Synthesis, labelling and biological characteristics of derivatives of mercaptoacetyltriglycine with two amide functions

Article abstract of DOI:10.1002/jlcr.538

Eight novel derivatives of MAG₃, containing in their structure two instead of three amide functions, have been synthesized and evaluated. The synthesized ligands were labelled with ^99mTc by exchange labelling at 100°C and/or by a dir

Full text of DOI:10.1002/jlcr.538

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 115–137.
DOI: 10.1002/jlcr.538
Research Article
Synthesis, labelling and biological
characteristics of derivatives of
Mercaptoacetyltriglycine with two amide
functions
S. M. Okarvi*, K. Verbekey, P. Adriaens and A. M. Verbruggen
Laboratory of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical
Sciences, K.U. Leuven, B-3000 Leuven, Belgium
Summary
Eight novel derivatives of MAG3, containing in their structure two instead of
three amide functions, have been synthesized and evaluated. The synthesized
ligands were labelled with 99mTcby exchange labelling at 100 8C and/or by a
direct labelling method at room temperature. After labelling with 99mTc, the
reaction mixtures were analyzed by radio-HPLC. Biological evaluation of
different 99mTc-diamide complexes in mice and a baboon indicated that
substitution in one amide function of MAG3 structure greatly influences the
renal handling of these compounds. It was found that the diamide derivatives
containing at least one inverted amide exhibit biological behavior comparable
to that of 99mTc-MAG3. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: technetium-99m; MAG3; diamides; renal agents; radiolabelling
Introduction
During the past three decades, a large number of radiolabelled
compounds have been developed and evaluated as potential tracer
*Correspondence to: S. M. Okarvi, King Faisal Specialist Hospital & Research Centre. MBC-03,
PO Box 3354, Riyadh 11211, Saudi Arabia. E-mail: sokarvi@kfshrc.edu.sa
ySenior research assistant of the Funds for Scientific Research, Belgium
Copyright # 2002 John Wiley & Sons, Ltd.
Received 17 September 2001
Revised 19 October 2001
Accepted 24 October 2001

116
S. M. OKARVI ET AL.
agents for renal function studies.1–8 The extensive search for a suitable
renal radiopharmaceutical can be explained from the viewpoint that the
kidney is a vital organ responsible for the maintenance of the
composition of blood and extracellular fluid and thus deserves an
optimal diagnostic agent for its precise evaluation. Since 1960, 131I-
Hippuran (OIH, Figure 1) has been used as the radiopharmaceutical of
choice for the determination of effective renal plasma flow (ERPF) and
functional evaluation of the kidneys by radioisotopic scintigraphy.9,10
Despite the drawbacks of the isotope 131I (i.e. high gamma-energy of
364 KeV, long physical half-life and the beta-component of its
radiation), OIH is still considered the golden standard in view of its
ideal biological characteristics.10–12 However, an agent labelled with
99mTc and having renal extraction characteristics comparable to or even
greater than OIH is highly desirable for practical and dosimetric
reasons. Over the last 15 years, several compounds labelled with 99mTc
have been proposed as alternatives to OIH.13–26 Until now, the most
appropriate and successful 99mTc-labelled tracer agent for renal function
studies is 99mTc-mercaptoacetyltriglycine (99mTc-MAG3, Figure 1). This
chelate is based on a N3S donor ligand system and can easily be
prepared by using a commercially available kit.27 The superior physical
characteristics of the 99mTclabel and its favorable biological properties
make 99mTc-MAG3, the agent of choice at present, especially for the
evaluation of transplant kidney, tubular necrosis and kidney function in
general.28–30
Nonetheless, 99mTc-MAG3 is still not the ideal replacement for OIH,
and improvements are still possible. The plasma-protein binding of
99mTc-MAG3 is high,30,31 and its plasma clearance in humans is not
higher than about 60–65% of the OIH value. Therefore, accurate
determination of the ERPF is rather difficult using 99mTc-MAG3,
although formulae have been proposed to calculate ERPF from the
Figure 1. Structure of 2-iodohippuric acid, OIH) and 99mTc-mercaptoacetyl-
triglycine 99mTc-MAG3)
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acquired data.31,32 Moreover, 99mTc-MAG3 shows some hepatobiliary
uptake.33 For these reasons, the development of a 99mTc-labelled renal
function agent that approaches OIH more closely (with respect to
biological properties) than 99mTc-MAG3 would still constitute an
improvement.
The present study was undertaken in an attempt to improve the renal
handling of 99mTc-MAG3-like agents, to evaluate the effect of
derivatization of MAG3 on the biological behavior, and to elucidate
the structural requirements for an efficient interaction of 99mTc-MAG3-
like agents with the tubular transport system. For this purpose, we have
synthesized a number of S-protected derivatives of MAG3 bearing in
their structures only two instead of three amide functions. In these
derivatives, one of the amides of MAG3 sequence has been replaced by
an amine function, and the remaining amides were also inverted (except
for the terminal amide). In this way, eight different diamide derivatives
of MAG3 are possible to synthesize (Figure 2).
Compounds B, C and D are called the ‘‘direct derivatives’’ of MAG3,
in which one amide has been replaced by an amine. Derivatives F, G, H
and I also contain one and derivative E two inverted amides, as
compared to MAG3. The new ligands were labelled with 99mTcby the
exchange reaction and/or direct labelling method and the labelling
mixtures were analyzed by reversed-phase HPLC. Their biodistribution
studies were performed in mice and a baboon. This paper presents the
chemistry, labelling and biological characteristics of diamide derivatives
of MAG3.
Results and discussion
Labelling with 99mTc
Exchange labelling of the studied ligands with 99mTcin the presence of
stannous chloride and sodium tartrate produced for five of the chelates
(B–G) one main radiochemical product with a retention time on HPLC
close to that of 99mTc-MAG3. However, the diamides B, H and I
showed poor labelling characteristics with 99mTc. The precursors of
these three S-protected ligands (27a, 41a and 32a) contain in their
structure an N-tosyl group for reasons of feasibility of the chemical
synthesis. Prior to labelling, the ligands were treated with sodium in
liquid ammonia in order to remove S-protecting (benzyl or trityl) and
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Figure 2. Structure of MAG3 derivatives with two amides and one amine
N-protecting (tosyl) groups simultaneously. After this complete
deprotection, labelling was performed under alkaline conditions (pH
12) at room temperature (direct labelling) and/or in the presence of
tartrate at 1008C (exchange labelling). Both the direct and exchange
labelling unexpectedly resulted in poor labelling efficiency for these
derivatives that can be characterized by a mixture of peaks as revealed
by HPLC analysis. The reason for this poor labelling is not yet clear. No
doubt exists about the identity of the precursors, as both NMR and
mass spectroscopy have confirmed this. One of the possibilities could be
that the treatment with sodium in liquid ammonia for removal of the
N-tosyl and S-benzyl or S-trityl group causes decomposition of the
tetraligand diamide. This deprotection method is, however, a common
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SYNTHESIS, LABELLING AND BIOLOGICAL CHARACTER
119
procedure in peptide chemistry and has been performed with high yields
by different authors.34,35 Another possibility is, of course, that a
combination of a thiol, two amides and one amine in the backbone of
these three ligands has no efficient Tc complexing properties and hence,
will not result in one single and/or stable radiochemical species.
Biodistribution in mice
The biodistribution studies of the 99mTc-labelled diamide derivatives
were performed in mice. For each 99mTc-compound, the main
component of the labelling reaction mixture was isolated by HPLC
analysis and after appropriate dilution with saline, it was co-injected
with [131I]-OIH in mice (n = 5) at 10 and 30 min post-injection. The
OIH was added as an internal biological standard with a well-known
biodistribution profile to allow rejection of results in animals with
impaired renal function. The results of the in vivo biodistribution of
different diamide derivatives (except for derivative I) are summarized in
Table 1.
To be a suitable tracer agent for renal function studies, a compound
should have biological characteristics that approach OIH as closely as
possible. This means high plasma clearance, a rapid uptake in the
kidneys with immediate transport to the urine and a negligible retention
in the liver, intestines and other body organs after the perfusion phase.
Compounds B, C and D (Figure 2) are the direct derivatives of MAG3,
in which one of the amides has been replaced by an amine, whereas the
other amides are not affected. They show poorer extraction from the
Table 1. Biodistribution of the different diamide derivatives of 99mTc-MAG3 in
mice (n=5) at 10 and 30 min p.i. The results for 99mTc-Iare not shown (see text)
per cent 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
OIH 81
A (Tc-MAG3) 77
91
89
34
63
78
85
84
82
66
3
8
4
6
5
3
3
4
3
1
2
3
5
1
1
1
1
2
2
3
4
3
6
5
2
2
4
1
2
6
4
5
2
5
1
3
1
2
4
3
2
2
2
2
3
1
5
10
4
3
8
9
4
3
3
2
21
8
5
2
3
3
13
1
1
48
4
2
1
1
2
14
B
C
D
E
F
36
47
64
80
74
75
44
G
H
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blood than the parent compound at both studied time points. At 10 min
post-injection, the percentage of injected dose remaining in the blood
was about 21, 8 and 5% for the 99mTc-complexes of B, C and D,
respectively, as compared to 2% for 99mTc-MAG3. At 10 min post-
injection, the activity in the urine was about 95, 44, 58 and 78%,
respectively, of the OIH value for A, B, C and D. As could be expected
from the slow blood clearance of 99mTc-complex B, this derivative also
shows slowest urinary excretion and significant elimination through the
hepatobiliary system. No major retention was observed for these
compounds in the kidneys, liver or intestines at 10 min post-injection.
For these three derivatives, it was found that the replacement of an
amide by an amine greatly impairs the renal excretion characteristics of
these derivatives in general and derivative B in particular, in which the
terminal amide is altered. It is well known that the presence of a
terminal carbonylglycine sequence (-CO–N–CH2–COOH) is necessary
in OIH for an efficient interaction of the compound with the renal
tubular transport proteins.36 The terminal carbonylglycine sequence is
also present in 99mTc-MAG3, while it is lacking in derivative B.
Furthermore, it is notable that the renal excretion characteristics
improve from B to D, the derivatives in which both the second and third
amide bond are unaffected. Although these results do not yet allow a
clear understanding of the structural requirements for an efficient
interaction of MAG3-like compounds with the tubular receptor
proteins, they nevertheless, seem to suggest that the presence of one
or more amide bonds close to the terminal glycine part is an important
requirement for the efficient tubular handling of MAG3-like agents.
The 99mTc-complexes E, F and G are different from the parent 99mTc-
MAG3 complex with respect to the presence of only two amide bonds.
In addition, one or more remaining amides are inverted, but they do not
contain an oxamide moiety in their structure. The renal excretion
characteristics of the complexes E, F and G are more comparable with
99mTc-MAG3, as appears from their more rapid clearance from the
blood and higher urinary excretion at both time points. At 10 min post-
injection, the percentage of injected dose in the urine was about 93, 92
and 91%, respectively, of the OIH value versus 98% for 99mTc-MAG3.
Compared to 99mTc-MAG3, their renal retention was slower and the
radioactivity remaining in the blood was almost the same (Table 1).
From the biodistribution data, it appears that the 99mTc-complex of
derivative E displayed superior biological characteristics than 99mTc-
MAG3 as a renal agent, except for the fact that its excretion to the
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SYNTHESIS, LABELLING AND BIOLOGICAL CHARACTER
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intestines at 30 min post-injection was somewhat higher. Also the
complexes F and G approach the parent compound closely and it may
be concluded that the differences in biological behavior between 99mTc-
MAG3 and its derivatives E, F and G are insignificant (Table 1).
It is striking that the derivative with the most favorable renal
excretion characteristics in mice is the 99mTc-complex of E, in which the
two-amide functions are reversed as compared to the structure of 99mTc-
MAG3. Both diamides E and F are lacking the terminal amide bond of
the parent compound, as is the case for compound B. Nevertheless, the
renal handling of E and F is very efficient, unlike that of B. This
indicates that the terminal amide is not indispensable for efficient
interaction at the tubular cells. Comparison of the structures of
compound B (poor clearance of the 99mTc-complex from the blood)
with E, F and G (efficient extraction and renal excretion) shows in B the
absence of a carbonyl group on the ethylene bridge connecting the
second and third nitrogen atom, whereas the other three derivatives do
contain such a carbonyl group in their structure. The only other diamide
derivative without such a carbonyl group in this part of the molecule is
compound I. As mentioned above, derivative I showed poor labelling
characteristics with 99mTc, combined with unfavorable biological
characteristics such as slow blood clearance, poor urinary excretion
and significant uptake in the liver and intestines. These results support
the hypothesis that the presence of at least one carbonyl group between
the second and third nitrogen atom is essential in MAG3-like
compounds for an efficient extraction and secretion by the tubular
system. It appears, however, that the presence of an amide carbonyl at
other sites of the molecule is also important. Derivative H has an
oxamide moiety just before the terminal glycine but no other carbonyl
groups in the remaining part of its structure. It also showed poor
extraction from the plasma and slow urinary excretion, but no
significant uptake or retention in the liver, intestines or kidneys. It thus
seems that for an efficient handling by the tubular system, 99mTc-
MAG3-like compounds need in their structure some specific distribution
of the amide bonds and at least one carbonyl group on the ethylene
bridge preceding the terminal glycine moiety.
Biodistribution in a baboon
On the basis of the results obtained in mice, five of the 99mTc-labelled
diamide derivatives (C, D, E, F and G) have also been evaluated in a
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Table 2. One-hour plasma clearance values in a baboon for 99mTc-MAG3 and
five of its diamide derivatives
Derivative
1-h plasma clearancea
A (MAG3)
51
6
13
38
53
30
C
D
E
F
G
a expressed as percentage of the clearance of co-injected 131I-OIH
baboon. The 1-h plasma clearance values of all tested compounds are
presented in Table 2. These results show that 99mTc-labelled F, which
also showed a favorable renal excretion in mice, has the highest plasma
clearance in baboon, comparable to that of 99mTc-MAG3. The same
was true for 99mTc-labelled E. On the other hand, the plasma clearance
was very low for the 99mTc-labelled derivatives C and D (Table 2), which
also exhibited poor renal excretion characteristics and slow blood
clearance in mice. It appears that the data obtained in the baboon
confirm the findings in mice although there are some inter-species
differences.
Materials and methods
General
Commercially available chemicals were of reagent grade and were used
without purification except for S-benzylthioglycolic acid (recrystallized
from ethyl acetate). Liquid ammonia was distilled from sodium into the
reaction flask. TLC was carried out using precoated-TLC silica gel
plates (Merck, 60F 254). Compounds were detected with UV light at
254 nm, or by exposing the TLC chromatograms to iodine vapors.
Column chromatography was run on a silica gel 60A (Merck 230–400
meshes). The structures of the synthesized ligands were confirmed with
1H NMR spectroscopy obtained on a Joel FX 90Q spectrometer (Joel,
Japan) using CDCl3, DMSO-d6 or D2O as solvents. The final products
were dried in a vacuum desiccator over phosphorus pentoxide. Animal
studies were performed according to the Belgian code of practice for the
care and use of experimental animals.
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Synthesis/chemistry
Detailed syntheticpathways of the ligands is described elsewhere. 37 For
reasons of simplicity only the chemistry part is mentioned here. The
chemistry necessary for the synthesis of the diamides was mainly based
on standard peptide chemistry techniques, e.g. use and choice of
protecting groups, standard activation, coupling and deprotection
procedures. The general chemistry for the preparation of different
diamides is described below.
N-Benzylmercaptoethyl-N0-acetic acid iminodiacetamide.HBr (6b)
The synthesis of compound 6b (derivative G) is given in Scheme 1.
Several methods have been described for the preparation of S-benzyl-
Scheme 1. Synthesis of N-benzylmercaptoethyl-N-acetic acid iminodiacetamide.
HBr (6b)
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cysteamine.38–42 For example, reaction of sodium S-benzylmercaptan
with bromoethylamine in boiling ethanol (yield 75%).39–41 Coupling of
benzyl chloride with mercaptoethylamine (1,cysteamine)40 in DMF in
the presence of potassium carbonate (yield 86%), or with the sodium
mercaptide of (1) in boiling ethanol38 (yield 75%) or in liquid
ammonia42 (yield 94%). An initial attempt to synthesize this compound
according to the method of Goldberg and Kelly38 was unsuccessful.
Finally, the procedure of Balasubramaniam et al.42 was adapted that
afforded a pure product with a high yield (94%). Condensation of
carbobenzyloxyimino acetic anhydride (3b) (prepared by warming the
corresponding acid (3a) with acetic anhydride) with S-benzylcysteamine
(2) resulted in (4), which was activated by reaction with N-
hydroxysuccinimide (NHS) and 1–3-dicyclohexylcarbodiimide (DCC)
to give the N-hydroxysuccinimidyl ester (5). Coupling of this active ester
with glycine yielded (6a), which was deprotected with 33% hydrogen
bromide in acetic acid (HBr/HOAc) to give (6b) in an overall yield of
43%.
Imino-N-acetic acid-N-acetylglycyl-S-benzylcysteamine.HBr (8b)
The preparation of 8b (derivative E) is outlined in Scheme 2. Reaction
of carbobenzyloxyglycine with S-benzylcysteamine (2) using DCC and
1-hydroxybenzotriazole (HBT) resulted in (7a), which was deprotected
to give glycyl-S-benzylcysteamine. HBr (7b). Condensation of N-
carbobenzyloxyimino acetic anhydride (3b) with the free amine of (7b)
gave (8a), which was deprotected to afford the hydrobromide (8b) in an
overall yield of 16%.
N1-Benzylmercaptoethyltriglycine (11)
The method used to prepare 11 (derivative D) is described in Scheme 3.
The first intermediate (9a), i.e., N-2-benzylmercaptoethylglycine has
previously been prepared from 2-benzylmercaptoethyl chloride and
glycine.43 Condensation of S-benzylcysteamine (2) with bromomalonic
acid at pH 10 in 50% ethanol,44 followed by thermal decarboxylation of
one carboxyl group resulted in (9a). This intermediate was N-protected
by reaction with carbobenzyloxy chloride in alkaline medium to give N-
2-benzylmercaptoethyl-N-carbobenzyloxyglycine (9b). Activation of
(9b) with DCC/HBT and subsequent coupling with glycylglycine ethyl
ester provided the fully protected benzylmercaptoethyl tripeptide ð10aÞ.
Alkaline hydrolysis produces the corresponding acid (10b), which was
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SYNTHESIS, LABELLING AND BIOLOGICAL CHARACTER
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Scheme 2. Synthesis of Imino-N-acetic acid-N0-acetylglycyl-S-benzylcysteamine.
HBr (8b)
Scheme 3. Synthesis of N1-Benzylmercaptoethyltriglycine (11)
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S. M. OKARVI ET AL.
converted to N-2-benzylmercaptoethyltriglycine (11) by acidolysis in
overall yield of 13%.
S-Benzylmercaptoacetamidoethylenediglycine (16)
For the preparation of 16 (derivative C, Scheme 4), commercially
available S-benzylthioglycolic acid (12) was reacted in the presence of
HBT/DCC with N-tert-butoxycarbonylethylenediamine (13). This
chemical was obtained from the reaction of ethylenediamine and di-
tert-butyldicarbonate in 1 : 9 ratios in the presence of triethylamine.45
N-S-benzylmercaptoacetyl-N0-tert-butoxycarbonylethylenediamine (14a)
was the first intermediate. Removal of the BOC group with acid resulted
in (14b), which was alkylated with bromoacetylglycine (15) (prepared
from glycine and bromoacetyl chloride in aqueous alkaline medium) to
give S-benzylmercaptoacetamidoethylenediglycine (16) in an overall
yield of 29%.
N-2-Benzylmercaptoacetyl-N0-imino (N00-acetic acid-N00-acetyl) ethylene
diamine (17b)
Compound 17b (derivative F) was prepared according to Scheme 5.
Coupling of (14b) (intermediate in the synthesis of 16) with carboben-
Scheme 4. Synthesis of S-Benzylmercaptoacetamidoethylenediglycine (16)
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Scheme 5. Synthesis of N-2-benzylmercaptoacetyl-N-imino (N 00-acetic acid-N 00
acetyl) ethylene diamine (17b)
zyloxyimino acetic anhydride (3b) gave (17a) The carbobenzyloxy
protecting group was removed by treatment with HBr/HOAc to afford
(17b) in an overall yield of 17%.
S-Benzylmercaptoacetylglycyl-2-aminoethylene-N-tosylglycine (27)
The method for the preparation of 27 is depicted in Schemes 6a and 6b.
S-Benzyl-mercaptoacetylglycine (19) was prepared from the N-hydro-
xysuccinimidyl ester (18a) of S-benzylmercaptoacetic acid (12) and
glycine and was then converted to its N-hydroxysuccinimidyl ester (19a,
Scheme 6a). Ethanolamine was converted to the N-BOC protected
compound (20), which was further transformed to the O-tosyl ester (21)
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S. M. OKARVI ET AL.
Scheme 6. a Synthesis of succinimidyl benzylmercaptoacetylglycinate (19a), and
N(2-tert-buytoxylcarbonylaminoethyl)-N-tosylglycine ethyl ester (24) Scheme
6b. Synthesis of S-Benzylmercaptoacetylglycyl-2-aminoethylene-N-tosylglycine
(27a)
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SYNTHESIS, LABELLING AND BIOLOGICAL CHARACTER
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Scheme 6. Continued.
using tosyl chloride in pyridine. Glycine ethyl ester was protected as the
N-tosyl derivative (22) from which the sodium salt (23) was prepared
with sodium ethoxide in ethanol. Reaction of (21) and (23) in DMF at
1058C following the method of Atkins et al.46 for the synthesis of
hexacyclen, resulted in (N-2-tert-butoxycarbonylaminoethyl)-N-tosyl-
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S. M. OKARVI ET AL.
glycine ethyl ester (24) (Scheme 6a). This product was deprotected by
TFA to give compound (25), which was isolated as the tosyl salt.
Coupling of (25) with the N-hydroxysuccinimidyl ester (19a) provided
(26) from which the ethyl ester was removed by alkaline hydrolysis to
give (27) in overall yield of 23%. Compound (27) was further
deprotected by sodium in liquid ammonia immediately before labelling
(27a, Scheme 6b, derivative B).
N-2-Tritylmercaptoethyl-N0-ethylene-N-tosylglycine oxamide (32)
Compound 32 was prepared according to Scheme 7. Different methods
for the preparation of S-tritylcysteamine (28) have been described,
starting either from bromoethylamine and sodium trityl mercaptan38
Scheme 7. Synthesis of N-2-Tritylmercaptoethyl-N 0-ethylene-N-tosylglycine
oxamide (32)
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(yield 93%) or from mercaptoethylamine and trityl chloride47 (yield
63%) or trityl alcohol48 (yield 70%). We performed however, the
reaction of trityl chloride with cysteamine and obtained in this way a
high yield of 76% of (28). Reaction of (28) with ethyl oxalyl chloride
resulted in (29) from which the acid (30) was obtained by alkaline
hydrolysis. Condensation of this acid with the 2-aminoethylglycine
derivative (25, intermediate of Scheme 6b) produced the oxamide ester
(31), which was hydrolyzed to the corresponding acid (32) in an overall
yield of 7%. Compound (32) was fully deprotected by the treatment of
sodium in liquid ammonia prior to radiolabelling to afford (32a)
(derivative I).
N-(2-Benzylmercaptoethyl-N-tosyl)-N0-(oxalylglycine) ethylenediamine
(41)
Compound 41 was obtained as represented in Scheme 8. Condensation
of the sodium salt (34) of S-benzyl-N-tosyl cysteamine (33) with N-tert-
butoxycarbonyl aminoethanol tosylate (21) (intermediate of Scheme 6a)
resulted in a fully protected derivative of ethylene diamine (35). The
BOC-group was removed with acid to give compound (36), which was
converted to the tosylate salt (37). Coupling of this salt with N-
oxalylglycine ethyl ester (39) (obtained from the corresponding mono-
tert-butyl ester by reaction with HBr/HOAc) afforded the oxamide ethyl
ester (40), which was hydrolyzed to the corresponding acid (41) in an
overall yield of 4%. This compound was fully deprotected by sodium in
liquid ammonia immediately before labelling (41a)(derivative H).
Overall yields were rather good for six end-products, especially
considering the lengthy and complex procedures required for the
syntheses. The other two diamides (with N-tosyl protective group) were
however, hampered with a low yield coupling step. On the other hand,
alkylation of the N-tosyl sodium salt with an O-tosyl leaving group
seems to be a useful approach especially when compared with direct
alkylation of the bromo compounds that resulted in impure mixtures.
It was our intention to use an S-benzyl protecting group for the
preparation of all the compounds in this study. However, one of the
intermediates, i.e. oxalyl-S-benzylcysteamine was found to be insoluble
in organic solvents and hence became completely useless in DCC-
mediated coupling reactions with amines. However, the solubility
problem was resolved by the utilization of a more lipophilic S-trityl
protecting group.
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Scheme 8. Synthesis of N-(2-benzylmercaptoethyl-N-tosyl)-N-(oxalylglycine)
ethylenediamine (41)
Initially, we intended to synthesize the N- and S-protected diamides
(B, H and I) in the form of precursors bearing an S-protective group
(but with no tosyl protection on the amine) as it has been done for the
five others studied derivatives. All our efforts were unsuccessful mainly
due to the formation of impure products, hence an alternative strategy
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133
to use tosyl and trityl protection on amine and thiol functions,
respectively, was employed that resulted in essentially pure products.
Labelling with 99mTc
Labelling of the synthesized diamide ligands was carried out by ligand
exchange and/or direct method. For exchange labelling, the ligand
(1–2 mg) and sodium tartrate (5 mg) were dissolved in 0.5 M phosphate
buffer pH 10–11 (0.5 ml) followed by the addition of 200 mg of
SnCl2 ꢀ 2H2O in 50 ml of 0.05N HCl. After the addition of 1–3 ml of
eluate (UltraTechnekow generator, Mallinckrodt Medical, The Nether-
lands) containing 370 to 740 MBq (10 to 20 mCi) of 99mTcO4ꢁ, the reac-
tion mixture was heated for 10 min at 1008C and cooled to room temp-
erature and filtered through a 0.2 mm pore membrane filter (Acrodisc,
Gelman Sciences). For the direct labelling, 1–2 mg of the ligand was
dissolved in 0.5 ml of 0.5 M phosphate buffer pH 12, followed by 200 mg
of SnCl2 ꢀ 2H2O and 1 ml of 99mTcOꢁ4 (370 MBq). The mixture was kept
at room temperature for 1 h prior to radio-HPLC analysis.
Analysis of 99mTc-labelled compounds
Analysis of labelled reaction mixtures was performed by RP-HPLC on a
Hypersil 5 mm ODS column (Shandon Scientific, UK) 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. The HPLC system consisted of a
Merck-Hitachi L6200 intelligent pump and a 250 mm ꢂ 4.6 mm reversed
phase column. Radioactivity in the column effluent was monitored using
a 2-inch NaI(Tl) scintillation detector coupled via a single channel
analyzer to a Rachel-5-LS integrator (Raytest).
Biological evaluation of 99mTc-labelled compounds
Evaluation in mice. Biodistribution of diamides was studied in 5 male
NMRI mice (body mass 25–35 g) at 10 and 30 min post-injection. The
HPLC purified product was diluted to a concentration of 144 kBq/ml
with sterile saline and 131I-OIH (Mallinckrodt Medical, The Nether-
lands) was added as an internal biological standard to a concentration
of 14.4 kBq/ml. The diluted tracer solution (0.1 ml) was injected via a
tail vein after sedation of the animals with Hypnorm1 (Duphar, The
Netherlands). The animals ware sacrificed by decapitation at fixed time
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134
S. M. OKARVI ET AL.
intervals. The organs were dissected and the radioactivity in each organ
was measured. Corrections were made for background radiation,
physical decay during counting and crossover of the 131I-radiation into
the 99mTc-channel. Radioactivity in each organ was expressed as a
percentage of the total radioactivity, which was equal to the sum of the
net counts in all body parts. To calculate the radioactivity in blood,
blood mass was assumed to be 7% of body mass.
Evaluation in a baboon. For baboon studies, a male baboon (body mass
about 12 kg) was anesthetized by intramuscular injection of 75 mg
1
"
#
!
ketamine (Imagene , Rhone Merieux). Anesthesia was sustained by
intravenous injection of 15 mg sodium pentobarbital (Nembutal1,
Sanofi) at time intervals of 15 min. Approximately 18.5 MBq (0.5 mCi)
of the 99mTc-complex was co-injected with 1.85 MBq (50 mCi) of 131I-
OIH via a limb vein. Blood samples (2 ml) were collected at 2, 4, 6, 8, 10,
15, 20, 30, 45 and 60 min post-injection in heparinized tubes
(Monoject1 H10, Sherwood Medical). After centrifugation, 500 ml
plasma samples were pipetted out in tubes, weighed and radioactivity
was measured using a g-counter. One-hour plasma clearance was
calculated using a double exponential fitting method.36
Conclusions
From the biodistribution studies in mice, it may be concluded that the
renal excretion properties of the direct derivatives of 99mTc-MAG3
(
99mTc-complexes of B, C and D) in which one amide is replaced by an
amine, are inferior to that of the parent compound. The results
suggested that these derivatives do not fulfill the requirements for a
suitable tracer agent for dynamic kidney function studies. On the other
hand, the diamide derivatives of 99mTc-MAG3, with at least one
inverted amide and without an oxamide structure (99mTc-complexes of
E, F and G), exhibited the most efficient renal handling in mice,
characterized by their rapid renal excretion along with their negligible
retention in other body organs. From these results, it seems that the
presence of three amide groups is not strictly required for an efficient
extraction of 99mTc-MAG3-like agents by the kidneys and its excretion
to the urine. Finally, it is evident that the position of the remaining
amides, and especially of the carbonyl functionality, greatly determines
the interaction of the tracer agent with the tubular receptor proteins.
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135
Acknowledgements
The authors thank H. Vanbilloen for help in NMR spectra and
M. Hoogmartens for animal experiments.
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