Synthesis and evaluation of a novel samarium-153 bifunctional chelating agent for radioimmunotargeting applications

Article abstract of DOI:10.1002/jlcr.1031

A new bifunctional chelating agent (BCA), 3-(4-isothiocyanatobenzyl) triethylenetetraaminehexaacetic acid (9), has been synthesized in fast and easy conditions. An improved synthesis of its position isomer 1-(4- isothiocyanatobenzyl)triethylenetetraaminehexaacetic acid (19) is also described. Stability in serum media of the two corresponding aminobenzyl derivatives-samarium-153 complexes, respectively, 3-(4-aminobenzyl) triethylenetetraaminehexaacetic acid-samarium-153 and 1-(4-aminobenzyl) triethylenetetraaminehexaacetic acid-samarium-153, have been evaluated. The 3-(4-aminobenzyl)triethylenetetraaminehexaacetic acid complex revealed excellent stability in serum media, and therefore 3-(4-isothiocyanatobenzyl) triethylenetetraaminehexaacetic acid (9) appears useful for future in vivo radioimmunotherapy investigations. Copyright

Full text of DOI:10.1002/jlcr.1031

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 109–123.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1031
Research Article
Synthesis and evaluation of a novel samarium-153
bifunctional chelating agent for radioimmunotargeting
applications
Laurence Morandeau1,*, Patricia Remaud-Le Saec2, Ali Ouadi3,
Karine Bultel-Riviere2, Marie Mougin-Degraef2, Agnes de France-Robert2,
Alain Faivre-Chauvet2 and Jean-Francois Gestin2
¸
1 Department of Medical Technology and Physics, Sir Charles Gairdner Hospital,
Nedlands 6009, Western Australia, Australia
2
´
Chimie des Bioconjugues, Institut de Biologie, INSERM U. 601, 9, Quai Moncousu,
44093 Nantes Cedex, France
3
´
Institut de Recherches Subatomiques, CNRS/IN2P3 and Universite L. Pasteur, B. P.
28, 67037 Strasbourg Cedex 2, France
Summary
A new bifunctional chelating agent (BCA), 3-(4-isothiocyanatobenzyl)triethylenete-
traaminehexaacetic acid ð9Þ, has been synthesized in fast and easy conditions. An
improved synthesis of its position isomer 1-(4-isothiocyanatobenzyl)triethylenetetraa-
minehexaacetic acid ð19Þ is also described. Stability in serum media of the two
corresponding aminobenzyl derivatives-samarium-153 complexes, respectively,
3-(4-aminobenzyl)triethylenetetraaminehexaacetic acid}samarium-153 and 1-(4-ami-
nobenzyl)triethylenetetraaminehexaacetic acid}samarium-153, have been evaluated.
The 3-(4-aminobenzyl)triethylenetetraaminehexaacetic acid complex revealed excel-
lent stability in serum media, and therefore 3-(4-isothiocyanatobenzyl)triethylenete-
traaminehexaacetic acid ð9Þ appears useful for future in vivo radioimmunotherapy
investigations. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: radioimmunotargeting; radioimmunotherapy; bifunctional chelating
agent; samarium-153
*Correspondence to: Laurence Morandeau, Department of Medical Technology and Physics, Sir Charles
Gairdner Hospital, Nedlands 6009, Western Australia, Australia. E-mail: Laurence.morandeau@
health.wa.gov.au
Contract/grant sponsor: La Ligue Departementale Contre le Cancer de Vendee, France
´ ´
Copyright # 2005 John Wiley & Sons, Ltd.
Received 18 March 2005
Revised 18 September 2005
Accepted 27 October 2005

110
L. MORANDEAU ET AL.
Introduction
Radiolabelled antibodies have been commonly used in order to target tumour-
cell antigens for application in immunoscintigraphy1 (IS) and radioimmu-
notherapy2,3 (RIT). However, this system often involves high irradiation of
normal tissues, due in part to the low rate of monoclonal antibody uptake at
the tumour site (0.01% of injected dose per gram of tumour) and the relatively
slow clearance of excess activity (several hours). In order to improve efficacy of
RIT, several strategies have been investigated. Among them are the develop-
ment of pretargeting methods4,5 and/or the replacement of iodine-131 by
radionuclides possessing better radiophysical and chemical characteristics.
Iodine-131 is the main radionuclide currently used in RIT because it
possesses satisfactory specific activity and is easy to link covalently to an
aromatic ring. However, its radiophysical properties: low-energy bꢀ radiation,
strong g emission and long half-life (Eb avg ¼ 182 keV, Eg ¼ 364 keV
(81.2%), T1=2 ¼ 8:0 days) do not make it the best b emitter for RIT
applications. A way to improve RIT applications is to replace iodine-131 by
radionuclides providing different LET (linear energy transfer) in order to
adapt the delivered energy to the target nature and size.6 Radionuclides
such as yttrium-90 (Eb avg ¼ 935 keV, T1=2 ¼ 2:67 days), rhenium-188
(Eb avg ¼ 764 keV, Eg ¼ 155 keV (15%), T1=2 ¼ 0:71 days), holmium-166
(Eb avg ¼ 666 keV, Eg ¼ 80:5 keV (6.3%), T1=2 ¼ 1:1 days), rhenium-186
(Eb avg ¼ 323 keV, Eg ¼ 137 keV (8.5%), T1=2 ¼ 3:8 days), lutetium-177
(Eb avg ¼ 133 keV, Eg ¼ 208 keV (11%), T1=2 ¼ 6:7 days) or samarium-153
(Eb avg ¼ 225 keV, Eg ¼ 103 keV (28%), T1=2 ¼ 1:9 days) are of great
interest. However, the replacement of iodine-131 by such radionuclides
requires the synthesis of different types of bifunctional chelating agents (BCA),
to ensure the formation of a stable complex in vivo with the selected
radionuclide. These BCAs also present an activated function allowing the
coupling with an antibody or other vector biomolecule.
Some BCAs are already known for their chelation properties towards a
specific radionuclide such as 4-aminobenzyl-DOTA7 (2-(4-aminobenzyl)-
1,4,7,10-tetraazacyclododecane-N,N0,N00,N000-tetraacetic acid) for yttrium-90
and lutetium-177, 4-aminobenzyl-HEHA8 (2-(4-aminobenzyl)-1,4,7,10,13,16-
hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid) for actinium-225 or
CHX-A00-DTPA9 (N-[(R)-2-amino-3-(4-aminophenyl)propyl]trans-(S,S)cyclo-
hexane-1,2-diamine-N,N,N0,N00,N00-pentaacetic acid) for bismuth-213. The
structural differences between these BCAs (linear, pre-organized, macrocyclic)
lead to their specificity towards a radionuclide. The common point between
these BCAs is their functionalization by a terminal aromatic amine easily
convertible in isothiocyanate, in order to react with a vector presenting an
amine function.
Copyright # 2005 John Wiley & Sons, Ltd.
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SAMARIUM-153 BIFUNCTIONAL CHELATING AGENT
111
CO2H
HO2C
HO2C
N
N
CO2H
CO2H
N
N
CO2H
Figure 1. TTHA (triethylenetetraaminehexaacetic acid)
HO2C
CO2H
PO(OH)2
CO2H
CO2H
N
H
N
N
PO(OH)2
N
N
N
PO(OH)2
CO2H
PO(OH)2
N
H
N
CO2H
CO2H
CO2H
NH2
Figure 2. cyclohexane-trans-1,2-bis(aminomethylphosphonic)-N,N0-bis(ethyl-2-
iminodiacetic) acid and 1-(4-aminobenzyl)-ethylenediamine-N,N0-bis(ethyl-2-
iminodiacetic)-N,N0-bis(methylphosphonic) acid
Prior studies on TTHA (triethylenetetraaminehexaacetic acid) (Figure 1)
and analogues10,11 indicate that this family of compounds is suitable for the
complexation of metallic ions such as Sm3+ and Ga3+. We have previously
described the synthesis of a series of different chelating agents for samarium-
153 based on a triethylenetetraamine structure, varying the pre-organization
of the chelating sites and the nature of the chelating functions (carboxylic and/
or phosphonic acids). Among them, cyclohexane-trans-1,2-bis(aminomethyl-
phosphonic)-N,N0-bis(ethyl-2-iminodiacetic) acid12,13 (Figure 2), a pre-orga-
nized monofunctionalized chelating agent based on a cyclohexyl diamine
backbone possessing two phosphonic and four carboxylic acid functions
showed promising results for future application to metastatic bone-cancer
therapy. Synthesis of its bifunctionalized analogue allowing coupling to
vectors is in progress in our laboratory. A similar mixed TTHA-like BCA was
also synthesized, where a 4-aminobenzyl group was linked to the ethylene
group in third position14 (Figure 2). Unfortunately, despite interesting
complexation studies with samarium-153, the resulting 153Sm-complex showed
to be unstable in serum media.
In addition, we investigated the synthesis of the non-mixed structural
analogue carrying six carboxylic acid functions.
Copyright # 2005 John Wiley & Sons, Ltd.
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112
L. MORANDEAU ET AL.
NH2
H
NH2
NH2
H
N
MeO
O
HO
O
MeO
O
N
O
O
O
NHBoc
NH2
NH2, HCl
NH2
N
H
N
H
N
H
a
f, g
b
c,d
e
NO2
NO2
NO2
NO2
NO2
1
2
4
5
ButO2C
CO2tBu
CO2tBu
HO2C
CO2H
N
HO2C
CO2H
CO2H
CO2H
N
N
N
N
CO2tBu
CO2H
CO2H
N
CO2H
N CO2H
CO2tBu
CO2H
CO2H
N
CO2tBu
N
N
N
N
S
h
i
NH2
NH2
N
C
7
8
9
Scheme 1. Synthetic scheme of 3-(4-isothiocyanatobenzyl) triethylenetetraami-
nehexaacetic acid 9. (a) MeOH, HCl(g), 91%; (b) (1) Et3N, MeOH; (2)
%
N(Boc)glycine, CDI, THF, 95%; (c) ethylenediamine, MeOH, 99%; (d) (1) HCl
3 M, (2) NaOH 3 M, 71%; (e) (1) BH3/THF, (2) NaOH(s), 78%; (f) KI, 1,8-bis-
(dimethylamino)naphthalene, tBu-bromoacetate, DMF, 62%; (g) H2, Pd/C,
MeOH, 100%; (h) HCl 6 M, acetone, 100%; (i) CSCl2, HCl 0.5 M, CHCl3,
97%
The present paper reports the synthesis of a new BCA, the 3-(4-
isothiocyanatobenzyl) triethylenetetraaminehexaacetic acid or (3-(4-isothio-
cyanatobenzyl)TTHA) (Scheme 1) and an improved synthesis of its position
isomer 1-(4-isothiocyanatobenzyl)TTHA15 (Scheme 2), potential candidates
for applications in radioimmunotargeting. Further studies on the aminobenzyl
precursor of these two BCAs were then carried on: 3-(4-aminobenzyl)TTHA
and 1-(4-aminobenzyl)TTHA have been radiolabelled with samarium-153 to
provide additional information on the influence of the position of the aromatic
ring on the TTHA backbone. Serum stability studies have been performed in
serum media at 378C at different time intervals in order to compare the in vitro
stability of these different 153Sm-radiolabelled chelating agents.
Results and discussion
The methyl ester of 4-nitrophenylalanine 1 was obtained after treatment of
%
4-nitrophenylalanine with HCl gas in methanol (Scheme 1). The amine
function of compound 1 was regenerated after treatment with triethylamine in
%
Copyright # 2005 John Wiley & Sons, Ltd.
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SAMARIUM-153 BIFUNCTIONAL CHELATING AGENT
113
a, b, c
OH
NH
H2N
BocHN
NH2
O
O
12
CO2H
CO2H
N CO2H
H
N
NH2
N
N
CO2H
NHBoc
N
H
N
NH2
CO2H CO2H
CO2H
d, e, f
g, h, i
NO2
NO2
NH2
15
j
18
CO2H
CO2H
N
N
N
CO2H
N
CO2H CO2H
CO2H
N
C
19
S
Scheme 2. Improved synthetic pathway for 1-(4-isothiocyanatobenzyl) triethy-
lenetetraaminehexaacetic acid 19. (a) MeOH, HCl(g), 94%; (b) Di-tert-butyl
dicarbonate, Et3N, 75%; (c) ethylenediamine, MeOH, 89%; (d) C.D.I.,
compound 12, 78%; (e) (1) HCl 3 M, (2) NaOH 3 M 92%; (f) (1) BH3/THF,
(2) NaOH(s), 79%; (g) KI, 1,8-bis-(dimethylamino)naphthalene, tBu-bromoa-
cetate, DMF, 78%; (h) H2, Pd/C, MeOH, 100%; (i) HCl 6 M, acetone, 100%;
(j) CSCl2, HCl 0.5 M, CHCl3, 98%
methanol. Compound 2 was obtained after activation of 1.2 equivalents of the
%
N-(Boc)-glycine with C.D.I. (carbonyldiimidazole) and amidic coupling with
the amine function of compound 1 in a 95% yield. Given that the purity of
%
commercial C.D.I. is variable due to its water sensitivity, a small excess of
activated N-(Boc)-glycine was used, in order to ensure complete reaction of
compound 1. Solution of N[N(Boc)glycyl]4-nitrophenylalanine methyl ester 2
%
%
in methanol was added dropwise to a large excess of ethylenediamine (40 eq.)
in order to avoid amidic coupling between two molecules of compound 2 on
%
ethylenediamine. The tertiobutoxycarbonyl group of compound 3 was
%
removed by acidic hydrolysis (HCl 3 M). Compound 4 was finally obtained,
%
after regeneration of the free amine functions, in a 71% yield. Reduction of the
carbonyl groups of compound 4 was achieved in BH3/THF. Dissolution of 4
%
%
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114
L. MORANDEAU ET AL.
was observed progressively while adding the BH3/THF solution. It was
followed by treatment with HCl 6 M under reflux affording 3-(4-nitrobenzyl)-
triethylenetetraamine 5 in a 77% yield. A silica gel column was necessary to
%
purify the tetraamine 5. Hexa-t-butylester 6 was then obtained in a 62% yield
%
%
by reacting 7 equivalents of 1,8-bis-(dimethylamino)naphthalene, 1.1 equiva-
lents of potassium iodide, 8 equivalents of t-butylbromoacetate16,17 with
1 equivalent of the tetraamine 5. The nitroaromatic function of compound 6
%
%
was reduced by catalytic hydrogenation in presence of palladium on active
carbon to give 7 in quantitative yield. Hydrolysis in acidic conditions (HCl
%
3 M) was then conducted to give 3-(4-aminobenzyl)-triethylenetetraaminehex-
aacetic acid 8 in quantitative yield. 3-(4-isothiocyanatobenzyl)-triethylenete-
%
traaminehexaacetic acid 9 was finally obtained after treatment with
%
thiophosgene.
In Scheme 2, glycine methyl ester hydrochloride 10 was obtained after
treatment of glycine with HCl gas in MeOH. The amine function was
protected with a Boc function in a 75% yield. Compound 11 was then reacted
with ethylene diamine as described for compound 3 for a 89% yield. Resulting
%
compound 12 was then coupled to the carboxylic acid function of N(Boc)4-
nitrophenylalanine after activation with C.D.I., with a 78% yield. The Boc
protective groups were cleaved after treatment with HCl 3 M and the amine
functions regenerated (92%). Carbonyl groups were reduced with BH3/THF
as described for compound 4, to yield 79% of compound 15. Tetraamine 15
%
was reacted with t-butylbromoacetate, as described for compound 6, leading
%
to compound 16 in 78% yield. The nitroaromatic function was then reduced
by catalytic hydrogenation (98%), followed with the acid hydrolysis of the
t-butylacetate ester functions to yield compound 18 (100%). Treatment of 18
with thiophosgene finally gave 1-(4-isothiocyanatobenzyl)-triethylenetetraa-
minehexaacetic acid 19 (98%).
We synthesized a new BCA, 3-(4-isothiocyanatobenzyl)TTHA 9, with a
%
28% global yield. We also improved the synthesis of its position isomer,
1-(4-isothiocyanatobenzyl)TTHA 19 with a global yield of 27%, in
comparison with the synthesis Bhargava et al.15 who obtained a global yield
of 17%. Serum media stability of 3-(4-aminobenzyl)TTHA-153Sm complex
(8-153Sm) has been compared to one of its position isomer 1-(4-aminoben-
%
zyl)TTHA- 153Sm complex (18-153Sm) (Figure 3).
Three solutions have been prepared, one as a control solution containing
153SmCl3 in a 0.02 M citrate buffer (pH5), the second one containing 153Sm-1-
(4-aminobenzyl)TTHA complex and the third one containing 153Sm-3-(4-
aminobenzyl)TTHA complex. These solutions were then transferred to human
serum and incubated at 378C. Aliquots of each were taken at various time
intervals (0, 1, 4, 24, and 144 h) and purified through a PD-10 column in order
to separate free samarium-153 from chelated samarium-153. As shown in
Copyright # 2005 John Wiley & Sons, Ltd.
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SAMARIUM-153 BIFUNCTIONAL CHELATING AGENT
1000000
115
t=0
800000
600000
400000
200000
0
t=1h
t=4h
t=24h
t=144h
1
2
3
4
5
6
7
8
9
10
(a)
Volume (mL)
Control : 153Sm-Cl3
250000
200000
150000
100000
50000
0
t=0
t=1h
t=4h
t=24h
t=144h
1
2
3
4
5
6
7
8
9
10
(b)
Volume (mL)
153Sm-1-(4-aminobenzyl)TTHA
500000
t=0
400000
300000
200000
100000
0
t=1h
t=4h
t=24h
t=144h
1 2
3
4
5
6
7
8
9
10
(c)
Volume (mL)
153Sm-3-(4-aminobenzyl)TTHA
Figure 3. PD-10 elutions of serum media solutions incubated at 378C: (a) control
153SmCl3; (b) 153Sm-1-(4-aminobenzyl)triethylenetetraaminehexaacetic acid and
(c) 153Sm-3-(4-aminobenzyl)triethylenetetraaminehexaacetic acid
Figure 3, free samarium-153 (control) is eluted in the first fractions, between 3
and 4.5 ml, while 153Sm-BCA complexes are eluted between 4.5 and 8 ml.
Serum stability of these two complexes at 378C showed significant difference.
While an important release (30%) of samarium-153 was observed for 153Sm-1-
(4-aminobenzyl)TTHA complex after 24 h in serum media, increasing to 42%
at 144 h, 153Sm-3-(4-aminobenzyl)TTHA complex was totally stable in serum
at 378C after 6 days. 153Sm-3-(4-aminobenzyl)TTHA complex was very stable
in serum media, unlike its isomer 153Sm-1-(4-aminobenzyl)TTHA complex.
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L. MORANDEAU ET AL.
The difference of stability of these two complexes could be explained by
the position of the 4-aminobenzyl group on the TTHA backbone. 3-(4-
aminobenzyl)TTHA appears to be more symmetrical than its structural isomer
1-(4-aminobenzyl)TTHA. Thus, the position of the 4-aminobenzyl group may
induce a stronger pre-organization of the TTHA backbone than the one
observed for 1-(4-aminobenzyl)TTHA.
This study shows once again that serum stability, and furthermore, in vivo
stability of a complex is not definitely predictable. Serum stability studies are
thus essential prior to any evaluation of in vivo stability and behaviour of
new radiopharmaceuticals. It is assumed that the amino precursor 8 or
%
isothiocyanato derivative 9, once coupled to an antibody or other vector
%
biomolecule would exhibit the same binding and serum stability properties
after labelling with samarium-153, as described in the present study for
compound 8. 3-(4-isothiocyanatobenzyl)TTHA 9 was successfully coupled to a
%
%
bivalent peptide and labelled with Indium-111 in our laboratory.18 However,
this needs to be confirmed in further experiments with samarium-153.
In summary, we synthesized a novel BCA, the 3-(4-isothiocyanatobenzyl)
TTHA with satisfactory yields, in fast and easy conditions. We also improved
the synthesis of its position isomer 1-(4-isothiocyanatobenzyl)TTHA by 10%.
Serum stability of the two amino precursors of these BCAs labelled with
samarium-153 were evaluated and compared. The 153Sm-1-(4-aminobenzyl)T-
THA complex (18-153Sm) was found to be very labile in serum media at 378C,
while the 153Sm-3-(4-aminobenzyl)TTHA complex (8-153Sm) showed remark-
%
able serum media stability.
Experimental
Materials and methods
All chemicals were of the highest purity commercially available (Sigma-
Aldrich company). The HPLC solvents were purchased from Carlo-Erba.
TLC was performed using precoated Kieselgel 60 plates F254 (TLC plates,
Carlo-Erba) and was visualized by UV or iodine. Silica gel (230–400 mesh,
Carlo-Erba) was purchased from VWR. High-performance liquid chromato-
graphy was carried out at 211 nm on a Waters 600 HPLC System using
analytical reverse-phase HPLC (SymmetryShield 5 mm RP-18 4.5 ꢁ 150 mm,
Waters). NMR spectra were recorded on a BRUKER AC 250 apparatus
(250.133 MHz for 1H). Chemical shifts are indicated in d values (ppm)
downfield from internal TMS, and coupling constants (J) are given in Hertz
(Hz). Multiplicities were recorded as s (singlet), d (doublet), t (triplet) and m
(multiplet). Mass spectra were recorded using a BRUKER Esquire LC
electrospray mass spectrometer with methanol or water as carrier solvent. IR
spectra were recorded on an Avatar 320 Thermonicolet FTIR-ATR, using a
Copyright # 2005 John Wiley & Sons, Ltd.
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SAMARIUM-153 BIFUNCTIONAL CHELATING AGENT
117
germanium crystal. Samarium-153 was graciously provided by Schering-
CisBio International in a solution of samarium-153 in HCl 0.04 M (volumetric
activity: 5.2 GBq/ml, specific activity: 40 GBq/mg). Radioactivity was counted
on a g counter (WIZARD3 480, automatic g counter, WALLAC).
4-Nitrophenylalanine hydrochloride methyl ester 1
%
A suspension of 5 g of 4-nitrophenylalanine monohydrate (21.9 mmol) in
100 ml of MeOH was saturated with HCl gas and maintained at room
temperature for 18 h. After cooling to 08C, a white precipitate was filtrated off
and washed with cold MeOH. Compound 1 (5.2 g, 91%) was obtained as a
%
white powder.
1H NMR (D2O) (d, ppm): 3.2–3.5 (m, 2H), 3.81 (s, 3H), 4.50 (t, 1H,
J ¼ 6:5 Hz), 7.51 (d, 2H, J ¼ 8:8 Hz), 8.23 (d, 2H, J ¼ 8:8 Hz).
Rf (MeOH): 0.82. MS (M þ Hþ): 225.
N{N(Boc)glycyl}4-nitrophenylalanine methyl ester 2
%
To 1.28 g (4.9 mmol) of 1 and 0.753 ml of triethylamine (5.4 mmol) in 3 ml
%
of MeOH, 20 ml of diethyl ether was added. After 4 h at 08C, salts of
triethylamine hydrochloride were removed by filtration. The filtrate was
evaporated to dryness, to give 1.1 g of yellow oil.
An amount of 0.955 g of C.D.I. (5.9 mmol) was added to a solution of 1.03 g
of N(Boc) glycine (5.9 mmol) in 5 ml of anhydrous THF, under nitrogen
atmosphere. After 10 min, the 4-nitrophenylalanine methyl ester in solution in
3 ml of anhydrous THF was added. The reaction mixture was stirred for 1 h at
room temperature. The THF was evaporated and the residue dissolved in
50 ml of CHCl3 and washed twice with 50 ml of HCl 0.5 M. The organic layer
was dried over Na2SO4, filtered and concentrated to dryness. Compound 2
%
(1.78 g, 95%) was obtained as a white powder.
1H NMR (CDCl3) (d, ppm): 1.45 (s, 9H), 2.16 (s, 2H), 3.16 (d.d, 1H,
J ¼ 13:7 Hz, and 5:8 Hz), 3.31 (d.d, 1H, J ¼ 13:7 Hz and J ¼ 5:8 Hz), 3.79
(s, 3H), 4.90 (d.d, 1H, J ¼ 13:4 Hz and J ¼ 5:8 Hz), 5.11 (s, 1H), 6.75 (d, 1H,
J ¼ 7:0 Hz), 7.30 (d, 2H, J ¼ 8:6 Hz), 8.14 (d, 2H, J ¼ 8:6 Hz). Rf (AcOEt):
0.75. MS (M+H+): 382.
N-(2-Aminoethyl)-{N(Boc)glycyl}4-nitrophenylalaninamide 3
%
An amount of 1.8 g of 2 (4.7 mmol) dissolved in 20 ml of MeOH was added
%
dropwise to 12.6 ml of ethylene diamine (188 mmol), under vigorous stirring.
The reaction mixture was kept for 3 h at room temperature. MeOH and the
excess of ethylene diamine were then evaporated under vacuum. The residue
was taken in a minimum of H2O and extracted five times with 25 ml of CHCl3.
The organic layers were dried over Na2SO4, filtrated and concentrated under
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118
L. MORANDEAU ET AL.
vacuum. Purification on silica gel column (MeOH/NH4OH 10/0.2) gave
compound 3 (1.94 g, 99%) as a white solid.
%
1H NMR (CDCl3) (d, ppm): 1.42 (s, 9H), 2.27 (s, 2H), 2.72 (m, 2H), 3.21
(m, 4H), 3.74 (m, 2H), 4.78 (m, 1H), 5.4 (s, 1H), 7.36 (d, 2H, J ¼ 8:5 Hz), 7.43
(m, 1H), 7.56 (m, 1H), 8.09 (d, 2H, J ¼ 8:5 Hz). Rf (MeOH/NH4OH, 10/0.2):
0.35. MS (M+H+): 410.
N-(2Aminoethyl)-glycyl 4-nitrophenylalaninamide 4
%
A solution of 1.89 g (4.6 mmol) of 3 in 46 ml of HCl 3 M (138 mmol) was
%
stirred for 8 h at room temperature. After evaporation of the mixture, the
residue was taken in 5 ml of water and the pH adjusted to 11–12 with a
solution of NaOH 3 M. This aqueous layer was extracted six times with 30 ml
of CHCl3. Organic layers were dried over Na2SO4, filtrated off and dried under
reduce pressure to yield compound 4 (1.4 g, 71%) as a white solid.
%
1H NMR (MeOD) (d, ppm): 2.56 (m, 2H), 3.1–3.3 (m, 6H), 4.53 (m, 1H),
7.41 (d, 2H, J ¼ 8:8 Hz), 8.09 (d, 2H, J ¼ 8:8 Hz). Rf (MeOH/NH4OH 10/1):
0.35. MS (M+H+): 310.
3-(4-Nitrobenzyl)triethylenetetraamine 5
%
An amount of 40 ml of a solution of BH3/THF 1 M (40 mmol) was added
dropwise to a suspension of 1.1 g of 4 (3.56 mmol) in 10 ml of anhydrous THF,
%
at 08C and under nitrogen atmosphere. The reaction mixture was kept under
reflux for 36 h. After cooling and slow addition of MeOH (5 ml), the solvent
was evaporated and the residue taken in 15 ml of a solution of HCl 6 M. The
mixture was maintained under reflux for 5 h and then concentrated under
vacuum. The residue was taken in 3 ml of water and solid sodium hydroxide
was added until the pH raised to 11–12. The aqueous layer obtained was then
extracted three times with 20 ml of CHCl3. The organic layers were dried over
Na2SO4, filtrated and concentrated. Purification on silica gel column (MeOH/
CHCl3/NH4OH, 7/1/3) led to compound 5 (0.776 g, 78%) as a yellow oil.
1H NMR (CDCl3) (d, ppm): 2.4–3.0 (m, 13H), 1.25 (s, 6H), 7.36 (d, 2H,
J ¼ 8:8 Hz), 8.15 (d, 2H, J ¼ 8:8 Hz). Rf (MeOH/CHCl3/NH4OH, 7/1/3):
0.15. SM (M+H+): 282.
3-(4-Nitrobenzyl)-triethylenetetraaminepentaacetyl tertiobutyl ester 6
%
An amount of 0.2 g of 5 (0.71 mmol), 0.13 g of potassium iodide (0.782 mmol),
%
1.1 g of 1,8-bis-(dimethylamino)naphthalene (5.1 mmol) and 30 ml of anhy-
drous DMF were stirred under nitrogen atmosphere. 0.95 ml of tertiobutyl-
bromoacetate (6.4 mmol) was added and the reaction mixture was maintained
at 658C for 16 h. After cooling, the DMF was removed under vacuum and the
residue was taken in 10 ml of CHCl3 and washed twice with 10 ml of HCl
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SAMARIUM-153 BIFUNCTIONAL CHELATING AGENT
119
0.6 M and once with 10 ml of water. The organic layer was dried over Na2SO4,
filtrated off and concentrated under vacuum. Compound 6 (0.424 g, 62%) was
%
finally obtained as a yellow oil after purification on silica gel column (Hexane/
AcOEt 2/1).
1H NMR (CDCl3) (d, ppm): 1.45 (s, 54H), 2.3–3.1 (m, 13H), 3.25 (s, 2H),
3.33 (s, 4H), 3.34 (s, 2H), 3.44 (s, 4H), 7.46 (d, 2H, J ¼ 8:8 Hz), 8.01 (d, 2H,
J ¼ 8:8 Hz). Rf (Hexane/AcOEt 2/1): 0.35. SM (M+H+): 966.
3-(4-Aminobenzyl)-triethylenetetraaminepentaacetyl tertiobutyl ester 7
%
Catalytic hydrogenation of 0.424 g of 6 (0.44 mmol) in 25 ml of MeOH was
%
carried out over 42 mg of 10% Pd/C (0.1eq.; w/w) at atmospheric pressure.
After 3 h, the catalyst was filtered off (Celite) and the filtrate was removed
under reduced pressure. Compound 7 (0.411 g, 100%) was obtained as a
%
yellow oil.
1H NMR (CDCl3) (d, ppm): 1.42 (s, 9H), 1.43 (s, 9H), 1.45 (s, 36H), 2.4–3.5
(m, 27H), 6.57 (d, 2H, J ¼ 8:2 Hz), 6.98 (d, 2H, J ¼ 8:2 Hz). Rf (AcOEt/
hexane 2/1): 0.4. MS (M+H+): 936.
3-(4-Aminobenzyl)-triethylenetetraaminepentaacetic acid 8
%
To 0.214 g of 7 (0.23 mmol) dissolved in 1 ml of acetone was added 1 ml of HCl
%
6 M. After 16 h under stirring at room temperature, the reaction mixture was
dried under vacuum, to give a white solid (0.178 g, 100%).
1H NMR (D2O) (d, ppm): 2.7–3.6 (m, 13H), 3.81 (s, 6H), 4.04 (s, 6H), 7.40
(m, 4H). MS (M+H+): 600.
3-(4-Isothiocyanatobenzyl)-triethylenetetraaminepentaacetic acid 9
%
To a solution of 12 ml of thiophosgene (0.16 mmol) in 1 ml of CHCl3 under
vigorous stirring was added dropwise a solution of 2.5 mg of 8 (3.2 mmol) in
1 ml of HCl 0.5 M. The reaction mixture was maintained at room temperature
%
for 12 h. The aqueous layer was then washed twice with 2 ml of CHCl3,
concentrated on a rotary vacuum and dried in vacuo. Compound 9 (2.5 mg,
%
97%) was obtained as a pale yellow solid.
MS (M+H+): 642. HPLC: solvent A: TFA 0.1%, Solvent B: CH3CN, flow
rate: 1 ml/min, gradient: 0–5 mn 100%A isocratic, 5–35 mn 100%A to 100%B.
Retention time: 19.1 min. IR: 2100 cmꢀ1 (SCN).
Glycine methyl ester hydrochloride 10
Synthesis was identical to 1 starting with 5 g of glycine (66.6 mmol).
%
Compound 10 was obtained (7.9 g, 94%) as a white powder.
1H NMR (D2O) (d, ppm): 3.81 (s, 3H), 3.91 (s, 2H). Rf (CHCl3/MeOH 4/1):
0.36. MS (M+H+): 90.
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120
L. MORANDEAU ET AL.
N(Boc) glycine methyl ester 11
An amount of 1.22 ml of triethylamine (8.75 mmol) was added to a suspension
of 1 g of 10 (7.96 mmol) in 2 ml of MeOH. 5 ml of ether was then added and the
mixture was placed in an ice bath. The salts were removed by filtration and the
filtrate evaporated. The resulting oil was dissolved in 10 ml of AcOEt. A
solution of 1.91 g of di-tert-butyl dicarbonate (8.75 mmol) in 10 ml of AcOEt
was added dropwise and the reaction maintained at room temperature for
18 h. The reaction mixture was washed twice with 10 ml of water. The organic
layer was dried over Na2SO4, filtrated off and concentrated to yield compound
11 (1.12 g, 75%) as a yellow oil.
1H NMR (CDCl3) (d, ppm): 1.45 (s, 9H), 3.75 (s, 3H), 3.92 (d, 2H,
J ¼ 5:5 Hz), 6.65 (s broad signal, 1H). Rf (MeOH): 0.65. MS (M+H+): 190.
N-(2-Aminoethyl) N(Boc)glycinamide 12
Synthesis was identical to compound 3 starting with 4.38 g of 11 (23.2 mmol) in
%
3 ml of MeOH and 62 ml (927 mmol) of ethylenediamine. After treatment, a
yellow oil (4.46 g, 89%) was obtained.
1H NMR (CDCl3) (d, ppm): 1.45 (s, 9H), 1.52 (s, 2H), 3.15 (m, 2H), 3.31
(m, 2H), 3.78 (d, 2H, J ¼ 5:8 Hz), 5.33 (s broad signal, 1H), 6.64 (s broad
signal), 1H). Rf (MeOH): 0.5. MS (M+H+): 218.
{N(Boc)glycyl-N-(2-aminoethyl)}4-N(Boc)nitrophenylalaninamide 13
An amount of 0.3 g of N(Boc)-4-nitro-L-phenylalanine (0.97 mmol) was
dissolved in 5 ml of anhydrous DMF under nitrogen atmosphere. 0.17 g of
C.D.I. (1.07 mmol) was added. After 10 min, 0.21 g of 12 (0.97 mmol) dissolved
in 5 ml of anhydrous DMF was added dropwise. The reaction was allowed to
proceed for 12 h at room temperature. DMF was evaporated and the residue
was purified on a silica gel column (AcOEt), to give compound 13 as a yellow
oil (0.38 g, 78%).
1H NMR (CDCl3) (d, ppm): 1.40 (s, 9H), 1.42 (s, 9H), 3.23 (m, 8H), 4.40
(s, 1H), 4.94 (s, 1H), 5.17 (m, 1H), 6.86 (s, 2H), 7.38 (m, 2H), 8.15 (m, 2H).
Rf (AcOEt/MeOH 1/1): 0.5. MS (M+H+): 510.
Glycyl-N-(2-aminoethyl)-4- nitrophenylalaninamide hydrochloride 14
Synthesis was identical to compound 4 starting with 0.38 g of 13 (0.746 mmol).
%
A white powder (0.21 g, 92%) was obtained.
1H NMR (D2O) (d, ppm): 3.27 (m, 6H), 3.76 (s, 2H), 4.23 (t, 1H,
J ¼ 7:4 Hz), 7.51 (d, 2H, J ¼ 8:5 Hz), 8.25 (d, 2H, J ¼ 8:6 Hz).
Rf (MeOH): 0.15. MS (M+H+): 310.
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SAMARIUM-153 BIFUNCTIONAL CHELATING AGENT
121
1-(4-Nitrobenzyl)triethylenetetraamine 15
Synthesis was identical to compound 5, starting with 0.16 g of compound 14
%
(0.51 mmol), 5.1 ml of BH3/THF 1 M (5.1 mmol). Compound 15 (0.115 g,
79%) was obtained as a yellow oil.
1H NMR (CDCl3) (d, ppm): 1.67 (s, 6H), 2.67–2.78 (m, 10H), 2.80–3.45
3
(m, 2H), 3.67 (t, 1H, J ¼ 7:5 Hz), 7.37 (d, 2H, J ¼ 8:7 Hz), 8.17 (d, 2H,
J ¼ 8:7 Hz). MS (M+H+): 282.
1-(4-Nitrobenzyl)-triethylenetetraaminepentaacetyl tertiobutyl ester 16
Synthesis was identical to compound 6, starting with 0.45 g of compound 15
%
(1.6 mmol), 0.29 g of potassium iodide (1.76 mmol), 2.4 g of 1,8-bis-(dimethy-
lamino)naphthalene (11.20 mmol). Compound 16 was obtained as a yellow oil
(1.2 g, 78%).
1H NMR (CDCl3) (d, ppm): 1.43 (s, 18H), 1.50 (s, 36H), 2.70–2.88 (m, 12H),
3.27–3.46 (m, 1H), 3.46 (s, 12H), 7.48 (d, 2H, J ¼ 8:82 Hz), 8.11 (d, 2H,
J ¼ 8:82 Hz). Rf (AcOEt/CHCl3 1/1): 0.5. MS (M+H+): 966.
1-(4-Aminobenzyl)-triethylenetetraaminepentaacetyl tertiobutyl ester 17
Catalytic hydrogenation of 0.5 g of compound 6 (0.52 mmol) in 25 ml of
%
MeOH was carried out over 50 mg of 10% Pd/C (0.1eq.; w/w) at atmospheric
pressure. After 3 h, the catalyst was filtered off (Celite) and the filtrate was
removed under reduced pressure. Compound 17 (0.48 g, 100%) was obtained
as an orange powder.
1H NMR (CDCl3) (d, ppm): 1.42 (s, 18H), 1.47 (s, 36H), 2.68–2.85 (m, 14H),
3.27–3.51 (m, 1H), 3.38 (s, 12H), 6.60 (d, 2H, J ¼ 8:2 Hz), 6.99 (d, 2H,
J ¼ 8:2 Hz). MS (M+H+): 936.
1-(4-Aminobenzyl)-triethylenetetraaminepentaacetic acid 18
To 0.45 g of compound 17 (4.81 mmol) dissolved in 2 ml of acetone was added
5 ml of HCl 6 M. After stirring for 16 h at room temperature, the reaction
mixture was dried under vacuum, to obtain a white solid (0.375 g, 100%).
1H NMR (D2O) (d, ppm): 3.0–3.4 (m, 13H), 3.92 (s, 12H), 7.40 (m, 4H).
1-(4-Isothiocyanatobenzyl)-triethylenetetraaminepentaacetic acid 19
Synthesis was identical to compound 9, starting with 20 ml of thiophosgene
%
(0.27 mmol) and 4.2 mg of 18 (5.3 mmol) in 1 ml of HCl 0.5 M. Compound 9
%
(4.4 mg, 98%) was obtained as a pale yellow solid.
MS (M+H+): 642. HPLC: solvent A: TFA 0.1%, Solvent B: CH3CN, flow
rate: 1 ml/min, gradient: 0–5 mn 100%A isocratic, 5–35 mn 100%A to 100%B.
Retention time: 19.1 min. IR: 2100 cmꢀ1 (SCN).
Copyright # 2005 John Wiley & Sons, Ltd.
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122
L. MORANDEAU ET AL.
Radiolabelling and serum media stability
The control solution contained, 5 ml of 153SmCl3 (0.370 nmol) and 595 ml of
citrate buffer (0.02 M, pH5). For the radiolabelling, 5 ml of 153SmCl3
(0.370 nmol), 550 ml of a solution at 135 nmol/ml of the two different chelating
agents in a citrate buffer (0.02 M, pH5) and 45 ml of citrate buffer (0.02 M,
pH5) were stirred for 1 h at room temperature.
Then 250 ml of each solution was added to 5 ml of human serum and
incubated at 378C, under stirring. At different time intervals (0, 1, 4, 24, and
144 h), 500 ml of each serum media were purified on a PD-10 column
and eluted with 10 ml of PBS. Twenty tubes of 0.5 ml were collected for each
time and their activity counted on a g counter.
Conclusion
We have described the synthesis of a novel bifunctional chelating agent, the 3-
(4-isothiocyanatobenzyl)TTHA that can be easily linked to any vector
biomolecules. The synthesis of its position isomer, the 1-(4-isothiocyanato-
benzyl)TTHA was also improved. Unlike the 153Sm-1-(4-aminobenzyl)TTHA
complex, the 153Sm-3-(4-aminobenzyl)TTHA complex showed remarkable
serum media stability. Future animal studies will be carried out to confirm the
interest of this new bifunctional chelating agent for in vivo applications in
radioimmunotherapy.
Acknowledgements
We thank Schering-Cis Bio International for supplying the 153SmCl3 solution.
We also want to thank ‘La Ligue departementale Contre le Cancer de Vendee’
´ ´
for a doctoral grant.
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