Convenient route for synthesis of bifunctional chelating agent: 1-(p-aminobenzyl)ethylenediaminetetramethylphosphonic acid-folate conjugate (Am-Bz-EDTMP-folate)

Article abstract of DOI:10.1246/cl.2005.1098

The present work describes a convenient and efficient approach for the synthesis of bifunctional chelating agent, 1-(p-aminobenzyl) ethylenediaminetetramethylphosphonic acid (Am-Bz-EDTMP). The key steps involved are functionalization of L-phenylalanine to

Full text of DOI:10.1246/cl.2005.1098

1098
Chemistry Letters Vol.34, No.8 (2005)
Convenient Route for Synthesis of Bifunctional Chelating Agent:
1-(p-Aminobenzyl)ethylenediaminetetramethylphosphonic acid–Folate Conjugate
(Am-Bz-EDTMP–Folate)
Anil Kumar Mishra,ꢀ Madhu Chopra,y and Viney Jain
Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Timarpur Delhi-110054, India
yDr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
(Received May 9, 2005; CL-050600)
The present work describes a convenient and efficient
consequently high specific activity of the radio-conjugate. They
should form stoichiometrically well-defined complexes that ex-
hibit thermodynamic and kinetic stability with respect to disso-
ciation and stabilization of the current oxidation state of the ra-
dionuclide.3 In this context, our group is involved in synthesis of
bifunctional chelating agents with prior knowledge of stability
constant of classical chelating agent such as EDTMP, well
known for its applications in nuclear medicine field.
Radiolabeling is mostly classified into three categories: di-
rect labeling,4 pre-labeling and post-labeling.5 In the post-label-
ing approach, the BFCA is first attached to the carrier-molecule
and then the radionuclide is coupled to the free chelating group
of the BFCA. Most suitable for clinical application might be the
post-labeling approach as it combines the ease and effectiveness
of direct labeling with the well-defined chemistry of pre-label-
ing. The objective of this study is to prepare a synthetically use-
ful chelate with higher coordinating ability and a reactive moiety
for conjugation chemistry with folate starting from an optically
active amino acid, L-phenylalanine, to form stable complexes
with 111In, 99mTc, and other metal ions of +3 or more oxidation
number, with lesser structural forms and more stable under
physiological conditions.
approach for the synthesis of bifunctional chelating agent,
1-(p-aminobenzyl)ethylenediaminetetramethylphosphonic acid
(Am-Bz-EDTMP). The key steps involved are functionalization
of L-phenylalanine to give ethylenediaminetetraacetic acid
derivative and its conversion to phosphonic acid followed by
the reaction with NHS-folate to give 1-(p-aminobenzyl)-
EDTMP–folate conjugate to be used for targeted radio imaging
and therapy. Radiolabeling with 111In and 153Sm of Folate–Bz-
EDTMP conjugate showed very high stability under physiolog-
ical conditions.
The folate receptors are over expressed in a wide variety of
human tumors. Conjugates of folate have been shown to be se-
lectively taken-up by tumor cells via the folate receptor.1 It
has been shown that the natural receptor mediated endocytosis
pathway for folic acid, can be exploited to selectively and non-
destructively deliver folate-conjugated small molecules, macro-
molecules and drug carriers into cultured tumor cells.2 The de-
velopment of a target specific radiopharmaceutical often re-
quires synthesis of bifunctional chelating agents. According to
the bifunctional approach the radionuclide is conjugated to the
carrier molecule by a bifunctional chelating agent (BFCA),
which consists of three parts: a chelating unit for strong coordi-
nation to the metallic radionuclide, a conjugation group for co-
valent attachment to the carrier-molecule and a linker as phar-
macokinetic modifier. The ideal BFCA must exhibit high label-
ing efficiencies to achieve good radiolabeling yields (>90%) and
1-(p-Nitrobenzyl)ethylenediaminetetraacetic acid (p-NO2-
Bz-EDTA, 2)6 was synthesized from 1-(p-nitrobenzyl)ethylene-
diamine 17 by standard amine alkylation technique employing
excess bromoacetic acid/NaOH. Purification of 2 was accom-
plished by HPLC by using 0.05% TFA and MeOH on C18-RP
column. Finally, in order to generate p-NO2-Bz-EDTMP, 38 car-
boxylic functions were converted into phosphonic functions ac-
O
O
P
Conjugation
Group
O
P
HO
HO
HO
HO
HO
HO
O
O
P
O
P
OH
OH
O2N
NH2BrCH2COOH
NH2
OH
OH
O2N
N
N
O2N
N
N
H2
H2N
N
N
OH
O
PCl3/H2PO3
O
OH
O
OH
Pd/C
P
OH
P
OH
HO
HO
1
HO
HO
OH
P
O
P
O
2
3
O
4 (BFCA)
HO
OH
O
HO
COOH
P
H
O
OH
OH
NH
COOH
H
O
NH
P
O
N
N
i. NHS/DCC in DMF
ii.
O
O
NH
O
P
4
NH
O
OH
HO
OH
O
HN
N
P
HO
Linker
NH
N
O
HN
N
N
H2N
N
6
Chelating Unit
5
N
H2N
Carrier Molecule
Scheme 1.
Copyright ꢀ 2005 The Chemical Society of Japan

Chemistry Letters Vol.34, No.8 (2005)
1099
cording to the method of Kruger and Bauer,9,10 by using H3PO3/
¨
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%.
A. K. Mishra, P. Panwar, M. Chopra, R. K. Sharma, and J.-F.
Chatal, New J. Chem., 7, 1054 (2003).
PCl3 in toluene. Reduction of NO2 group with H2/Pd–C in basic
media unmasked the amino group of p-NH2-Bz-EDTMP with a
purity of above 97%. 1-(p-Aminobenzyl)ethylenediaminetetra-
methylenephosphonic acid (4, BFCA) thus obtained was reacted
with NHS-ester of folic acid 511 in anhydrous dimethyl-
formamide to give p-NH2-Bz-EDTMP–folate conjugate 6
(Scheme 1).11 The compound was purified by HPLC and charac-
terized by NMR and mass spectrometry. Complexation of
folate–EDTMP 6 (aq solution, 0.01 mM, 20 mL) was carried
out with 111InCl3 (carrier free, 20 mL in sodium acetate buffer)
and the pH was marked to 7 with sodium acetate buffer. Radio-
chemical purity was checked after 3 h on a TLC system (1:1
MeOH–10% NH4OAc) and was above 97%. 6 was radiolabled
with 153Sm under the same conditions as 111In except the incuba-
tion time samarium was 5 h at room temperature.
The rates of decomplexation of the metal chelate (com-
plexes of 111In and 153Sm) were studied in serum under physio-
logical conditions over a 7-day period. 5.7 mL of the 111In-
Folate–EDTMP complex solution was mixed with 2 mL of
healthy human serum to study the transchelation of indium metal
ion. The rate of transchelation was determined by polyacryl-
amide gel electrophoresis under physiological conditions.12
Over 7-day period no measurable loss (less than 0.5% per day) of
metal ion from the EDTMP–folate conjugate was observed.
In conclusion, we described a simple, efficient synthesis
pathway to a new bifunctional chelating agent having tetraphos-
phonic ligands. The BFCA was successfully linked to folic acid
and a 1:1 conjugation was achieved. The key steps in synthesis
were the dramatizations of simple amino acid to synthesize first
bifunctional EDTA followed by introduction of phosphonic
functions and finally conjugation with NHS-ester of folic acid.
Complexation studies concerning indium-111 and samarium-
153 showed that the chelating agent thus developed has excellent
stability in human serum and deserves further investigation as a
potential therapeutic bone agent for targeted therapy.
7
8
Preparation of compound 4 (p-NH2-Bz-EDTMP): To a solution
of p-NO2-Bz-EDTA (2, 0.5 g; 1.17 mmol) dissolved in 10 mL of
toluene phosphorous acid (0.42 g; 5.15 mmol) was added with
stirring. The reaction mixture was refluxed while phosphorous
trichloride (0.94 g; 6.14 mmol) was added drop wise to the re-
fluxing mixture at 80 ꢁC. After 3 h, toluene was removed follow-
ing addition of deionised water. The filtrate was concentrated
under vacuum. The concentrated product was precipitated by
addition of methanol/ethanol to give p-NO2-Bz-EDTMP, 3.
1H NMR (250 MHz, D2O) ꢀ 7.55 (d, 2H, ArH), 7.12 (d, 2H,
ArH), 3.75 (d, 1H), 3.53 (dd, 1H, J ¼ 15:0, 7.5 Hz), 3.46 (dd,
1H, J ¼ 15:0, 7.5 Hz), 3.39 (dd, 1H, J ¼ 14:0, 7.5 Hz), 3.26
(dd, 1H, J ¼ 14, 7.5 Hz), 3.0842–3.0153 (m, 8H); FAB-MS:
found: 572 [M þ Hþ] (calcd m=e 571) Elemental Anal. Calcd
for C13H25N3O14P4: C, 27.33; H, 4.41; N, 7.36%. Found: C,
27.30; H, 4.44; N, 7.38%. p-NO2-Bz-EDTMP was converted
to p-NH2-Bz-EDTMP, 4, by reduction of NO2 group with H2
using Pd/C in aqueous sodium hydroxide (pH 11.0) to give
p-NH2-Bz-EDTMP with a purity of above 97%. The product
was characterized by mass spectrometry. FAB-MS: found: 542
[M þ Hþ] (calcd: m=e 541) Elemental Anal. Calcd for
C13H27N3O12P4: C, 28.85; H, 5.03; N, 7.76%. Found: C,
28.81; H, 5.06; N, 7.72%.
9
T. Bailya and R. Burgada, Phosphorus, Sulfur Silicon, 101, 131
(1995).
10 F. Kruger and L. Bauer, Chem.-Ztg., 36, 691 (1972).
¨
11 Preparation of compound 6 (p-NH2-Bz-EDTMP–Folate) 6: 10 g
(22.65 mmol) of folic acid 5, were dissolved in 150 mL of
dimethylformamide (DMF). A slight excess of NHS (2.67 g,
24.94 mmol) and dicyclohexylcarbodiimide (DCC, 4.67 g,
22.65 mmol) were then added. The reaction was allowed to stir
at room temperature for 4 h. The dicyclohexylurea (DHU) was
removed by filtration. The DMF solution of the NHS-folate
was stored at ꢃ20 ꢁC until use. Conjugation of folate with com-
pound 4: To a solution of folate-NHS ester (obtained above) in
anhydrous DMF (78.74 mL containing 2 g NHS ester; 3.7 mmol)
compound 4 (2 g, 3.69 mmol) was added slowly within 15 min
and reaction was further stirred at room temperature for 2 h.
The progress of the reaction was followed by analytical HPLC.
After full consumption of NHS-folate (2 h) pH was set to 8.5
with 2 M Na2CO3 and the resultant solution was filtered to re-
move and the clear orange solution was concentrated under vac-
uum and purified by preparative HPLC on C-18 column (5 m,
20 ꢂ 250 mm2) with a gradient (eluent A, 0.1 M ammonium
acetate buffer at pH 6.6, eluent B acetonitrile; gradient 0 min
at 4% B, 10 min at 12% B and 15 min at 15% B at a flow rate
of 10 mL/min; tR 6.3 min) to remove bis-conjugated side
product and to obtain EDTMP–folate conjugate in a yield of
49%. Analytical HPLC on C-18 reversed phase column (5 m,
4:6 ꢂ 250 mm2) revealed a single peak with a retention time
of 18.5 min (eluent, 10 mM ammonium acetate buffer (pH 6.0)
at 75% and acetonitrile 25%; flow rate of 0.7 mL/min) 1H NMR
(250 MHz, D2O): ꢀ 8.46 (s, 1H), 7.59 (d, 2H, ArH), 7.41 (d,
J ¼ 8:3 Hz, 2H, ArH), 7.10 (d, 2H, ArH), 6.34 (d, J ¼ 8:3 Hz,
2H, ArH), 4.24 (dd, J ¼ 4:4, 8.4 Hz, 1H), 4.15 (s, 2H), 3.75
(d, 1H), 3.53 (dd, 1H, J ¼ 15:0, 7.5 Hz), 3.46 (dd, 1H, J ¼
15:0, 7.5 Hz), 3.39 (dd, 1H, J ¼ 14:0, 7.5 Hz), 3.26 (dd, 1H,
J ¼ 14, 7.5 Hz), 3.0842–3.0153 (m, 8H), 2.28–1.84 (m, 4H);
31P NMR: (D2O): 17.0448, 15.7118; FAB-MS: found: 965
[M þ Hþ] (calcd for C32H44N10O17P4: m=e 964). Elemental
Anal. Calcd for C32H44N10O17P4: C, 39.84; H, 4.60; N,
14.52%. Found: C, 39.81; H, 4.63; N, 14.50%.
References and Notes
1
S. Wang, J. Luo, D. A. Lantrip, D. J. Waters, C. J. Mathias,
M. A. Green, P. L. Fuchs, and P. S. Low, Bioconjugate Chem.,
9, 673 (1997).
2
S. Wang, R. J. Lee, C. J. Mathias, M. A. Green, and P. S. Low,
Bioconjugate Chem., 7, 56 (1996).
3
4
S. Liu and D. S. Edwards, Chem. Rev., 99, 2235 (1999).
M. Langer and A. G. Beck-Sickinger, Curr. Med. Chem.
Anti-Cancer Agents, 1, 71 (2001).
5
6
W. C. Eckelman, Eur. J. Nucl. Med., 22, 249 (1995).
Preparation of compound 2 (p-NO2-Bz-EDTA): compound 1
(1.0 g, 3.73 mmol) was dissolved in H2O (5 mL) and allowed
to stir at 70 ꢁC. An aqueous solution of bromoacetic acid
(2.10 g, 15.1 mmol) was added in equal portions over 3 h, while
the reaction mixture was maintained at pH 10.2 using pH stat
(10 M sodium hydroxide solution). The reaction mixture was
further allowed to stir for 5 h at 60 ꢁC thereafter it was neutral-
ized by 3 M hydrochloric acid and solvent was removed under
reduced pressure to dryness. Obtained yellowish product 2
was purified by preparative HPLC (C18, 5 m, 20 ꢂ 250 mm2).
The major peak at retention time 13.94 min. was collected using
gradient solvent: NH4OAc (pH 6.0, 0.1 M) solution and metha-
nol as solvent. 1H NMR (250 MHz, D2O), ꢀ 8.23 (d, 2H,
J ¼ 8:0 Hz), 7.64 (d, 2H, J ¼ 8:0 Hz), 4.12 (s, 8H), 3.92 (d,
2H), 3.88 (m, 1H), 3.20 (t, 2H). FAB-MS: found: 196
[M þ Hþ] (calcd for C9H13N3O2: m=e 195) Elemental Anal.
12 M. Li and C. F. Mears, Bioconjugate Chem., 4, 275 (1993).
Published on the web (Advance View) July 2, 2005; DOI 10.1246/cl.2005.1098