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Chloro → hydroxy substitution on technetium BATO [TcCl(dioxime)3BR] complexes
0883-2897/91 $3.00 + 0.00 Copyright 0 1991 Pergamon Press plc
NW/. Med. Biol. Vol. 18, No. 7, pp. 735-744, 1991 Inr. J. Radiat. Appl. Instrum. Part B Printed in Great Britain. All rights reserved
Chloro +Hydroxy Substitution on Technetium BAT0 [TcCl(Dioxime), BR] Complexes S. S. JURISSON,
W. HIRTH, K. E. LINDER, R. J. Di ROCCO, D. P. NOWOTNIK* and A. D. NUNN
R. K. NARRA,
The Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903, U.S.A. (Received 3 December 1990) The neutral, seven coordinate complexes of technetium known as the BAT0 Qoronic acid Adducts of Technetium dioximes) complexes have shown their utility as myocardial and cerebral perfus& agents. The axial chloride ligand of the BAT0 complexes pTcCl(dioxime),BR] is labile to substitution by a competitive anion; under physiological conditions, the axial chloride ligand can be replaced by a hydroxy group. The chloro and hydroxy analogs have different biodistributions and single-pass cerebral extraction efficiencies. The influence of structure on the rate of the in vitrochloro/hydroxy exchange process has been
studied. The mechanism of axial ligand exchange was found to be S, l-CB, which proceeds by way of a transient, neutral six coordinate complex. Evidence is presented which indicates that chloro/hydroxy exchange is not the mechanism by which BAT0 complexes are retained in the brain.
Introduction Seven coordinate technetium complexes of the general formula TcCl(dioxime),BR, where BR is a boronic acid adduct, have shown their utility as myocardial and cerebral perfusion imaging agents (Linder et al., 1987; Narra et al., 1989, 1990; Seldin er al., 1989; Zielonka et al., 1990). These complexes, better known as BATOs @oronic Acid Adducts of Technetium d@xime) have been well characterized on the macroscopic scale (Treher et al., 1989); the general structure of these complexes is shown in Fig. 1. BAT0 complexes are prepared by template synthesis (Cotton and Wilkinson, 1980); TcO; is reduced with stannous ion in the presence of a vicinal dioxime, a boronic acid, and chloride ion. The mechanism of formation of BATOs has been reported previously (Linder et al., 1990). ‘A large number of 99mTc-BAT0 complexes have been prepared by varying the dioxime ligand and boronic acid employed in the template reaction, giving rise to the determination of structuredistribution relationships for BATOs with a chloride axial ligand (Nunn et al., 1989). x-Ray photoelectron spectroscopy of the BAT0 complexes has shown that the axial chloride ligand has a binding energy (197.1 + 0.3 eV) between that of ___ *Author for correspondence.
a covalent (197.9 f 0.25 eV) chlorine and ionic (195.9 f 0.2 eV) chloride, and so, could be subject to exchange by a competitive anion (Thompson et al., 1986). This paper describes studies which investigate the rate and mechanism of the exchange of the axial chloride ligand by hydroxide for some of the BAT0 complexes. The effect of this exchange process on the biodistribution properties of these compounds is also discussed. Methods General chemicals
Ammonium acetate, citric acid, sodium chloride, sodium phosphate (monobasic) and HPLC grade acetonitrile were obtained from Fisher Scientific. Cyclohexanedione dioxime (CDO) and dimethylglyoxime (DMG) were obtained from Eastern Chemicals, The Nucleosil CB and Hamilton PRP-1 HPLC columns were obtained from Alltech Associates. Sample preparation (a) *TcCl(BATO)s. The 99”TcCl(BATO)s were prepared from lyophilized kits which contained dioxime, boronic acid, and stannous chloride as reductant. 1 mL of *TcO; generator eluent was added to the kits. The kits were heated at 100°C for 15 min, cooled, and the BAT0 complex was 135
736
S. S. JURISSON et
R
R
Fig. 1. The general structure of BATOs.
separated from nonradioactive kit components by the following process: A small volume (approx. 0.5 mL) of a 20% v/v suspension of PRP-1 resin (10 pm dia) in a 25% v/v ethanol/saline mixture was drawn into a 1 mL syringe. A Millex HV, 0.45 micron filter was attached to the end of the syringe and the suspension filtered, causing the PRP-1 resin to pack into a plug on the Millex filter. The contents of the kit were drawn into a 1 mL syringe and loaded onto the top of the syringe containing the PRP-1 resin. The kit contents were passed through the PRP-1 resin, and the filtrate was discarded. The PRP-1 resin was washed twice with 0.5 mL of 25% v/v ethanol/water solution and the filtrate again discarded. The product radioactivity was eluted from the PRP-1 resin with 0.8 mL of absolute ethanol into a siliconized glass vial. (b) ‘“TcOH(BATO)s. Following isolation of the 99mTcC1(BATO)as an ethanolic solution (as described above), the solvent was removed by evaporation under a stream of nitrogen. The BAT0 was redissolved in 1 mL of chloride-free phosphate buffer (pH 8.0) and heated at 37°C for 30 min. The axial ligand exchange process was halted by the addition of 20 p L of cont. HJP04, and the 99mTcOH(BATO) was isolated by same PRP-1 resin procedure as described for the chloro analogs. (c) 9gTcCl(DMG),2MP. This complex was prepared from 99mTcCl(DMG), and 2-methylpropylboronic acid by the method described previously (Linder et al., 1990). (d) “TcOH(DMG),ZMP. Three drops of 2 M aqueous sodium hydroxide and distilled water (2 mL) were added to a solution of TcCl(DMG),2MP (50 mg, 0.091 mmol) in acetronitrile (2 mL). The solution was gently heated for 5 min. Water (2 mL) was added, and the product extracted into ether (3 x 5 mL). The combined ether extracts were washed until the washings were no longer basic and gave a negative silver nitrate test for Cl-. Ether was removed by rotary evaporation at room temperature, and the resultant red-orange oil was dissolved in acetonitrile (2 mL). Water (2 mL) and one drop of aqueous 1 M
al.
trifluoromethanesulfonic acid were added, and the acetronitrile was slowly removed with a gentle stream of nitrogen. The red-orange product crystals which formed were isolated by centrifugation, washed well with water, and air dried. Yield 29 mg (60%). Anal. calcd for C,, H,, N6 BOs Tc (product monohydrate): C, 35.18; H, 5.90; N, 15.38. Found: C, 35.12; H, 5.69; N, 15.34. Optical spectrum (acetronitrile) A,,,,,, nm, (Lmol-’ cm-‘): 450 (3850), 395 (3950), 294 (6700), 258 (sh), 234 (22,000). ‘H NMR (CD,Cl,; 270MHz) 6 0.6 (2H, d, B-CH,), 1.0 (6H, d, CH(CI&),), 1.8-2.0 (2H, bm, CH and Tc-OHJ, 2.35 (12H, s, DMG CH,), 2.4 (6H, s, unique DMG CI-I,), 14-15 (lH, bs, oxime 0I-J. Conductivity (acetronitrile): 5.44 ohm-’ M-l cm2. (e) Phosphate b@ered saline (PBS). The PBS buffers used for the Cl-OH exchange experiments were prepared to contain 6.49 g/L sodium chloride (111 mequiv/L Cl-) and 0.05 M sodium phosphate monobasic, and adjusted to the appropriate pH with NaOH. HPLC analysis
The HPLC system used for monitoring the rate of axial ligand exchange and for comparison of 99Tcand 99mTccomplexes consisted of two Altex 110A pumps (controlled by a model 420 microprocessor controller) and a Rheodyne model 7125 injection valve. The flow-through y detector, which utilized a Tennelec NaI crystal, was built in-house. A U.V. detector set at 265 nm was used to detect the 99Tc material. The percent radioactivity in the eluting peaks was determined using a Spectra-Physics model SP 4270 integrator connected to an IBM PC running chromatographic Labnet software. Two column/eluent combinations were used to measure the rate of axial ligand exchange: (i) Hamilton PRP- 1 column (150 x 4.6 mm, Alltech Associates) with a mobile phase of 65 : 35 acetonitrile : 0.1 M ammonium acetate (pH 4.6), and flowrate of 2.0 mL/min. (ii) Nucleosil Cs column (150 x 4.6 mm, Alltech Associates) with a mobile phase of 70: 30 acetonitri1e:O.l M citric acid (pH 2.4) and flowrate of 1.5 mL/min. Conversion rate of chloro to hydroxy
A 1 mL aliquot of PBS with pH adjusted to predefined values in the range 3.0-l 1.4 was placed into a 16 x 125 mm siliconized Vacutainer tube and either allowed to remain at room temperature (20°C) or incubated at 37°C. An aliquot (25-50 pL, approx. 2OOpCi) of the isolated 99mTcCl(BATO) in ethanol was added and the resultant solution was mixed by shaking. The percent of radioactivity in the form of the chloro complex over time was determined by HPLC analysis. All exchange rates were determined in duplicate. Linear regression analysis of In [fraction of 99mTcCl(BATO)] versus time (in min) gave the rates and half times of Cl -+ OH conversion.
131
Chloro -+ hydroxy substitution Conversion rate of hydroxy to chloro
An aliquot (50 pL, approx. 100 FCi) of the purified hydroxy BAT0 in ethanol was added to 1 mL of PBS buffer at either pH 7.4, 6.4, 5.0 or 3.0, containing 0.5% Tween 80. The half time for conversion to the chloro analog was determined by HPLC, measuring the fraction of the hydroxy and chloro forms at several times after mixing. As minor (unidentified) radioactive products were formed in addition to the chloro BATO, the fraction of the hydroxy form was determined from: fraction *TcOH(BATO)
=
% %TcOH(BATO) % 99”TcOH(BATO) + % *TcCl(BATO)’ (The minor radioactive products were formed immediately on mixing the isolated hydroxy BAT0 with buffer, and the proportion of these components did not increase with time. The surfactant, Tween 80, was required to prevent loss of the minor products.
(20 PCi) of radioactivity via the jugular vein. After 15 s, 1 min, 5 min and 60 min the animals were sacrificed by exsanguination, and the target tissues removed, weighed, and assayed for radioactivity. Cerebral extraction studies
The cerebral blood flow (CBF) was varied in rats anesthetized with Nembutal (50 mg/kg, i.p.) by controlling respiration. A 0.2 mL mixture of ?Sr-labeled microspheres and test compound was rapidly injected into the left ventricle of the heart (using a 1 cm’ Tuberculin syringe with a 27 gage needle) while blood was withdrawn from the femoral artery at a known rate. Six seconds later the rat was decapitated and simultaneously, the arterial withdrawal pump was stopped. Radioactivity in the brain and blood was measured with a y counter which corrected for cross over counts. The cerebral extraction fraction (E) was calculated using the formula (Irwin and Preskorn, 1982): E = (cpm z:;;;nm (cpm
;;;;;.;; 00
Study of the Cl/OH exchange process by ‘H NMR spectroscopy
%TcCl(DMG), 2MP (16 mg) was dissolved in CD,CN (1 mL), and the ‘H NMR spectrum recorded on a JEOL GX-400 spectrometer. A 1% solution of KOD in D,O (0.1 mL) was added to the BAT0 solution, and the ‘H NMR spectra recorded at 10, 60 and 1080 min post mixing. The time dependence of the proportion of chloro and hydroxy WT~X(DMG),2MP complexes under these reaction conditions was determined by HPLC analysis, using u.v./visible detection at 400 nm. Biodistribution studies
Sprague-Dawley rats were anesthetized with Nembutal (50 mg/kg), and injected with 0.1 mL
gemTcCl (CDO)
3
(cpm = counts per min).
Results Treatment of 99TcCl(DMG)Y2MP with base provided a single product in good yield, which was identified from analytical data as *TcOH(DMG),2MP. The same product was obtained when this reaction was conducted with no carrier added 99mTcCl(DMG),2MP, demonstrated by HPLC analysis [both 9Tc and 99mTc complexes of Tc0H(DMG)92MP provided a single peak on HPLC analysis with a R, of 2.1 min, k’ = 2.8, on the PRP-1 analytical column]. Figure 2 shows the HPLC chromatograms recording the conversion of 99mT~Cl(CDO)3EtB
EtB
Q9mTcOH (CDO)
3
EtB
\
l*
-
0 1 2 ,_3 ‘, -5 Retrntion Urn*(minutt3r) t-7
min
1
-Al t-14
min
t-24
min
i
t-30
min
t-45
min
Fig. 2. Series of HPLC chromatograms showing Cl/OH exchange. NMB 11117-E
!_L
1 t-100
min
738
S. S. JURISSON et al, Table 1. Cl -OH
half times of BATOs Cl -+ OH
Dioxime
Boronic acid
DMG
OH
DMG
MC
DMG
Et
DMG
2MP
DMG
Ph
CD0
OH
CD0
Me
CD0
Et
?,,2 (min)
Temp. (“C)
pH 6.4
pH 1.4
pH 8.4
20 37 20
29 IS 59 14 63 24 100 28
27 II 50 II 45 II 63 21 36
II 4 32 8 21 6 43 10
58 14
43 9 41
25
:; 23
365 7.5
37 20 37 37 20 3-l 20 37 20 37
to 99mTcOH(CDO), EtB over time (EtB = ethylboronic acid). These demonstrate that the hydroxy analog is the only reaction product. For the listed complexes, there is a slight increase in the void volume peaks (typically 2-5%) which occurs immediately after mixing with the buffer. The percentage of total radioactivity associated with the void volume peaks remains constant throughout the remainder of the experiment. Table 1 shows the half lives for the disappearance of the chloro analog for some of the BAT0 complexes, demonstrating the influence of pH on rate (also shown in Fig. 7), and showing that the nature of the dioxime and boronic acid cap influence the exchange rate. Figure 3 provides an example graph of the natural log of the fraction of a chloro BAT0 remaining versus time. The linear plots indicate that the disappearance of the chloro analog over this pH range follows pseudo first order kinetics. Since there is no significant conversion to any radioactive product other than the hydroxy analog (as detectable by HPLC chromatography), this suggests that the replacement of the chloro group by the hydroxy
263
group results from a pseudo first order reaction. The poor water solubility of the more lipophilic BAT0 complexes, for example, TcCl(CDO),2MP and TcCl(CDO), BuB (2MP = 2-methylpropylboronic acid; BuB = n -butylboronic acid) prevented the determination of their axial ligand exchange rates under the conditions described (no carrier added levels), due to appreciable deposition of the complexes onto the sides of the vessel. Results showing the conversion of hydroxyl axial ligand to chloride are listed in Table 2. This conversion process occurs to a significant extent only below pH 5. No conversion of a hydroxy BAT0 to its chloro analog could be detected for up to 1 h at the physiological pH of 7.4. The ‘H NMR spectra of VcCl(DMG),2MP before and 10 min after addition of 1% KOD in D,O are shown in Fig. 4 (Note: the conditions used for the determination of spectra caused the signal from the oxime protons, normally at 15 6 to appear at 0.1 6.) 10 min after the addition of KOD/D,O, the exchange of the oxime protons for deuterium was complete. All remaining peaks are doubled, showing conversion
-0.5
-1 In (fraction Co -1.5
-2
-25
i
J
0
20
40
60
00
100
120
140
160
tlmo (minkhs) Fig. 3. Graph
of In (chloro fraction) vs time for *TcCl(DMG),EtB
at 20°C.
180
139
Chloro + hydroxy substitution Table 2. OH -+ Cl conversion half times of BATOs at 37°C OH + Cl r,,2 (min) Dioxime CD0 CD0 DMG
Boronic acid OH Me 2MP
DH 6.4
DH 5.0
DH3.0
k-1000 >looO >lcNIo
42.2 147.8 182.5
30.1 63.0 55.84
Table 3. LiDODhikitY of technetium corn&x&s Technetium complex
log k’
Calculated log P
@%OH(DMG)32MP WmTcCI(DMG),2MP P9mTcOH(CDO),MeB ‘=‘“TcCI(CDO), MeB
OS38 1.181 1.342 1.56
1.72 3.51 3.96 4.56
99”Tc-d,l-HM-PA0 99”Tc-ECD
0.259 0.315
0.95 1.11
to some other species; HPLC analysis indicated that
the conversion product was ?cOR(DMG)~ 2MP (R = D, H). Even 18 h after the addition of KOD/D*O, none of the carbon-bound hydrogen atoms showed any exchange for deuterium. The retention of complexes on reverse phase HPLC can be used to provide an estimate of the lipophilicity of these complexes (for example, Braumann, 1986). Lipophilicity estimates of BATOs have been conducted on the PRP-1 HPLC system described in the Methods section (Feld and Nunn, 1989). From the measured retention time of the complex (R,, in min), the capacity factor (k’) was calculated by the relationship: k’=-
R, - v t, (v = R, of a non-retained
standard;
nitrate was used for this purpose).
By calibration of the HPLC system with compounds of known lipophilicity (Feld and Nunn, 1989), the relationship between log k’ and log P (log of the octanol/water partition ratio) was found to be: log k’ log P=- o 36 + 0.23. Table 3 lists the determined log k’ values and estimated log P (from the above equation) for four BAT0 complexes, and, for comparison, two other complexes, 99mTc-d,l-HM-PA0 and %Tc-L,L-ECD; neutral complexes which are reported to cross the intact blood-brain barrier (Nowotnik et al., 1985; Walovitch et al., 1988). The rat biodistribution data for both the chloro and hydroxy analogs of 99”TcX(CDO),MeB and
Spectrum 10 minutoa r&r
of KODW (omt by 0.65 ppm).
the add&n
Fig. 4. ‘H NMR spectra of TcCl(DMG),2MP
in CD,CN with and without KOD/D,O.
740
S. S. JURISSONet al. Table 4. Rat biodistribution
of %TcX(DMG),2MP
and *TcX(CDO),MeB
(X = Cl, OH)
% I.D./organ W”TcCI(CDO),MeB
@“‘TcOH(CDO),MeB
60m
5m
60m
5m
60m
5m
60m
Blood Heart Brain Lung Liver Kidneys
2.90 1.71 0.30 2.05 28.5 4.93
1.79 0.39 0.24 0.91 14.1 1.74
5.30 1.19 0.03 2.83 36.3 3.49
2.30 0.45 0.02 I .47 19.9 2.24
2.92 1.44 0.81 1.31 28.6 3.32
1.99
3.38 1.16 0.05 3.04 28.9 4.21
2.00 0.24 0.03 0.99 10.0 I .49
Discussion Factors which influence axial Iigand exchange
While %Tc-BAT0 camp lexes with chloride axial ligands are stable for several hours when stored in the presence of kit components, the axial ligand in the isolated complex is responsive to exchange by competing monodentate anionic ligands (Nowotnik et al., 1989). The present study establishes the following points: (a) The rate of conversion from the Cl to the OH analog increases with increasing pH (increasing OH- concentration) suggesting an exchange mechanism which is not solely dependent on the loss of the Cl- from the BAT0 complex. (b) As expected, the rate of the reaction increases with increasing temperature (20 vs 37°C). (c) For BATOs with the same boronic acid cap, the exchange process is generally faster for DMG BATOs than CD0 BATOs. (d) For BATOs derived from alkyl boronic acids, the rate of Cl-OH exchange decreases with increasing size and/or branching of the alkyl group. While steric factors could account for the observed exchange rate differences between CD0 and DMG BATOs (the greater bulk of the cyclohexane rings, compared to the size of two methyl groups, inhibiting 5.
Initial
rat
biodistribution of (X = OH, Cl)
“mTcX(DMG),2MP
% I.D./organ 99mTcCcCI(DMG),2MP WmTcOH(DMG),2MP Brain Blood Heart Lungs Liver Kidneys
%TcOH(DMG),2MP
sm
99mT~X(DMG)J2MP are listed in Tables 4 and 5. When compared to the chloro compounds, the hydroxy analogs display slightly increased liver, lung, and blood activity and only slightly decreased heart activity. Brain uptake of the hydroxy BATOs is much lower than observed for the corresponding chloro analogs, and this appears to be a result of a lower cerebral extraction efficiency (Fig. 5).
Table
%TcCI(DMG),2MP
Organ
15s
60s
15s
60s
0.20 33.38 1.84 9.07 7.05 5.78
0.10 18.74 1.68 5.74 4.08 6.76
0.66 20.62 1.88 11.79 8.87 6.76
0.61 11.93 2.01 3.80 13.50 5.96
0.54 0.45 0.41 12.1 1.13
the approach of OH-), a steric effect due to the more distant boron substituent seems less likely. The effect of the boron substituent on conversion rate might be a reflection of differences in electron-donor abilities of these substituents; long range inductive effects involving a similar R-B-O-N-metal pathway have been demonstrated in the clathrochelate iron(I1) complexes, Fe(oxime),(BR)* (Robbins et al., 1985). The hydroxy analogs can be prepared in close to quantitative yields from the chloro analogs in high pH buffers containing no chloride. The conversion rates for hydroxy- to- chloro BATOs can be measured in PBS buffers made up in saline. Only below pH 5 is there appreciable conversion to the chloro analog; there appears to be an equilibrium reached between the chloro and hydroxy analogs at pH
The mechanism by which the chloro to hydroxy exchange takes place may play a significant role in the resulting biodistributions of these complexes; for example, there may be chemical intermediates whose biological behavior varies markedly from that of either the chloro or hydroxy analogs. There are three possible mechanisms by which this chloro- to- hydroxy exchange may take place, as shown in Fig. 6. The dependence of the exchange rate on the OHconcentration is evidence against an S, 1 mechanism. In this case, the rate controlling step would be the loss of Cl-, which should be independent of the OHconcentration. The lack of any chromatographic intermediates, even at low pH where the OH- attack should be slowest, is evidence against a rapid chloride loss followed by slow addition of the OH- group. The SN2 mechanism and a variation of the S, 1 mechanism, SNI-CB (Wilkins, 1974) (in which the rate controlling step is a base-catalyzed removal of a nearby proton preceding the rapid elimination of an anion) are possible alternatives. To distinguish between an SN I-CB and an SN2 mechanism, the rate of disappearance of 99mT~Cl(CD0)3MeB was plotted against the OHconcentration. An “S”-shaped curve was obtained (Fig. 7). If an SN2 mechanism were followed, the rate of the reaction should be directly related to the OHconcentration and a linear plot of rate versus pH would be obtained. The S-shaped curve is indicative of an SNl-CB mechanism in which the OH- concen-
Chloro --thydroxy substitution ’
8smT~CI (DMG),
2MP b-0.34
741
addition of HX (where X is a suitable, anionic, monodentate ligand) to give the BAT0 product.
(CBF) +0.07. R.O.BI
The influence of the axial ligand on myocardial uptake
q
0.2
0.01 0.0
.
0.2
0.4
-
0 0.8 CBF
0.0
1.0
1.2
1.4
I.6
(mc/mfn/g)
Fig. 5. Graph of CEE of TcX(DMG),2MP; X = Cl, OH.
tration controls the rate of the reaction by deprotonation of one proton from the BAT0 backbone. The rate of reaction appears to be controlled by this deprotonation step, as judged by the S-shaped curve. The point of inflection in the curve probably reflects the pK, of the deprotonation step. The plateau region at high pH is delimited by essentially complete monodeprotonation of the BAT0 backbone, while that at low pH, by minimal deprotonation. Increasing the concentration of chloride in the buffer to 1 M at pH 7.4 does not affect the rate of exchange. This suggests that the rate is not determined by a competitive exchange between the chloride and hydroxide anions, as would be expected with either an SN1 or S,2 mechanism. Therefore it must depend upon a secondary reaction, which can have a direct influence on the removal of the axial ligand, as would be the case in the deprotonation of the BAT0 backbone in an S, l-CB mechanism. A consequence of the S, l-CB mechanism is that the intermediate is an uncharged, six coordinate complex (S, 1 or f&.,2 mechanisms would provide cationic and anionic intermediates, respectively), and not a positively charged complex, as has been suggested (Kung, 1990). There is no evidence for this intermediate by HPLC analysis, indicating that the intermediate is transient, with rapid conversion back to a neutral, seven coordinate complex. As shown in Fig. 8, it is possible that the proton eliminated from the BAT0 backbone during the S, I-CB axial ligand exchange process may originate from either a free oxime group, or from one of the carbon atoms adjacent to an oxime group. To discriminate between these two possibilities, an NMR study was undertaken in which axial ligand exchange in y9”TcCI(DMG),2MP was studied in CD3CN/KOD/D,0. Under these conditions, the proton lost during the formation of the intermediate complex should be replaced by a deuterium atom. The spectra showed that only the oxime protons underwent deuterium exchange; there was no deuterium exchange of any of the carbon bound hydrogen atoms. Therefore, the S, 1-CB process proceeds by way of oxime deprotonation and loss of the axial ligand to give the transient, neutral, 6-coordinate intermediate complex, which is followed by rapid
A comparison of the biodistribution data for the hydroxy and chloro analogs of TcX(CDO), MeB and TcX(DMG)r2MP (Table 4) shows two interesting features. First, conversion of a chloro BAT0 to its hydroxy analog results in a 20-30% reduction of heart uptake in rats at 5 min post injection. When the heart uptake of a large number of BATOs was examined in rats, it was found that a parabolic relationship exists between heart uptake at 5 min post injection and the lipophilicity (log k’) of the complex (Nunn et al., 1989). The maximum of that parabola occurs at log k’ 1.65 (estimated log P of 4.8). Based on this structure-distribution relationship, the relative heart uptake values at 5 min post injection shown in Table 4 can be predicted solely from the lipophilicity of these complexes, shown in Table 3. The influence of the axial ligand on cerebral uptake and retention
As freely-diffusible cerebral perfusion tracers such as Xe-133 (Lassen et al., 1981) and 9ymTc-PnA0 (Volkert et al., 1984) display rapid washout (t,lz < 5 min) from the brain, any tracer which has a long half-time of cerebral washout must be provided with some mechanism which reduces normal washout kinetics. For two technetium tracers which display appreciable cerebral retention, 99mTc-d,l-HM-PA0 and 99”Tc-L,L-ECD, it has been proposed that the mechanisms of retention are the result of conversion of the administered freely-diffusible, lipophilic tracers to non-diffusible, less lipophilic forms once the compounds have crossed the blood-brain barrier (Nowotnik et al., 1985; Walovitch et al., 1988). As demonstrated in this paper, chloro-to-hydroxy exchange in BATOs is a facile process at physiological pH which results in the production of a less lipophilic complex. This led us to speculate (Nowotnik et al., 1989) that, like Y9”Tc-d,l-HM-PA0 and smTc-L,L-ECD, the cerebral retention of 99”TcCl(DMG),2MP (a BAT0 complex which has been extensively studied as a cerebral perfusion tracer; Narra et al., 1990) results from intracerebral conversion from a diffusible form to a non-diffusible form; in this case, intra-cerebral conversion of the diffusible chloro BAT0 to its non-diffusible hydroxy analog. This hypothesis was supported by preliminary biodistribution data (Table 4) which demonstrated that the percentage of injected dose of 9ymTcX(DMG),2MP in the brain (at 5 min post i.v. administration) is far greater for the chloro compound (0.81%) than its hydroxy analog (0.05%). However, examination of the initial (15 and 60 s) biodistribution of the chloro and hydroxy analogs of TcX(DMG),2MP (Table 5) confirms that both
S. S. JURISSONet al.
742
Possible mechanisms for axial ligand exchange. S, 1 mechanism
Initial loss of the Cl ligand to form a cationic intermediate followed by addition of the OH ligand to form the neutral OH analog. S, I-CB mechanism
Base attack of the dioxime backbone with removal of a proton to form an anionic complex. This is followed by rapid loss of the Cl ligand to the neutral 6 coordinated complex. The final step involves attack by a water molecule, placing an OH group in the axial coordination site and reprotonating the dioxime backbone. S, 2 mechanism
Initial attack by the OH group to form an eight coordinate, anionic intermediate followed by rapid loss of the Cl group to form the neutral, seven coordinate OH analog. Fig. 6. S, 1, S, I-CB, and S,2 mechanisms.
compounds are taken up by the brain, although uptake by the hydroxy analog is poor. The cerebral extraction data presented in Fig. 5 confirm that
I'
' ? g
60 40
al z 20 a 3 600 L 456769
Ldd-C~
/:/
10
11
12
PH Fig. 7. Graph of rate of Cl/OH exchange for TcCl(CDO)r MeB.
the poor cerebral uptake of TcOH(DMG),2MP compared with TcCl(DMG),2MP is a result of a low single-pass cerebral extraction efficiency, rather than some other factor, such as binding to blood components. Thus, it appears that TcCl(DMG),2MP cannot be trapped as its hydroxy analog as the latter compound displays significant cerebral washout. Another factor which negates the conversion trapping theory is the rate of conversion. To achieve cerebral retention by this mechanism, an in vivo chloro-to-hydroxy conversion half time of l-2 min would be required (Neirinckx, 1986), well in excess of the rate observed at pH 7.4/37”C in vitro. In addition, tissue homogenate and ex viva studies of Cl/OH conversion of 9hnT~CI(DMG),2MP have shown that the rate of chloro- to- hydroxy conversion is not
143
Chloro -+ hydroxy substitution Possible mechanisms for the S, l-CB axial ligand exchange process. (1) Initial removal of a hydrogen atom from an oxime group.
,OH
(2) Initial removal of a hydrogen atom from the carbon backbone.
The diagrams display the partial structure of BATOs, indicating two possible mechanisms for chloro-tohydroxy axial Iigand exchange by way of an SNI-CB process, involving hydrogen atom abstraction from either one of the free (uncapped) oxime groups or a carbon atom adjacent to one of the oxime groups. Fig. 8. Possible mechanisms for the abstraction of a proton in the S, I-CB axial ligand exchange process.
sufficiently rapid (t,,, > 5 min) to account for cerebral trapping of this radiopharmaceutical (Nowotnik et al., 1989). At this stage, it is unclear why the chloro and hydroxy derivatives of TcX(DMG),2MP and TcX(CDO),MeB display markedly different levels of uptake in rat brain. While the hydroxy compounds are less lipophilic than the corresponding chloro BATOs, their lipophilicity values are well above the minimum (log P of 0.5) and close to the presumed optimum (log P of 2.0) for blood-brain barrier penetration by physical diffusion (Hansch and Clayton, 1973). In addition, the lipophilicities of the hydroxy BATOs are greater than those for gQmTc-d,l-HM-PAO and wmTc-ECD (Table 3), complexes which also demonstrate high cerebral uptake in rats. Molecular size (implied by molecular weight) has also been shown to influence brain uptake, with uptake inversely related to size (Levin, 1980). However, models of these complexes have demonstrated that chloro and hydroxy analogs differ only marginally in size (Di Rocco et al., 1989). While other mechanisms, such as hydrogen bonding (Young ef al., 1988), have been shown to influence the interaction of substrates with the blood-brain barrier, and might explain the differences in the brain uptake of the chloro and hydroxy BATOs, further studies will be required to determine why the nature of the axial ligand has such a profound influence on brain uptake.
Conclusions There appears to be little difference in the rates of chloro-to-hydroxy exchange among the BAT0 complexes examined in this study with the half lives of these complexes ranging from 9 to 21 min under physiological conditions. The exchange between the axial chloro and hydroxy groups appears to proceed by an SNl-CB mechanism. Axial ligand exchange by this mechanism will provide a neutral six coordinate technetium complex, which rapidly converts back to a neutral seven coordinate complex. The observed differences in conversion rate among the BATOs studied probably result from steric and electronic effects of substituents on the deprotonation of the BAT0 backbone. While heart uptake values of the chloro and hydroxy BATOs are predictable from their lipophilicities (based on previous SDR studies; Nunn et al., 1989), the hydroxy BATOs display far lower brain uptake in rats than their chloro analogs. The reason for this difference is unclear. A hypothesis was tested, that slow cerebral washout of 99mTcCl(DMG),2MP results from intra-cerebral conversion of diffusible chloro complex to its “non-diffusible” hydroxy analog. It was found that 99mTcOH(DMG)32MP was taken up by rat brain, and is washed out, indicating that in viva chloro-to-hydroxy conversion cannot account for the cerebral retention of *TcCI(DMG)~~MP.
S. S. JUR:ls.WN et al.
744 Acknowledgemenu. -The
authors wish to thank Lillian Belnavis, Maryann Homack, and Christine Hood for technical assistance with the biodistribution and cerebral extraction studies, and Dr Michael Porubcan for the deuterium exchange ‘H NMR study.
References Braumann T. (1986) Determination of hydrophobic parameters by reversed-phase liquid chromatography: theory, experimental techniques, and application in studies on quantitative structure-activity relationships. J. Chromatogr. 373, 191-225.
499-506.
Cotton F. A. and Wilkinson G. (1980) Advanced Inorganic Chemistry, p. 80 (and references therein). Wiley, New York. Di Rocco R. J., Silva D., Narra R. K., Hirth W., Nunn A. D. and Ecklman W. C. (1989) Effect of lipophilicity and hydrogen bonding on the single-pass cerebral extraction of SQ 32,097. J. Cereb. Bid Flow Metab. 9, S399. Feld T. and Nunn A. D. (1989) A chromatographic method for the measurement of the lipophilicity of lipophilic technetium complexes. J. Labelled Compd. Radiophnrm. 26, 274.
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Hansch C. and Clayton J. M. (1973) Lipophilic character and bioloaical activitv of druas. II. Parabolic case. J. Pharm. SC? 62, l-21 .-
New Radiopharmaceuticals. Presented at the Society of Nuclear Medicine Annual Meeting. Nowotnik D. P., Hirth W., Jurisson S., Linder K. E., Narra R. K., Nunn A. D. and Eckelman W. C. (1989) Studies of the in vivo chemistry of BATOs. J. Nucl. Med. Allied Sci. 33, 310. Nowotnik D. P., Canning L. R., Cumming S. A., Harrison R. C., Higley B., Nechvatal G., Pickett R. D., Piper I. M., Bayne V. J., Foster A. M., Weisner P. S., Neirinckx R. D., Volkert W. A., Troutner D. E. and Holmes R. A. (1985) Development of a Tc-99m-labelled radiophamaceutical for cerebral blood flow imaging. Nucl. Med. Commun. 6,
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Irwin G. H. and Peskom S. H. (1982) A dual label radiotracer technique for the simultaneous measurement of cerebral blood Row and the single transit extraction of diffusion-limited compounds in the rat. Bruin Res. 249, 23-30. Kung H. F. (1990) New technetium 99m-labeled brain perfusion imaging agents. Semin. Nucl. Med. 20, 150-158. Lassen N. A., Henriksen L. and Paulson 0. (1981) Regional blood flow in stroke by 133-Xenon inhalation and emission tomography. Stroke 12, 284-288. Levin V. A. (1980) Relationship of octanol/water partition coefficient and molecular weight to rat brain capillary permeability. J. Med. Chem. 23, 682-684. Linder K., Feld T., Juri P. N., Nunn A. D. and Treher E. H. (1987) The svnthesis of a new technetium agent. SO 32,097, which can be used to assess regional-cerebral blood flow. J. Nucl. Med. 28, 592-593. Linder K. E., Malley M. F., Gougoutas J. Z., Unger S. E. and Nunn A. D. (1990) Neutral, seven-coordinate dioxime complexes of technetium(JI1): synthesis and characterization. Inorg. Chem. 29, 2428-2434. Narra R. K., Nunn A. D., Kuczynski B. L., Feld T., Wedeking P. and Eckelman W. C. (1989) A neutral technetiumd9m complex for myocardial imaging. J. Nucl. Med. 30, 1830-1837. Narra R. K., Nunn A. D., Kuczynski B. L., Di Rocco R. J., Feld T., Silva D. A. and Eckelman W. C. (1990) A neutral lipophilic Tc-99m complex for regional cerebral blood Bow imaging. J. Nucl. Med. 31, 1370-1377. Neirinckx R. D. (1989) Development and trapping mechanism of Tc-99m dl-HM-PAO. In Funcfional Bruin Imaging:
Nunn A. D., Feld T. and Narra R. K. (1989) Quantitative structure distribution relationships (QSDRs) of BATOs. J. Nucl. Med. Allied Sci. 33, 310.
Robbins M. K., Naser D. W., Heiland J. L. and Gryzbowski J. J. (1985) Synthesis and electrochemistry of iron (II) clathrochelates. Inorg. Chem. 24, 3381-3387. Seldin D. W., Johnson L. L., Blood D. K., Muschel M. J., Smith K. F., Wall R. M. and Cannon P. J. (1989) Myocardial perfusion imaging with technetium-99m SQ30217: comparison with thallium-201 and coronary anatomy. J. Nucl. Med. 30, 312-319. Thompson M., Nunn A. D. and Treher E. H. (1986) X-ray photoelectron spectroscopy of potential tcchnetium-based organ imaging agents. Anaiyt. Chem. 58, 3100-3103. Treher E. H., Francesconi L. C., Gougoutas J. Z., Malley M. F. and Nunn A. D. (1989) Monocapped Tris(dioxime) complexes of technetium(II1): synthesis and structural characterization of TcX(dioxime), B-R (X = Cl, Br; dioxime = dimethylglyoxime, cyclohexanedione dioxime; R = CH,, C,H,). Inorg. Chem. 28, 3411-3416. Volkert W. A., Hoffman T. J., Seger S. M., Troutner D. E. and Holmes R. A. (1984) Tc-99m propylene amine oxime (Tc-99m PnAO); a potential brain radiopharmaceutical. Eur. J. Nucl. Med. 9, 51 l-516. Walovitch R. C., Makuch J., Knapik G., Watson A. D. and Williams S. J. (1988) Brain retention of Tc-99m-ECD is related to in vivo metabolism. J. Nucl. Med. 29, 747. Wilkins R. G. (1974) The Study of Kinetics and Mechanism of Reactions of Transitional Metal Complexes. Allyn & Bacon, Boston, Mass. Young R. C., Mitchell R. C., Brown T. H., Ganellin C. R., Griffiths R., Jones M., Rana K. K., Saunders D., Smith I. R., Sore N. E. and Wilks T. J. (1988) Development of a new physicochemical model for brain penetration and its application to the design of the centrally acting H, receptor histamine antagonists. J. Med. Chem. 31, 656 671. Zielonka J. S., Cannon P., Johnson L., Seldin D., Bellinger R. L., Chua E., Coleman R. E., Reba R. C., Wasserman A,, Drane W., Williams C., Goris M. L., Leppo J. A., Akhtar R., Bamrah V. S. and Krubsack A. J. (1990) Multicenter trial of 99mTc-teboroxime (Cardiotec): a new myocardial perfusion agent. Nukfear Medizine (Suppl.) 26, 21 I-212.
NW/. Med. Biol. Vol. 18, No. 7, pp. 735-744, 1991 Inr. J. Radiat. Appl. Instrum. Part B Printed in Great Britain. All rights reserved
Chloro +Hydroxy Substitution on Technetium BAT0 [TcCl(Dioxime), BR] Complexes S. S. JURISSON,
W. HIRTH, K. E. LINDER, R. J. Di ROCCO, D. P. NOWOTNIK* and A. D. NUNN
R. K. NARRA,
The Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903, U.S.A. (Received 3 December 1990) The neutral, seven coordinate complexes of technetium known as the BAT0 Qoronic acid Adducts of Technetium dioximes) complexes have shown their utility as myocardial and cerebral perfus& agents. The axial chloride ligand of the BAT0 complexes pTcCl(dioxime),BR] is labile to substitution by a competitive anion; under physiological conditions, the axial chloride ligand can be replaced by a hydroxy group. The chloro and hydroxy analogs have different biodistributions and single-pass cerebral extraction efficiencies. The influence of structure on the rate of the in vitrochloro/hydroxy exchange process has been
studied. The mechanism of axial ligand exchange was found to be S, l-CB, which proceeds by way of a transient, neutral six coordinate complex. Evidence is presented which indicates that chloro/hydroxy exchange is not the mechanism by which BAT0 complexes are retained in the brain.
Introduction Seven coordinate technetium complexes of the general formula TcCl(dioxime),BR, where BR is a boronic acid adduct, have shown their utility as myocardial and cerebral perfusion imaging agents (Linder et al., 1987; Narra et al., 1989, 1990; Seldin er al., 1989; Zielonka et al., 1990). These complexes, better known as BATOs @oronic Acid Adducts of Technetium d@xime) have been well characterized on the macroscopic scale (Treher et al., 1989); the general structure of these complexes is shown in Fig. 1. BAT0 complexes are prepared by template synthesis (Cotton and Wilkinson, 1980); TcO; is reduced with stannous ion in the presence of a vicinal dioxime, a boronic acid, and chloride ion. The mechanism of formation of BATOs has been reported previously (Linder et al., 1990). ‘A large number of 99mTc-BAT0 complexes have been prepared by varying the dioxime ligand and boronic acid employed in the template reaction, giving rise to the determination of structuredistribution relationships for BATOs with a chloride axial ligand (Nunn et al., 1989). x-Ray photoelectron spectroscopy of the BAT0 complexes has shown that the axial chloride ligand has a binding energy (197.1 + 0.3 eV) between that of ___ *Author for correspondence.
a covalent (197.9 f 0.25 eV) chlorine and ionic (195.9 f 0.2 eV) chloride, and so, could be subject to exchange by a competitive anion (Thompson et al., 1986). This paper describes studies which investigate the rate and mechanism of the exchange of the axial chloride ligand by hydroxide for some of the BAT0 complexes. The effect of this exchange process on the biodistribution properties of these compounds is also discussed. Methods General chemicals
Ammonium acetate, citric acid, sodium chloride, sodium phosphate (monobasic) and HPLC grade acetonitrile were obtained from Fisher Scientific. Cyclohexanedione dioxime (CDO) and dimethylglyoxime (DMG) were obtained from Eastern Chemicals, The Nucleosil CB and Hamilton PRP-1 HPLC columns were obtained from Alltech Associates. Sample preparation (a) *TcCl(BATO)s. The 99”TcCl(BATO)s were prepared from lyophilized kits which contained dioxime, boronic acid, and stannous chloride as reductant. 1 mL of *TcO; generator eluent was added to the kits. The kits were heated at 100°C for 15 min, cooled, and the BAT0 complex was 135
736
S. S. JURISSON et
R
R
Fig. 1. The general structure of BATOs.
separated from nonradioactive kit components by the following process: A small volume (approx. 0.5 mL) of a 20% v/v suspension of PRP-1 resin (10 pm dia) in a 25% v/v ethanol/saline mixture was drawn into a 1 mL syringe. A Millex HV, 0.45 micron filter was attached to the end of the syringe and the suspension filtered, causing the PRP-1 resin to pack into a plug on the Millex filter. The contents of the kit were drawn into a 1 mL syringe and loaded onto the top of the syringe containing the PRP-1 resin. The kit contents were passed through the PRP-1 resin, and the filtrate was discarded. The PRP-1 resin was washed twice with 0.5 mL of 25% v/v ethanol/water solution and the filtrate again discarded. The product radioactivity was eluted from the PRP-1 resin with 0.8 mL of absolute ethanol into a siliconized glass vial. (b) ‘“TcOH(BATO)s. Following isolation of the 99mTcC1(BATO)as an ethanolic solution (as described above), the solvent was removed by evaporation under a stream of nitrogen. The BAT0 was redissolved in 1 mL of chloride-free phosphate buffer (pH 8.0) and heated at 37°C for 30 min. The axial ligand exchange process was halted by the addition of 20 p L of cont. HJP04, and the 99mTcOH(BATO) was isolated by same PRP-1 resin procedure as described for the chloro analogs. (c) 9gTcCl(DMG),2MP. This complex was prepared from 99mTcCl(DMG), and 2-methylpropylboronic acid by the method described previously (Linder et al., 1990). (d) “TcOH(DMG),ZMP. Three drops of 2 M aqueous sodium hydroxide and distilled water (2 mL) were added to a solution of TcCl(DMG),2MP (50 mg, 0.091 mmol) in acetronitrile (2 mL). The solution was gently heated for 5 min. Water (2 mL) was added, and the product extracted into ether (3 x 5 mL). The combined ether extracts were washed until the washings were no longer basic and gave a negative silver nitrate test for Cl-. Ether was removed by rotary evaporation at room temperature, and the resultant red-orange oil was dissolved in acetonitrile (2 mL). Water (2 mL) and one drop of aqueous 1 M
al.
trifluoromethanesulfonic acid were added, and the acetronitrile was slowly removed with a gentle stream of nitrogen. The red-orange product crystals which formed were isolated by centrifugation, washed well with water, and air dried. Yield 29 mg (60%). Anal. calcd for C,, H,, N6 BOs Tc (product monohydrate): C, 35.18; H, 5.90; N, 15.38. Found: C, 35.12; H, 5.69; N, 15.34. Optical spectrum (acetronitrile) A,,,,,, nm, (Lmol-’ cm-‘): 450 (3850), 395 (3950), 294 (6700), 258 (sh), 234 (22,000). ‘H NMR (CD,Cl,; 270MHz) 6 0.6 (2H, d, B-CH,), 1.0 (6H, d, CH(CI&),), 1.8-2.0 (2H, bm, CH and Tc-OHJ, 2.35 (12H, s, DMG CH,), 2.4 (6H, s, unique DMG CI-I,), 14-15 (lH, bs, oxime 0I-J. Conductivity (acetronitrile): 5.44 ohm-’ M-l cm2. (e) Phosphate b@ered saline (PBS). The PBS buffers used for the Cl-OH exchange experiments were prepared to contain 6.49 g/L sodium chloride (111 mequiv/L Cl-) and 0.05 M sodium phosphate monobasic, and adjusted to the appropriate pH with NaOH. HPLC analysis
The HPLC system used for monitoring the rate of axial ligand exchange and for comparison of 99Tcand 99mTccomplexes consisted of two Altex 110A pumps (controlled by a model 420 microprocessor controller) and a Rheodyne model 7125 injection valve. The flow-through y detector, which utilized a Tennelec NaI crystal, was built in-house. A U.V. detector set at 265 nm was used to detect the 99Tc material. The percent radioactivity in the eluting peaks was determined using a Spectra-Physics model SP 4270 integrator connected to an IBM PC running chromatographic Labnet software. Two column/eluent combinations were used to measure the rate of axial ligand exchange: (i) Hamilton PRP- 1 column (150 x 4.6 mm, Alltech Associates) with a mobile phase of 65 : 35 acetonitrile : 0.1 M ammonium acetate (pH 4.6), and flowrate of 2.0 mL/min. (ii) Nucleosil Cs column (150 x 4.6 mm, Alltech Associates) with a mobile phase of 70: 30 acetonitri1e:O.l M citric acid (pH 2.4) and flowrate of 1.5 mL/min. Conversion rate of chloro to hydroxy
A 1 mL aliquot of PBS with pH adjusted to predefined values in the range 3.0-l 1.4 was placed into a 16 x 125 mm siliconized Vacutainer tube and either allowed to remain at room temperature (20°C) or incubated at 37°C. An aliquot (25-50 pL, approx. 2OOpCi) of the isolated 99mTcCl(BATO) in ethanol was added and the resultant solution was mixed by shaking. The percent of radioactivity in the form of the chloro complex over time was determined by HPLC analysis. All exchange rates were determined in duplicate. Linear regression analysis of In [fraction of 99mTcCl(BATO)] versus time (in min) gave the rates and half times of Cl -+ OH conversion.
131
Chloro -+ hydroxy substitution Conversion rate of hydroxy to chloro
An aliquot (50 pL, approx. 100 FCi) of the purified hydroxy BAT0 in ethanol was added to 1 mL of PBS buffer at either pH 7.4, 6.4, 5.0 or 3.0, containing 0.5% Tween 80. The half time for conversion to the chloro analog was determined by HPLC, measuring the fraction of the hydroxy and chloro forms at several times after mixing. As minor (unidentified) radioactive products were formed in addition to the chloro BATO, the fraction of the hydroxy form was determined from: fraction *TcOH(BATO)
=
% %TcOH(BATO) % 99”TcOH(BATO) + % *TcCl(BATO)’ (The minor radioactive products were formed immediately on mixing the isolated hydroxy BAT0 with buffer, and the proportion of these components did not increase with time. The surfactant, Tween 80, was required to prevent loss of the minor products.
(20 PCi) of radioactivity via the jugular vein. After 15 s, 1 min, 5 min and 60 min the animals were sacrificed by exsanguination, and the target tissues removed, weighed, and assayed for radioactivity. Cerebral extraction studies
The cerebral blood flow (CBF) was varied in rats anesthetized with Nembutal (50 mg/kg, i.p.) by controlling respiration. A 0.2 mL mixture of ?Sr-labeled microspheres and test compound was rapidly injected into the left ventricle of the heart (using a 1 cm’ Tuberculin syringe with a 27 gage needle) while blood was withdrawn from the femoral artery at a known rate. Six seconds later the rat was decapitated and simultaneously, the arterial withdrawal pump was stopped. Radioactivity in the brain and blood was measured with a y counter which corrected for cross over counts. The cerebral extraction fraction (E) was calculated using the formula (Irwin and Preskorn, 1982): E = (cpm z:;;;nm (cpm
;;;;;.;; 00
Study of the Cl/OH exchange process by ‘H NMR spectroscopy
%TcCl(DMG), 2MP (16 mg) was dissolved in CD,CN (1 mL), and the ‘H NMR spectrum recorded on a JEOL GX-400 spectrometer. A 1% solution of KOD in D,O (0.1 mL) was added to the BAT0 solution, and the ‘H NMR spectra recorded at 10, 60 and 1080 min post mixing. The time dependence of the proportion of chloro and hydroxy WT~X(DMG),2MP complexes under these reaction conditions was determined by HPLC analysis, using u.v./visible detection at 400 nm. Biodistribution studies
Sprague-Dawley rats were anesthetized with Nembutal (50 mg/kg), and injected with 0.1 mL
gemTcCl (CDO)
3
(cpm = counts per min).
Results Treatment of 99TcCl(DMG)Y2MP with base provided a single product in good yield, which was identified from analytical data as *TcOH(DMG),2MP. The same product was obtained when this reaction was conducted with no carrier added 99mTcCl(DMG),2MP, demonstrated by HPLC analysis [both 9Tc and 99mTc complexes of Tc0H(DMG)92MP provided a single peak on HPLC analysis with a R, of 2.1 min, k’ = 2.8, on the PRP-1 analytical column]. Figure 2 shows the HPLC chromatograms recording the conversion of 99mT~Cl(CDO)3EtB
EtB
Q9mTcOH (CDO)
3
EtB
\
l*
-
0 1 2 ,_3 ‘, -5 Retrntion Urn*(minutt3r) t-7
min
1
-Al t-14
min
t-24
min
i
t-30
min
t-45
min
Fig. 2. Series of HPLC chromatograms showing Cl/OH exchange. NMB 11117-E
!_L
1 t-100
min
738
S. S. JURISSON et al, Table 1. Cl -OH
half times of BATOs Cl -+ OH
Dioxime
Boronic acid
DMG
OH
DMG
MC
DMG
Et
DMG
2MP
DMG
Ph
CD0
OH
CD0
Me
CD0
Et
?,,2 (min)
Temp. (“C)
pH 6.4
pH 1.4
pH 8.4
20 37 20
29 IS 59 14 63 24 100 28
27 II 50 II 45 II 63 21 36
II 4 32 8 21 6 43 10
58 14
43 9 41
25
:; 23
365 7.5
37 20 37 37 20 3-l 20 37 20 37
to 99mTcOH(CDO), EtB over time (EtB = ethylboronic acid). These demonstrate that the hydroxy analog is the only reaction product. For the listed complexes, there is a slight increase in the void volume peaks (typically 2-5%) which occurs immediately after mixing with the buffer. The percentage of total radioactivity associated with the void volume peaks remains constant throughout the remainder of the experiment. Table 1 shows the half lives for the disappearance of the chloro analog for some of the BAT0 complexes, demonstrating the influence of pH on rate (also shown in Fig. 7), and showing that the nature of the dioxime and boronic acid cap influence the exchange rate. Figure 3 provides an example graph of the natural log of the fraction of a chloro BAT0 remaining versus time. The linear plots indicate that the disappearance of the chloro analog over this pH range follows pseudo first order kinetics. Since there is no significant conversion to any radioactive product other than the hydroxy analog (as detectable by HPLC chromatography), this suggests that the replacement of the chloro group by the hydroxy
263
group results from a pseudo first order reaction. The poor water solubility of the more lipophilic BAT0 complexes, for example, TcCl(CDO),2MP and TcCl(CDO), BuB (2MP = 2-methylpropylboronic acid; BuB = n -butylboronic acid) prevented the determination of their axial ligand exchange rates under the conditions described (no carrier added levels), due to appreciable deposition of the complexes onto the sides of the vessel. Results showing the conversion of hydroxyl axial ligand to chloride are listed in Table 2. This conversion process occurs to a significant extent only below pH 5. No conversion of a hydroxy BAT0 to its chloro analog could be detected for up to 1 h at the physiological pH of 7.4. The ‘H NMR spectra of VcCl(DMG),2MP before and 10 min after addition of 1% KOD in D,O are shown in Fig. 4 (Note: the conditions used for the determination of spectra caused the signal from the oxime protons, normally at 15 6 to appear at 0.1 6.) 10 min after the addition of KOD/D,O, the exchange of the oxime protons for deuterium was complete. All remaining peaks are doubled, showing conversion
-0.5
-1 In (fraction Co -1.5
-2
-25
i
J
0
20
40
60
00
100
120
140
160
tlmo (minkhs) Fig. 3. Graph
of In (chloro fraction) vs time for *TcCl(DMG),EtB
at 20°C.
180
139
Chloro + hydroxy substitution Table 2. OH -+ Cl conversion half times of BATOs at 37°C OH + Cl r,,2 (min) Dioxime CD0 CD0 DMG
Boronic acid OH Me 2MP
DH 6.4
DH 5.0
DH3.0
k-1000 >looO >lcNIo
42.2 147.8 182.5
30.1 63.0 55.84
Table 3. LiDODhikitY of technetium corn&x&s Technetium complex
log k’
Calculated log P
@%OH(DMG)32MP WmTcCI(DMG),2MP P9mTcOH(CDO),MeB ‘=‘“TcCI(CDO), MeB
OS38 1.181 1.342 1.56
1.72 3.51 3.96 4.56
99”Tc-d,l-HM-PA0 99”Tc-ECD
0.259 0.315
0.95 1.11
to some other species; HPLC analysis indicated that
the conversion product was ?cOR(DMG)~ 2MP (R = D, H). Even 18 h after the addition of KOD/D*O, none of the carbon-bound hydrogen atoms showed any exchange for deuterium. The retention of complexes on reverse phase HPLC can be used to provide an estimate of the lipophilicity of these complexes (for example, Braumann, 1986). Lipophilicity estimates of BATOs have been conducted on the PRP-1 HPLC system described in the Methods section (Feld and Nunn, 1989). From the measured retention time of the complex (R,, in min), the capacity factor (k’) was calculated by the relationship: k’=-
R, - v t, (v = R, of a non-retained
standard;
nitrate was used for this purpose).
By calibration of the HPLC system with compounds of known lipophilicity (Feld and Nunn, 1989), the relationship between log k’ and log P (log of the octanol/water partition ratio) was found to be: log k’ log P=- o 36 + 0.23. Table 3 lists the determined log k’ values and estimated log P (from the above equation) for four BAT0 complexes, and, for comparison, two other complexes, 99mTc-d,l-HM-PA0 and %Tc-L,L-ECD; neutral complexes which are reported to cross the intact blood-brain barrier (Nowotnik et al., 1985; Walovitch et al., 1988). The rat biodistribution data for both the chloro and hydroxy analogs of 99”TcX(CDO),MeB and
Spectrum 10 minutoa r&r
of KODW (omt by 0.65 ppm).
the add&n
Fig. 4. ‘H NMR spectra of TcCl(DMG),2MP
in CD,CN with and without KOD/D,O.
740
S. S. JURISSONet al. Table 4. Rat biodistribution
of %TcX(DMG),2MP
and *TcX(CDO),MeB
(X = Cl, OH)
% I.D./organ W”TcCI(CDO),MeB
@“‘TcOH(CDO),MeB
60m
5m
60m
5m
60m
5m
60m
Blood Heart Brain Lung Liver Kidneys
2.90 1.71 0.30 2.05 28.5 4.93
1.79 0.39 0.24 0.91 14.1 1.74
5.30 1.19 0.03 2.83 36.3 3.49
2.30 0.45 0.02 I .47 19.9 2.24
2.92 1.44 0.81 1.31 28.6 3.32
1.99
3.38 1.16 0.05 3.04 28.9 4.21
2.00 0.24 0.03 0.99 10.0 I .49
Discussion Factors which influence axial Iigand exchange
While %Tc-BAT0 camp lexes with chloride axial ligands are stable for several hours when stored in the presence of kit components, the axial ligand in the isolated complex is responsive to exchange by competing monodentate anionic ligands (Nowotnik et al., 1989). The present study establishes the following points: (a) The rate of conversion from the Cl to the OH analog increases with increasing pH (increasing OH- concentration) suggesting an exchange mechanism which is not solely dependent on the loss of the Cl- from the BAT0 complex. (b) As expected, the rate of the reaction increases with increasing temperature (20 vs 37°C). (c) For BATOs with the same boronic acid cap, the exchange process is generally faster for DMG BATOs than CD0 BATOs. (d) For BATOs derived from alkyl boronic acids, the rate of Cl-OH exchange decreases with increasing size and/or branching of the alkyl group. While steric factors could account for the observed exchange rate differences between CD0 and DMG BATOs (the greater bulk of the cyclohexane rings, compared to the size of two methyl groups, inhibiting 5.
Initial
rat
biodistribution of (X = OH, Cl)
“mTcX(DMG),2MP
% I.D./organ 99mTcCcCI(DMG),2MP WmTcOH(DMG),2MP Brain Blood Heart Lungs Liver Kidneys
%TcOH(DMG),2MP
sm
99mT~X(DMG)J2MP are listed in Tables 4 and 5. When compared to the chloro compounds, the hydroxy analogs display slightly increased liver, lung, and blood activity and only slightly decreased heart activity. Brain uptake of the hydroxy BATOs is much lower than observed for the corresponding chloro analogs, and this appears to be a result of a lower cerebral extraction efficiency (Fig. 5).
Table
%TcCI(DMG),2MP
Organ
15s
60s
15s
60s
0.20 33.38 1.84 9.07 7.05 5.78
0.10 18.74 1.68 5.74 4.08 6.76
0.66 20.62 1.88 11.79 8.87 6.76
0.61 11.93 2.01 3.80 13.50 5.96
0.54 0.45 0.41 12.1 1.13
the approach of OH-), a steric effect due to the more distant boron substituent seems less likely. The effect of the boron substituent on conversion rate might be a reflection of differences in electron-donor abilities of these substituents; long range inductive effects involving a similar R-B-O-N-metal pathway have been demonstrated in the clathrochelate iron(I1) complexes, Fe(oxime),(BR)* (Robbins et al., 1985). The hydroxy analogs can be prepared in close to quantitative yields from the chloro analogs in high pH buffers containing no chloride. The conversion rates for hydroxy- to- chloro BATOs can be measured in PBS buffers made up in saline. Only below pH 5 is there appreciable conversion to the chloro analog; there appears to be an equilibrium reached between the chloro and hydroxy analogs at pH
The mechanism by which the chloro to hydroxy exchange takes place may play a significant role in the resulting biodistributions of these complexes; for example, there may be chemical intermediates whose biological behavior varies markedly from that of either the chloro or hydroxy analogs. There are three possible mechanisms by which this chloro- to- hydroxy exchange may take place, as shown in Fig. 6. The dependence of the exchange rate on the OHconcentration is evidence against an S, 1 mechanism. In this case, the rate controlling step would be the loss of Cl-, which should be independent of the OHconcentration. The lack of any chromatographic intermediates, even at low pH where the OH- attack should be slowest, is evidence against a rapid chloride loss followed by slow addition of the OH- group. The SN2 mechanism and a variation of the S, 1 mechanism, SNI-CB (Wilkins, 1974) (in which the rate controlling step is a base-catalyzed removal of a nearby proton preceding the rapid elimination of an anion) are possible alternatives. To distinguish between an SN I-CB and an SN2 mechanism, the rate of disappearance of 99mT~Cl(CD0)3MeB was plotted against the OHconcentration. An “S”-shaped curve was obtained (Fig. 7). If an SN2 mechanism were followed, the rate of the reaction should be directly related to the OHconcentration and a linear plot of rate versus pH would be obtained. The S-shaped curve is indicative of an SNl-CB mechanism in which the OH- concen-
Chloro --thydroxy substitution ’
8smT~CI (DMG),
2MP b-0.34
741
addition of HX (where X is a suitable, anionic, monodentate ligand) to give the BAT0 product.
(CBF) +0.07. R.O.BI
The influence of the axial ligand on myocardial uptake
q
0.2
0.01 0.0
.
0.2
0.4
-
0 0.8 CBF
0.0
1.0
1.2
1.4
I.6
(mc/mfn/g)
Fig. 5. Graph of CEE of TcX(DMG),2MP; X = Cl, OH.
tration controls the rate of the reaction by deprotonation of one proton from the BAT0 backbone. The rate of reaction appears to be controlled by this deprotonation step, as judged by the S-shaped curve. The point of inflection in the curve probably reflects the pK, of the deprotonation step. The plateau region at high pH is delimited by essentially complete monodeprotonation of the BAT0 backbone, while that at low pH, by minimal deprotonation. Increasing the concentration of chloride in the buffer to 1 M at pH 7.4 does not affect the rate of exchange. This suggests that the rate is not determined by a competitive exchange between the chloride and hydroxide anions, as would be expected with either an SN1 or S,2 mechanism. Therefore it must depend upon a secondary reaction, which can have a direct influence on the removal of the axial ligand, as would be the case in the deprotonation of the BAT0 backbone in an S, l-CB mechanism. A consequence of the S, l-CB mechanism is that the intermediate is an uncharged, six coordinate complex (S, 1 or f&.,2 mechanisms would provide cationic and anionic intermediates, respectively), and not a positively charged complex, as has been suggested (Kung, 1990). There is no evidence for this intermediate by HPLC analysis, indicating that the intermediate is transient, with rapid conversion back to a neutral, seven coordinate complex. As shown in Fig. 8, it is possible that the proton eliminated from the BAT0 backbone during the S, I-CB axial ligand exchange process may originate from either a free oxime group, or from one of the carbon atoms adjacent to an oxime group. To discriminate between these two possibilities, an NMR study was undertaken in which axial ligand exchange in y9”TcCI(DMG),2MP was studied in CD3CN/KOD/D,0. Under these conditions, the proton lost during the formation of the intermediate complex should be replaced by a deuterium atom. The spectra showed that only the oxime protons underwent deuterium exchange; there was no deuterium exchange of any of the carbon bound hydrogen atoms. Therefore, the S, 1-CB process proceeds by way of oxime deprotonation and loss of the axial ligand to give the transient, neutral, 6-coordinate intermediate complex, which is followed by rapid
A comparison of the biodistribution data for the hydroxy and chloro analogs of TcX(CDO), MeB and TcX(DMG)r2MP (Table 4) shows two interesting features. First, conversion of a chloro BAT0 to its hydroxy analog results in a 20-30% reduction of heart uptake in rats at 5 min post injection. When the heart uptake of a large number of BATOs was examined in rats, it was found that a parabolic relationship exists between heart uptake at 5 min post injection and the lipophilicity (log k’) of the complex (Nunn et al., 1989). The maximum of that parabola occurs at log k’ 1.65 (estimated log P of 4.8). Based on this structure-distribution relationship, the relative heart uptake values at 5 min post injection shown in Table 4 can be predicted solely from the lipophilicity of these complexes, shown in Table 3. The influence of the axial ligand on cerebral uptake and retention
As freely-diffusible cerebral perfusion tracers such as Xe-133 (Lassen et al., 1981) and 9ymTc-PnA0 (Volkert et al., 1984) display rapid washout (t,lz < 5 min) from the brain, any tracer which has a long half-time of cerebral washout must be provided with some mechanism which reduces normal washout kinetics. For two technetium tracers which display appreciable cerebral retention, 99mTc-d,l-HM-PA0 and 99”Tc-L,L-ECD, it has been proposed that the mechanisms of retention are the result of conversion of the administered freely-diffusible, lipophilic tracers to non-diffusible, less lipophilic forms once the compounds have crossed the blood-brain barrier (Nowotnik et al., 1985; Walovitch et al., 1988). As demonstrated in this paper, chloro-to-hydroxy exchange in BATOs is a facile process at physiological pH which results in the production of a less lipophilic complex. This led us to speculate (Nowotnik et al., 1989) that, like Y9”Tc-d,l-HM-PA0 and smTc-L,L-ECD, the cerebral retention of 99”TcCl(DMG),2MP (a BAT0 complex which has been extensively studied as a cerebral perfusion tracer; Narra et al., 1990) results from intracerebral conversion from a diffusible form to a non-diffusible form; in this case, intra-cerebral conversion of the diffusible chloro BAT0 to its non-diffusible hydroxy analog. This hypothesis was supported by preliminary biodistribution data (Table 4) which demonstrated that the percentage of injected dose of 9ymTcX(DMG),2MP in the brain (at 5 min post i.v. administration) is far greater for the chloro compound (0.81%) than its hydroxy analog (0.05%). However, examination of the initial (15 and 60 s) biodistribution of the chloro and hydroxy analogs of TcX(DMG),2MP (Table 5) confirms that both
S. S. JURISSONet al.
742
Possible mechanisms for axial ligand exchange. S, 1 mechanism
Initial loss of the Cl ligand to form a cationic intermediate followed by addition of the OH ligand to form the neutral OH analog. S, I-CB mechanism
Base attack of the dioxime backbone with removal of a proton to form an anionic complex. This is followed by rapid loss of the Cl ligand to the neutral 6 coordinated complex. The final step involves attack by a water molecule, placing an OH group in the axial coordination site and reprotonating the dioxime backbone. S, 2 mechanism
Initial attack by the OH group to form an eight coordinate, anionic intermediate followed by rapid loss of the Cl group to form the neutral, seven coordinate OH analog. Fig. 6. S, 1, S, I-CB, and S,2 mechanisms.
compounds are taken up by the brain, although uptake by the hydroxy analog is poor. The cerebral extraction data presented in Fig. 5 confirm that
I'
' ? g
60 40
al z 20 a 3 600 L 456769
Ldd-C~
/:/
10
11
12
PH Fig. 7. Graph of rate of Cl/OH exchange for TcCl(CDO)r MeB.
the poor cerebral uptake of TcOH(DMG),2MP compared with TcCl(DMG),2MP is a result of a low single-pass cerebral extraction efficiency, rather than some other factor, such as binding to blood components. Thus, it appears that TcCl(DMG),2MP cannot be trapped as its hydroxy analog as the latter compound displays significant cerebral washout. Another factor which negates the conversion trapping theory is the rate of conversion. To achieve cerebral retention by this mechanism, an in vivo chloro-to-hydroxy conversion half time of l-2 min would be required (Neirinckx, 1986), well in excess of the rate observed at pH 7.4/37”C in vitro. In addition, tissue homogenate and ex viva studies of Cl/OH conversion of 9hnT~CI(DMG),2MP have shown that the rate of chloro- to- hydroxy conversion is not
143
Chloro -+ hydroxy substitution Possible mechanisms for the S, l-CB axial ligand exchange process. (1) Initial removal of a hydrogen atom from an oxime group.
,OH
(2) Initial removal of a hydrogen atom from the carbon backbone.
The diagrams display the partial structure of BATOs, indicating two possible mechanisms for chloro-tohydroxy axial Iigand exchange by way of an SNI-CB process, involving hydrogen atom abstraction from either one of the free (uncapped) oxime groups or a carbon atom adjacent to one of the oxime groups. Fig. 8. Possible mechanisms for the abstraction of a proton in the S, I-CB axial ligand exchange process.
sufficiently rapid (t,,, > 5 min) to account for cerebral trapping of this radiopharmaceutical (Nowotnik et al., 1989). At this stage, it is unclear why the chloro and hydroxy derivatives of TcX(DMG),2MP and TcX(CDO),MeB display markedly different levels of uptake in rat brain. While the hydroxy compounds are less lipophilic than the corresponding chloro BATOs, their lipophilicity values are well above the minimum (log P of 0.5) and close to the presumed optimum (log P of 2.0) for blood-brain barrier penetration by physical diffusion (Hansch and Clayton, 1973). In addition, the lipophilicities of the hydroxy BATOs are greater than those for gQmTc-d,l-HM-PAO and wmTc-ECD (Table 3), complexes which also demonstrate high cerebral uptake in rats. Molecular size (implied by molecular weight) has also been shown to influence brain uptake, with uptake inversely related to size (Levin, 1980). However, models of these complexes have demonstrated that chloro and hydroxy analogs differ only marginally in size (Di Rocco et al., 1989). While other mechanisms, such as hydrogen bonding (Young ef al., 1988), have been shown to influence the interaction of substrates with the blood-brain barrier, and might explain the differences in the brain uptake of the chloro and hydroxy BATOs, further studies will be required to determine why the nature of the axial ligand has such a profound influence on brain uptake.
Conclusions There appears to be little difference in the rates of chloro-to-hydroxy exchange among the BAT0 complexes examined in this study with the half lives of these complexes ranging from 9 to 21 min under physiological conditions. The exchange between the axial chloro and hydroxy groups appears to proceed by an SNl-CB mechanism. Axial ligand exchange by this mechanism will provide a neutral six coordinate technetium complex, which rapidly converts back to a neutral seven coordinate complex. The observed differences in conversion rate among the BATOs studied probably result from steric and electronic effects of substituents on the deprotonation of the BAT0 backbone. While heart uptake values of the chloro and hydroxy BATOs are predictable from their lipophilicities (based on previous SDR studies; Nunn et al., 1989), the hydroxy BATOs display far lower brain uptake in rats than their chloro analogs. The reason for this difference is unclear. A hypothesis was tested, that slow cerebral washout of 99mTcCl(DMG),2MP results from intra-cerebral conversion of diffusible chloro complex to its “non-diffusible” hydroxy analog. It was found that 99mTcOH(DMG)32MP was taken up by rat brain, and is washed out, indicating that in viva chloro-to-hydroxy conversion cannot account for the cerebral retention of *TcCI(DMG)~~MP.
S. S. JUR:ls.WN et al.
744 Acknowledgemenu. -The
authors wish to thank Lillian Belnavis, Maryann Homack, and Christine Hood for technical assistance with the biodistribution and cerebral extraction studies, and Dr Michael Porubcan for the deuterium exchange ‘H NMR study.
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