH,), 4.09 ppm (s, 2H, C2-H, and C3-H). (2S,3S)-( -)dimethoxybutane-
1,4-diol
To a stirred slurry of lithium aluminum hydride (35.2 g, 0.94 mol) in ether (600 mL) was added drop-
e/ ul.
wise a solution of diethyl-(2S,3S)-( - )2.3-dimethoxysuccinate (83 g. 0.35 mol) in dry ether (400 tnL). The reaction mixture was refluxed for 6 h and then allowed to stir overnight at room temperature. Excess lithium aluminum hydride was carefully destroyed by the successive addition of water (35.0 mL), 15% sodium hydroxide (35 mL) and water (150 mL). The reaction mixture was filtered, the filter cake was washed with acetone (6 x 200 mL), and the combined filtrate was evaporated on a rotary evaporator. The oil obtained was distilled under reduced pressure to give 36 g of (2S,3S)-( -)2,3-dimethoxybutane-I .4diol (75%). b.p. 92-93‘CO.l mm. Lit b.p. (Mori. 1974): 92297”C/O. 1 mm. ‘HNMR (CDCI,): 3.54 ppm (s, 8H, OCH, and OH), 3.78 ppm (m, 4H, CH2), 4.05 ppm (s, 2H, C2-H and C3-H). (2S,3S)-( -)2,3-dimethoxybutanetoluenesulphonate
1.4-dial
di-p-
To a cooled (OC) stirring solution of (2&3S)(-)dimethoxybutane-1,4-diol (36.0 g, 0.24 mol) in dry pyridine was added p-toluenesulfonyl chloride (112 g, 0.56 mol) and the resulting mixture was left at 5°C for 24 h. The reaction mixture was poured into ice-water and the oil that separated was extracted with ether (4 x 200 mL). The ether solution was washed with 5% HCI (2 x 200 mL) and dried with sodium sulfate. Evaporation of the ether afforded a viscous oil. Yield 70.0 g (64%). The compound was used for the next step without further purification. ‘HNMR (CDCI,): 2.42 ppm (s, 6H, -CH,), 3.27 ppm (s, 6H, OCH,), 3.5 ppm (t, 2H, C2-H and C3-H), 7.41 ppm (d, 4H, ArH). 7.64 ppm (d, 4H, ArH). (3S,4S)-(-)3,4-Dimethoxyhexane-l,&dinitrile To a solution of (2S,3S)-( -)dimethoxybutane-l,4diol di-p-toluenesulfonate (70 g. 0.153 mol) in dry DMSO (150 mL) at room temperature was added NaCN (18.5 g, 0.38 mol) in portions over a period of 3 days. The reaction mixture was stirred for a further 72 h at which time the clear brown solution was poured into water (2 L) and extracted with methylene
MeO
OMe
\f
I
MC?0
Cl
‘R’
TcC~(DMDMG)~BR Fig. 1. General structures of the BATOs derived from methoxy-substituted
dioximes
BAT0
complexes derived from dimethoxy dioximes
chloride (6 x 500 mL). The methylene chloride solution was washed with water (3 x 500 mL) and dried with sodium sulfate. Evaporation of the methylene chloride afforded a yellow solid. Yield 15.8 g (61.8%). m.p. 71-72°C. Lit m.p. (Cope and Mehta, 1964) 71.8-72.2”C. ‘HNMR (CDCI,): 2.66ppm (d, 4H, CH,), 3.5 ppm (s, 6H, OCH,), 3.72 ppm (2H, t, C3-H and C4-H). Dimethyl (3S,4S)-3,4-dimethoxyadipate A stirred solution of (2S,3S)-( -)3,4-dimethoxyhexane- 1.6-dinitrile (15.8 g, 0.94 mol) in dry methanol was saturated with dry HCl. The mixture was refluxed for 2 h and then the methanol was removed on a rotary evaporator. The remaining solution was diluted with water and the oil that formed was extracted with ether (3 x 300mL). Removal of the solvent afforded a brown oil, which was added to a solution of thionyl chloride in methanol (20 mL of thionyl chloride was added to 150 mL of methanol at - 78°C) and kept overnight in a refrigerator. The methanol was evaporated on a rotary evaporator and the residue was dissolved in ether (200mL). The ether solution was washed with sodium bicarbonate solution (2 x 200 mL) and water (2 x 300 mL) and dried with sodium sulfate. The solvent was removed and the residue was distilled to give 17.8 g of dimethyl(3S,4S)-(-)3,4-dimethoxy adipate (81.5%). b.p. 12&22C/0.5mm. Lit. b.p. (Cope and Mehta, 1964) 85’C/O.O6mm. ‘H-NMR (CDCl,): 2.53 ppm (m, 4H, CH,), 3.42 ppm (s, 6H, COCH,), 3.76 ppm (s, 6H, OCH,), 3.96 ppm (m, 2H, C‘3-H and C4-H). 1,2-Bis(trimethylsilylo.~y)-4,5-dimetho.~ycyclohexene A 500mL three necked flask was fitted with a stirrer, addition funnel and a reflux condenser and maintained under nitrogen atmosphere. The flask was charged with 200 mL of dry toluene and 1.84 g (0.08 mol) of sodium. The solvent was brought to gentle reflux and the stirrer was operated until the sodium was fully dispersed. A mixture of dimethoxy adipate (4.68 g, 0.02 mol) and trimethylsilylchloride (8.68 g, 10.1 mL, 0.08 mol) in 50 mL of dry toluene was added over 3 h. The reaction was exothermic and a dark purple precipitate formed. A gentle reflux was maintained throughout the addition. After 5 h of additional stirring, the contents of the flask were cooled and then filtered under a nitrogen atmosphere. The pale yellow filtrate was transferred to a distilling flask, the solvent was evaporated under reduced pressure and residue was distilled. b.p. 75-78’C/ 1 mm. Yield 2.8 g (47%).
1,4Cyclohexadiene (21.4 g, 0.27 mol), potassium acetate (0.066 mol) and iodine (0.134 mol) in acetic acid (400 mL) were stirred at RT for 12 h. Potassium acetate (30.0 g, 0.3 mol) was added and the mixture was refluxed for 7 h. After cooling, water (5 mL) was
627
added and the solvent was removed in vacua. The residue was taken up in ether (250 mL), and washed with aqueous sodium thiosulfate solution to remove the brown color. After drying, solvent was removed in vacua and cyclohex-4-ene-cis- 1,2-diol monoacetate was distilled. Yield 18.0 g, b.p. 94C/2mm. MS: (M + H)+ = 157. The cis-monoacetate (5.0 g) and sodium methoxide (1 .Og) in dry methanol (60 mL) were heated to reflux for 1 h, cooled, and neutralized with carbon-dioxide. The solvent was removed in vacua and the residue was extracted with ether. Removal of ether afforded cyclohex-4-ene-cis - 1,2diol. m.p. 8&82’C. Lit m.p. (Ali and Owen, 1958): 80-8 I (C. 2-lodo-4,5-dimethoxycyclohexanone Iodine (93.2 g, 0.0125 mol) and a solution of pyridine (0.4 g) in 5 mL of methylene chloride at 0 ‘C were added to a suspension of silver chromate (3.3 1 g, 0.01 mol) and 4A molecular sieves (5 g) in 25 mL of methylene chloride and stirred for 10 min. A solution of dimethoxy cyclohexene (1.46 g, 0.01 mol) in 15 mL of methylene chloride was added dropwise over 5 min to the ice-cooled suspension, and then stirred for 30min at 0°C. The cooling bath was removed and the reaction mixture was stirred for an additional hour at room temperature. The dark-brown mixture was filtered through a pad of celite, washed with aqueous sodium thiosulfate (5%) and dried with sodium sulfate. The crude product obtained after concentration was purified by column chromatography (silica gel, hexane-ethyl acetate 7: 3) to give the title compound as a light yellow oil. IR (neat) 17lOcm~‘. ‘H-NMR (CDCI,): 2.12-3.12 ppm (m. 4H, CH?), 3.42 and 3.48 ppm (s, 6H, OCH,), 3.82-3.98 ppm (m, 2H, CH), 4.75 ppm (m, IH, CHI). MS: (M + NH,)’ = 302. 4.5Dimethoxycycloheuanone-2-nitrate
ester
To a solution of 2-iodo-4,5-dimethoxycyclohexanone (1 g, 3.5 mmol) in acetonitrile (2 mL) was added a solution of silver nitrate (0.67 g, 4 mmol) in 2 mL of acetonitrile. After stirring for 24 h at RT the mixture was filtered, the silver bromide was washed with ether, and the combined filtrate and washings were evaporated at 30mm (3OC). The residue was taken up in ether, washed with water, and dried. Removal of the solvent afforded the nitrate ester. Yield 0.5 g (65%). ‘H-NMR (CDCI,): 2.28-2.84 ppm (m. H, CH,), 3.42 and 3.48 ppm (s, 6H, OCH,). 3.7553.85 ppm (m, 2H, CH), 4.05 ppm (m. 1H, CHONO,). MS: (M + H)+ = 237. cis-Dimethoxycyclohe.uane-1,2-dione
dioxime
To a mixture of hydroxylamine hydrochloride (1.05 g, 0.015 mol) and sodium acetate (1.64 g, 0.02 mol) in methanol (10 mL) was added a solution of the nitrate ester (1.14 g, 0.005 mol) in methanol (5 mL) and the mixture was stirred at RT for 12 h. Silica gel (5.0 g) was added to this reaction mixture
Kondareddiar Ramalingam et crl
62X
and the methanol was removed on a rotary evaporator to afford a free flowing powder. The free flowing powder was loaded on to a silica gel column (50 g) and eluted first with 9.5:0.5 methylene chloride-methanol followed by 9: 1 methylene chloride-methanol. Fractions containing the product were collected and evaporated to give a tan yellow solid. Yield 0.61 g (60%). The product was crystallized from acetone. m.p. 180-181 ‘C. ‘H-NMR (DMSOd6): 2.66 ppm (m, 4H, CH,), 3.29 ppm (s, 6H, OCH,), 3.64 ppm (t, J = 6.0 Hz, lH, CH), 11.23 ppm (s, 2H, OH). MS: (M + H)+ = 203; (M + NH,)+ = 220. Anal. Calcd. for: C,H,,N20,: C, 47.51, H, 6.98, N, 13.86. Found: C, 47.62, H, 7.13, N, 13.87. 1,4-Dimethoxy-2-butene Sodium hydride (24g, 60% in mineral oil) was washed with anhydrous ether (100 mL) and then suspended in dry THF (200 mL). 2-butene-1,4-diol (22 g, 0.25 mol) was added, the mixture was cooled to 0°C and then stirred at 0°C for 20 min. Methyl iodide (20 mL) was added and the reaction mixture was allowed to warm to RT while stirring. The reaction mixture became exothermic (+ 10 min) at which time it was cooled with an ice bath. After cooling for 20 min the reaction mixture was allowed to warm to room temperature and stirred at RT overnight. The THF was evaporated and the residue was extracted with ether (3 x 150 mL) and dried with sodium sulfate. Evaporation of the ether afforded an oil which was distilled under diminished pressure. Yield 14 g (48%). b.p. 85-87”C/25 mm. ‘H-NMR (CDCI,): 3.31 ppm (s, 6H, OCH,), 3.98 ppm (d, 4H, CH?). 5.82 ppm (t, 2H, CH = CH). 2-Iodo-4,Sdimethoxljbutanone 1,4-Dimethoxy-2-butene (5.8 g, 0.05 mol) was reacted with silver chromate (16.55 g, 0.05 mol) and iodine (16.51 g, 0.065 mol) and pyridine (2 g) in methylene chloride in the presence of 4 8, molecular sieves (25 g). The reaction mixture was filtered, the methylene chloride solution was washed with sodium thiosulfate and water, and then dried with sodium sulfate. Evaporation of the methylene chloride afforded a yellow oil. Yield 6.5 g. ‘H-NMR (CDCI,): 3.31 and 3.33ppm (s, 6H, OCH,), 3.64.4ppm (m, 4H, CH2). 4.8 ppm (m, lH, CHI). MS: (M+H)+ =261. 4,5-Dimethoxy-1,2-butanedione
product formed was purified by column chromatography (silica gel, methylene chloride--methanol~8:2). Yield 1.5 g. The product was crystallized from water, m.p. 165-66°C. ‘HNMR (CDCI,): 6 3.2 ppm (s, 6H, OCH,), 4.25ppm (s, 4H, CHJ. I I.8 ppm (s, 2H, NOH). MS: (M + H)+ = 177. Anal. Calcd. for: C,H,,N,O,: C, 40.90, H, 6.87, N, 15.9. Found: C. 40.80, H. 7.09, N, 15.81.
NH,99Tc0, (0.05142 g; 0.284 mmol) was dissolved of H,O. DMCDO (0.18113g; in ca 15mL 0.897 mmol) and methylboronic acid (0.02093 g; 0.340 mmol) dissolved in 5 mL of EtOH were added to the stirring NH,TcO, solution. The reaction mixture was heated gently; SnCI, (0.125 18 g; 0.659 mmol) dissolved in I mL of cone HCl and diluted with 1 mL of H,O was added dropwise over 5 min to the stirring reaction mixture. The reaction mixture became an intense reddish-brown in color. The reaction was stirred with heat for ca 1 h. An equal volume of CH,Cl, (ca 25 mL) was added and the reaction mixture was allowed to sit at room temperature overnight. The CH2Cl, fraction was collected and the aqueous fraction then exhaustively extracted with CHJl,. The combined CH,C& fractions were dried through anhydrous Na,SO, and concentrated to UI 2 mL. The CH,Cl, fraction was purified by silica gel chromatography (1 x 10 cm;. Four green and one orange band (unidentified) were eluted with CH2ClI. CHCI, eluted the orange product band. partially separating the diastereomers (Vide @a). The product was collected in five fractions, The five fractions were analyzed by HPLC [Nucleosil C, (15 cm), 50150 ACN/O. 1 M citric acid (pH 2.4). 1 mL/min, 400 nm], and showed that a partial separation of diastereomers had been achieved (fractions l-5 from the column had retention times in the range 6.9-7.6 min). Crystallization from CH$I,/MeOH/ HCI yielded solid from two of the fractions, The orange solids from fractions 1 and 2 were collected. washed with MeOH/HCl and vacuum dried. Mass Spectra (FAB): Fraction
1:
Fraction 2:
M +/(M - H) (M - Cl)+/(M - HCI) M+/(M-H)(M - Cl)+/(M - HCl)
= 762,:761 = 727/726 =762!‘761 = 727.‘726
dioxime
2-Iodo-4,5-dimethoxybutanone (6.5 g, 0.0256 mol) was reacted with a solution of silver nitrate (8.7 g, 0.044 mol) in acetonitrile (25 mL) at RT for 24 h. The silver iodide formed was filtered, and the solvent was removed under vacuum to afford the nitrate ester. Yield 2.5 g. ‘HNMR (CDCI,): fi 3.31 and 3.42 ppm (s, 6H, OCH,), 3.742 ppm (m, 4H, CH,), 5.7 ppm (m, lH, CHNO,). The crude nitrate ester obtained above (2.0 g) was reacted with hydroxylamine hydrochloride and sodium acetate in methanol and the
FTIR Specta (KBr pellets): Fraction
1: C=N B-O Fraction 2: C=N B-O
1633cm-‘, N-O 1048cm.‘, 1096 cm ’ 1633 cm ’ , N-O 1049 cm I, 1099 cm ’
The following were added to a 6 mL glass vial: methylboronic acid (2-3 mg, 33-50 mmol), DMCDO
BAT0
complexes derived from dimethoxy dioximes
(0.24.4 mg, IO-20 mmol), 0.2 mL of aqueous citric acid (100 mg/mL, 95.1 mmol), 0.1 mL of DTPA (20 mg/mL 0.5 M NaOH, 5.1 mmol), 0.2 mL of saturated NaCl, 5 p L of &Cl, solution (0.1 g/mL in cone HCl diluted to 4 ml, 2.6 mmol). The vial was sealed and 0.5 mL of 99mTc04m/saline (21.8 mCi generator eluent) was added. The vial was heated at 70°C for 15 min and cooled. The reaction mixture was purified by a solid-phase extraction procedure described previously (Jurisson et al., 1991). The lipophilic product was adsorbed onto a plug of PRP-1 resin. The resin was washed three times with 0.5 mL of 25% EtOH/saline to remove the non-radioactive constituents, then the product eluted by washing the resin with 0.5 mL of EtOH. The RCP of the purified complex was found to be 90.3%. The ethanolic solution was diluted with saline to obtain a sample (24 mCi/O.l mL) suitable for rat biodistribution studies. The product had the same retention time on HPLC analysis as “9TcCI(DMCDO),BMe.
Table I. Reaction conditions and HPLC results in the attempted preparation of 99mTcCl(DMDMG)1BBu Peak retention times (min)
RCP (%)
(a) pH 2, no non-essential ingredients
0.90 I .39
x7.3 12.7
(2) pH 4 with NH,OAc, no non-essential ingredients
0.X6 0.98 I .39
20. I 50.0 2Y.9
(3) Kit 2 conditions, heated at 7o’C for ISmin
0.99 1.39
62.3 37.7
(4) pH 4 with Na,citrate, no non-essential ingredients
0.76 I .42
66.9 33.1
(5) Kit 4 conditions heated at 7o’C for I5 min
0.75 I .40
52.2 47.8
Kit conditions
Purification of kit 4 (Table 1) on a plug of PRP-1 resin by adsorbing the complex of interest, washing once with 0.5 mL of 25% EtOH-saline and eluting with 0.5 mL of EtOH yielded the 1.44 min peak in 73% RCP. Biodistribution
This uncapped tris(dioxime) complex (Linder et al., 1990) was prepared following the procedure described above for 99mTcCl(DMCDO),BMe but omitting the methylboronic acid. HPLC analysis (same system as above; retention time of 3.03 min) gave an RCP of ca 80% for the desired complex. The complex was purified by solid-phase extraction; the complex was adsorbed onto the PRP-1 resin, washed three times with 25% EtOH-saline and eluted with 0.5 mL of EtOH. After purification, the RCP of the complex was 97%. To insure that this complex was the uncapped TcCl(DMCDO),, ca 2mg of (HO),BMe was added to the purified complex in EtOH, followed by two drops of 2 M HCl. This resultant solution was heated at 70’ C for 10 min. ‘““TcCI(DMCDO).,B-pTBA A complex was prepared following the procedure described above for 99mTcCl(DMCDO),BMe, substituting the equivalent molar quantity of p-tolueneboronic acid @-TBA) for MeB. EtOH (0.2 mL) was also added to the kit to help dissolve the pTBA. HPLC analysis (as above) showed four pairs of diastereomers (retention times of 12.4, 13.1, 13.6 and 14.3min) with a combined RCP of ca 83%. The complex was purified on a plug of PRP-1 resin; the compound was adsorbed onto the PRP-1 resin, washed twice with 0.5 mL of 25% EtOH-saline, once with 0.5 mL of 50% EtOH/saline and eluted with 0.8 mL of EtOH. This gave the desired complex with an RCP of cu 96%. The ethanolic solution was diluted with saline for a rat biodistribution study. Attempted synthesis (BBu = n-hutylhoronic
OJ’ yHnTcCI(DMDMG)lBBu, acid)
Several reaction conditions were examined for the preparation of this complex, as listed in Table 1.
629
studies
Biodistribution studies were conducted using male rats anesthetized with sodium Nembutal (50 mg/kg, i.p.). The agents were injected into the external jugular vein as a 0.1 mL bolus and the rats were sacrificed 5 and 60 min later. The organs and blood were collected and the radioactivity in each sample was counted to determine the injected dose per organ and per gram of organ. Five rats were used for each time point. Gamma cameru stud?) An anesthetized male rat was positioned (anterior view) for gamma camera acquisition and then injected i.v. with 2.1 mCi of 99mTcCl(DMCDO),BMe (prepared as above). Immediately following the injection, a 30 min (1 frame/min) dynamic acquisition was initiated using a pinhole collimator. For each frame of the dynamic acquisition, counts in the liver region of interest (ROI) were obtained using the gamma camera histogram function. The liver washout data (liver ROI counts) was extrapolated from percent injected dose at 30 min and compared to a typical Cardiotec histogram of liver washout (Narra et a/.. 1989).
Results and Discussion Syntheses of‘ the dimethoxy dioximes Syntheses of cis-4,5-dimethoxycyclohexanedione dioxime (DMCDO) and 1,4-dimethoxy-2,3-butanedione dioxime (DMDMG), were undertaken for the purpose of producing more hydrophilic BAT0 complexes. There are a few examples of the synthesis of substituted cyclohexanedione dioximes in the literature, so some preliminary studies had to be undertaken to develop a successful method of synthesis cis-4,5-dimethoxycyclohexanedione dioxime (DMCDO).
Kondareddiar Ramalingam CI trl
MICAH,CH,I
CO,C,Hs
CH,OH
UAIH, b diethyl ether
TsCl
CH,OTs
b
pyridine
NaCN / DMSO
m30, b
C&OH / HCI
I,
CH,OH
CH,CO,CH,
WCY), QCW,
CH,OTs
WP
JI
1
CY%c=H,
0
Na / xylene
HOAc
W,),Si~
0
OSi(CH,),
Scheme 1. Attempted synthesis of 4,5-dimethoxycyclohexane- I ,2-dione via the reduction of diethyl 3,4-dimethoxyadipate. Based on Ruhlmann’s report (Ruhlmann, 1971) on the successful synthesis of 1,2-cyclopentanedione using an acyloin condensation, the route shown in Scheme 1 was devised. Dimethyl (3S,3S)-3,4dimethoxyadipate (1) was prepared according to the published report (Mori, 1974) from diethyl-L-tartrate. Acyloin condensation of this diester in the presence of trimethylsilyl chloride produced a complex product mixture. Purification of this mixture by column chromatography afforded 15% of the desired 4,5-dimethoxy-1,2-bis(trimethylsilyloxy)cyclohexene (2). However, oxidation of this compound with acetic acid and cupric acetate did not yield the desired diketone (3). Therefore, this route was abandoned due to the difficulty in isolating the silyl ether and oxidizing the ether to diketone. Instead a five step route (Scheme 2) was investigated. The first step of
the procedure was that of Krow et al. (1977), whereby cyclohexene- 1,4-diene was converted to the diol. The diol was smoothly converted to the methyl ether with methyl iodide and sodium hydride. The next step involved the oxidation of the olefin to a a-iodocyclohexanone. The method was based on a literature report (Cardillo and Shimizu, 1977) of the synthesis of a-iodocyclohexanone from cyclohexene. Oxidation of 4,5-dimethoxycyclohexene with silver chromate and iodine in methylene chloride afforded the iodoketone in 46% yield. The iodoketone was readily converted to the nitrate ester by reaction with silver nitrate in acetonitrile. Conversion of the nitrate ester to the 1,2-diketone proceeded smoothly. However, isolation of the diketone product from the reaction mixture proved difficult. One reason for this is the fact that the 1,2-diketone may exist as an enol
CH30
HO NELH/CH,I CH3CN
+ CH30
HO
12/CH2C12
I
Ag2Cr04
NH20H. cn3c0pa
c-I30
5LizYic-t30 5
HCl
CH30
CH30
AgN03/CH3CN CH30
CH30 4
Scheme 2. Synthesis of 4,5-dimethoxycyclohexane- I ,2-dione dioxime.
3
BAT0
Ho
Ho
NaH, CH,I
MeO
THF
MeO
l3
4W, CH,CN
complexes derived from dimethoxy
*
MeO
2
631
AS,CrO, 1 I,
ME!0 32
cw4
3
N&OH. HCI
I
Me0
I-
04
Mt30
dioximes
MeO M&)
b
NaOAc, -OH 0
0 NOH
NOH
Scheme 3. The synthesis of DMDMG.
tautomer. The highly polar enolic product partitions into the aqueous layer rather than the organic layer which may make isolation of the product in the diketo form difficult. Since the diketone is ultimately needed as its dioxime derivative, the nitrate ester was reacted with hydroxylamine hydrochloride and sodium acetate in ethanol to afford the dioxime. DMDMG was prepared from butene-1,4-diol by a procedure similar to that described above for DMCDO (Scheme 3).
developing a linear plot of log k’ and log P (octanol/water) for a set of standard organic compounds. The mathematical equation relating to k’ and log P was found to be log P = (log k’/0.36) + 0.23. The log P values estimated for 99”TcCI(DMCDO),BMe and 99mTcC1(CDO),BMe using this system are shown in Table 2. As expected the lipophilicity of 99mTcCI(DMCDO),BMe is much less than that of 99”TcC1(CDO),BMe. While DMCDO BAT0 complexes appear as a broad peak on the PRP-1 HPLC system, each comFormation of technetium complexes plex can be resolved into four peaks on a Nucleosil BAT0 complexes were formed from DMCDO and C8 reversed-phase column. As shown in the schematic representation in Fig. 2, the DMCDO BATOs two boronic acids, methylboronic acid (MeB), and p-tolylboronic acid (p-TBA), and a number of reacare a mixture of four enantiomeric pairs of diastereomers, which are formed as a result of the cis tion conditions were examined for the formation of a complex derived from DMDMG and n-butylconfiguration of the methoxy groups on the cyclohexane ring and the presence of the seventh ligand about boronic acid (BBu). For the DMCDO complexes, the 99Tc and 99mTc the Tc. For 99mTcCI(DMCDO),BMe, the retention times of this quartet are 6.5, 6.9, 7.1 and 7.5 min, BAT0 complexes were formed under conditions while for 99mTcCl(DMCDO),-p-TBA the retention similar to that described previously for CD0 complexes (Linder et al., 1990; Jurisson et al., 1991). times are 12.4, 13.1, 13.6 and 14.2min. Purification of 99TcCl(DMCDO),BMe by silica The initial radiochemical purities of the DMCDO gel chromatography partially separated two of complexes were lower than had been observed for the four diastereomers. These were isolated and their CD0 analogs, but RCPs could be improved each analyzed by FTIR and mass spectroscopy. by using a solid-phase extraction procedure. 99mTcC1(DMCDO)JBMe can also be formed in a The mass specta were consistent with the assignment of structure (Fig. 1) while FTIR spectrum of two-step process involving the isolation of the 99TcCl(DMCDO),BMe was similar to that reported intermediate uncapped complex 99mT~Cl(DMCDO)1 previously for 99TcCl(CDO),BMe (Treher et a/., followed by capping with methylboronic acid, which 1989). One of the two isolated diastereomers was also has been reported for other BAT0 complexes (Linder characterized by ‘H and ‘)C-NMR spectroscopy. The et al., 1990). The 99mTcDMCDO BAT0 complexes appeared as analytical data are consistent with the structural assignment shown in Fig. 1, indicating that the two a broad single peak on the Hamilton PRP-1 HPLC system. This system has been used previously for the isolated complexes are diastereomers. Attempts to prepare 99mT~Cl(DMDMG)3BB~ reestimation of log P. The system is calibrated by sulted in mixtures of products. Reaction conditions which were investigated are listed in Table 1. Based *The expected retention time of DMDMG-BBu was calcuon the retention times of 99mTcCI(DMCDO)3BMe, lated based on the observed difference in log k’ values for CDO-MeB and DMCDO-MeB of 0.97 (1.52 and 0.55, ““‘TcCl(CDO),BMe, and 99”TcCI(DMG),BBu on respectively). If we assume that the log k’ difference for DMG-BBu and DMDMG-BBu is comparable (0.97), we can calculate an approximate retention time for DMDMG-BBu on PRP-I (65/35 ACN/O.l M NH,OAc, 2 mL/min). The log k’ for DMG-BBu is 1.20. If the log k’ difference between DMG-BBu is ca 0.97, then log k’ for DMDMG-BBu is ca 0.2. A log k’ value of 0.2 indicates a retention time of ca 1.4 min when the void volume is 0.55 min.
Table 2. Log P values of BAT0 complexes estimated from HPLC lee k’ data log P Compound
Compound TcCI(CDO),MeB TcCI(DMCDO),MeB *(Estimated
4.6 I .7
TcCI(DMG),BBu TcCI(DMDMG),BBu*
as described in the text).
log P 3.x 1.4
Kondareddiar Ramalingam el al.
bMe
OMe
Fig. 2. Schematic representation of the four diastereomers of TcCl(cis-DMCDO),BR.
the PRP-1 system, the retention time of 99mTcCl(DMDMG),BBu was estimated to be 1.4 min*. A product with this retention time was observed, but could not be isolated from the other reaction products. The 0.75 min peak observed in kits 4 and 5 is believed to be a Tccitrate complex. Based on our prior experience with the BAT0 complexes (Treher et al., 1989; Linder et al., 1990; Jurisson et al., 1991) it is clear that complex yields with DMCDO and DMDMG are inferior to their corresponding CD0 and DMG complexes, In the case of DMCDO, it is possible that the two ether oxygen atoms can compete with the oximes when binding to technetium, so lowering the yield(s) of the BAT0 reaction precursors. In the caseof DMDMG, while the separation and flexible link between the ether groups make it less likely that these groups can compete with the oximes compared to DMCDO, it is possible that the formation of a six-membered ring resulting from intramolecular hydrogen-bond formation between an ether group and its adjacent oxime group reduces the chelating ability of this dioxime. Biodistribution
studies
The results of biodistribution studies of 99mTcCl(DMCDO),BMe and 99mTcCl(DMCDO),-pTBA in rats are shown in Tables 3 and 4. Part of the motivation for developing these less lipophilic agents was to overcome our concern that the BATOs might be so lipophilic that they would readily penetrate into a phospholipid bilayer but remain trapped in the hydrophobic membrane interior. We reasoned that an agent that was somewhat less lipophilic, might retain enough water solubility that it would be able to move back out of the membrane and into the
aqueous cytoplasmic compartment. The biodistribution data show, however, that the brain uptake of these agents is quite low relative to the DMG and CD0 based BATOs [reported previously (Narra et al., 1989)]. Whereas reduced lipophilicity of the methoxy BATOs should increase the ability of an agent to leave the hydrophobic membrane interior once therein, it would also make it more difficult to get in since there would be less free energy available to penetrate the initial membrane resistancepresented by the rigid spacing between phospholipid polar head groups (Pirro et al., 1994). In addition, the increase in size of these agents would increase this resistance. These factors may be related to the reduction of brain “uptake” seen in the biodistribution data compared to their unsubstituted analogs (Narra et al., 1989). As shown in Fig. 4, the liver washout profile of 99mTcCl(DMCDO),BMe is considerably more rapid than that of 99mTcCl(CDO),BMe. In addition, the renal clearance of 99mTcCl(DMCDO),BMe is more rapid than that for 99mTcCl(CDO),BMe, resulting in lower ‘background’ levels for the methoxy BAT0 compared to the unsubstituted compound. However, heart uptake of 99”TcCl(DMCDO),BMe is less than adequate at 0.32% ID at 5 min for imaging.
Conclusions Two routes to 4,5-dimethoxycyclohexane- 1,2dione dioxime (DMCDO) were investigated. This compound was prepared in good overall yield via an iodo nitrate ester intermediate. A similar route was used to prepare 1,4-dimethoxybutane-2,3-dione dioxime (DMDMG). The 99Tccomplex, TcCl(DMCDO),BMe was prepared and characterized. HPLC analyses of the ““TcCl(cis-DMCDO),BR complexes Table 4. Biological
Table 3. Biological
distribution
of 99mTcCI(DMCDO),MeB
distribution
in rats
% i.d./organ 5 min
of ‘gmTcC1(DMCDO)~B-p-TBA rats % i.d./orean
60 min
5 min
Tissue
Mean
SD
Mean
SD
Brain Blood Heart Lungs Kidneys Liver Muscle Bone Stomach Upper intestine Lower intestine
0.03 1.22 0.32 0.49 3.39 Il.96 IS.48 5.36 2.92 10.98 3.09
0.004 0.816 0.016 0.066 1.503 1.142 2.728 0.699 3.61 6.991 2.037
0.02 2.24 0.12 0.19 0.85 I .73 7.77 2.13 0.55 35.47 1.51
0.009 0.153 0.019 0.029 0.169 0.510 I.619 0.305 0.713 3.639 0.313
Tissue
Mean
SD
Mean
SD
Brain Blood Heart Lungs Kidneys Liver Muscle Bone Stomach Upper intestine Lower intestine
0.05 10.49 0.55 0.89 4.69 17.4s 15.80 8.01 2.33 12.93 2.17
0.013 1.274 0.052 0.047 0.835 2.302 2.413 0.853 1.180 2.860 0.345
0.02 3.24 0.31 0.67 I .89 5.14 10.78 3.94 0.44 40.14 1.15
0.002 0.359 0.045 0.163 0.3 I3 0.434 1.573 0.460 0.118 2.845 0.101
in
BAT0
complexes derived from dimethoxy
h3?
The DMCDO dioxime ligand does produce BAT0 complexes which are more hydrophilic than unsubstituted analogs, and the preliminary data reported here indicates that methoxy substitution increases liver and renal clearance. However, despite a reduction in lipophilicity, there is no evidence for a significant improvement in membrane permeability. Therefore, BATOs based on “hydrophilic” dioximes merit further study because of their potential for faster hepatic clearance, although applications for these BATOs may be limited to targetting sites which do not require physical diffusion across lipid membranes.
BAT0 complexes
I Hydrophilic impurities
References
J I
I
I
I
0
5
10
15
Rt (min) Fig. 3. Reversed-phase HPLC chromatogram of TcCl(DMCDO),-p-TBA. HPLC analysis (Nucleosil C, (15 cm), 50.‘50 ACN/O.I M citric acid (pH 2.4) Flow-l mlimin).
indicated that the product is a mixture of four diastereomers. Had trans-DMCDO been used instead. a similar mixture of complexes would have resulted. However, trans-DMCDO is itself a mixture of D- and L-enantiomers, and use of a single enantiomer should result in the formation of a single stereoisomeric BAT0 complex. The initial radiochemical yields of methoxy-substituted BAT0 complexes was lower than previously obtained with their unsubstituted analogs. Lowering of radiochemical yields is attributed to interaction of the ether group with either the metal and/or the oxime groups. In the case of the dioxime DMDMG, a product with the HPLC retention time expected for the BAT0 complex was observed, but this product could only be obtained in low yield.
0
dioximes
5
IO
15
20
25
30
35
40
Time (min) Fig. 4. Comparison TcCl(CDO),MeB
of the liver uptake and washout of and TcCl(DMCDO),MeB in rats.
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