Synthesis and biological characteristics of the technetium-99m triamide derivatives of mercaptoacetyltriglycine (MAG3)

Article abstract of DOI:10.1002/jlcr.645

A number of MAG₃-derivatives, containing the three-amide functions modified by inverting the sequence -CO-NH- to -NH-CO- in the first-and/or second-amide functions, have been labelled with ^99mTc in order to study the renal characteristics of the resulting MAG₃-derivatives versus the reference ^99mTc-MAG₃. The ^99mTc-MAG₃-derivatives displayed HPLC-profiles similar to that of ^99mTc-MAG₃. Furthermore, in mice they exhibited biological behaviour comparable or even superior to ^99mTc-MAG₃, which indicates that the sequence of the first- and/or second-amide bond in ^99mTc-MAG₃ affects the biological behaviour only to a limited degree. However, in baboon the plasma clearance of the ^99mTc-MAG₃-derivatives was relatively slower than ^99mTc-MAG₃, which underlines the inter-species variability. Copyright

Full text of DOI:10.1002/jlcr.645

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 73–84.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.645
Research Article
Synthesis and biological characteristics of
the technetium-99m triamide derivatives of
mercaptoacetyltriglycine ðMAG3Þ
S. M. Okarvi*, P. Adriaens, and A. M. Verbruggen
Laboratory of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical
Sciences, University Hospital Gasthuisberg, K.U. Leuven, B-3000 Leuven,
Belgium
Summary
A number of MAG3-derivatives, containing the three-amide functions
modified by inverting the sequence –CO–NH– to –NH–CO– in the first-
and/or second-amide functions, have been labelled with 99mTc in order to study
the renal characteristics of the resulting MAG3-derivatives versus the reference
99mTc-MAG3. The 99mTc-MAG3-derivatives displayed HPLC-profiles similar
to that of 99mTc-MAG3. Furthermore, in mice they exhibited biological
behaviour comparable or even superior to 99mTc-MAG3, which indicates that
the sequence of the first- and/or second-amide bond in 99mTc-MAG3 affects the
biological behaviour only to a limited degree. However, in baboon the plasma
clearance of the 99mTc-MAG3-derivatives was relatively slower than
99mTc-MAG3, which underlines the inter-species variability. Copyright #
2002 John Wiley & Sons, Ltd.
Key Words: Technetium-99m; MAG3; renal agents; radiolabelling
Introduction
The introduction of the technetium-99m complex of mercaptoacetyl-
triglycine ð99mTc-MAG3Þ into clinical use as a potential replacement of
*Correspondence to: S. M. Okarvi, King Faisal Specialist Hospital & Research Centre, Cyclotron
and Radiopharmaceuticals Department, MBC-03, PO Box 3354, Riyadh 11211, Saudi Arabia.
E-mail: sokarvi@kfshrc.edu.sa
Copyright # 2002 John Wiley & Sons, Ltd.
Received 10 April 2002
Revised 24 June 2002
Accepted 24 July 2002

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S. M. OKARVI ET AL.
123=131I-labelled-Hippuran (OIH) has opened up a new era in the field of
renal radiopharmaceuticals.1 99mTc-MAG3 has played an unchallenged
role as a radiopharmaceutical of choice for dynamic kidney function
studies in routine clinical use. However, the wide acceptance of
99mTc-MAG3 in clinical nuclear medicine is mainly due to the excellent
scintigraphic imaging qualities of 99mTc rather than its biological
properties.2; 3 Since, compared to 131I-OIH, 99mTc-MAG3 displays lower
extraction efficiency, a significantly higher protein binding, a smaller
volume of distribution and consequently slower plasma clearance
(about 60–65% of OIH clearance),225 efforts have continued to
develop a 99mTc-labelled renal agent with a more comparable
biological behaviour to 131I-OIH. As a result of these efforts, many
derivatives of MAG3 with a slight structural modification have been
prepared and evaluated in animals and humans.6212 These efforts have
made clear that subtle changes in the structure of MAG3 can have
profound effects on the routes and rates of excretion of 99mTc-MAG3-
like agents.3
Design of an improved 99mTc-labelled renal agent requires a
systematic identification of the physical properties which are directly
associated with optimal tubular transport. For a long time it was
believed that the carbonylglycine moiety was required for recognition by
the tubular proteins.13 However, it has now become evident that the
interaction of 99mTc-MAG3-like compounds with the tubular transport
system is not determined by only one part of the molecule, but is
dependent on several important factors such as the sequences of the
different functionalities, the presence of a free carboxylate group and its
relationship to the metal-oxo-group, the distribution of polar and
less polar structural moieties and partial charges.14–17 In view of
these findings, it seemed of interest to prepare the derivatives of
MAG3 in which the three-amide functions are still present, but
modified by an inversion of the –CO–NH– sequence to the –NH–CO–
in order to investigate the renal characteristics of their 99mTc-labelled
derivatives.
99mTc-MAG3 is a mercaptoacetyltripeptide complex and contains
three-amide bonds. However, in this study we investigated the renal
characteristics of MAG3-derivatives with modifications to the first and
second amide but not the terminal amide bond (as the terminal
carbonylglycine sequence is assumed to be essential for maintaining its
efficient renal handling characteristics and hence is the subject of a
separate study9). In this way, three different MAG3-derivatives
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SYNTHESIS AND BIOLOGICAL CHARACTERISTICS
75
Figure 1. Structure of MAG3 and its studied triamide derivatives (shown without
an S-benzyl protective group)
(5, 13 and 18, Figure 1) are possible in which the first or second or both
amide functions are inverted compared to MAG3. These novel ligands
were labelled with 99mTc by an exchange method and analysed by
reversed-phase HPLC. The biological behaviour of these complexes was
studied in mice and a baboon. This paper describes the chemistry,
labelling and biological characteristics of the MAG3-derivatives.
Results and discussion
Chemistry/synthesis
The chemistry necessary for the synthesis of the MAG3-derivatives was
mainly based on standard peptide chemistry techniques.18; 19 Due to the
poor chemical stability of the thiol-group, MAG3 and its derivatives
were synthesized as the thiol-protected precursors, the S-benzyl
protecting group being split off during chelation with 99mTc at elevated
temperature.20 Since all the derivatives (5, 13 and 18) have an oxamide
moiety with different substituents on the nitrogen atoms, ethyloxalyl
chloride (Schemes 1 and 2) and oxalylglycine ethyl ester (Schemes 2 and
3) were used for the introduction of the oxamide moieties. The ethyl
ester protection was selectively removed in the last step of the synthesis
by alkaline hydrolysis without affecting the integrity of the molecules.
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S. M. OKARVI ET AL.
Scheme 1. Synthesis of MAG3 derivative 5
The overall yield of the preparation of different derivatives of MAG3
was rather low (see chemistry section) because many steps were required
for the synthesis of these compounds, which often required extensive
purification by recrystallization and/or by column chromatography.
However, a sufficient amount of each ligand was obtained, permitting
identification and confirmation of the structure, radiolabelling and
animal experiments.
Labelling with 99mTc
The 99mTc complexes of the MAG3-derivatives were obtained by the
exchange labelling method in the presence of stannous tartrate at
different pH values (pH 10–12). Since the deprotonation of the amide
nitrogens and removal of the S-protecting group in MAG3-like agents is
facilitated under alkaline conditions, a relatively high pH (10 or above)
for the labelling reaction mixtures was preferred in order to maximize
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SYNTHESIS AND BIOLOGICAL CHARACTERISTICS
77
Scheme 2. Synthesis of MAG3 derivative 13
the labelling yield as well as to minimize the formation of side
products.20 Radio-HPLC analysis of 99mTc-MAG3-derivatives revealed
that labelling with 99mTc resulted in the formation of one radiochemical
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S. M. OKARVI ET AL.
Scheme 3. Synthesis of MAG3 derivative 18
species ð> 95%Þ with similar HPLC-profiles and comparable
retention times to that of the parent 99mTc-MAG3, except for
99mTc-13, which showed a slightly shorter retention time (about 13
min versus 15 min). The comparable retention times of the
99mTc-MAG3-derivatives indicate that the lipophilicity has not been
drastically changed by the inversion of one- or two-amide functions in
the MAG3 structure.
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SYNTHESIS AND BIOLOGICAL CHARACTERISTICS
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Biodistribution in mice
The results of the biodistribution study of 99mTc-MAG3 and its
derivatives are summarized in Table 1. The differences in biological
behaviour between 99mTc-MAG3 and its derivatives (99mTc-5, 99mTc-13,
and 99mTc-18) are rather limited. All three complexes showed an
efficient clearance from the blood, a rapid excretion to the urine, a low
retention in the liver and kidneys and negligible radioactivity in the
intestines, except for 99mTc-5, which showed a slightly higher uptake in
intestines (7% versus 4% for 99mTc-MAG3 at 30 min post-injection).
On the basis of these results, it seems that 99mTc-13 has the most
favourable properties as a renal function agent. At both studied times
radioactivity in the urine was the highest (up to 92%) and radioactivity
in the liver and intestines was clearly lower than that of the parent
compound (Table 1). As mentioned above, the 99mTc-13 showed a
slightly shorter retention time on RP-HPLC and thus relatively higher
polarity. Nonetheless, it has been shown that the polar characteristics of
99mTc-compounds are one of the factors, but certainly not the most
important one, for determining the renal handling of the
compounds.8; 11 The results seem to suggest that an arrangement of
the amides as in 99mTc-13 is more suitable in 99mTc-MAG3-like
compounds for an efficient handling by the tubular cells. In this
context, it is surprising to see that the inversion of the first amide in
99mTc-13 (resulting in 99mTc-18) clearly retarded the clearance from the
blood and subsequent excretion to the urine. It is not clear at this point
whether this was due to the slightly higher lipophilicity of this
compound or to a less efficient fit with the tubular receptor system, or
a combination of both factors. From these results it appears that the
Table 1. Biodistribution in mice ðn ¼ 5Þ of 99mTc-MAG3 and its derivatives at 10
and 30 min post-injection
% of injected dose in the organs
Urinary system Kidneys
Liver
Intestines
Blood
10 min 30 min 10 min30 min10 min30 min10 min30 min10 min30 min
99mTc-MAG3 76
89
7
2
4
2
2
4
2
1
99mTc-5
99mTc-13
99mTc-18
78
82
71
84
92
86
5
4
5
1
1
1
5
1
2
2
1
1
2
1
1
7
1
1
2
2
5
1
1
3
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S. M. OKARVI ET AL.
specific position of the amide-carbonyls in 99mTc-MAG3 is important
for an efficient fit of the tracer agent with tubular transport proteins and
cannot be modified without impairing the interaction at this site.
Biological evaluation in a baboon
The 1-h plasma clearance of the 99mTc-MAG3-derivatives is presented in
Table 2. The results are expressed as a percentage of the relative
clearance of 131I-OIH, which was co-injected as an internal biological
standard in each study to permit an inter-study comparison. From these
results, it is clear that only 99mTc-5 is extracted from the plasma as
efficiently as 99mTc-MAG3, whereas the disappearance rates were found
to be much slower for the derivatives (99mTc-13 and 99mTc-18) (Table 2).
Materials and methods
Commercially available chemicals were of reagent grade and were used
without purification except for S-benzylmercaptoacetic acid, which was
recrystallized from ethyl acetate. TLC analysis with different solvent
mixtures was carried out using precoated-TLC silica gel plates (Merck,
60F 254) to verify the purity of the products. Column chromatography
was run on a silica gel 60 A (Merck 230–400 mesh). All melting points
.
were determined on a Buchi–Tottoli apparatus. The structures of the
synthesized ligands were confirmed by 1H NMR spectroscopy on a Jeol
FX 90Q spectrometer (Joel, Japan) using CDCl3, DMSO-d6 or D2O as
solvents. The intermediates and final products were dried in a vacuum
desiccator over phosphorus pentoxide. Animal experiments were
performed according to the Belgian code of practice for the care and
use of experimental animals.
Table 2. One-hour plasma clearance values in a baboon for 99mTc-MAG3 and its
triamide derivatives
Derivative
1 h plasma clearancea
99mTc-MAG3
99mTc-5
99mTc-13
99mTc-18
51
54
23
9
a Expressed as percentage of the clearance of co-injected 131I-OIH.
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SYNTHESIS AND BIOLOGICAL CHARACTERISTICS
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Chemistry
Detailed syntheses of the triamide derivatives are reported elsewhere,21
only the synthetic schemes are described here. Compound names do not
necessarily follow IUPAC system.
S-Benzylcysteamido-oxalyl-glycylglycine 5
The synthesis of compound 5 is outlined in Scheme 1. S-benzylcystea-
mine hydrochloride 1 (prepared from cysteamine hydrochloride
dissolved in liquid ammonia and benzyl chloride) was reacted with
ethyloxalyl chloride in the presence of diisopropylethylamine (DIPEA)
to afford 2, which was saponified to oxalyl-S-benzylcysteamine 3.
Condensation of this compound with glycylglycine ethyl ester hydro-
chloride in the presence of 1-hydroxybenzotriazole (HOBT)/1; 3-
dicyclohexylcarbodiimide (DCC) and triethylamine ðEt3NÞ resulted in
the formation of ester 4, which was hydrolyzed to the corresponding
acid 5 in an overall yield of 5%.
S-Benzylmercaptoacetyl-ethylenediamido-N-oxalylglycine 13
Preparation of the compound 13 is shown in Scheme 2. Condensation of
benzyl-mercaptoacetic acid and tert-butyloxycarbonyl-ethylenediamine
(BOC-EDA) 6 (obtained by the reaction of ethylenediamine and di-tert-
butyldicarbonate in 1:9 molar ratios in Et3N) in the presence of HOBT/
DCC provided compound 7, which was deprotected by HBr/HOAc to
give the hydrobromide 8. Reaction of this compound with ethyloxalyl
chloride resulted in the formation of ethyl ester derivative 9, which after
alkaline hydrolysis gave the corresponding acid 10. It was then coupled
with glycine ethyl ester hydrochloride to afford 12. The same compound
12 was obtained by the treatment of the intermediate 8 with
oxalylglycine ethyl ester 11 (obtained by the reaction of equimolar
amounts of tert-butyl alcohol, oxalyl chloride and glycine ethyl ester
hydrochloride in the presence of DIPEA followed by the removal of
tert-butyl group with TFA) in the presence of HOBT/DCC and Et3N.
Alkaline hydrolysis of 12 resulted in the formation of the desired
derivative 13. The overall yield for this preparation was about 9%.
S-Benzyl-N-(glycinamidooxalylglycyl)cysteamine 18
Compound 18 was prepared according to Scheme 3. The intermediate
N-carbobenzyloxyglycyl-S-benzylcysteamine 14 was produced by the
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reaction of N-carbobenzyloxyglycine (CBZ-Gly) with S-benzylcystea-
mine hydrochloride 1. Deprotection of the CBZ-group with HBr/HOAc
provided 15, which was condensed with N-hydroxysuccinimidyl
oxalylglycine ethyl ester 16 (prepared from oxalylglycine ethyl ester 11
in the presence of NHS/DCC) to provide the ethyl ester 17 of the desired
derivative. This ester was then hydrolyzed to the corresponding acid 18
in an overall yield of 31%.
Labelling with 99mTc
To 1–2 mg of the ligand, dissolved in 0:5 M phosphate buffer ð500 mlÞ of
the desired pH (10 or above) was added in succession: 10 mg sodium
potassium tartrate dissolved in 0:25 ml of water, 100 mg of SnCl2 ꢀ 2H2O
in 25 ml of 0:05 N HCl, and 1–3 ml of generator eluate (UltraTechne-
kow generator, Mallinckrodt Medical, The Netherlands) containing
370–740 MBq (10–20 mCi) of 99mTcOꢁ4 . The reaction mixture was
heated in a boiling water-bath for 10 min, cooled to room temperature,
and filtered through a 0:22mm pore membrane filter (Acrodisc, Gelman
Sciences, USA) prior to radio-HPLC analysis.
Radio-HPLC analysis
The HPLC analysis of labelled reaction mixtures was performed on a
250 ꢂ 4:6 mm reversed-phase column filled with Hypersil ODS 5 mm
(Shandon Scientific, UK) and eluted with a ternary gradient mixture of
ethanol, 0:025 M phosphate buffer pH 5.85 and water at a flow rate of
1 ml=min (Merck-Hitachi L6200 intelligent pump). Radioactivity in the
column effluent was monitored using a 2-in NaI(Tl) scintillation
detector coupled to a single-channel analyser and a Rachel analysis
program.
Biological evaluation in mice
The biodistribution of the 99mTc-MAG3-derivatives was studied in male
NMRI mice (body mass 25–35 g) at 10 and 30 min post-injection,
according to a published procedure.8 Briefly, the HPLC-purified
product was diluted to a concentration of 144 kBq=ml with saline and
to this 131I-OIH (Mallinckrodt Medical, The Netherlands) was added as
an internal biological standard to a concentration of 15 kBq=ml. The
mice were weighed and 0:1 ml of the diluted tracer solution was injected
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SYNTHESIS AND BIOLOGICAL CHARACTERISTICS
83
via a tail vein after sedation of the animals with Hypnorm1 (Duphar,
The Netherlands). Five mice were sacrificed by decapitation at 10 and
30 min post-injection. Blood was collected and mice were dissected.
Organs were isolated and radioactivity in all organs and body parts
were measured using a g-counter. Radioactivity in each organ is
expressed as a percentage of the injected dose. For total blood
radioactivity calculation, blood mass is assumed to be 7% of total
body mass.
Evaluation in a baboon
The plasma clearance of HPLC-isolated peaks of the 99mTc-MAG3-
derivatives was studied in a male baboon according to a published
procedure.8 Briefly, a male baboon (body mass about 12 kg) was
1
"
anesthetized by intramuscular injection of 75 mg ketamine (Imalgene ,
#
!
Rhone Merieux). Anesthesia was sustained by intravenous injection of
15 mg sodium pentobarbital (Nembutal1, Sanofi) at 10-min intervals.
Approximately 18:5 MBq ð0:5 mCiÞ of the 99mTc-labelled complex was
co-injected with 1:85 MBq ð50 mCiÞ of 131I-OIH via a limb vein. Blood
samples ð2 mlÞ were collected at 2, 4, 6, 8, 10, 15, 20, 30, 45 and 60 min
post-injection in heparinized tubes. After centrifugation, 500 ml plasma
samples were pipetted, weighed and radioactivity was measured using a
g-counter. The 1-h plasma clearance was calculated using a double
exponential fitting method based on a two-compartment model.22
Conclusions
The data seem to suggest that the inversion of the first and/or second
amide bond in 99mTc-MAG3 has no significant influence on the
biological behaviour of 99mTc-MAG3-like compounds, as these mod-
ified derivatives displayed biological behaviour comparable to
99mTc-MAG3 in mice. The results from the baboon study, nevertheless,
demonstrate that the data obtained in mice are poorly predictive for the
worth of a compound as a tracer agent. The very high and rapid urinary
excretion of 99mTc-13 in mice did suggest that this compound is equal or
even superior to 99mTc-MAG3 for evaluation of the renal function;
however, it showed clearly a less efficient renal extraction in a baboon.
The inter-species variations observed between the mice and the baboon
suggest that the real potential of these compounds as useful renal tracer
agents would ultimately be determined by evaluating them in humans.
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