Synthesis and initial evaluation for in vivo chelation of Pu(IV) of a mixed octadentate spermine-based ligand containing 4-carbamoyl-3-hydroxy-1-methyl-2(1H)-pyridinone and 6-carbamoyl-1-hydroxy-2(1H)-pyridinone

Article abstract of DOI:10.1021/jm010564t

An improved synthesis for a series of 1-hydroxy-2(1H)-pyridinone-based octadentate ligands is reported. The mixed chelate, octadentate ligand, 3,4,3-LI(1,2-Me-3,2-HOPO), was designed, synthesized, and tested for in vivo chelation of Pu in a mouse model. This ligand incorporates both 1,2-HOPO and Me-3,2-HOPO metal chelating units; the latter has higher affinity toward actinide ions than does 1,2-HOPO at physiological pH. Injected or administered orally to fasted or normally fed mice at the standard clinical dose 30 μmol/kg, both 3,4,3-LI(1,2-HOPO) and 3,4,3-LI(1,2-Me-3,2-HOPO) remove significantly more Pu than injected CaNa₃DTPA. Injected doses of 0.1 μmol/kg of these HOPO ligands are as effective as 30 μmol/kg of injected CaNa₃-DTPA. Ten daily injections of 30 μmol/kg of a HOPO ligand did not induce detectable acute toxicity in mice. The mixed HOPO ligand is somewhat more effective than 3,4,3-LI(1,2-HOPO) when given orally, and the enhanced reduction of liver Pu by the mixed ligand is statistically significant. Thus, both octadentate HOPO ligands meet the criterion of low toxicity at doses that are more effective than the standard dose of CaNa₃DTPA. Their improved effectiveness at low dose along with great oral activity (despite low gastrointestinal absorption) implies that new treatment regimens can be developed using the HOPO ligands alone or as adjuncts to CaNa₃DTPA therapy, which will greatly exceed the amount of Pu excretion that is achievable with CaNa₃DTPA alone.

Full text of DOI:10.1021/jm010564t

J . Med. Chem. 2002, 45, 3963-3971
3963
Syn th esis a n d In itia l Eva lu a tion for In Vivo Ch ela tion of P u (IV) of a Mixed
Octa d en ta te Sp er m in e-Ba sed Liga n d Con ta in in g
4-Ca r ba m oyl-3-h yd r oxy-1-m eth yl-2(1H)-p yr id in on e a n d
6-Ca r ba m oyl-1-h yd r oxy-2(1H)-p yr id in on e
J ide Xu, Patricia W. Durbin, Birgitta Kullgren, Shirley N. Ebbe, Linda C. Uhlir, and Kenneth N. Raymond*
Department of Chemistry and Glenn T. Seaborg Center, Chemical Sciences Division, Lawrence Berkeley National Laboratory,
University of California, Berkeley, California 94720
Received December 11, 2001
An improved synthesis for a series of 1-hydroxy-2(1H)-pyridinone-based octadentate ligands
is reported. The mixed chelate, octadentate ligand, 3,4,3-LI(1,2-Me-3,2-HOPO), was designed,
synthesized, and tested for in vivo chelation of Pu in a mouse model. This ligand incorporates
both 1,2-HOPO and Me-3,2-HOPO metal chelating units; the latter has higher affinity toward
actinide ions than does 1,2-HOPO at physiological pH. Injected or administered orally to fasted
or normally fed mice at the standard clinical dose 30 µmol/kg, both 3,4,3-LI(1,2-HOPO) and
3,4,3-LI(1,2-Me-3,2-HOPO) remove significantly more Pu than injected CaNa3DTPA. Injected
doses of 0.1 µmol/kg of these HOPO ligands are as effective as 30 µmol/kg of injected CaNa3-
DTPA. Ten daily injections of 30 µmol/kg of a HOPO ligand did not induce detectable acute
toxicity in mice. The mixed HOPO ligand is somewhat more effective than 3,4,3-LI(1,2-HOPO)
when given orally, and the enhanced reduction of liver Pu by the mixed ligand is statistically
significant. Thus, both octadentate HOPO ligands meet the criterion of low toxicity at doses
that are more effective than the standard dose of CaNa3DTPA. Their improved effectiveness
at low dose along with great oral activity (despite low gastrointestinal absorption) implies that
new treatment regimens can be developed using the HOPO ligands alone or as adjuncts to
CaNa3DTPA therapy, which will greatly exceed the amount of Pu excretion that is achievable
with CaNa3DTPA alone.
In tr od u ction
istered by injection, because it is not effective when
given orally.8,11-14
Plutonium is an industrial and environmental hazard.
Nuclear weapons tests, military and power reactor
accidents, and inadequate waste disposal practices have
contaminated radiation workers1,2 and the environ-
ment.3,4 After intake via inhalation, ingestion, or depo-
sition in a wound, Pu enters the blood stream where
complexation by the nonfilterable iron transport protein
transferrin (TF) severely inhibits its renal excretion.5
The Pu-TF complex circulating in the blood dissociates,
and the displaced Pu forms more stable long-lived
complexes with bioligands in the skeleton and liver.
Inhaled Pu is released very slowly from the respiratory
tract. The accumulated radiation doses damage tissue
and induce cancers in the three target organs: bone,
liver, and lung.6,7 Aggressive, and often protracted,
treatment with chelating agents that form excretable
low molecular weight Pu complexes is the only practical
way to reduce the health consequences of internally
deposited Pu.2,8-10 Diethylenetriaminepentaacetic acid
(DTPA), the only clinically accepted Pu removal agent,
has clinical defects. In particular, CaNa3DTPA or less
potent ZnNa3DTPA must be given to avoid depletion of
essential divalent metal ions. Furthermore, clinically
acceptable doses of DTPA do not remove Pu from bone
mineral or liver ferritin. Finally, DTPA must be admin-
The similar coordination properties of Fe(III) and Pu-
(IV)5,9,11,15,16 suggested that synthetic actinide seques-
tering agents containing the iron-binding units of
siderophores (microbial iron chelators) would be effec-
tive complexing agents for Pu(IV).5,9,15,16 Our initial
synthetic actinide sequestering agents contained cat-
echol, the functional group of the potent siderophore
enterobactin.9,14,17 However, no catecholate ligand tested
to date significantly improves in vivo Pu chelation with
acceptably low toxicity at an effective dose when com-
pared to CaNa3DTPA.10,18-21
The heterocyclic hydroxypyridinone (HOPO) isomers
are highly selective for “hard” metal ions with large
charge/radius ratios, such as Fe(III) and Pu(IV). The
HOPO monoanions have zwitterionic resonance forms
that are isoelectronic with the catechol dianion. The
HOPO metal binding units, which are monoprotic
(deprotonated) at physiological pH, were expected to
form stable Pu(IV) complexes at physiological pH.22-25
Among the multidentate 1,2-HOPO ligands synthesized
to date, spermine-based octadentate 3,4,3-LI(1,2-HOPO)
(Figure 1, 1) is the most effective Pu chelator. (The
abbreviation of 1 stands for the 1,2-HOPO ligand with
a linear tetramine backbone, abbreviated as LI, in which
the four amine nitrogen atoms are separated by 3, 4,
and 3 carbon atoms, respectively.) Given by injection
or orally, its efficacies for in vivo chelation of Pu(IV),
Am(III), and Th(IV) greatly exceed those of CaNa3DTPA
* To whom correspondence should be addressed. Tel: 510-486-6145.
Fax: 510-486-5283. E-mail: raymond@socrates.berkeley.edu.
10.1021/jm010564t CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/31/2002

3964 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Xu et al.
F igu r e 2. Functionalized HOPOs and the metal complexes
of these predisposed ligands.
protected species 1-benzyloxy-2(1H)-pyridinone-6-car-
boxylic acid (6) could be activated and coupled to an
amine scaffold. It was shown that like other aromatic
carboxylic acids, the HOPO carboxyl group could be
activated by various activation procedures used widely
in peptide synthesis.29,30 Those procedures include the
N-hydroxysuccinimide (NHS)/1,3-dicyclohexylcarbodi-
imide (DCC), HOBT/DCC, and 2-mercaptothiazoline/
DCC, as well as acid chloride activations. Bailly and
Burgada31 independently improved the synthesis of 1
by using 1-benzyloxy-2(1H)-pyridinone-6-carbonyl chlo-
ride (7) as the activated species, which was reacted with
the spermine to afford the benzyl-protected ligand in
good yield. We developed the new synthesis for 5
reported herein, synthesized a series of multidentate
1,2-HOPO ligands with different topologies, and evalu-
ated these ligands as Pu(IV) sequestering agents in Pu-
injected mice.26,32 Transition metal complexes of these
multidentate ligands were also synthesized and struc-
turally characterized.32
F igu r e 1. Possible octadentate 1,2-HOPO and Me-3,2-HOPO
ligands based on spermine scaffolds.
in all low dose evaluations in small laboratory ani-
mals.18,23,26,27 Subsequently, we developed a strategy25
for the design and synthesis of powerful HOPO-based
metal sequestering agents that functionalizes the HOPO
ring carbon atom adjacent to the HOPO hydroxyl group
with a carboxyl group and then attaches the HOPO
units through amide linkages to a suitable molecular
scaffold. Structural characterization of these function-
alized 1,2-HOPO and 3,2-HOPO ligands as well as their
metal complexes indicated that these ligands are not
only predisposed toward complexation, but their metal
complexes are also further stabilized by the amide
hydrogen bonding as shown in Figure 2.22,24,28
Concerns about the acute toxicity of 1 at high injected
doses and its difficult original synthesis23 stimulated the
design and synthesis of multidentate HOPO ligands
based on a new set of ligand scaffolds and containing
the less acidic Me-3,2-HOPO isomer.33
In initial evalu-
ations in mice at the standard ligand dose (30 µmol/
kg), most of the Me-3,2-HOPO ligands, when promptly
injected and nearly all when orally administered, were
significantly more effective for in vivo Pu chelation than
an equimolar amount of CaNa3DTPA. When injected
promptly, six of the Me-3,2-HOPO ligands were as
effective as 1.33 It was then reasoned that introduction
of the Me-3,2-HOPO unit into a spermine-based octa-
dentate 1,2-HOPO ligand such as 1 would result in new
ligands 2-4 with enhanced affinity for Pu(IV) (Figure
1). In this paper, we present the improved syntheses
(Scheme 1) of the precursor 5 and of the octadentate
ligand 1 and the synthesis (Scheme 2) and initial
biological evaluation of a new octadentate mixed HOPO
ligand, 3,4,3-LI(1,2-Me-3,2-HOPO) (Figure 1, 2). The
mixed ligand 2 has been evaluated in mice for acute
toxicity and for in vivo Pu chelation with the same Pu
removal protocols used to evaluate and compare 3,4,3-
LI(1,2-HOPO) and CaNa3DTPA.26
Because the 1,2-HOPO ligands, in particular 3,4,3-
LI(1,2-HOPO) 1, are potentially useful sequestering
agents for hard metal ions including the actinides,
research was directed toward improving the initial
synthesis. The original synthesis of multidentate 1,2-
HOPO ligands, reported a decade ago,23 involves several
low yield steps and difficult purifications: 2,6-dibro-
mopyridine was converted into 6-bromopicolinic acid,
which was oxidized and then hydrolyzed to 1-hydroxy-
2(1H)-pyridinone-6-carboxylic acid (6-carboxy-1,2-HO-
PO) 5. Compound 5 was activated by phosgene and then
coupled to a suitable amine scaffold.23 Separation and
purification of the multidentate 1,2-HOPO products are
tedious, often requiring high-performance liquid chro-
matography (HPLC) purification.
In 1992, Uhlir and Raymond29 reported that following
benzyl protection of the N-hydroxyl group of 5, the

Chelation of Pu(IV) of a Mixed Octadentate Ligand
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3965
Sch em e 1. Improved Synthesis of 3,4,3-(LI-1,2-HOPO)
Sch em e 2. Synthesis of 3,4,3-LI(1,2-Me-3,2-HOPO)
Resu lts
benzyl-protected 3,4,3-LI(1,2-HOPOBn) (8), which is
deprotected under acidic conditions to give an 81% yield
of ligand 1 (as compared to the 15% reported for the
original synthesis).23
Liga n d Syn th eses. The improved synthesis of 1 is
shown in Scheme 1. The protection of 1,2-HOPO-6-
carboxylic acid (5) with benzyl chloride (Scheme 1) is
easily accomplished if potassium carbonate is used as
the base in methanol. The benzyl-protected acid (1,2-
HOPOBn acid 6) is now isolated in 91% yield after
removing the solvent and acidifying the aqueous solu-
tion of the residue.31
The mixed HOPO ligand, 3,4,3-LI(1,2-Me-3,2-HOPO)
(2), is synthesized in three steps (Scheme 2): spermine
is reacted with 2 equiv of 3,2-HOPOBn-thiazolide (9)
to form only the Ν1,Ν14-disubstituted species 10, as
confirmed by NMR spectroscopies since 9 only reacts
with primary amine moieties. Compound 10 is then
coupled with 1,2-HOPOBn acid chloride to yield the
protected mixed ligand 11. Because hydrogenation
would reduce the 1,2-HOPO moieties, acidic deprotec-
tion is used to produce the free mixed HOPO ligand 2.
The yield, 73% based on spermine, was also favorable.
Efficient syntheses of these octadentate chelators strongly
indicate that useful amounts of these promising com-
pounds can be prepared at reasonable cost.
Liga n d E ffica cy for P u R em ova l. In ject ed
Liga n d s. Excretion and distribution of retained Pu are
shown in Table 1 for mice treated with the octadentate
HOPO ligands or CaNa3DTPA (30 µmol/kg, ip). In these
trials, 1 was as effective for reducing Pu retention in
the skeleton and soft tissue as in the original mouse
trials and significantly more effective for reducing Pu
in liver and whole body.18,23 Both octadentate HOPO
ligands 1 and 2, injected at 1 h, greatly reduce Pu in
the skeleton and all soft tissues, significantly compared
with Pu-injected controls, and with minor exceptions,
1,2-HOPOBn acid (6) is a versatile compound for
synthesizing multidentate 1,2-HOPO ligands. Com-
pound 6 can be converted to a number of activated
intermediates and then coupled to a variety of amines.
For example, 6 reacts with 2-mercaptothiazoline in the
presence of DCC and DMAP (1,4-(dimethylamino)-
pyridine) to give 1,2-HOPO/thiazolide,29,32 which selec-
tively reacts with aliphatic primary amines, like its 3,2-
HOPO analogue, 3,2-HOPO-thiazolide.33 Compound 6
can also be converted to NHS or other activated esters
for amine coupling. In most cases, the active esters are
not separated but are reacted with amines in situ.29,32
Like 2,3-dibenzyoxybenzoic acid35 and 1-methyl-3-ben-
zyloxy-2(1H)-pyridinone-4carboxylic acid,28 6 can be
converted to the acid chloride, a very active species. The
acid chloride 7 reacts with not only aliphatic primary
and secondary amines but also with aromatic amines.
Selective coupling with a variety of amines can be easily
accomplished by using different activated 1,2-HOPOBn
acid species. Coupling of spermine with 7 yields the

3966 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Xu et al.
Ta ble 1. Removal of 238Pu(IV) from Mice by Injected and Orally Administered Octadentate HOPO Ligands and CaNa3DTPA
percent of injected 238Pu ( SDa
excretab
tissues
kidneys
Ligand Injected at 1 hd,f
feces and GI
contents
ligand
no. of mice
skeleton
liver
soft tissue whole body
urine
3,4,3-LI(1,2-Me-3,2-HOPO)
3,4,3-LI(1,2-HOPO) olde
3,4,3-LI(1,2-HOPO) new
CaNa3DTPA
10
5
20
14
9.1 ( 1.2
7.5 ( 0.7
8.0 ( 1.9
14 ( 1.6
11 ( 2.6
8.9 ( 1.7
5.4 ( 1.6g
19 ( 5.8
0.2 ( 0.1
1.0 ( 0.9
1.6 ( 0.7
22 ( 2.4
18 ( 1.7
14 ( 2.3g
37 ( 5.1
27
25
21( 0.7
55 ( 6.1
51
57
65 ( 2.6
8.4 ( 2.9
0.2b
0.1 ( 0.06 0.8 ( 0.7
1.2 ( 0.9d 2.9 ( 0.6
Ligand Orally at 3 min, Normally Fedd,f
3,4,3-LI(1,2-Me-3,2-HOPO)
3,4,3-LI(1,2-HOPO)
10
10
7.4 ( 2.5
8.8 ( 3.7
2.4 ( 1.7,g
5.9 ( 3.2
0.1 ( 0.06 0.9 ( 0.5
11 ( 4.3
16 ( 5.9
31
27
58
57
0.2 ( 0.1
1.5 ( 0.7
Pu-injected fed controls
35
37 ( 7.6
45 ( 7.2
1.5 ( 05
7.2 ( 1.4
90 ( 1.1
6.5 ( 0.8
3.7 ( 0.6
Ligand Orally at 3 min, Fastedd,f
3,4,3-LI(1,2-Me-3,2-HOPO)
3,4,3-LI(1,2-HOPO) olde
3,4,3-LI(1,2-HOPO) new
CaNa3DTPA
20
5
20
5
7.8 ( 1.3
12 ( 2.4
8.0 ( 2.0
40 ( 6.8d
2.2 ( 1.6g 0.09 ( 0.04 0.8 ( 0.6
11 ( 2.7
24 ( 7.7
14 ( 4.6
79 ( 1.3
28 ( 6.4
26
26 ( 3.2
15
61 ( 6.1
51
60 ( 1.4
6.5
11.0 ( 4.9
4.2 ( 2.7
33 ( 5.4
0.1b
1.3 ( 0.7
0.1 ( 0.05 1.1 ( 0.7
0.7 ( 0.1
5.1 ( 1.3d,
5.4 ( 1.3
Pu-injected fasted controls
20
38 ( 5.2
46 ( 5.0
1.3 ( 0.3
90 ( 1.1
5.6 ( 0.8
4.0 ( 0.4
a
b
Data for each mouse, expressed as %ID, were normalized to 100% material recovery; discrepancies are due to rounding. No SD is
shown for kidney (pooled for each five mouse group in the original 3,4,3-LI-(1,2-HOPO) studies) or excreta of single experiments (five
mice) or experiments replicated only once (10 mice), because the excreta in each cage of five mice were pooled for radioanalysis; SDs were
calculated for excreta in multiple replicated experiments, where n ) one-fifth (number of mice). c Ligands (30 µmol/kg) were injected ip
at 1 h or given orally (gastric tube) at 3 min after iv injection of 238Pu(IV) citrate; kill at 24 h. Means for ligand-treated mice are significantly
d
less than for Pu-injected controls (t-test, p e 0.01),29 except five designated means. e Previously reported.18 f Mean for HOPO ligand-
g
treated mice is significantly less than for mice similarly treated with CaNa3DTPA. Mean is significantly less than for mice given the
different HOPO ligand.
also significantly compared with mice similarly treated
with CaNa3DTPA. Injected 2 reduces skeleton, kidney,
and bulk soft tissue Pu as much as 1, but significantly
more Pu remained in the livers of mice treated with the
mixed ligand (2).
Or a lly Ad m in ister ed Liga n d s. Excretion and dis-
tribution of retained Pu are shown in Table 1 for mice
gastrically intubated with the octadentate HOPO ligands
or CaNa3DTPA (30 µmol/kg, by gastric tube (po) at 3
min to fasting or normally fed mice). In these trials, 1,
given orally, is as effective for reducing kidney and soft
tissue Pu and significantly more effective for reducing
skeleton, liver, and whole body Pu than was observed
in the original animal testing.18,23 Both octadentate
HOPO ligands, given orally, markedly reduce Pu reten-
tion in the skeleton and all soft tissues of fasting mice,
significantly, as compared with fasted Pu-injected con-
trols, and also, significantly, as compared with mice
F igu r e 3. Dose-dependent removal of 238Pu from mice with
3,4,3-LI(1,2-HOPO), 3,4,3-LI(1,2-Me-3,2-HOPO), and CaNa3-
DTPA. Key: sbs, 3,4,3-LI(1,2-Me-3,2-HOPO), ip; ‚‚‚9‚‚‚,
treated orally or injected intraperitoneally (ip) with 30
µmol/kg of CaNa3DTPA (Table 1). Given orally to fasting
mice, 1 is as effective for reducing tissue Pu as the same
3,4,3-LI(1,2-HOPO), ip; sOs, 3,4,3-LI(1,2-Me-3,2-HOPO), oral;
ligand dose injected ip at 1 h. Orally administered 2 is
‚‚‚0‚‚‚ 3,4,3-LI(1,2-HOPO), oral; - ‚ -2- ‚ -, CaNa3DTPA, ip.
as effective in fasting mice for reducing skeleton, kidney,
and soft tissue Pu as the same ligand dose injected at 1
h and significantly more effective for reducing liver Pu.
In contrast to their relative efficacies when injected
(Table 1), 2 given orally to fasting mice is somewhat
more effective overall than 1 and significantly more
effective for reducing liver Pu.
expectations (Table 1). The presence of food in the upper
GI tract somewhat reduced the oral efficacy of 1 but did
not lessen the effectiveness of orally administered 2. The
greater reductions of liver and whole body Pu obtained
with the mixed ligand, as compared with 1, are signifi-
cant at the 95% level of confidence (p ) 0.05).
The great efficacies of the orally administered octa-
dentate HOPO ligands for reducing tissue Pu in fasting
mice (empty upper gastrointestinal (GI) tract) suggested
that those ligands might also remove useful amounts
of Pu, if given orally to normally fed mice (variable mass
of contents in upper GI tract). The efficacies of both
orally administered octadentate HOPO ligands for
reducing tissue Pu in normally fed mice exceeded
Liga n d Dose-Effectiven ess. Figure 3 shows, in
comparison with injected CaNa3DTPA, the efficacies for
reducing total body Pu of 1 and 2, injected ip or
administered orally to fasting mice, over the dose range
of 0.003-100 µmol/kg. Ligand dose-effectiveness is
described by pairs of intersecting straight lines relating
Pu retention (%ID) to the logarithm of the ligand dose
(µmol/kg). Each curve segment for the individual ligands

Chelation of Pu(IV) of a Mixed Octadentate Ligand
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3967
and treatment modes was fitted independently by
regression analysis.26 The remarkable efficacy, as com-
pared with CaNa3DTPA, of low doses of injected 1 has
been reported.18,26,36 Similarly, 2, injected at doses as
low as 0.3 µmol/kg, reduces body Pu significantly more
than the injected standard clinical dose of CaNa3DTPA
(30 µmol/kg). Injected 1 is most efficient (%ID Pu
removed/µmol/kg ligand) at doses e0.4 µmol/kg; larger
doses promote little additional Pu excretion. The mixed
HOPO ligand is most efficient at injected doses e0.1
µmol/kg; larger doses gradually reduce skeletal Pu, but
there is no additional reduction of body Pu. At injected
doses of e0.3 µmol/kg, the mixed HOPO ligand (2) is
significantly more effective for reducing whole body Pu
than 1.
Even though their GI absorption is estimated to be
less than 5%ID,18,27,37 oral doses of the octadentate
HOPO ligands as low as 3 µmol/kg reduce body Pu of
fasting mice as much as or more than 30 µmol/kg of
injected CaNa3DTPA. The mixed HOPO ligand 2 is
somewhat more effective than 1, when given orally at
doses from 0.1 to 100 µmol/kg, but the differences were
statistically significant only for the reduction of liver
Pu.
Liga n d Toxicity. The acute toxicity of injected
multidentate ligands containing 1,2-HOPO or Me-3,2-
HOPO is caused primarily by chemical tubular neph-
rosis, the severity of which depends on both ligand
structure and dose.23,25,34 There was no evidence of
toxicity in mice given 1 in 10 daily 30 µmol/kg injec-
tions26 or in baboons given eight 30 µmol/kg injections
over 26 days.37 In mice given 10 daily 100 µmol/kg
injections, mortality was 2.5%, and the BUN (blood urea
nitrogen) was consistently and significantly elevated.
On day 11, liver weight was elevated and body weight
was depressed (both significant); 13 of 19 kidneys were
moderately nephrotic. By day 21, body and tissue
weights were normal; 10 of 20 kidneys contained foci
and/or radial strands of regenerating tubular epithe-
lium.26
There was no evidence of acute toxicity in mice given
10 daily 30 µmol/kg injections of 2. There were no deaths
among mice given 10 daily 100 µmol/kg injections, and
body, spleen, and kidney weights and the BUN were
consistently within control limits. On day 11, liver
weight was significantly elevated; three of 10 kidneys
were moderately nephrotic, and five were mildly neph-
rotic. On day 21, liver weight was the same as the
controls; five of 10 kidneys contained rare small foci or
radial strands of regenerating tubular epithelium. The
acute toxicity of the mixed ligand appears to be some-
what less than that of 1.
F igu r e 4. Multidentate 1,2-HOPO and Me-3,2-HOPO ligands.
tially the same as those for Ce(IV) complexes with Me-
3,2-HOPO ligands.24 Because the affinities of Me-3,2-
HOPO for Fe(III) and Th(IV) at physiological pH exceed
those of 1,2-HOPO, multidentate Me-3,2-HOPO ligands
were expected to be more effective in vivo Pu chelators
than 1,2-HOPO ligands of the same denticity.22,33 How-
ever, direct comparisons could not be made, because the
backbones of the available 1,2-HOPO and Me-3,2-HOPO
ligands differed.23,33 To allow for direct comparison of
1,2-HOPO and Me-3,2-HOPO ligands, four pairs of
HOPO ligands with identical backbones containing the
different HOPO units were synthesized as shown in
Figure 4.26 The new multidentate 1,2-HOPO ligands
12-15 were prepared with the same backbones as the
four most effective Me-3,2-HOPO ligands 16-19 (Figure
4). Each set included an octadentate ligand with the
H-shaped PENTEN backbone. Compound 1 was syn-
thesized in the revised procedure reported here. Com-
pound 1 and CaNa3DTPA were evaluated in mice for
in vivo Pu chelation, with emphasis on oral activity and
effectiveness at low dose.26 The four Me-3,2-HOPO
ligands were overall more effective than their structural
1,2-HOPO analogues, in agreement with their relative
Discu ssion
Design of th e Octa d en ta te Mixed HOP O Liga n d .
The design of mixed ligands was motivated by the desire
to develop new highly effective chelators by introducing
Me-3,2-HOPO moieties into a multidentate 1,2-HOPO
chelator. Our solution thermodynamic studies reveal
extraordinarily high pM values (≈37) for Ce(IV) com-
plexes with tetradentate Me-3,2-HOPO ligands. With
the same ionic charge and radius, Ce(IV) is generally
accepted as a good model for Pu(IV). The stabilities for
Pu(IV) complexes are therefore expected to be substan-

3968 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Xu et al.
metal binding affinities. However, octadentate 1, with
its linear spermine backbone, was more effective than
either the octadentate H(2,2)-Me-3,2-HOPO (15) or the
octadentate H(2,2)-1,2-HOPO ligand (19) with the H-
shaped branched backbone. Superior in vivo Pu com-
plexation by linear 1 is attributed mainly to the linear
backbone, which appears to be more suitable for eight
coordination as required by Pu(IV).26
Com p a r ison of t h e Mixed H OP O Liga n d w it h
3,4,3-LI(1,2-HOP O). The stability of the Pu(IV) com-
plex with 2 was predicted to be somewhat greater than
that of Pu(IV) with 1. Therefore, the mixed ligand could
be expected to be more effective for in vivo chelation of
Pu, especially in the low dose range. The biological
results agree with that expectation. Compound 2 is more
effective than 1 when injected in mice at doses e0.3
µmol/kg, given orally to fasting mice from 0.3 to 100
µmol/kg, or given orally to normally feeding mice at the
standard dose of 30 µmol/kg. This mixed ligand 2 also
appears to be somewhat less acutely toxic than 1.
Compounds 2 (0.1 µmol/kg injected or 5 µmol/kg orally)
and 1 (0.3 µmol/kg injected or 10 µmol/kg orally) remove
significantly more Pu from mice than 30 µmol/kg of
injected CaNa3DTPA. The effective injected doses of the
two HOPO ligands are about 1% of the standard dose
(30 µmol/kg ip), a dose level that did not induce
detectable acute toxicity in mice when given daily for
10 days. Thus, both HOPO ligands meet the criterion
of low toxicity at doses that are more effective than the
standard dose of CaNa3DTPA. Their great effectiveness
at low dose and great oral effectiveness (despite low GI
absorption) imply that effective treatment regimens can
be developed using the HOPO ligands alone or as
adjuncts to CaNa3DTPA therapy, which will greatly
exceed the amount of Pu excretion that is achievable
with CaNa3DTPA alone.
On the basis of initial evaluation, the effectiveness
for in vivo Pu chelation, in particular oral activity, could
be improved if Me-3,2-HOPO are introduced into the
spermine backbone, thereby incrementally increasing
the stability of the Pu complex and the lipophilicity of
the ligand.34 Because 4-LI(Me-3,2-HOPO) (12) (Figure
4) has poor solubility in water and is toxic in mice, one
would expect 3,4,3-LI(Me-3,2-1,2-HOPO) (3) and 3,4,3-
LI(Me-3,2-HOPO) (4) (Figure 1) be less soluble and
probably toxic, because they contain the 4-LI(Me-3,2-
HOPO) moiety as the central part. On the other hand,
4-LI(1,2-HOPO) (16) (Figure 4) is much more soluble
in water and much less toxic than 4LI(Me-3,2-HOPO).26
The combination of the 4-LI(1,2-HOPO) moiety with Me-
3,2-HOPO moieties, 3,4,3-LI(1,2- Me-3,2-HOPO) (Figure
1, 2), was likely to be superior overall to that of the 4-LI-
(Me-3,2-HOPO) moiety with 1,2-HOPO moieties, 3,4,3-
LI(Me-3,2-1,2-HOPO) (3) (Figure 1). Therefore, the
mixed ligand, 2, was prepared, while mixed ligands 3
and 4 (Figure 1) were not synthesized. In ligand 2, the
Me-3,2-HOPOs are attached by amide linkages to the
two terminal primary amines and 1,2-HOPO is similarly
attached to the two secondary amines of the spermine
scaffold (Figure 1).
Exp er im en ta l Section
Syn th esis. Gen er a l. All chemicals were obtained from
commercial suppliers (Aldrich or Fisher) and were used as
received. 6-Hydroxypicolinic acid was purchased from Fluka.
Me-3,2-HOPOBn-thiazolide (9) was prepared as previously
described.33 Reactions were carried out under an atmosphere
of nitrogen. Flash silica gel chromatography was performed
using Merck 40-70 mesh silica gel. Unless otherwise specified,
all NMR spectra were recorded at ambient temperature on
Bruker DRX 500, AMX 400, or AMX 300 spectrometers in the
University of California, Berkeley, NMR laboratory. HPLC
analyses were performed on a Varian Pro Star System with a
Dynamax-60A C18-reversed phase column using a mobile
phase of 65% methanol in water. Microanalyses were per-
formed by the Microanalytical Services Laboratory, College of
Chemistry, University of California, Berkeley. Mass spectra
were recorded at the Mass Spectrometry Laboratory, College
of Chemistry, University of California, Berkeley.
P r eviou s E xp er ien ce w it h Oct a d en t a t e Mixed
Liga n d s. Several ligands containing different metal
binding units (mixed ligands) have been synthesized in
this laboratory. When ligand denticity was increased,
for example, by addition of various bidentate binding
units to hexadentate desferrioxamine B (DFO B) to
make octadentate ligands, the formation constants (log
Kf) of the respective Th(IV) and Pu(IV) complexes38 and
their abilities to remove Pu from mice20,23 were also
greatly increased. In contrast, when binding units with
greatly differing pH-dependent formation constants for
“hard” metal complexes were combined in a single
macromolecule with denticity held constant, Pu removal
efficacies were usually disappointing. The Pu removal
efficacy of DTPA-DX, a mixed ligand containing two
hydroxamic acid and three amino acetate groups, was
only marginally greater than that of native CaNa3-
DTPA.18 In vivo Pu chelation by TREN-bisacetate-
bisMe-3,2-HOPO, a mixed octadentate ligand containing
two Me-3,2-HOPO and two amino acetate groups,33 or
by 3,4,3-LI(diCAM-diHOPO), a mixed octadentate ligand
containing two 1,2-HOPO and two catecholamide (CAM)
groups,39 was significantly less than that of the struc-
tural analogues containing only HOPO units. Clearly,
the contributions of all of the individual binding units
to the overall pH-dependent formation constant of the
Pu(IV) complex must be taken into account. An increase
in the overall formation constant and improved ef-
fectiveness for Pu chelation can be achieved only by
replacing an effective binding group (e.g., 1,2-HOPO)
with a more effective unit (e.g., Me-3,2-HOPO).
6-Ca r boxy-1-h yd r oxy-2(1H)-p yr id in on e (5). Acetic an-
hydride (100 mL) was mixed with 30% H2O2 solution (25 mL)
in an ice bath. The mixture was stirred for 4 h until a
homogeneous peracetic acid solution formed. This peracetic
acid solution was added slowly with stirring to a solution of
6-hydroxy-picolinic acid (Fluka, 25.0 g, 0.18 mol) in a mixture
of trifluoroacetic acid (150 mL) and glacial acetic acid (100 mL).
(CAUTION! Any solid particles in the mixture will cause
vigorous oxygen evolution and lead to an uncontrolled reac-
tion.) The mixture was stirred at room temperature for 1 h
and then heated slowly to 80 °C and kept at 80 °C for 10 h. A
white precipitate formed during this period, which was col-
lected by filtration, washed with cold methanol, and dried. It
was dissolved in aqueous 10% KOH, heated to 80 °C for 6 h,
and reprecipitated with concentrated HCl. The product was
collected by filtration, washed with water, and dried in a
vacuum oven to yield 20.5 g (0.132 mol, 73%); mp 176-177
°C. Anal. Calcd (Found) for C6H5NO4: C, 46.46 (46.31); H, 3.25
1
(3.45); N, 9.03 (9.12). H NMR (500 MHz, DMSO-d6): δ 6.63
(dd, J ) 7.0, 1.5 Hz, 1H), 6.71 (dd, J ) 9.2, 1.5 Hz, 1H), 7.43
(dd, J ) 9.0, 7.1 Hz, 1H). 13C NMR (125 MHz, DMSO-d6): δ
106.9, 120.5, 134.9, 137.3, 157.4, 163.3. IR (KBr pellet): ν 1734
cm-1 (br, CdO); 1616 cm-1 (m, CdO).

Chelation of Pu(IV) of a Mixed Octadentate Ligand
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3969
1-Ben zyloxy-6-ca r b oxy-2(1H)-p yr id in on e (6).29 Com-
pound 5 (15.50 g, 0.10 mol) and anhydrous potassium carbon-
ate (27.60 g, 0.20 mol) were mixed with benzyl chloride (15.20
g, 0.12 mol) in methanol (250 mL). The mixture was refluxed
for 16 h and filtered, and the filtrate was evaporated to
dryness. The residue was dissolved in water (50 mL) and
acidified with 6 N HCl to pH 2. The white precipitate was
isolated by filtration, washed with cold water, and dried in
vacuo to yield 22.3 g (91%) of compound 6; mp 176-177 °C.
Anal. Calcd (Found) for C13H11NO4: C, 63.66 (63.75); H, 4.53
night and then washed with 1 M KOH solution (30 mL × 3).
The organic phase was then dried in vacuo to afford a pale
brown oil as raw product (yield 85%). 1H NMR (300 MHz,
CDCl3): δ 1.43 (m,br, 4H), 1.50 (qint, J ) 6.7 Hz, 4H), 2.44 (t,
J ) 6.7 Hz, 8H), 3.25 (q, J ) 6.7 Hz, 4H), 3.57 (s, 6H), 5.34 (s,
4H, OCH2), 6.72 (d, J ) 7.2 Hz, 2H), 7.10 (d, J ) 7.2 Hz, 2H),
7.21-7.55 (m, 10H), 8.09 (t, J ) 5.3 Hz, 2H). 13C NMR (125
MHz, CDCl3): δ 27.2, 28.7, 37.4, 37.6, 46.8, 49.2, 74.5, 104.6,
128.5, 128.6, 128.8, 130.7, 132.0, 136.1, 146.0, 159.4, 163.1.
MS (FAB+): 685.3 (MH+).
1
(4.55); N, 5.71 (5.52). H NMR (500 MHz, DMSO-d6): δ 5.26
3,4,3-LI(1,2-Me-3,2-HOP O)Bn (11). The raw 3,4,3-LI-Bis-
(Me-3,2-HOPO Bn) (10) (0.82 g, 1.2 mmol) was dissolved in
dry THF (50 mL) containing triethylamine (1.2 mL) and slowly
added to a solution of raw 1,2-HOPOBn acid chloride (1.7 g,
6.4 mmol) in dry THF (60 mL) over 4 h while stirring. The
reaction mixture was heated at 60 °C overnight. After the
solvents were removed, the residue was partitioned into a
mixture of water (50 mL) and dichloromethane (50 mL). The
organic phase was washed successively with 1 M NaOH (100
mL), 1 M HCl (100 mL), and saline water (100 mL) and loaded
onto a flash silica column. Elution with 3-8% methanol in
dichloromethane allowed the separation of the benzyl-protect-
(s, 2H, CH2), 6.54 (dd, J ) 6.7, 1.1 Hz, 1 H), 6.73 (dd, J ) 9.2,
1.6 Hz, 1 H), 7.39-7.51 (m, 6 H). 13C NMR (125 MHz, DMSO-
d6): δ 77.9, 106.0, 124.1, 128.5, 129.1, 129.6, 133.8, 138.7,
140.4, 157.6, 161.7.
1,2-HOP OBn Acid Ch lor id e (7). To a suspension of 1,2-
HOPOBn acid (5.0 g, 20 mmol) in toluene or benzene (50-70
mL), excess oxalyl chloride (5.0 g) was added while stirring.
Gas bubbles evolved, and the suspension became clear upon
the addition of a drop of dimethyl formamide (DMF) as a
catalyst. The mixture was then warmed to 60 °C for 4 h, and
the solvent was removed by rotary evaporation to leave a pale
brown oil. After it was coevaporated twice with toluene (5 mL),
the residue was dissolved in dry tetrahydrofuran (THF),
passed through a silica gel plug, and eluted with dry THF.
The 1,2-HOPOBn acid chloride was obtained as a thick pale
yellow oil after the solvent was removed under reduced
pressure with a raw yield of 5.0 g (95%). It was used directly
for the next reaction without further purification. 1H NMR (300
MHz, CDCl3): δ 5.32 (s, 2H, CH2), 6.88 (d, J ) 7.0 Hz, 1 H),
6.726 (d, J ) 9.0 Hz, 1 H), 7.32-7.51 (m, 6 H). 13C NMR (125
MHz, DMSO-d6): δ 78.5, 112.2, 128.5, 128.6, 129.4, 130.3,
132.7, 136.4, 140.1, 158.1, 158.8.
1
ed precursor as white foam (yield 75%). H NMR (500 MHz,
CDCl3): δ 0.91-1.64 (m, 16H), 2.65-3.45 (m, 24H), 3.54 (s,
6H), 4.90-5.02 (m, 4H), 5.20-5.6 (m, 8H), 5.80-6.10 (m, 4H),
6.54-6.80 (m, 8H), 7.07-7.11 (m, 4H), 7.15-7.51 (m, 20H),
7.75 (m, br, 2H), 7.99 (m,br, 1H). 13C NMR (125 MHz, CDCl3):
δ 24.9, 26.3, 26.6, 27.6, 36.4, 36.6, 37.3, 41.8, 43.6, 45.6, 47.8,
74.3, 74.4, 74.7, 74.8, 78.8, 79.0, 104.1, 104.2, 104.3, 104.4,
122.5, 128.1, 128.2, 128.4, 128.5, 128.7, 129.8, 129.9, 131.9,
133.1, 142.8, 157.8 157.9, 159.1, 159.2, 161.4, 163.0, 163.12.
MS (FAB+): 1139.8.
3,4,3-LI(1,2-Me-3,2-HOP O) (2). The benzyl-protected 11
was deprotected in the same manner as 3,4,3-LI(1,2-HOPO).
The raw product was dissolved in a minimum amount of
distilled water, pure 3,4,3-LI(1,2-Me-3,2-HOPO) was precipi-
tated as beige solid upon cooling, and it was filtered and dried
under vacuum (yield 73% based on spermine). Analytical
purity (g99%) was determined by HPLC (Dynamax RP-C18,
15 cm × 4.6 mm ID, 8 µm particles, mobile phase 65% MeOH/
35% H2O). 1H NMR (500 MHz, DMSO-d6): δ 1.21-1.85 (m,
16H), 2.85-3.35 (m, 24H), 3.45 (s, 6H), 6.10-6.52 (m, 6H),
7.10-7.41 (m, 4H), 8.32 (m, 1H), 8.48 (m, 1H). 13C NMR (125
MHz, D2O/NaOD): δ 23.8, 24.0, 24.1, 24.5, 24.8, 25.0, 26.8,
27.0, 28.1, 28.3, 35.6, 35.7, 36.4, 36.5, 37.5, 42.9, 43.0, 43.1,
44.7, 44.9, 45.1, 46.7, 48.3, 48.4, 48.6, 105.7, 106.1, 106.2, 106.4,
106.9, 107.1, 114.8, 114.9, 115.0, 115.8, 116.1, 119.6, 119.9,
132.7, 132.8, 133.0, 142.8, 143.1, 160.3, 161.9, 162.2, 164.4,
164.4, 165.6, 165.7, 169.4, 169.6. MS (FAB+): 779.4 (MH+).
Anal. for C36H42N8O12‚3H2O‚2HCl (905.76), Calcd (Found): C,
47.74 (48.05); H, 5.56 (5.22); N, 12.37 (12.16).
Biologica l Eva lu a tion . Liga n d Effica cy. Under anes-
thesia, groups of five female Swiss-Webster mice (87 ( 9 days
old, 34 ( 2 g)40 were injected intravenously (iv) in a warmed
lateral tail vein with 0.2 mL of a solution containing 925 Bq
(0.025 µCi) of 238Pu(IV) in 0.008 M sodium citrate plus 0.14 M
NaCl, pH 4. Solutions of the HOPO 1igands (0.002 M) were
prepared in 0.14 M NaCl, pH 7.4. CaNa3DTPA was obtained
as a 25% solution. The standard ligand dose (30 µmol/kg) and
dilutions for dose-effectiveness studies were delivered to a 35
g mouse in 0.5 mL of ligand solution. Ligand-to-metal molar
ratio at standard ligand dose was 2 × 105 for 238Pu. Ligand
doses were standardized by adjusting the injected volume to
the body weight of each mouse.
3,4,3-LI(1,2-HOP O)Bn (8). To a solution of crude 1,2-
HOPOBn acid chloride (7) (5.0 g, 19 mmol) and triethylamine
(2.5 mL) in dry THF (60 mL), spermine (0.8 g, 4 mmol) was
added in three portions while stirring. The mixture was heated
to 60 °C (oil bath temperature) in a stoppered 100 mL round
flask overnight. The solvent was then removed with a rotary
evaporator, and the residue was partitioned into a mixture of
water (50 mL) and dichloromethane (50 mL). The organic
phase was then washed successively with 1 M NaOH (100 mL),
1 M HCL (100 mL), and saline solution (100 mL) and loaded
onto a flash silica column. Elution with 2-6% methanol in
dichloromethane allowed separation of the benzyl-protected
precursor 3,4,3-LI(1,2-HOPOBn) as white foam (yield 70%).
1H NMR (500 MHz, CDCl3): δ 0.40-1.85 (m, 16H), 2.81-3.66
(m, 24H), 4.81-5.13 (m, 2H), 4.88-5.05 (m, 2H), 5.15-5.30
(m, 4H), 5.30-5.45 (m, 2H), 6.00-6.46 (m, 4H), 6.55-6.70 (m,
4H), 7.25-7.55 (m, 24H), 8.72-8.95 (m, 2H, NH). 13C NMR
(125 MHz, DMSO-d6): δ 23.4, 24.2, 24.5, 24.6, 26.4, 26.6, 26.7,
27.3, 27.7, 36.6, 36.7, 41.8, 42.0, 42.4, 43.7, 46.2, 47.4, 47.7,
48.1, 79.2, 102.7, 104.7, 123.1, 128.3, 128.4, 128.7, 129.2, 129.3,
129.4, 130.0, 130.1, 130.2, 130.3, 130.4, 132.8, 132.9, 133.0,
133.1, 138.2, 142.0, 142.5, 142.6, 143.3, 157.9, 158.1, 158.3,
160.4, 160.6, 161.2, 161.3. MS (FAB+): 1111.5 (MH+).
3,4,3-LI(1,2-HOP O) (1). The precursor 8 was deprotected
at room temperature with 1:1 HCl (37%)/glacial HOAc for 4
days. All of the volatiles were removed in vacuo. The residue
was dissolved in a minimum amount of water, filtered, and
evaporated to dryness (yield 81%). 1H NMR (400 MHz, DMSO-
d6): δ 0.25-1.87 (m, 8H), 2.81-3.63 (m, 24H), 6.11-6.22 (m,
3H), 6.29-6.34 (m, 2H), 6.48-6.58 (m, 4H), 7.31-7.42 (m, 4H),
8.82 (q, J ) 7.2 Hz,1H), 8.91 (q, J ) 7.2 Hz, 1H). 13C NMR
(125 MHz, D2O): δ 23.1, 23.7, 24.2, 25.4, 26.7, 36.6, 36.8, 41.9,
44.0, 45.8, 47.7, 48.1, 106.2, 106.5, 108.7, 109.0, 118.9, 120.1,
138.8, 139.0, 139.6, 140.0, 140.5, 159.2, 161.1, 161.2, 162.2.
MS (FAB+): 751 (MH+). Anal. for C34H38N8O12‚H2O‚2HCl (FW
) 841.68), Calcd (Found): C, 48.52 (48.16); H, 5.03 (4.82); N,
13.31 (13.23).
In parenteral injection studies, ligands were injected ip at
1 h after iv injection of Pu, and the mice were killed at 24 h.
In oral studies, ligands were administered po 3 min after iv
injection of Pu to mice previously fasted for 16 h or to normally
fed mice, and the mice were killed at 24 h. In studies of dose-
effectiveness for Pu removal, Pu-injected mice were injected
ip at 1 h or intubated orally at 3 min (mice fasted 16 h) with
ligand at doses ranging from 0.001 to 100 µmol/kg, achieving
ligand:Pu molar ratios from 7.0 to 7 × 105, and the mice were
killed at 24 h. Groups of five normally fed or fasted Pu-injected
3,4,3-LI-Bis(Me-3,2-HOP OBn ) (10). To a solution of sper-
mine (0.50 g, 2.5 mmol) in dry dichloromethane (60 mL), Me-
3,2-HOPO-thiazolide (9) (2.0 g, 5.5 mmol) was added while
stirring. The mixture was stirred at room temperature over-

3970 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Xu et al.
(13) Bruenger, F. W.; Neilson, E. R.; Stevens, W. In Vitro Test System
for Evaluating the Effectiveness of Chelators. In Research in
Radiobiology; J ee, W. S. S., Ed.; University of Utah College of
Medicine: Salt Lake City, 1976; Report C00-119-251, pp 203-
212.
(14) Durbin, P. W.; J ones, E. S.; Raymond, K. N.; Weitl, F. L. Removal
of 238Pu(IV) from Mice by Sulfonated Tetrameric Catechoyl
Amides. Radiat. Res. 1980, 81, 170-187.
(15) Shannon, R. D. Revised Effective Ionic Radii and Systematic
Studies of Interatomic Distances in Halides and Chalcogenides.
Acta Crystallogr. 1976, A32, 751-767.
(16) Cotton, F. A.; Wilkinson, G. Advanced Inorganic Chemistry 4th
ed.; J ohn Wiley: New York, 1980; pp 61-106.
mice were given 0.5 mL of 0.14 M NaCl ip or orally and killed
at 24 h to define the control distribution and excretion of Pu.
Each five mouse experimental or control group was housed
together in a plastic cage lined with a 0.5 cm layer of highly
absorbent low ash pelleted cellulose bedding. Water was
available ad lib, and mouse chow was given 4 h after the Pu
injection. The combined control groups were representative of
the several Pu solutions used and the several shipments of
mice. The replicated Pu removal studies are, in addition,
representative of the several synthetic batches of the ligands.
Details of autopsy procedures, tissue and excreta collection
and processing, and R particle detection (liquid scintillation
counting, Packard Tri-Carb model B4430), and methods of data
managenent have been previously reported.14,17,26,33 The Pu
distribution data are expressed as mean ( SD percent of
(17) Durbin, P. W.; J eung, N.; J ones, E. S.; Weitl, F. L.; Raymond,
K. N. Enhancement of 238Pu Elimination from Mice by Poly-
(catechoylamide) Ligands. Radiat. Res. 1984, 99, 85-105.
(18) Durbin, P. W.; J eung, N.; Rogers, S. J .; Turowski, P. N.; Weitl,
F. L.; White, D. L.; Raymond, K. N. Removal of 238Pu(IV) from
Mice by Polycatecholate, Hydroxamate or Hydroxypyridinonate
Ligands. Radiat. Prot. Dosim. 1989, 26, 351-358.
injected Pu (%ID) in individual tissues and the whole body
1/2 41
(sum of tissues), where SD ) [∑dev2(n - 1)-1
]
.
For tissue
and body Pu, n is the number of mice in the group. Excreta
were pooled for each five mouse group, and SD was calculated
for excreta only for experiments and control groups replicated
more than twice. “Significant” is used throughout in the
statistical sense (Student’s t-test, p ‚ 0.01).41 That criterion of
statistical significance is sufficiently conservative that ap-
plication of the Bonferroni correction42 was not needed for
comparing multiple individual test groups to the same group
of controls.
(19) Durbin, P. W.; White, D. L.; J eung, N.; Weitl, F. L.; Uhlir, L. C.;
J ones, E. S.; Bruenger, F. W.; Raymond, K. N. Chelation of 238
-
Pu(IV) In Vivo by 3,4,3-LICAM(C): Effects of Ligand Methyla-
tion and pH. Health Phys. 1989, 56, 839-855.
(20) Durbin, P. W.; Kullgren, B.; J eung, N.; Xu, J .; Rodgers, S. J .;
Raymond, K. N. Octadentate Catecholamide Ligands for Pu(IV)
Based on Linear or Preorganized Molecular Backbones. Hum.
Exp. Toxicol. 1996, 15, 352-360.
(21) Kappel, M. J .; Nitsche, H.; Raymond, K. N. Complexation of
Plutonium and Americium by Catechoylate Ligands. Inorg.
Chem. 1985, 24, 605-611.
(22) Scarrow, R. C.; Riley, P. E.; Abu-Darij, K.; White, D. L.;
Raymond, K. N. Synthesis, Structures and Thermodynamics of
Complexation of Cobalt(III) and Iron(III) Tris Complexes of
Several Chelating Hydroxypyridinones. Inorg. Chem. 1985, 24,
954-967.
(23) White, D. L.; Durbin, P. W.; J eung, N.; Raymond, K. N. Synthesis
and Initial Biological Testing of Polydentate Oxohydroxy-pyri-
dine-carboxylate Ligands. J . Med. Chem. 1988, 31, 11-18.
(24) Xu, J .; Radkov, E.; Ziegler, M.; Raymond, K. N. Plutonium(IV)
Sequestration: Structural and Thermodynamic Evaluation of
the Extraordinarily Stable Cerium(IV) Hydroxypyridinonate
Complexes. Inorg. Chem. 2000, 39, 4156-4164.
(25) Raymond, K. N.; Xu, J . Siderophore-based Hydroxypyridonate
Sequestering Agents. In The Development of Iron Chelators for
Clinical Use; Bergeron, R. J ., Brittenham, G. M., Eds.; CRC
Press: Boca Raton, FL, 1994; pp 307-327.
(26) Durbin, P. W.; Kullgren, B.; Xu, J .; Raymond, K. N. Multidentate
Hydroxypyridinonate Ligands for Pu(IV) Chelation In Vivo;
Comparative Efficacy and Toxicity in Mouse of Ligands Contain-
ing 1,2-HOPO or Me-3,2-HOPO. Int. J . Radiat. Biol. 2000, 76,
199-214.
(27) Stradling, G. N. Recent Progress in Decorporation of Plutonium,
Americium and Thorium. Radiat. Prot. Dosim. 1994, 53, 297-
304.
(28) Xu, J .; Whisenhunt, D. W., J r.; Veeck, A. C.; Raymond, K. N.
Structural and Thermodynamic Evaluation of A Thorium(IV)
Hydroxpyridinonate Complex. Manuscript to be submitted to
Inorg. Chem.
Acu te Liga n d Toxicity. The acute toxicity of 1 in mice
has been reported.26,34 Using the reported methods, 20 mice
were injected subcutaneously daily for l0 days with 30 or 100
µmol/kg of 2. Toxicity was assessed from the combined data,
and observations were obtained for 10 mice each on days 11
and 21: survival; body, liver, spleen, and kidney weight; BUN;
gross pathology of internal organs; microscopic pathology of
liver, kidneys, and injection site.
Ack n ow led gm en t. This research was supported by
the Director, Office of Energy Research, Office of Basic
Energy Science, Chemical Sciences Division, U.S. De-
partment of Energy under Contract No. DE-AC-03-
76F00098 and a grant from the National Institute of
Environmental Health Science ES.-02698. We thank
Emily Dertz for helpful discussions.
Refer en ces
(1) Kornberg, H. A., Norwood, W. D., Eds. Diagnosis and Treatment
of Deposited Radionuclides; Excerpta Medica: Amsterdam, 1967.
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