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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1384-1389
Tumor Cell Cytotoxicity of a Novel Metal Chelator
By
S.V. Torti,
F.M. Torti,
S.P. Whitman,
M.W. Brechbiel,
G. Park, and
R.P. Planalp
From the Departments of Biochemistry, Cancer Biology, and Internal
Medicine, Wake Forest University School of Medicine and the
Comprehensive Cancer Center of Wake Forest University, Winston-Salem,
NC; the Radiation Oncology Branch, National Institutes of Health,
Bethesda, MD; and the Department of Chemistry, University of New
Hampshire, Durham, NH.
 |
ABSTRACT |
We have synthesized a novel six-coordinate metal chelator from the
triamine cis-1,3,5-triaminocyclohexane by the addition of a
2-pyridylmethyl pendant arm on each nitrogen, which we term tachpyr.
The experiments described here were designed to explore whether this
compound exhibits potential antitumor activity. When added to MBT2 or
T24 cultured bladder cancer cells, tachpyr was profoundly cytotoxic,
with an IC50 of approximately 4.6 µmol/L compared with 70 µmol/L for desferioxamine. To explore the mode of action of tachpyr,
several metal complexes were prepared, including Fe(II), Ca(II),
Mn(II), Mg(II), Cu(II), and Zn(II) tachpyr complexes. Of these, the
Zn(II), Cu(II), and Fe(II) complexes were without toxic effect, whereas
the Ca(II), Mn(II), and Mg(II) complexes remained cytotoxic. To further
probe the role of Zn(II) and Cu(II) chelation in the
cytotoxicity of tachpyr, sterically hindered tachpyr derivatives were
prepared through N-alkylation of tachpyr. These derivatives were unable
to strongly bind Fe(III) or Fe(II) but were able to bind Zn(II) and
Cu(II). When added to cells, these sterically hindered tachpyr
derivatives were nontoxic, consistent with a role of iron depletion in
the cytotoxic mechanism of tachpyr. Further, the addition of tachpyr to
proliferating cultures resulted in an early and selective inhibition of
ferritin synthesis, an iron storage protein whose translation is
critically dependent on intracellular iron pools. Taken together, these
experiments suggest that tachpyr is a cytotoxic metal chelator that
targets intracellular iron, and that the use of tachpyr in cancer
therapy deserves further exploration.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
IRON IS ESSENTIAL FOR the catalytic
activity of numerous critical enzymes, including respiratory chain
enzymes and ribonucleotide reductase, the rate limiting step in DNA
synthesis. Thus, iron is absolutely required for mammalian cell growth.
The close linkage between cell proliferation and iron has suggested
that iron deprivation strategies may be useful in inhibiting tumor cell
growth. For example, antitransferrin receptor antibodies, which inhibit
a major pathway of cellular iron uptake, are being explored for their
potential to inhibit growth of hematopoietic and nonhematopoietic tumors.1,2 Desferioxamine, a potent iron chelator, is
currently in clinical trials in combined chemotherapy of
neuroblastoma3,4 and prostate cancer. Similarly, gallium
nitrate, which blocks iron incorporation by binding to the transferrin
receptor, is being used in combined chemotherapy of bladder
cancer5,6 and lymphoma.7
The design of iron chelators for use in tumor treatment is a relatively
unexplored field. Desferioxamine, a bacterial siderophore and potent
iron chelator used in conditions of iron overload, has been used in
some studies of antiproliferative effects of iron
chelation.8-10 However, desferioxamine suffers from several limitations.11 Systematic examination of the ability of
alternative iron chelators to function as antiproliferative agents has
largely been limited to chelators of the pyridoxyl isonicotonyl
hydrazide (PIH) family.12 This group of compounds has been
shown to be effective in mobilizing iron in vitro and in vivo. A recent
study of the antiproliferative effect of PIH and its analogs on
cultured human neuroblastoma cells identified several analogs with an
IC50 lower than that of desferioxamine and a potential
correlation between lipophilicity and cytotoxic effects.13
Cytotoxicity was not uniformly correlated with iron chelation efficacy,
suggesting that the ability to permeate cells and target selected
essential pools of iron may be critical components of the
antiproliferative activity of iron chelators.13
We have synthesized a novel metal chelator based on the framework
triamine cis-1,3,5-triaminocyclohexane, which we term
tachpyr.14 Tachpyr has three aminopyridine arms attached to
a cyclohexyl ring (Fig 1). It is a
lipophilic molecule that undergoes a cyclohexyl ring flip (Fig 1a) to
give a chelator with hexadentate octahedral geometry. Tachpyr accepts
metals of radius from 0.76 Å to 0.94 Å with minimal
distortion.15
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| Fig 1.
Structure of tachpyr in the open and closed conformations
(A), its metal complexes (B), and N-alkylated derivatives (C).
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Tachpyr differs from desferioxamine and PIH in chemical structure and
properties. For example, unlike either of these chelators, tachpyr
reacts with Fe(III) to yield an Fe(II) complex in a redox process that
oxidizes tachpyr (G. Park et al, manuscript in
preparation). The experiments described here were designed to explore
whether this compound exhibits potential antitumor activity. We report that tachpyr shows cytotoxic activity consistent with a potential application in antitumor therapy.
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MATERIALS AND METHODS |
Preparation of tachpyr and its derivatives.
Tachpyr, (N-Me)3tachpyr, and
(N-Et)3tachpyr were synthesized from
cis-1,3,5-triaminocyclohexane14 (G. Park et al,
manuscript in preparation). To purify and facilitate handling of
chelators, their nitrate salts were prepared through treatment of the
chelator with excess nitric acid in ethanol. Typically, 8 molar
equivalents of concentrated nitric acid was added to a solution of
approximately 5 × 10 3 mol of tachpyr in 1 mL
of anhydrous ethanol resulting in the formation of a pale green
precipitate. This precipitate was washed twice with approximately 8 mL
of anhydrous diethyl ether and dried under reduced pressure (5 × 102 Torr). The compositions of the nitrate salts
were determined by elemental analysis and were found to be
tachpyr.5HNO3.H2O,
(N-Me)3tachpyr.6HNO3, and
(N-Et)3tachpyr.6HNO3.
Metal complexes of tachpyr were prepared from reaction of the
appropriate metal salt with the chelator in methanol solution, followed
by slow diffusion of diethyl ether into the reaction solution.
Subsequently, recrystallization was conducted to achieve sufficient
purity as determined by combustion analyses16 (G. Park et
al, manuscript in preparation). They are represented as M[tachpyr][X]2 (M = Ca(II), Mg(II), Cu(II), X = Cl; M = Mn(II), Zn(II), X = ClO4). Further description of the
structures and reactivity of these complexes will appear elsewhere;
however, their formulations are represented by the leftmost structure
of Fig 1b in all cases except for the Fe(II) complex, for which the
rightmost two structures of Fig 1b apply. Compounds were dissolved in
phosphate buffered saline, pH 7.4, before use in cytotoxicity assays.
Cytotoxicity assays.
MBT2 and T24 are mouse and human bladder cancer cell lines,
respectively. MBT2 cells were a generous gift from Dr N. Lattime (Thomas Jefferson University, Philadelphia, PA), and T24 cells were
obtained from the American Type Culture Collection. MRC-5 cells were a
gift from H. Blau (Stanford University, Stanford, CA). Cells were grown
in RPMI medium (MBT2) or DME medium (T24 and MRC-5) containing 10%
fetal bovine serum in a humidified chamber containing 5%
CO2. A total of 5 × 103 cells were plated
in 96-well tissue culture dishes and allowed to attach overnight before
test compounds were added. Six replicate cultures were used for each
point. After 72 hours (or less, in time course studies), viable cells
remaining were assessed using a methytetrazolium (MTT)
assay, in which (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide is added to the medium and the formation of a reduced product
assayed by measuring the optical density at 560/650 nm after 3 hours.
Color formation is proportional to viable cell number.17
IC50 was defined as the concentration required to inhibit
viability by 50% and was calculated by using the Multiple Drug-Effect
Analysis Program (Biosoft, Ferguson, MO). The
IC50 of tachpyr was not significantly affected by the
addition of 100 µg/mL iron-saturated transferrin to the medium (not
shown).
In some experiments, cell survival was assessed by using a clonegenic
assay, which measures the ability of cells to divide and form visible
colonies of approximately 50 or more cells. For these experiments,
replicate plates of cells were prepared and various concentrations of
tachpyr added to the growth medium (control cells were plated at the
same time and received no drug). At the end of the incubation period,
surviving cells were removed from the dish by trypsin, diluted, and
replated in growth medium without drug. Colonies were allowed to grow
for 7 days. The growth medium was removed, the plates fixed with 0.5%
gluteraldehyde, stained with 1% crystal violet, and colonies were
counted.
Ferritin synthesis.
Ferritin synthesis was assessed after 7 or 16 hours of exposure of
cells to media containing either no additions (control), tachpyr,
desferioxamine, or ferric nitrolotriacetate. Cells were metabolically
labeled for the last 2 hours of incubation with [35]S-translabel
(ICN), and ferritin visualized by immunoprecipitation and sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as
previously described.18
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RESULTS |
Tachpyr is toxic to proliferating bladder cancer cells.
To explore the fundamental biological properties of tachpyr, and to
assess whether these were consistent with an iron-depleting mode of
action, we measured the effects of tachpyr on growing cells in culture
and compared them with desferioxamine, a known iron chelator. MBT2, a
mouse bladder tumor cell line, was incubated with varying doses of
tachpyr or desferioxamine for 72 hours, and effects on viability were
assessed using an MTT dye reduction assay.17 As seen in
Fig 2, cell growth was inhibited by both tachpyr and desferioxamine. Tachpyr was more potent than
desferioxamine: In four independent experiments, the average
IC50 of tachpyr was 4.6 ± 2.0 µmol/L (standard
error), compared with 70 µmol/L for desferioxamine.
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| Fig 2.
Comparative cytotoxic effect of tachpyr and
desferioxamine on cultured MBT2 bladder cancer cells. Cells were
incubated with varying doses of desferioxamine mesylate or tachpyr for
72 hours and viability was assessed by using an MTT assay as described in the Materials and Methods. Calculated IC50s from this
data are 3.6 µmol/L for tachpyr and 70 µmol/L for desferioxamine.
Mean IC50 of tachpyr in four independent experiments was
4.6 ± 2.0 µmol/L (standard error).
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The MTT assay is widely used to measure drug effects on cell
viability.13,19,20 It is generally believed to measure
mitochondrial function. To confirm that the results of the MTT assay
did not reflect an interference with mitochondrial activity, but a true effect of tachpyr on cell viability, we also measured effects of
tachpyr using a clonegenic assay. As seen in
Fig 3, this assay confirmed that viability,
as measured by colony-forming ability, was also sharply reduced by
tachpyr.
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| Fig 3.
Tachpyr inhibits colony-forming ability of treated cells.
MBT2 cells were incubated for 72 hours with tachpyr and viability was
assessed by using a clonegenic assay.
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As shown in Fig 4, a time course study
showed that tachpyr was not immediately cytotoxic to cells. Rather, a
delay of approximately 24 hours was observed before the cytotoxic
action of tachpyr became manifest. This time course exactly paralleled
that of desferioxamine, a known iron chelator (Fig 4).
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| Fig 4.
Time course of cytotoxicity of tachpyr and
desferioxamine. MBT2 cells were incubated with 150 µmol/L (dfo) or 25 µmol/L tachpyr for various lengths of time and survival was measured
by using an MTT assay. Viability at time 0 was defined as 100%.
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Effects of tachpyr/metal complexes on cell viability.
To explore whether iron deprivation contributed to tachpyr
cytotoxicity, a number of metal complexes of tachpyr were prepared and
their effects on MBT2 cells were compared with those of tachpyr. As
shown in Figs 5 and
6, the Ca(II), Mn(II), and Mg(II) complexes of tachpyr were toxic, whereas the Zn(II), Fe(II), and Cu(II) complexes
were not. Initially, these findings appear consistent with the
hypothesis that tachpyr functions via the complexation of Zn(II),
Fe(II), or Cu(II). However, it is also possible that the association
between Zn(II), Cu(II), or Fe(II) and tachpyr may be sufficiently
strong to prevent the formation of free ligand. This in turn would
restrict the amount of tachpyr available to deplete intracellular iron
pools. Further support of this hypothesis comes from studies with
sterically hindered chelators that bind Zn(II) and Cu(II) but not
Fe(II) (see below). In either case, results with tachpyr/metal
complexes suggest that depletion of Ca(II), Mn(II), or Mg(II) does not
play a major role in the cytotoxicity of tachpyr.
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| Fig 5.
Comparative cytotoxicity of tachpyr and
Fe[tachpyr]Cl2. MBT2 cells were treated for 72 hours with
either tachpyr ( ) or Fe[tachpyr]Cl2 ( ) before
viability was assessed using an MTT assay.
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| Fig 6.
Comparative cytotoxicity of tachpyr and its metal
derivatives. Viability was assessed after 72 hours exposure to the
indicated compounds as described in the Materials and Methods. ( )
tachpyr; ( ) [Mg]tachpyr; ( ) [Mn]tachpyr; ( ) [Cu]tachpyr;
() [Zn]tachpyr; ( ) [Ca]tachpyr.
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Tachpyr derivatives unable to bind iron are not toxic.
To assess further whether iron chelation was involved in the cytotoxic
mechanism of tachpyr, we prepared alkylated derivatives of tachpyr in
which methyl or ethyl groups were substituted for hydrogen at the amino
nitrogens N1, N2, and N3 (Fig 1c).
To probe the chemical and structural effects of this change on
complexation properties, the single-crystal X-ray structures of the
Ni(II) complexes Ni[tachpyr]Cl2 and
Ni[(N-Me)3tachpyr][ClO4]2 were
compared. The bond distance between the metal and the amine nitrogens
of the respective tachpyr chelator were lengthened from 2.102(4) to
2.168(4) Å on methylation of tachpyr (G. Park et al, manuscript in
preparation). This bond lengthening shows the steric effect of the
methyl group on nitrogen, an effect that weakens the association of
(N-Me)3tachpyr with metal ions, and in particular affects
the reaction of tachpyr with iron. Thus, we have been able to prepare
the complexes
Cu[(N-Me)3tachpyr][ClO4]2,
Zn[(N-Me)3tachpyr][ClO4]2, and
Zn[(N-Et)3tachpyr][ClO4]2,
although it has not been possible to observe a strong association
between Fe(II) or Fe(III) and (N-Me)3tachpyr (G. Park et
al, unpublished observations). Figure 7
compares the growth-inhibitory ability of these alkylated tachpyr derivatives with the parent compound. In contrast to tachpyr, which was
cytotoxic, these derivatives were completely without effect on the
viability of MBT2 cells. This observation is consistent with a role of
iron chelation, as opposed to zinc or copper chelation, in the
cytotoxic effects of tachpyr.
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| Fig 7.
N-alkylated derivatives of tachpyr are not cytotoxic.
Viability was assessed after 72 hours treatment with the indicated
compounds.
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| Fig 8.
Tachpyr inhibits ferritin synthesis. Cells were treated
with 20 µmol/L tachpyr (tach), 150 µmol/L desferioxamine (def) or 200 µmol/L ferric nitrilotriacetate (iron) in growth medium for 16 hours. Cells were labeled with [35]S amino acids for the last 2 hours
of treatment. Ferritin was isolated by immunoprecipitation and analyzed
by SDS-PAGE. The H and L subunits of ferritin are shown (lower and
upper bands, respectively).
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Tachpyr inhibits ferritin synthesis.
To directly examine whether iron represented an important intracellular
target of tachpyr, we assessed whether tachpyr affected synthesis of
ferritin, a major iron storage protein whose synthesis is
critically dependent on intracellular concentrations of
iron.21 Desferioxamine, an iron chelator known to inhibit
ferritin synthesis, was used as a positive control. Cells were treated
for 16 hours, labeled with [35]S amino acids, and ferritin isolated
by immunoprecipitation and SDS-PAGE. As shown in Fig 8, under these
conditions tachpyr repressed ferritin synthesis as effectively as
desferioxamine. This was a specific effect and did not reflect a
generalized inhibition of protein synthesis, because equal numbers of
trichloroacetic acid-precipitable counts were
immunoprecipitated in all cases. A similar inhibition of ferritin
synthesis was seen after 7 hours of treatment (not shown), a timepoint
that precedes all cytotoxic effects of tachpyr (Fig 2). These results
suggest that intracellular iron depletion is an early, proximal event
initiated by tachpyr.
Effects of tachpyr are not restricted by cell type.
To ensure that effects of tachpyr were not restricted to a particular
cell type, the effect of tachpyr on the viability of T24 bladder cancer
cells and MRC-5 normal human diploid fibroblasts was also assessed. As
shown in Table 1, both cell types were sensitive to tachpyr. However, the IC50 of T24 cells was
4.3 µmol/L, whereas that of MRC-5 cells was 30.5 µmol/L,
approximately sevenfold higher than observed for the two tumor cell
lines.
| |
DISCUSSION |
Tachpyr is a metal ligand with a unique chemical structure relative to
other ligands that have been explored as antitumor therapeutics. It
differs from the PIH chelator family13 in containing exclusively nitrogen donor ligands (Fig 1). In this regard, tachpyr also differs from natural siderophores, such as desferioxamine, as well
as most chemical chelators (eg, L1, CP94, DTPA, DBED, and
EDTA22), which generally contain oxygen donor atoms.
Additionally, tachpyr differs from PIH in denticity, being hexadentate
rather than bidentate. Our preliminary results suggest that the
chemical features of tachpyr may allow it to both bind and reduce iron, properties that may profoundly influence its biological activity (G. Park et al, manuscript in preparation). Because it is chemically unrelated to previously studied metal chelators, the biological properties of tachpyr were unknown. The experiments described here were
designed to assess biological targets of tachpyr and its potential as
an antitumor therapeutic.
Our results show a striking cytotoxic effect of tachpyr. In the cells
studied, the IC50 of 4.6 µmol/L was lower than that of
desferioxamine (70 µmol/L), suggesting an effective dose in the range
reported for the most effective PIH analogs (1-7 µmol/L).13 We have measured the lipophilicity
(n-octanol-water partition coefficient; log Poct/H2O) of
tachpyr as  0.10 (F. Lu et al, manuscript in
preparation), a value consistent with an ability to penetrate biological membranes. Thus, the relative decrease in IC50
of tachpyr relative to desferioxamine may in part be attributable to
its enhanced permeability relative to desferioxamine. Alternatively or
additionally, tachpyr may have greater access to key intracellular iron
pools, enabling it to achieve equivalent cytotoxicity at lower doses.
A number of lines of evidence point to iron as a major target of
tachpyr. Tachpyr has the potential to chelate several biologically important metals including Ca(II), Mg(II), Mn(II), Fe(II), Cu(II), and
Zn(II). Although we cannot definitively exclude a role of chelation of
other metals in the effects of tachpyr, our toxicity studies of tachpyr
complexes of the above metals are consistent with the suggestion that
iron is a major target of tachpyr. Thus Ca[tachpyr]Cl2,
Mg(tachpyr)Cl2, and
Mn(tachpyr)[ClO4]2 retained their
toxicity, whereas Fe[tachpyr]Cl2,
Cu(tachpyr)Cl2, and
Zn(tachpyr)[ClO4]2 were nontoxic (Figs 5 and
6). Assuming that metal-tachpyr complexes do not cross the cell
membrane because they are cationic,23 we postulate that the
toxicity of the Ca(II), Mn(II), and Mg(II) complexes arises from the
liberation of free tachpyr by transmetallation of the metal ion to a
ligand with greater affinity for these metals (such as apotransferrin).
We further hypothesize that this reaction does not occur appreciably
with Zn(II), Cu(II), and Fe(II) complexes of tachpyr because of their
greater inherent stability. These hypotheses are in accord with the
well-established theoretical affinities of nitrogen-donor ligands (such
as tachpyr) for metal ions, which predict that Ca(II), Mn(II), and
Mg(II) are less tightly bound than Fe(II), Zn(II), or
Cu(II).24 Toxicity studies with N-alkylated derivatives of
tachpyr extend these conclusions by showing that N-alkylation blocks
the cytotoxicity of tachpyr. Because N-alkylation inhibits the reaction
of tachpyr with iron but not with zinc or copper, these results suggest
that Zn(II) or Cu(II) are not major cellular targets of tachpyr (Fig
7). Taken together, these experiments are consistent with a mechanism
in which iron chelation leads to cytotoxicity.
The lipophilicity constant of uncomplexed tachpyr is consistent with a
mode of action involving penetration of the cell and chelation of an
intracellular iron pool. A reduction of the intracellular labile iron
pool is also consistent with the observed ability of tachpyr to inhibit
ferritin synthesis. The translation of ferritin mRNA is regulated by an
intracellular pool of chelatable iron.21 Thus, when iron
levels are low, ferritin synthesis is decreased; conversely, when iron
levels are high, ferritin synthesis increases. This regulatory response
to iron is posttranscriptional and is caused by the recruitment of
stored message from monosomes to polysomes in the presence of
iron,21 a process mediated by iron regulatory
element-binding proteins (IRPs), proteins that bind to a region (the
iron regulatory element [IRE]) of the 5 UTR of the ferritin mRNA and
regulate its translation.25-27 The observation that
treatment of MBT2 cells results in an early and selective repression of
ferritin synthesis is consistent with iron depletion as a major
consequence of tachpyr activity.
Based on previous results suggesting that bladder cancer may be
particularly susceptible to iron depletion therapy,9,28-30 the experiments described here assessed the cytotoxic potential of
tachpyr on cultured murine bladder cancer cells. The observed cytotoxic
effects were not restricted to these MBT2 cells, but were also seen in
other cell types examined, including the human bladder cancer cell line
T24 (Table 1) as well as breast cancer cell lines (not shown). Normal
diploid fibroblasts were sixfold to sevenfold more resistant to tachpyr
than the tumor cell lines we tested (Table 1). Although the
significance of this difference will remain unclear until more cell
lines are examined, it is tempting to speculate that despite the
cytotoxic effects of tachpyr on normal cell types, cancer cells may
exhibit a heightened sensitivity to tachpyr that can be exploited
therapeutically.
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FOOTNOTES |
Received November 17, 1997; accepted April 10, 1998.
Supported in part by Grant No. DK 42412 from the National Institutes of
Health (F.M.T. and S.V.T.) and by Grant No. DK42412-09S1 (S.P.W.).
Address reprint requests to S.V. Torti, PhD, The Wake Forest University
School of Medicine, Department of Biochemistry, Wake Forest University,
Medical Center Boulevard, Winston-Salem, NC 27157-1082.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank F.H. Lu for preparation of the N-alkyl tachpyr
ligands, N. Ye for the preparation of tachpyr derivatives, and R. Ma
for superb technical assistance. We thank the NIH for the support of
F.H. Lu through the STRP (student training research program) fellowship
program during 1997.
| |
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Abstract
Introduction
Abstract