Synthesis of novel bifunctional Schiff-base ligands derived from condensation of 1-(p-nitrobenzyl)ethylenediamine and 2-(p-nitrobenzyl)-3-monooxo-1,4,7-triazaheptane with salicylaldehyde

Article abstract of DOI:10.1039/b300621m

Two potentially tetradentate (N₂O₂) and pentadentate (N₃O₃) bifunctional Schiff-base ligands, N,N′-bis(2-hydroxybenzyl)-1-(p-aminobenzyl)ethylenediamine (7) and N,N′-bis(2-hydroxybenzyl)-2-(p-aminobenzyl)-3-monooxo-1,4,7- triazaheptane (5′) have been prepared and characterized by various spectroscopic methods (IR, FAB-MS, NMR). They are derived from the condensation reactions of the C-functionalized diamines 1-(p-nitrobenzyl)ethylenediamine and 2-(p-nitrobenzyl)-3-monooxo-1,4,7-triazaheptane with 2.1 equiv. of salicylaldehyde. The first complexation trials with ^99mTc are reported.

Full text of DOI:10.1039/b300621m

View Article Online / Journal Homepage / Table of Contents for this issue
Synthesis of novel bifunctional Schiff-base ligands derived
from condensation of 1-(p-nitrobenzyl)ethylenediamine and
2-(p-nitrobenzyl)-3-monooxo-1,4,7-triazaheptane with
salicylaldehydey
a
Anil Kumar Mishra,*z Puja Panwar,a Madhu Chopra,a Rakesh Kumar Sharmaa and
Jean-Francois Chatalb
a
Department of Radiopharmaceutical Chemistry, Institute of Nuclear Medicine and Allied
Sciences, Brig. S. K. Mazumdar Road, Delhi 110 054, India. E-mail: akmishra@inmas.org
INSERM-U463, Institut de Biologie, 9 quai Moncousu, 44093, Nantes cedex, France
b
Received (in Montpellier, France) 15th January 2003, Accepted 21st February 2003
First published as an Advance Article on the web 2nd June 2003
Two potentially tetradentate (N2O2) and pentadentate (N3O3) bifunctional Schiff-base ligands,
N,N0-bis(2-hydroxybenzyl)-1-(p-aminobenzyl)ethylenediamine (7) and N,N0-bis(2-hydroxybenzyl)-
2-(p-aminobenzyl)-3-monooxo-1,4,7-triazaheptane (50) have been prepared and characterized by various
spectroscopic methods (IR, FAB-MS, NMR). They are derived from the condensation reactions of the
C-functionalized diamines 1-(p-nitrobenzyl)ethylenediamine and 2-(p-nitrobenzyl)-3-monooxo-1,4,7-
triazaheptane with 2.1 equiv. of salicylaldehyde. The first complexation trials with 99mTc are reported.
Radiolabeled (bio)molecules are potentially useful tools for
cancer diagnosis and therapy.1–3 Some radioactive metals, such
as 99mTc(VII), have physical properties that are well-suited for
tumor imaging with monoclonal antibodies, while others, such
as 186Re(VII) and 188Re(VII), have cytotoxic properties that can
be exploited for therapy by antibody-directed tumor target-
ing.2–7 To achieve targeted imaging and therapy the radio-
isotope should be irreversibly bound to the biological
molecules. If this condition is met then the tissue specificity
and the biological half-life of the antibody in vivo and the phy-
sical half-life of the decaying radioisotope will determine the
total radiation dose delivered to both cancerous and healthy
cells.8 If this condition is not met, most importantly with
b-emitting radioisotopes, then the premature release of radio-
nuclide invariably leads to localization of the activity in sensi-
tive organs, such as the kidney, liver, and the bone marrow,
which can result in lethal radiotoxic effects. The most common
approach used to radiolabel (bio)molecules has involved the
use of bifunctional chelating agents.9
In the literature several Schiff-base ligands have been
reported and most recently, heptadentate (N3O4) ones with
various ring-substituted salicylaldehydes have been prepared
and their coordination chemistry with a number of metal
ions has been extensively investigated.10–26 Another class of
N-functionalized aminephenol Schiff-base ligands such as
1,7-bis(2-hydroxybenzyl)-4-(p-aminobenzyl)diethylenetriamine
(Bhabdt)27 has been developed and used for the conjugation of
proteins to label with radionuclides.
Schiff-base ligands and preliminary conjugation and 99mTc
labeling studies. The specific ligands that we have developed
are for conjugation of biologically active molecules for clinical
applications and are derived from the optically active amino
acid L-phenylalanine. The synthesis of potentially tetradentate
and pentadentate ligands by the reaction of the C-substituted
diamines 1-(p-nitrobenzyl)ethylenediamine and 2-(p-nitro-
benzyl)-3-monooxo-1,4,7-triazaheptane with salicylaldehyde
(see Schemes 1 and 2 below) provides a new class of optically
active Schiff-base ligands.
Experimental
Materials and methods
The corresponding diamine derivative of the optically active
amino acid was synthesized in our laboratory by the standard
procedure.29 Salicylaldehyde, sodium borohydride, stannous
chloride dihydrate and other solvents (Aldrich) were used as
received. Reduction of amide was performed under argon
atmosphere. TLC was run on plastic-backed silica gel plates
(0.2 mm thick silica gel 60 F-254, E. Merck, Germany) using
a 10% w/v aqueous ammonium acetate–CH3OH (1:1 v/v)
solution as eluent. HPLC was carried out on a Waters 600E
System (Millipore Corporation, Milford, MA, USA) equipped
with titanium piston washing pump heads. Solvents were
mixed using a Dynamax dual-chamber dynamic mixer (Tita-
nium). UV absorbance was measured using an absorbance/
fluorescence monitor (Waters 2487) at 254 or 354 nm. A
Gilson model 201 fraction collector was used. Reversed-phase
HPLC was performed at room temperature with a C18
reversed-phase column, generally using a gradient of CH3OH
and 0.1 M ammonium acetate (pH 6) or 0.05% trifluoroacetic
acid at a flow rate of 1 mL minꢀ1. All the solvents for HPLC
and reaction mixtures were filtered through a nylon 66 Milli-
pore filter (0.45 mm) prior to use. NMR data were recorded
on a Bruker 400 MHz apparatus operating near 400 (1H) or
As part of our efforts to develop bifunctional chelating
agents,28 this paper describes a new class of bifunctional
y Electronic supplementary information (ESI) available: mass spectra
of compounds 5–7 and 30–50. See http://www.rsc.org/suppdata/nj/
b3/b300621m/.
z Max-Planck-Institute for Biological Cybernetics, Spemannstrasse
38, D-72076 Tubingen, Germany. Tel: +49 707 1601 1679; Fax: +49
¨
707 1601 652; E-mail: anil.mishra@tuebingen.mpg.de.
1054
New J. Chem., 2003, 27, 1054–1058
DOI: 10.1039/b300621m
This journal is # The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2003

View Article Online
Scheme 1 Reagents: (i) H2SO4(96%)–HNO3 . (ii) MeOH/HCl(g) . (iii) NH3(g) . (iv) BH3ꢂTHF. (v) C7H6O2 . (vi) NaBH4 . (vii) SnCl2ꢂ2H2O.
100 (13C) MHz or a Brucker AC 200 operating near 200 (1H)
or 50 (13C) MHz. Chemical shifts were relative to either HDO
(4.70 ppm) or residual CHCl3 (7.24 ppm).
water and neutralized with 30% ammonium hydroxide. Dur-
ing neutralization precipitation started; the reaction mixture
was kept at room temperature for 45 min and then filtered
to separate out the precipitate. The precipitate was washed
with water until the washings were neutral to litmus. The
solid product was recrystallized with hot water to yield (L)-
p-nitrophenylalanine. IR (KBr) n/cmꢀ1: 1591 and 1347 (NO2);
1H NMR (250 MHz, CDCl3): d 7.87 (d, 2H), 7.23 (d, 2H),
4.20 (t, 1H) 3.12 (m, 2H); anal. calcd. for C9H10N2O4 : C,
51.43; H, 4.80; N, 13.33; O, 30.45; found: C, 51.60; H, 4.78;
N, 13.28; O, 30.60; FAB-MS: Found: m/z 211 [M + H]+; calcd
for C9H10N2O4 : 210.
Syntheses
(L)-p-Nitrophenylalanine (1). L-Phenylalanine (99.0 g, 0.6
mol) was dissolved in 96% sulfuric acid (240 mL) with vigorous
stirring at 10 ꢁC and concentrated nitric acid (30 mL) was
added over a period of 35 min with vigorous stirring at 0–5 ꢁ;
the reaction mixture was then stirred for an additional 1 h.
The reaction mixture was poured on 800 mL of ice cold
Scheme 2 Reagents: (i) C2H8N2 . (ii) C7H6O2 . (iii) NaBH4 . (iv) SnCl2ꢂ2H2O.
New J. Chem., 2003, 27, 1054–1058
1055

View Article Online
Synthesis of the methyl ester of p-nitrophenylalanine (2). p-
Nitrophenylalanine (10.0 g, 47.6 mmol) was treated with dry
methanol (150 mL) and saturated with HCl gas at 0 ꢁC till dis-
solution and left to stir at room temperature for 2 h. The clear
solution thus obtained was left at 0 ꢁC for 3 h; during this time
most of the product precipitated out. The product was filtered,
washed with chilled methanol and dried under reduced pres-
sure (1 mm Hg) for 3 h, which yielded 10.9 g (88.3%). TLC
of the free amino ester developed in CHCl3–MeOH (4:1)
12.92 (br s, 1H), 8.53 (s, 1H), 8.23 (d, 2H, J ¼ 8.0), 8.04 (s,
1H), 6.82–7.30 (m, 10H), 4.12 (m, 1H), 3.53 (dd, 1H, J ¼
15.0, 7.50), 3.46 (dd, 1H, J ¼ 15.0, 7.50), 3.39 (dd, 1H,
J ¼ 14.0, 7.50), 3.26 (dd, 1H, J ¼ 14.0, 7.50); FAB-MS:
Found: m/z 404 [M + H]+; calcd for C23H21N3O4 : 403; anal.
calcd. for C23H21N3O4 : C, 68.47; H, 5.25; N, 10.42; O,
15.86; found: C, 68.52; H, 5.32; N, 10.51; O, 15.93.
N,N0-bis(2-hydroxybenzyl)-1-(p-nitrobenzyl)ethylenediamine (6).
To a solution of Schiff base 5 (3.2 g, 7.86 mmol in 100 mL
methanol), NaBH4 (5.0 g, 132.17 mmol) was added in several
portions and the reaction mixture was allowed to stir at 60 ꢁC
for 2 h. Solvent was removed under reduced pressure and the
resulting solid was dissolved in CHCl3 (200 mL). The organic
phase was separated with water (5 ꢃ 200 mL), dried over anhy-
drous MgSO4 , filtered and the solvent evaporated to dryness
under vacuum to yield 6 as a yellow brown oil in 90% yield.
Reverse-phase C18 HPLC: solvent A, 0.01% TFA; solvent B,
MeOH; 15–65% B, 0–25 min.; 65–100% B, 30–35 min.; 100–
15% B, 35–40 min.; product peak at 17.67 min.1H NMR
(250 MHz, CDCl3): 8.23 (d, 2H, J ¼ 8.0), 6.82–7.30 (m, 10H),
3.75–3.92 (m, 5H), 3.53 (dd, 1H, J ¼ 15.0, 7.50), 3.46 (dd, 1H,
J ¼ 15.0, 7.50), 3.39 (dd, 1H, J ¼ 14.0, 7.50), 3.26 (dd, 1H,
J ¼ 14.0, 7.50); 13C NMR: a total of 23 peaks have been
obtained at 38.57, 48.31, 48.35, 50.16, 50.46, 52.74, 57.76,
116.37, 116.51, 119.49, 119.58, 122.17, 122.28, 123.96, 128.50,
128.70, 129.12, 129.18, 129.94, 145.75, 146.85, 157.51 and
157.62; FAB-MS: Found: m/z 408 [M + H]+; calcd. for
C23H25N3O4 : 407; anal. calcd. for C23H25N3O4 : C, 67.80; H,
6.18; N, 10.31; O, 15.71; found: C, 67.85; H, 6.22; N, 10.40;
O, 15.79.
1
revealed an Rf value of 0.85–0.88. H NMR (250 MHz, D2O):
d 8.19 (d, 2H, J ¼ 10.0), 7.48 (d, 2H, J ¼ 10.0), 4.48 (t, 1H,
J ¼ 5.0), 3.78 (s, 3H), 3.36 (m, 2H); FAB-MS: Found: m/z
225 [M + H]+; calcd for C10H12N2O4 : 224; anal. calcd. for
C10H12N2O4 : C, 53.57; H, 5.39; N, 12.49; O, 28.54; found:
C, 53.61; H, 5.40; N, 12.60; O, 28.65.
p-Nitrophenylalanine amide (3). A slurry of 2 (10.97 g, 42.1
mmol) in dry methanol (5 mL) was treated with triethylamine
(6.45 mL). Anhydrous ether (200 mL) was added and the solu-
tion was stirred at ꢀ10 ꢁC for 2 h. The triethylamine hydro-
chloride was filtered off and the filtrate concentrated to a
viscous orange oil. The oil was added to dry methanol (250
mL) and saturated with NH3 gas at ꢀ5 ꢁC in a fume hood,
tightly stoppered and kept at 0–5 ꢁC for 24 h. The precipitated
product was collected and dried under reduced pressure. The
filtrate was concentrated to dryness and the remaining solid
combined with the precipitate gave the amide in 92% yield.
1H NMR (250 MHz, Me2SO-d6): d 8.19 (d, 2H, J ¼ 8.0),
7.48 (d, 2H, J ¼ 8.0), 7.41 (s, 1H), 7.00 (s, 1H), 3.50 (t, 1H,
J ¼ 3.0), 3.09 (dd, 1H, J ¼ 8.0, 300), 2.81 (dd, 1H, J ¼ 8.0,
300); FAB-MS: Found: m/z 210 [M + H]+; calcd for C9H11N3-
O3 : 209; anal. calcd. for C9H11N3O3 : C, 51.67; H, 5.30; N,
20.09; O, 22.94; found: C, 51.02; H, 5.38; N, 21.03; O, 23.01.
N,N0-bis(2-hydroxybenzyl)-1-(p-aminobenzyl)ethylenediamine
(7). To a suspension of product 6 (0.330 g, 0.81 mmol in 200
mL ethanol), SnCl2ꢂ2H2O (0.914 g, 4.05 mmol) was added in
small amounts. The reaction mixture was refluxed for 6 h.
The reaction was monitored by HPLC using a C18 column;
the reaction only went to 80% completion to the amino deriva-
tive. One additional equivalent of SnCl2ꢂ2H2O was added and
the mixture allowed to stir at the ethanol boiling point for an
additional 3 h. The reaction mixture was cooled to room tem-
perature and poured onto ice. The pH was adjusted to 8 using
NaOH (2 M) and the reaction mixture extracted with ethyl
acetate (4 ꢃ 100 mL). The combined ethyl acetate layers was
dried over anhydrous MgSO4 and evaporated under reduced
pressure. The brownish solid was purified on HPLC and lyo-
philized on Lyovac to give 7 (yield 60%).1H NMR (250
MHz, CDCl3): d 8.23 (d, 2H, J ¼ 8.0), 6.82–7.30 (m, 10H),
3.75–3.92 (m, 5H), 3.53 (dd, 1H, J ¼ 15.0, 7.50), 3.46 (dd,
1H, J ¼ 15.0, 7.50), 3.39 (dd, 1H, J ¼ 14.0, 7.50), 3.26 (dd,
1H, J ¼ 14.0, 7.50); 13C NMR: a total of 23 peaks have been
obtained at 38.57, 42.31, 48.81, 50.16, 50.46, 52.74, 57.76,
116.37, 116.51, 119.49, 119.58, 122.17, 122.28, 123.96, 128.50,
128.70, 129.12, 129.18, 129.94, 145.75, 146.85, 157.51, and
157.62; FAB-MS: Found: m/z 378 [M + H]+; calcd. for C23-
H27N3O2 : 377; anal. calcd. for C23H27N3O2 : C, 73.18; H, 7.21;
N, 11.13; O, 8.48; found: C, 73.22; H, 7.24; N, 10.93; O, 8.77.
1-(p-Nitrobenzyl)ethylenediamine (4). To a suspension of 3
(8.12 g, 38.9 mmol in 150 mL dry THF), 200 mL of 1 M dibor-
ane in THF at 0 ꢁC was added with a syringe under argon
atmosphere and the reaction mixture stirred for 30 min. The
temperature of the reaction was raised to 60 ꢁC and the reac-
tion mixture was refluxed for 24 h. After cooling, 20 mL of
water was carefully added into the solution to destroy the
excess of diborane. The solvent was evaporated under reduced
pressure to half the volume and about 100 mL of 18% aqueous
hydrochloric acid was added. The reaction mixture was stirred
overnight at room temperature and then at 80–100 ꢁC for 15
min. The mixture was cooled, filtered and sufficient ammo-
nium hydroxide was added to the filtrate to give pH > 12.
The solution was extracted with chloroform (5 ꢃ 100 ml).
The combined chloroform extracts were dried over anhydrous
magnesium sulfate, filtered and evaporated under reduced
pressure, yielding 88% product as a viscous orange oil. 1H
NMR (250 MHz, D2O): d 8.23 (d, 2H, J ¼ 8.0), 7.64 (d, 2H,
J ¼ 8.0), 4.12 (m, 1H), 3.53 (dd, 1H, J ¼ 15.0, 7.50), 3.46
(dd, 1H, J ¼ 15.0, 7.50), 3.39 (dd, 1H, J ¼ 14.0, 7.50), 3.26
(dd, 1H, J ¼ 14.0, 7.50); FAB-MS: Found: m/z 196 [M +
H]+; calcd for C9H13N3O2 : 195; anal. calcd. for C9H13N3O2 :
C, 55.37; H, 6.71; N, 21.52; O, 16.39; found: C, 55.40; H,
6.73; N, 21.60; O, 16.50.
2-(p-Nitrobenzyl)-3-monooxo-1,4,7-triazaheptane (20). The
methyl ester of p-nitrophenylalanine (2; 4.4 g, 17.9 mmol)
was dissolved in neat ethylenediamine at 0–5 ꢁC and the reac-
tion mixture was stirred for 24 h at ambient temperature. By
the end of this time, the reaction was completed (as monitored
by TLC) and remaining ethylenediamine was removed under
reduced pressure. The obtained oily viscous product was
dissolved in chloroform (100 mL) and allowed to evaporate
slowly at room temperature; the brown solid thus precipitated
was filtered and washed with chilled chloroform to give 20
in 70% yield. 1H NMR (250 MHz, D2O): d 8.23 (d, 2H,
J ¼ 8.0), 7.54 (d, 2H, J ¼ 8.0), 4.01 (t, 1H, J ¼ 7.00), 3.27
N,N0-bis(salicylidene)-1-(p-nitrobenzyl)ethylenediamine (5).
1-(p-Nitrobenzyl) ethylenediamine 4 (0.450 g, 2.30 mmol)
was taken up in 50 mL acetonitrile. Salicylaldehyde (0.917 g,
4.84 mmol) in 40 mL acetonitrile was added at room tempera-
ture and the reaction mixture stirred for 20 h at ambient
temperature. The reaction was monitored by TLC. After
completion of the reaction, the solvent was removed to dryness
and the residue was dissolved in methanol (50 mL), filtered,
and the methanol evaporated off to dryness. The yellow solid
obtained was dried under vacuum to give the desired product
1
in 85% yield. H NMR (250 MHz, CDCl3): d 3.05 (br s, 1H),
1056
New J. Chem., 2003, 27, 1054–1058

View Article Online
(m, 1H), 3.04 (m, 3H), 2.77 (m, 2H); FAB-MS: Found: m/z
253 [M + H]+; calcd for C11H16N4O3 : 252; anal. calcd. for
C11H16N4O3 : C, 52.37; H, 6.39; N, 22.21; O, 19.03; found:
C, 52.42; H, 6.43; N, 22.17; O, 19.11.
On the basis of chromatographic analysis the radiolabeling
efficiency was found to be > 98%. Labeled complex was incu-
bated at 37 ꢁC in fresh human serum at a concentration of 100
nM mLꢀ1. Stability as assessed by TSK 3000 gel filtration
HPLC showed that the metal ion was intact under physio-
logical conditions and not taken up by the protein. Unbound
radioactivity was less than 0.5% in 7 h.
N,N0-Bis(salicylidene)-2-(p-nitrobenzyl)-3-monooxo-1,4,7-triaza-
heptane (30). To a solution of 20 (0.232 g, 0.92 mmol in 50 mL
methanol), salicylaldehyde (0.254 g, 2.4 mmol) in 60 mL of
methanol was added dropwise over 30 min. The reaction mix-
ture was allowed to stir at 40 ꢁC for 18 h and monitored by
TLC. After completion of the reaction solvent was removed
to dryness and the residue dissolved in methanol (50 mL), fil-
tered, and methanol evaporated to reduce the volume to half.
The obtained yellow solid was filtered to give the desired pro-
Conjugation
The amino group of the Schiff-base ligand was converted to the
isothiocyanato group by reacting with thiophosgene at pH 2.
Compounds 7 and 50 (1.5 mmol) in 5 mL of 3 M HCl were
added to 3 mL of CSCl2 (85% in CCl4) at room temperature.
The reaction mixture was stirred vigorously for 4 h. The aqu-
eous phase was washed with CHCl3 (4 ꢃ 5 mL) in a fume hood
to remove excess CSCl2 and then purified by reverse phase
HPLC. Conjugation of EGFr monoclonal antibody was per-
formed by adding 25 mL of the 20 mM chelate solution to
300 mL of a solution containing 3 mg of antibody in 0.1 M
sodium phosphate, pH 7. Saturated trisodium phosphate solu-
tion (40 mL) was added to make the pH 8.5. The reaction mix-
ture was incubated at 37 ꢁC for 60 min and then subjected to
centrifuged column gel chromatography, which removed the
unreacted chelate, and the buffer changed to 0.1 M sodium
acetate, pH 5.5. UV absorbance at 280 nm for the centrifuged
column effluent was used to determine the antibody concentra-
tion and 57Co assay used to obtain the bound chelate concen-
tration. In short, 1 nmol of conjugated antibody, 2 nmol
of 59Co and a tracer of 57Co were mixed and incubated at
pH 5.5 at room temperature for 30 min. An aliquot was
applied to thin layer chromatography plates with ITLC-SG
(instant thin layer chromatography-silica gel; Gelman
Sciences, Ann Arbor, MI, USA) with ammonium acetate–
CH3OH (1:1 v/v) solution as eluent. Free cobalt migrates to
Rf ¼ 1.0 while labeled cobalt with immuniconjugate stays at
Rf ¼ 0. The 57Co binding assay indicates 2.1 ꢄ 0.1 chelate
molecules were conjugated per antibody molecule.
1
duct in 88% yield. H NMR (250 MHz, CDCl3): d12.85 (br s,
1H), 12.03 (br s, 1H), 8.28 (s, 1H), 8.12 (d, 2H,), 8.02 (s, 1H),
6.81–7.39 (m, 10H), 6.13(t, 1H), 4.12 (t, 1H), 3.23–3.72 (m,
6H); FAB-MS: Found: m/z 461 [M + H]+; calcd for C25H24-
N4O5 : 460; anal. calcd. for C25H24N4O5 : C, 65.21; H, 5.25; N,
12.17; O, 17.37; found: C, 65.25; H, 5.30; N, 12.26; O, 17.43.
N,N0-Bis(2-hydroxybenzyl)-2-(p-nitrobenzyl)-3-monooxo-1,4,7-
triazaheptane (40). To a solution of Schiff base 30 (1.5 g, 3.2
mmol in 150 mL methanol), NaBH4 (2.5 g, 66 mmol) was
added in several portions and the reaction mixture was allowed
to stir at 60 ꢁC for 2 h. Solvent was removed under reduced
pressure and the resulting solid was dissolved in CHCl3 (200
mL) and washed with water (5 ꢃ 200 mL). The organic layers
were separated, dried over anhydrous MgSO4 , filtered and the
solvent was removed to dryness under reduced pressure to give
an oily yellow brown product (yield 90%). Reverse-phase C18
HPLC: solvent A, 0.01% TFA; solvent B, MeOH; 15–65% B,
0–25 min.; 65–100% B, 30–35 min.; 100–15% B, 35–40 min.;
product peak at 17.9 min. 1H NMR (250 MHz, CDCl3): d
8.12 (d, 2H), 6.73–7.31 (m, 10H), 6.34 (t, 1H), 4.00 (t, 1H),
3.23–3.72 (m, 10H); FAB-MS: Found: m/z 465 [M + H]+;
calcd for C25H28N4O5 : 464; anal. calcd. for C25H28N4O5 : C,
64.64; H, 6.08; N, 12.06; O, 17.22; found: C, 64.24; H, 6.11;
N, 12.03; O, 17.32.
Results and discussion
N,N0-Bis(2-hydroxybenzyl)-2-(p-aminobenzyl)-3-monooxo-1,4,7-
triazaheptane (50). To a suspension of 40 (0.390 g, 0.84 mmol in
ethanol 200 mL), SnCl2ꢂ2H2O (1.59 mg, 7.05 mmol) was added
at room temperature. The reaction mixture was refluxed for
6 h with the reaction being monitored by HPLC using a C18
column. One additional equivalent of SnCl2ꢂ2H2O was added
and the mixture allowed to stir at the boiling point of ethanol
for an additional 3 h. The reaction mixture was cooled to room
temperature and poured onto ice. The pH was adjusted to 8
using NaOH (2 M) and the mixture was extracted with ethyl
acetate (400 mL). The ethyl acetate layer was dried over anhy-
drous MgSO4 and evaporated under reduced pressure. The
brownish solid was purified on HPLC and lyophilized on Lyo-
The preparation of diamines has been achieved in high yields
starting from optically active L-(ꢀ)-phenylalanine followed
by nitration, esterification, aminolysis and reduction with
diborane and SnCl2ꢂ2H2O. The reported method29 for the
reduction of amide with diborane to the corresponding amine
followed by HCl gas often gives lower yields, which can be
increased by passing HCl gas in several installments, while in
the present studies we have used 6 M HCl, which provides bet-
ter yield and easy workup. The previously described method29
for the reduction of the nitro group uses 10% Pd/C under
hydrogen atmosphere at 4 ꢁC in basic media while we have
followed the method reported in the literature30 using
SnCl2ꢂ2H2O in absolute ethanol, which gave quantitative
yields. All the products were fully characterized on the basis
of spectral studies.
1
vac (yield 70%). H NMR (250 MHz, CDCl3): d 8.12 (d, 2H),
6.73–7.31 (m, 10H), 6.34 (t, 1H), 4.00 (t, 1H), 3.23–3.72 (m,
10H); FAB-MS: Found: m/z 435 [M + H]+; calcd for C25H30-
N4O3 : 434; anal. calcd. for C25H30N4O3 : C, 69.10; H, 6.96; N,
12.89; O, 11.05; found: C, 69.16; H, 7.01; N, 12.93; O, 11.13.
The Schiff-base complexing agents (7 and 50) obtained by the
condensation of a salicylaldehyde with the corresponding func-
tionalized amines are bifunctional in nature and can be used
for conjugation and radiolabeling as reported in the litera-
ture,27 with better stability under physiological conditions by
conversion of primary amines to the isothiocyanato group. It
has been noted that carbon-backbone-substituted chelating
agents form more stable radiolabeled complexes or immuno-
conjugates, which have higher serum stability than the N-
attached (including amide-attached) derivatives. The ligand
in the –NCS derivative was found to be stable when stored
at ꢀ20 ꢁC in 0.3 M HCl.
Complexation studies
Preliminary complexation studies with technetium-99m metal
ion withꢀcompounds 7 and 50 were carried out by the reduction
of TcO4 (500–740 MBq) with stannous chloride dihydrate
(3.4 mM) in the presence of ligands (10 mM) in the pH range
5.5 to 6.0 at 25 ꢁC. Strip chromatography with ITLC-SG (Gel-
man Sciences, Ann Arbor, MI, USA) using acetone eluent, in
which pertechnetate migrates, and using saline eluent, in which
both labeled chelate and pertechnetate migrate, were used to
determine the radiochemical purity of the labeled complexes.
Monoclonal antibodies against abundantly expressed
antigens could prove to be effective tracer molecules for
New J. Chem., 2003, 27, 1054–1058
1057

View Article Online
R. W. Kozak, A. Raubitschek, S. Mirzadeh, M. W. Brechbiel,
R. Junghaus, O. A. Gansow and T. A. Waldmann, Cancer Res.,
1989, 49, 2639.
J. L. Humm, Nucl. Med., 1986, 27, 1490.
7
radioimmunodiagnosis. The mAb ior egf/r3 recognizes EGFr
and inhibits the binding to its ligand. This antigen has been
used successfully as a target for imaging and therapy because
of its over-expression in tumors of epithelial origin.31
Although normal cells also express EGFr, the elevated number
of receptors on tumor cells confers a degree of targeting speci-
ficity. Therefore, the tumor cells can proportionally bind more
radiolabeled mAbs.
Conjugation was performed in the ratio of 1:20. UV absor-
bance and the 57Co binding assay indicated 2.1 ꢄ 0.1 chelate
molecules per monoclonal antibody, which was labeled with
a specific activity of 20–30 mCi mgꢀ1 of protein. Labeling effi-
ciency was measured by ascending paper chromatography on
ITLC-SG strips. Preliminary complexation with 99mTc was
found to give sufficiently stable complexes under physiological
conditions. In conclusion, the preliminary studies with these
novel Schiff-base ligands are encouraging to carry out further
in vivo experiments for targeted imaging of human tumors in
animal models.
8
9
M. K. Moi, C. F. Meares, M. J. McCall, W. C. Cole and S. J.
DeNardo, Anal. Biochem., 1985, 148, 249.
10 K. Bertoncello, G. D. Fallon, K. S. Murray and E. R. Tiekink.,
Inorg. Chem., 1991, 30, 3562.
11 W. Mazurek, K. J. Berry, K. S. Murray, M. J. O’Connor, M. R.
Snow and A. G. Wedd, Inorg. Chem., 1982, 21, 3071.
12 F. L’Eplattenier, I. Murase and A. E. Martell, J. Am. Chem. Soc.,
1967, 89, 837.
13 M. C. Kuchta, J. M. Hahn and G. Parkin, J. Chem. Soc. Dalton
Trans., 1999, 3559.
14 R. Kasahara, M. Tsuchimoto, S. Ohba, K. Nakajima, H. Ishida
and M. Kojima, Inorg. Chem., 1996, 35, 7661.
15 C. J. Mathias, Y. Sun, J. M. Connett, G. W. Philpot, M. Welch
and A. E. Martell, Inorg. Chem., 1990, 29, 1475.
16 Z. Liu and F. Anson, Inorg. Chem., 2000, 39, 274.
17 H. Keypour, S. Salehazadeh and R. V. Parish, Molecules, 2002,
7, 140.
18 J.-P. Costes, A. Dupuis, G. Commenges, S. Lagrave and J.-P.
Laurent, Inorg. Chim. Acta, 1999, 285, 49.
19 J.-P. Costes, F. Dahan, A. Dupuis, S. Lagrave and J.-P. Laurent,
Inorg. Chem., 1998, 37, 153.
20 P. Bhattacharyya, J. Parr, A. T. Ross and A. M. Z. Slawin,
J. Chem. Soc, Dalton Trans., 1998, 3149.
Acknowledgements
21 M. Kanesato, T. Yokoyama, O. Itabashi, T. M. Suzki and
M. Shiro, Bull. Chem. Soc. Jpn., 1996, 69, 1297.
22 A. Aguiari, E. Bullia, U. Casellato, P. Guerriero, S. Tamburini
and P. A. Vigato, Inorg. Chim. Act., 1994, 219, 135.
23 (a) R. Robson, Inorg. Nucl. Chem. Lett., 1970, 6, 125; (b) R.
Doedens, Prog. Inorg. Chem., 1976, 21, 209.
We are grateful to Professor Roger Guilard (Universite de
Bourgogne, Dijon, France) for helpful discussions. This work
is supported by DRDO, Ministry of Defence, Government of
India.
24 D. A. Atwood, M. S. Hill, J. A. Jegier and D. Rutherford,
Organometallics, 1997, 16, 2659.
25 D. A. Atwood, Coord. Chem. Rev., 1997, 165, 267.
26 M. E. Marmion, S. R. Woulfe, W. L. Newman, G. Pilcher and
D. L. Nosco, Nucl. Med. Biol., 1996, 23, 567.
27 M. R. A. Pillai, C. S. John and D. E. Toroutner, Bioconjugate
Chem., 1990, 1, 191.
28 A. K. Mishra and J. F. Chatal, New J. Chem., 2001, 25, 336.
29 M. W. Brechbiel, O. A. Gansow, R. W. Atcher, J. Schlom,
J. Esteban, D. E. Simpson and D. Colcher, Inorg. Chem., 1986,
25, 272.
30 S. Morrone, D. Guillon and D. W. Bruce, Inorg. Chem., 1996, 35,
7041.
References
1
2
C. F. Meares, Int. J. Radiat. Appl. Instrum., Part B: Nucl. Med.
Biol., 1986, 13, 311.
O. A. Gansow, Int. J. Radiat. Appl. Instrum. Part B: Nucl. Med.
Biol., 1991, 18, 369.
3
4
5
Y. Liu and C. Wu, Pure Appl. Chem., 1991, 63, 427.
B. W. Wessels and R. D. Rogus, Med. Phys., 1984, 11, 638.
W. J. Wolf and J. Shani, Int. J. Radiat. Appl.Instrum. Part B:
Nucl. Med. Biol., 1986, 13, 319.
6
J. Scholm, K. Siler, D. E. Milenic, D. Eggensperger, D. Colcher,
L. S. Miller, D. Houchens, R. Cheng, D. Kaplan and W.
Goeckeler, Cancer Res., 1991, 51, 2889.
31 M. A. Rios, A. Fernandez, B. R. Tormo and S. Quintero,
Anticancer Res., 1992, 12, 205.
1058
New J. Chem., 2003, 27, 1054–1058