Organometallic 99mTc-technetium(I)- and Re-rhenium(I)-folate derivatives for potential use in nuclear medicine

Article abstract of DOI:10.1016/j.jorganchem.2004.08.045

The folate receptor (FR) is a high affinity membrane protein which is overexpressed on a wide variety of tumor cells, but highly restricted in normal tissues. Therefore folate derivatives labeled with short living isotopes such as ^99mTc (γ, t_1/2 = 6 h) or ¹⁸⁸Re (β^-, t_1/2 = 17 h) could be used for tumor diagnosis and therapy. In this respect there is a great interest to develop organometallic technetium(I) and rhenium(I) modified folate radiopharmaceuticals. For this purpose folic acid was functionalized with a tridentate picolylamine monoacetic acid chelating system. The chelating system was selectively coupled via an aminohexane spacer to the γ- or α-carboxyl group of the glutamate moiety of folic acid to obtain the corresponding γ- or α-folate derivative or - if directly attached to pteroic acid - the pteroate derivative. The derivatives were reacted with the precursor [M(OH₂) ₃(CO)₃]⁺ (M = ^99mTc, Re) to form uniform organometallic folate complexes under mild reaction conditions. All compounds were chemically characterized by means of NMR, MS, IR and HPLC. The determination of the IC₅₀-values for the PAMA-γ-folate derivative (100 nM) and the corresponding organometallic rhenium complex (110 nM) proved retained receptor binding properties. The radiolabeling with [ ^99mTc(OH₂)₃(CO)₃]⁺ was achieved in excellent yield (>95%) at low ligand concentration (10 ^-4 M). The cell binding (>45% of total activity) and internalization (>15% of total activity) of all ^99mTc-complexes was very high and specificity for the FR was proved by their complete displacement with excess folic acid. The ^99mTc-complexes were positively tested for their plasma stability and for the absence of binding to plasma proteins.

Full text of DOI:10.1016/j.jorganchem.2004.08.045

Journal of Organometallic Chemistry 689 (2004) 4712–4721
www.elsevier.com/locate/jorganchem
Organometallic 99mTc-technetium(I)- and Re-rhenium(I)-
folate derivatives for potential use in nuclear medicine
a
Cristina Muller , Cecile Dumas , Ute Hoffmann , P. August Schubiger
¨
a
a
a,b
´
,
a,b,
*
Roger Schibli
a
Center for Radiopharmaceutical Science ETH-PSI-USZ, Paul Scherrer Institute, OIPA, 5232 Villigen, Switzerland
b
Department of Chemistry and Applied Biosciences of the ETH, 8056 Zu¨rich, Switzerland
Received 13 August 2004; accepted 27 August 2004
Available online 1 October 2004
Abstract
The folate receptor (FR) is a high affinity membrane protein which is overexpressed on a wide variety of tumor cells, but highly
restricted in normal tissues. Therefore folate derivatives labeled with short living isotopes such as 99mTc (c, t1/2 = 6 h) or 188Re (bꢀ,
t1/2 = 17 h) could be used for tumor diagnosis and therapy. In this respect there is a great interest to develop organometallic
technetium(I) and rhenium(I) modified folate radiopharmaceuticals. For this purpose folic acid was functionalized with a tridentate
picolylamine monoacetic acid chelating system. The chelating system was selectively coupled via an aminohexane spacer to the c- or
a-carboxyl group of the glutamate moiety of folic acid to obtain the corresponding c- or a-folate derivative or – if directly attached
to pteroic acid – the pteroate derivative. The derivatives were reacted with the precursor [M(OH2)3(CO)3]+ (M = 99mTc, Re) to form
uniform organometallic folate complexes under mild reaction conditions. All compounds were chemically characterized by means of
NMR, MS, IR and HPLC. The determination of the IC50-values for the PAMA-c-folate derivative (100 nM) and the corresponding
organometallic rhenium complex (110 nM) proved retained receptor binding properties. The radiolabeling with
[
99mTc(OH2)3(CO)3]+ was achieved in excellent yield (>95%) at low ligand concentration (10ꢀ4 M). The cell binding (>45% of total
activity) and internalization (>15% of total activity) of all 99mTc-complexes was very high and specificity for the FR was proved by
their complete displacement with excess folic acid. The 99mTc-complexes were positively tested for their plasma stability and for the
absence of binding to plasma proteins.
ꢀ 2004 Elsevier B.V. All rights reserved.
Keywords: Rhenium(I); Technetium(I); Folic acid derivatives; Folate radiopharmaceutical; Folate receptor
1. Introduction
uptake of folic acid by endocytosis [1,2]. The reduced fo-
late carrier (RFC) is a low affinity membrane spanning
protein that transports preferentially reduced folates di-
rectly into the cell cytosol. In normal cells the RFC is
ubiquitary existing [3,4] whereas the FR is known to
be overexpressed in various neoplastic tissues, including
ovarian, breast, cervical, colorectal and nasopharyngeal
tumors, but highly restricted in most normal human tis-
sues [5,6]. Folic acid is small in size (441.4 Da), non-
immunogenic and has proven its affinity for the FR even
after site specific modification. This makes it an ideal
tool to be used as a Trojan horse for tumor targeting
The vitamin folic acid is involved in diverse processes
during cell proliferation and therefore plays an essential
role in the growth of tumors. To cope with this increased
requirement for folic acid, tumor cells often overexpress
the folate receptor (FR, a high affinity membrane an-
chored protein, KD = 10ꢀ9 M), which mediates the cell
*
Corresponding author. Tel.: +41 56 310 2837; fax: +41 56 310
2849.
E-mail address: roger.schibli@psi.ch (R. Schibli).
0022-328X/$ - see front matter ꢀ 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2004.08.045

C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
4713
[7–9]. Folic acid is structurally composed of pteroic acid
and glutamic acid connected via an amide linkage (Fig.
1). Both of the carboxyl groups (a- and c-carboxyl) of
the glutamate moiety are accessible for a potential reac-
tion with an amino-group of any functional ligand. Sev-
eral studies in recent years describe the derivatization of
folic acid at the c-carboxyl group of the glutamate moi-
ety [10]. To date folate conjugates of chemotherapeutic
agents [11,12], antisense oligonucleotides and ribozymes
[13,14], proteins and protein toxins [15–17], immuno-
therapeutic agents [18,19] as well as liposomes with
entrapped drugs [20–22], plasmids [23–25] and radio-
pharmaceutical agents [10,26–28] have been synthesized
and successfully tested in cancer cells overexpressing the
FR on their surface.
The most thoroughly investigated folate radiophar-
maceutical is the 111In-labeled diethylenetriamine penta-
acetate-(DTPA)-functionalized folate [29–31]. However
99mTc-technetium-based radiopharmaceuticals for diag-
nostic imaging purposes are of great interest because
of the more ideal physical decay properties (99mTc: c,
140 keV, t1/2 = 6 h) than for 111In (111In: c, 245 keV,
t1/2 = 2.8 d), lower costs and a generator system which
makes it readily available on-site in any hospital. In
addition its bꢀ-radiation emitting counterpart 188Re-
rhenium with a likewise availability shows the required
decay properties for therapy (188Re: bꢀ, 2.1 MeV,
t1/2 = 17 h). Previous studies describe folate conjugates
for radiolabeling with 99mTc(V) [10]. Compared to
common Tc(V)/Re(V) complexes the use of the high
kinetically inert and relatively small Tc(I) or Re(I)-tri-
carbonyl core is favorable due to an increased in vivo
stability and a reduced interference with biological activ-
ity [32–35]. Therefore, currently there is a high interest
to develop a folate derivative functionalized with the
organometallic 99mTc(I)/Re(I)-tricarbonyl core for
potential use in nuclear medicine [36,37].
sequent separating step of the two isoforms – resulting
from unspecific coupling procedures – superfluous.
2. Experimental
Solvents for syntheses were purchased from Aldrich
Chemical Co. or Fluka, Buchs, Switzerland and were
dried according to standard methods. The chemical sub-
stances were purchased from Aldrich Chemical Co. or
Fluka, Buchs, Switzerland. The Boc-protected a- and c-
glutamate methyl esters were purchased from Bachem
AG, Bubendorf, Switzerland. The N2,N10-protected pte-
roic acid precursor was generously provided by Merck
Eprova
AG,
Schaffhausen,
Switzerland
and
[30,50,7,9-3H]Folic acid potassium salt (1 mCi/ml, 24 Ci/
mmol) was purchased from Amersham Biosciences, Buc-
kimghamshire, UK. The scintillation solution Ultima
GoldTM high flash-point LSC-cocktail was purchased
from Packard Company, Groningen, Netherlands. The
organometallic precursor (NEt4)2[ReBr3(CO)3] and the
radioactive precursor [99mTc(H2O)3(CO)3]+ were pre-
pared as previously reported [32,38]. [Na][99mTcO4] was
eluted from a 99Mo/99mTc generator (Mallinckrodt-Tyco,
Petten, Netherlands) using a 0.9% saline solution. KB
cells (CCL-17) were purchased from ATCC (American
Type Culture Collection, Manassas, USA). Special
RPMI cell culture medium (without folic acid, vitamin
B12, phenol red) was purchased from Cell Culture Tech-
nologies GmbH, Gravesano. HPLC analyses were per-
formed on a Merck-Hitachi L-6200A-system equipped
with an L-3000 tunable absorption detector and a Bert-
hold LB 508 radiometric detector using an uptisphere
C-18 reversed phase column (5 lm, 250 · 4.0 mm). HPLC
solvents: Aqueous 0.05 M TEAP (triethylammonium
phosphate) buffer, pH 7.0 (solvent A), MeOH (solvent
B). The HPLC system started with 100% A with a linear
gradient to 20% A and 80% B over 30 min followed by
10 min of 100% A. (If the HPLC analyses were performed
with the XterraTM MS C-18 reversed phase column (5 lm,
150 · 4.6 mm) the program was shortened to 20 min
(Fig. 2)). The flow rate was 1 ml/min for both programs.
Plasma protein binding studies were performed using a
Superdex 200 size-exclusion column (13–15 lm, 300 ·
10 mm) with PBS pH 7.4 containing 0.05% Tween20 as
the mobile phase and a flow rate of 0.9 ml/min. If not else
defined TLC analyses of organic synthesis were per-
formed with silica gel 60 aluminium sheets (5 · 7.5 cm),
F254, Merck, using CH2Cl2/MeOH = 9/1 as an eluent.
TLC analyses for plasma stability of the 99mTc-complexes
were performed with silica gel plates (5 cm · 20 cm),
F254, Merck, using MeOH/conc. HCl = 99/1 as an eluent
and the activity was measured by a RAYTEST RITA-
3200 radioanalyzer. Nuclear magnetic resonance spectra
were recorded on a 300 MHz Varian Gemini 2000 spec-
In the present work we describe the derivatization of
folic acid and pteroic acid by a picolylamine monoacetic
acid (PAMA) chelating system for tridentate labeling
with 99mTc(I)/Re(I)-tricarbonyl and their in vitro inves-
tigation. We established a direct method to synthesize
uniform c- and a-folate derivatives which made the sub-
O
OH
O
OH
O
N
N
H
γ
α
O
N
N
HN
H2N
N
H
glutamic acid
pteroic acid
folic acid
1
Fig. 1. Structure of folic acid.
trometer. The H and 13C chemical shifts were reported

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C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
eq.) were added to a solution of compound 1 (400 mg;
1.85 mmol; 1 eq.) in dry toluol (15 ml) and stirred for
30 min at rt. After 4 h reflux the solution was filtered,
evaporated and dried in vacuum. The yellow oil of the
imine was dissolved in methanol (15 ml) and the reduc-
tion was performed by NaBH4 (70 mg; 1.85 mmol; 1
eq.), stirred at rt over night. After addition of water to
destroy residual NaBH4, the organic solvent was re-
moved in vacuum. The aqueous solution was extracted
three times with EtAc, the organic layer dried over
Na2SO4, evaporated and dried in vacuum to obtain a
1
68% yield of a yellow oil 2. Analytical data for 2: H
NMR (CDCl3): d 1.38–1.66 (m, 8H), 1.55 (s, 9H), 2.79
(t, J = 7.1 Hz, 2H), 3.16–3.29 (m, 2H), 4.01 (s, 2H),
4.78 (s, 1H), 7.23–7.30 (m, 1H), 7.39–7.42 (m, 1H),
7.75 (ddd, 1H), 8.64–8.69 (m, 1H); 13C NMR (CDCl3):
d 26.8, 27.1, 28.6 (3C), 30.1, 30.2, 40.6, 49.7, 55.5,
79.1, 122.0, 122.4, 136.5, 149.4, 156.1, 160, 0, 171.3;
IR (KBr): 1697 cmꢀ1; MS (ESI): m/z (%) 308
[M+ + H] (100).
2.3. Synthesis of compound 3
The following procedure is based on literature proce-
dures with a slight modification [40]. Compound 2 (386
mg; 1.26 mmol; 1 eq.) was dissolved in 20 ml dry THF.
Methyl bromoacetate (350 ll; 3.78 mmol; 3 eq.) and
NEt3 (530 ll; 3.78 mmol; 3 eq.) were added and refluxed
for 4 h. After cooling to rt the reaction solution was fil-
tered and evaporated. The crude product was purified
by chromatography on silica gel using CH2Cl2/MeOH
(9/1) as an eluent. After drying in high vacuum the prod-
uct 3 was obtained in a 70% yield. Analytical data for 3:
1H NMR (CDCl3): d 1.34–1.44 (m, 4H), 1.50–1.66 (m,
4H), 1.53 (s, 9H), 2.75 (t, J = 7.2 Hz, 2H), 3.12–3.22
(m, 2H), 3.51 (s, 2H), 3.79 (s, 3H), 4.03 (s, 2H), 7.26
(ddd, 1H), 7.58–7.64 (m, 1H), 7.77 (ddd, 1H), 8.60–
8.64 (m, 1H); IR (KBr): 1742, 1708 cmꢀ1; MS (ESI):
m/z (%) 402 [M+ + Na] (100).
Fig. 2. HPLC traces (XterraTM MS C-18 column): (a) UV-trace (254
nm) of compound 8; (b) UV-trace (254 nm) of the rhenium complex
9a; (c) c-trace of the radioactive 99mTc-complex 9b.
relative to residual solvent protons as a reference. IR
spectra were recorded on a Perkin–Elmer FT-IR 16PC
using KBr pellets. Mass and elemental analyses were per-
formed at the Swiss Federal Institute of Technology,
3
ETH, Zurich. Radioactivity (99mTc, H) was measured
with a c-counter (CobraTM II, Model B 5003, Packard)
or a b-counter (TRI-CARB, 1900 TR, Liquid Scintilla-
tion Analyzer, Packard). Protein concentrations for the
in vitro experiments were measured with a microplate
reader (Bio-Rad, Model 550), using a Micro BCATM Pro-
tein Assay Kit (Prod # 23235), Socochim.
2.4. Synthesis of compound 4
2.1. Synthesis of compound 1
The Boc-deprotection of compound 3 (334 mg; 0.88
mmol; 1 eq.) was perfomed quantitatively in 6 N HCl
(10 ml) at rt, monitored by TLC/ninhydrine. The solvent
was evaporated and the compound 4 Æ HCl was dried in
Compound 1 has been synthesized in a 80% yield by a
previously published procedure and the analyses were in
agreement with the literature [39]. Analytical data for 1:
1H NMR (CDCl3): d 1.38–1.50 (m, 6H), 1.50–1.66 (m,
2H), 1.55 (s, 9H), 2.79 (t, J = 6.9 Hz, 2H), 3.16–3.29
(m, 2H).
1
vacuum. Analytical data for 4: H NMR (CD3OD): d
1.52–1.66 (m, 4H), 1.79–1.92 (m, 2H), 1.92–2.09 (m,
2H), 3.09 (t, J = 7.2 Hz, 2H), 3.42–3.48 (m, 2H), 3.97
(s, 3H), 4.53 (s, 2H), 5.06 (s, 2H), 8.11–8.21 (m, 1H),
8.31–8.41 (m, 1H), 8.61–8.70 (m, 1H), 9.03–9.10 (m,
1H); 13C NMR (CD3OD): d 23.7, 25.5, 26.8, 39.3,
52.8, 53.0, 53.4, 54.6, 56.4, 128.3, 130.2, 143.9, 144.4,
146.7, 166.5; IR (KBr): 1747 cmꢀ1; MS (ESI): m/z (%)
280 [M+ + H] (100).
2.2. Synthesis of compound 2
The following procedure is based on literature proce-
dures with a slight modification [40]. Molecular sieve
and pyridine-2-carboxaldehyde (198 mg; 1.85 mmol; 1

C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
4715
2.5. Synthesis of compound 5
organic layer was dried over Na2SO4 and filtered to ob-
tain a transparent slightly yellow colored solution. The
formation of the activated protected pteroic acid was
monitored by HPLC (Rt = 25 min). Compound 6 Æ TFA
(354 mg; 0.66 mmol; 1 eq.) was dissolved in dry CH2Cl2,
neutralized by N-ethyldiisopropylamine (115 ll; 0.66
mmol; 1 eq.) (HPLC: Rt = 28 min) and added in two
portions. The coupling reaction, monitored by HPLC
(Rt = 29.9 min), was stirred at rt over night. The reac-
tion solution was washed once with water, the organic
layer dried over Na2SO4, filtered and evaporated for di-
rect use of the following deprotection reaction. The fully
protected PAMA-aminohexane-c-folate was dissolved
in 1 N NaOH (10 eq.) and stirred for 5 h, monitored
by HPLC (Rt = 18.9 min). After neutralization with 1
N HCl, the pH was decreased to 3 by adding 0.1 N
HCl. The yellow product precipitated by adding EtOH.
After centrifugation the solid was washed three times
with EtOH and dried in vacuum to obtain an orange
powder of the product 8 in a 10% yield. Analytical data
for 8: 1H NMR (DMSO): d 1.10–1.62 (m, 8H), 1.92–2.35
(m, 4H), 2.68–2.81 (m, 2H), 3.01–3.19 (m, 2H), 3.43 (s,
2H), 4.04 (s, 2H), 4.30–4.45 (m, 1H), 4.61 (s, 2H),
6.65–6.82 (m, 2H), 7.24–7.46 (m, 2H), 7.70–7.82 (m,
2H), 7.84–8.00 (m, 1H), 8.54–8.66 (m, 1H), 8.77 (s,
1H); IR (KBr): 1687, 1651, 1632, 1608 cmꢀ1; MS
(ESI): m/z (%) 687 [M+ ꢀ H] (100); 688 [M+] (40).
HOBt Æ H2O (142 mg; 1.93 mmol; 1 eq.) was added to
a solution of Boc–Glu–OMe (243 mg; 0.93 mmol; 1 eq.)
in dry CH2Cl2 to form a suspension and after addition
of EDC Æ HCl (194 mg; 1.01 mmol; 1.1 eq.) a transparent
solution. After 4 h stirring at rt, the UV-active product
was detected by TLC analysis (Rf = 0.9). The reaction
solution was washed three times with water to remove
impurities, the organic layer was dried over Na2SO4,
evaporated and dried in vacuum to obtain a white solid
in a 95% yield, directly processed in the further coupling
reaction. Compound 4 Æ HCl (278 mg; 0.88 mmol; 1 eq.)
and N-ethyldiisopropylamine (300 ll; 1.76 mmol; 2 eq.)
dissolved in DMF were added in two portions to a solu-
tion of the activated Boc–Glu–OMe (333 mg; 0.88
mmol; 1 eq.) in dry DMF. After a night at 50 ꢁC, the
solution was distilled to remove DMF and the crude
product was purified by chromatography on silica gel
using CH2Cl2/MeOH (9/1) to obtain the product 5 in a
1
75% yield. Analytical data for 5: H NMR (CDCl3): d
1.26–1.43 (m, 4H), 1.48–1.61 (m, 2H), 1.51 (s, 9H),
1.96–2.12 (m, 1H), 2.16–2.30 (m, 1H), 2.35 (t, 2H),
2.46–2.56 (m, 2H), 2.66–2.76 (m, 2H), 3.27 (q, 2H),
3.48 (s, 2H), 3.80 (s, 3H), 3.81 (s, 3H), 4.00 (s, 2H),
4.25–4.50 (m, 1H), 7.44–7.62 (m, 1H), 7.59–7.65 (m,
1H), 7.73–7.85 (m, 1H), 8.59–8.65 (m, 1H); 13C NMR
(CDCl3) d 14.5 (3C), 26.5 (2C), 27.0, 28.0, 30.2, 52.2,
52.3, 53.0, 54.1, 54.8, 59.8, 61.0, 122.0, 123.1, 136.6,
148.9, 171.0, 171.8, 172.0; IR (KBr): 1711, 1737 cmꢀ1
MS (ESI): m/z (%) 545 [M+ + Na] (100).
;
2.8. Synthesis of compound 10
Boc–Glu(OMe)–OH Æ DCHA (530 mg; 1.20 mmol)
was dissolved in CH2Cl2 (40 ml) and washed twice
with KHSO4 10% (15 ml). The organic layer was
dried over Na2SO4, filtered and dried in vacuum to
obtain a 80% yield of the free Boc–Glu(OMe)–OH.
The activation of Boc–Glu(OMe)–OH (243 mg; 0.93
mmol; 1 eq.) with HOBt Æ H2O (142 mg; 0.93 mmol;
1 eq.) and EDC Æ HCl (194 mg; 1.01 mmol; 1.1 eq.)
was performed as described for the Boc–Glu–OMe
in a 95% yield. Compound 10 has been synthesized
according to the above described procedure for com-
2.6. Synthesis of compound 6
Compound 5 (345 mg; 0.66 mmol; 1 eq.) was quan-
titatively deprotected in CH2Cl2/TFA (9/1) (10 ml) at rt
over night, monitored by TLC/ninhydrine. The reac-
tion solution was evaporated and the crude product 6
dried in vacuum. Analytical data for 6: 1H NMR
(CD3OD): d 1.44–1.70 (m, 6H), 1.78–1.94 (m, 2H),
2.18–2.48 (m, 3H), 2.50–2.78 (m, 2H), 3.26–3.42 (m,
3H), 3.95 (s, 3H), 3.98 (s, 3H), 4.18–4.30 (m, 1H),
4.32 (s, 2H), 4.74 (s, 2H), 7.68–7.83 (m, 2H), 8.11–
8.25 (m, 1H), 8.80–8.88 (m, 1H); 13C NMR (CDCl3):
d 26.5 (2C), 27.0 (2C), 27.4, 29.1, 51.4, 51.6, 53.0,
54.3, 54.8, 59.8, 60.1, 122.0, 123.1, 136.6, 148.9,
pound
5 using the activated Boc–Glu(OMe)–OH
(333 mg; 0.88 mmol; 1 eq.) to obtain the purified
product 10 in a 75% yield. Analytical data for 10:
1H NMR (CDCl3): d 1.28–1.60 (m, 8H), 1.51 (s,
9H), 1.92–2.10 (m, 1H), 2.10–2.28 (m, 1H), 2.40–2.62
(m, 2H), 2.78 (t, 2H), 3.24–3.34 (m, 2H), 3.57 (s,
2H), 3.76 (s, 3H), 3.79 (s, 3H), 4.12 (s, 2H), 4.18–
4.35 (m, 1H), 7.32–7.40 (m, 1H), 7.67–7.73 (m, 1H),
7.76–7.82 (m, 1H), 8.58–8.64 (m, 1H); 13C NMR
(CDCl3): d 26.7, 26.8, 27.3, 28.2, 28.4 (3C), 29.4,
30.4, 39.5, 51.6, 51.8, 53.7, 54.2, 54.7, 60.0, 68.2,
122.3, 123.3, 136.8, 148.9, 155.8, 159.1, 171.5, 171.7,
173.8; IR (KBr): 1737, 1715, 1667, cmꢀ1; MS (ESI):
m/z (%) 545 [M+ + Na] (100), 523 [M+ + H] (20).
171.0, 171.8, 171.0; IR (KBr): 1741, 1678, 1653 cmꢀ1
MS (ESI): m/z (%) 423 [M+ + H] (100).
;
2.7. Synthesis of compound 8
N2-N,N-dimethylaminomethylene-10-formylpteroic
acid 7 (261 mg; 0.66 mmol; 1 eq.) was suspended in dry
CH2Cl2. CDI (428 mg; 2.64 mmol; 4 eq.) was added and
stirred under N2 for 2 h to obtain a yellow solution. The
reaction solution was washed four times with water, the

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C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
2.9. Synthesis of compound 11
eq.) and the ligands 8, 12 or 14 (1 eq.) were dissolved
in a H2O/MeOH (2/1) and stirred at rt for 2 h. An aque-
ous solution of 1 M NaHCO3 was dropwise added
increasing the pH value to 8–9. The formation of the
rhenium-complex was monitored by HPLC (9a:
Rt = 26.3 min; 15a: Rt = 27.2 min). The reaction solu-
tion was acidified with 1 N HCl to a pH of 3 to precip-
itate an orange solid which was centrifuged, washed
several times with H2O and dried in vacuum. Analytical
The deprotection reaction of compound 10 (345 mg;
0.66 mmol; 1 eq.) to obtain compound 11 has been per-
formed according to the above described deprotection
reaction of compound 5. Analytical data for 11: 1H
NMR (CD3OD): d 1.44–1.59 (m, 4H), 1.59–1.75 (m,
2H), 1.75–1.93 (m, 2H), 2.18–2.45 (m, 2H), 2.50–2.78
(m, 2H), 3.25–3.45 (m, 4H), 3.81 (s, 3H), 3.93 (s, 3H),
4.02–4.11 (m, 1H), 4.29 (s, 2H), 4.74 (s, 2H), 7.68–7.89
(m, 2H), 8.16–8.28 (m, 1H), 8.80–8.90 (m, 1H); MS
(ESI): m/z (%) 423 [M+ + H] (100), 445 [M+ + Na] (95).
1
data for 9a: H NMR (DMSO): d 1.25–1.95 (m, 8H),
1.95–2.13 (m, 1H), 2.13–2.26 (m, 1H), 2.26–2.44 (m,
2H), 3.00–3.6 (m, 5H), 3.94 (d, J = 16.8 Hz, 1H), 4.33–
4.55 (m, 1H), 4.61 (s, 2H), 4.65 (d, J = 16.2 Hz, 1H),
4.88 (d, J = 15.9 Hz, 1H), 6.68–6.88 (m, 2H), 7.65–7.90
(m, 3H), 7.90–8.08 (m, 1H), 8.22–8.40 (m, 1H), 8.78 (s,
1H), 8.88 (d, J = 5.4 Hz, 1H); IR (KBr): 2024, 1899,
1638 cmꢀ1; MS (ESI): m/z (%) 957 [M+ ꢀ H], 958
2.10. Synthesis of compound 12
Compound 12 has been synthesized and purified
according to the procedure described for compound 8,
but using compound 11 Æ TFA (354 mg; 0.66 mmol; 1
eq.) as a ligand for coupling with the N2-N,N-dimethyl-
aminomethylene-10-formylpteroic acid 7 (261 mg; 0.66
mmol; 1 eq.), which was previously activated with
CDI. The coupling reaction and the in situ deprotection
to obtain the product 12 in a 7% yield were monitored
by HPLC (Rt = 29.9 min, Rt = 18.9 min (12), respec-
1
[M+] (40). Analytical data for 13a: H NMR (DMSO):
d 1.17–1.85 (m, 8H), 3.15–3.65 (m, 5H), 3.82 (d,
J = 16.2 Hz, 1H), 4.52 (d, J = 16.0 Hz, 1H), 4.08–4.16
(m, 2H), 4.76 (d, J = 16.5 Hz, 1H), 6.50–6.95 (m, 2H),
7.50–7.68 (m, 3H), 7.68–7.80 (m, 1H), 7.96–8.20 (m,
1H), 8.69 (s, 1H), 8.72–8.80 (m, 1H); IR (KBr): 2025,
1898, 1610 cmꢀ1; MS (ESI): m/z (%) 852 [M+ + Na]
(100), 874 [M+ + 2 Na] (50). Analytical data for 15a:
1H NMR (DMSO): d 1.15–1.85 (m, 8H), 3.10–3.60 (m,
5H), 3.80 (d, J = 16.5 Hz, 1H), 4.50 (d, J = 15.9 Hz,
1H), 4.06–4.16 (m, 2H), 4.75 (d, J = 16.5 Hz, 1H),
6.48–6.90 (m, 2H), 7.50–7.66 (m, 3H), 7.66–7.75 (m,
1H), 7.94–8.20 (m, 1H), 8.68 (s, 1H), 8.70–8.78 (m,
1H); IR (KBr): 2025, 1897, 1608 cmꢀ1; MS (ESI): m/z
(%) 852 [M+ + Na] (100), 874 [M+ + 2 Na] (50).
1
tively). Analytical data for 12: H NMR (DMSO): d
1.10–1.70 (m, 8H), 1.79–2.15 (m, 4H), 2.25–2.50 (m,
2H), 2.86–3.25 (m, 4H), 4.33 (s, 2H), 4.40–4.51 (m,
1H), 4.62 (s, 2H), 6.65–6.85 (m, 2H), 7.02–7.40 (m,
2H), 7.72–7.89 (m, 2H), 7.93–8.05 (m, 1H), 8.65–8.75
(m, 1H), 8.78 (s, 1H); MS (ESI): m/z (%) 689 [M+ + H]
(40); 710 [M+ ꢀ H + Na] (50).
2.11. Synthesis of compound 14
2.13. Synthesis of the complexes 9b, 13b, 15b
Compound 14 has been synthesized and purified by
the above described procedure for compound 8 using di-
rectly compound 4 Æ HCl (208 mg; 0.66 mmol; 1 eq.) as a
ligand for coupling with the N2-N,N-dimethylamino-
methylene-10-formylpteroic acid 7 (261 mg; 0.66 mmol;
1 eq.), which was previously activated. The coupling
reaction and the in situ deprotection to obtain the prod-
uct 14 in a 15% yield were monitored by HPLC
(Rt = 32.8 min, Rt = 20.0 min (14), respectively). Analyt-
The complexes 9b, 13b, 15b were prepared according
to the following general procedure: fac-[99mTc(OH2)3-
(CO)3]+ (100 ll; ꢁ1 GBq/ml), PBS pH 7.4 (350 ll) and
the stock solution (10ꢀ3 M) of the compound (8, 12,
14) in PBS buffer (50 ll) were placed in a sealed glass
vial. After 30 min reaction time at 75 ꢁC the vial was
cooled on ice. The formation of the complexes were
monitored by HPLC (9b: Rt = 26.3 min, 13b: Rt = 26.3
min, 15b: Rt = 27.2 min).
1
ical data for 14: H NMR (DMSO): d 1.10–1.80 (m,
8H), 3.08–18 (m, 2H), 3.18–3.32 (m, 2H), 4.06 (s, 2H),
4.50 (s, 2H), 4.59 (s, 2H), 6.68–6.80 (m, 2H), 6.88–7.20
(m, 1H), 7.26–7.36 (m, 1H), 7.72–7.78 (m, 2H), 7.96–
8.06 (m, 1H), 8.68–8.80 (m, 2H); IR (KBr): 1703,
1688, 1605 cmꢀ1; MS (ESI): m/z (%) 560 [M+ + H]
(100), 582 [M+ ꢀ H + Na] (30).
2.14. Plasma stability and protein binding of the 99mTc-
complexes
The 99mTc-radiolabeled complexes 9b, 13b and 15b
were purified by HPLC to separate them from the
unlabeled compounds 8, 12, 14. Each of the collected
solutions was diluted in PBS pH 7.4 to a concentration
of ꢂ65 MBq/ml and mixed with 100 ll human plasma
(1:1). The samples were incubated at 37 ꢁC and analyzed
after 1, 4 and 24 h either by TLC to determine decom-
position rate or by HPLC on a Superdex size-exclusion
2.12. Synthesis of the complexes 9a, 13a, 15a
The complexes 9a, 13a and 15a were prepared
according to the following general procedure: A slight
excess of the precursor (NEt4)2[ReBr3(CO)3] (1.1–1.5

C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
4717
column to determine the fraction of the complexes
bound to plasma proteins. For the TLC analyses it is
known that pertechnetate migrates with an Rf ꢂ 0.7,
whereas the pure 99mTc-complexes (9b, 13b, 15b) were
detected with an Rf ꢂ 0.2. No decomposition could be
observed up to 4 h incubation time and only <10% of
the 99mTc-complexes were decomposed to pertechnetate
after 24 h. For the HPLC analyses it is known that the
plasma proteins are detected after 12 and 18 min in the
UV trace. The 99mTc-complexes 9b and 13b were de-
tected after a retention time of 25 min, whereas the com-
plex 15b was detected after 55 min. HPLC analysis for
9b: after 1 h, Rt = 25 min (100%); after 4 h, Rt = 25
min (100%); after 24 h, Rt = 15 min (1%) and Rt = 25
scintillation flacons to be counted for radioactivity using
a b-counter.
2.17. Cell binding experiment
Cell binding experiments were performed according
to the following general procedure: The monolayers
were rinsed with ice-cold PBS pH 7.4. Pure special
RPMI medium (without FCS/L-glutamine/antibiotics)
only (975 ll), or medium (475 ll) and a folic acid solu-
tion (200 lM, 500 ll) were added into the corresponding
wells. The well plates were pre-incubated at 37 ꢁC for 40
min. The solutions of the complexes 9b, 13b, 15b (25 ll,
1 MBq/ml), purified by HPLC, were added and the well
plates incubated again at 37 ꢁC for 1 h and then rinsed
three times with ice-cold PBS pH 7.4 or twice with PBS
pH 7.4 and once with a stripping buffer (aqueous solu-
tion of 0.1 M acetic acid and 0.15 M NaCl), respectively,
to remove bound complex from the FR on the cell sur-
face [11]. The monolayers were dissolved in 1 N NaOH
(1000 ll) and after transfer in 4 ml-tubes homogenized
by vortex. They were counted for radioactivity using a
CobraTM II c-counter. The concentration of proteins
for each sample was determined.
min (99%); HPLC analysis for 13b: after
1 h,
Rt = 25 min (100%); after 4 h, Rt = 25 min (100%); after
24 h, Rt = 15 min (1%) and Rt = 25 min (99%); HPLC
analysis for 15b: after 1 h, Rt = 55 min (100%); after 4
h, Rt = 55 min (100%); after 24 h, Rt = 15 min (10%)
and Rt = 55 min (90%).
2.15. Cell culture
KB cells, a human nasopharyngeal epidermal carci-
noma cell line, overexpressing the folate receptor (FR),
were cultured continuously as a monolayer at 37 ꢁC in
a humidified atmosphere containing 5% CO2 in a special
RPMI 1640 medium without folic acid, vitamin B12 and
phenol red, supplemented with 10% fetal calf serum
3. Results and discussion
3.1. Syntheses
(FCS), L-glutamine and antibiotics (penicillin 100 IU/
Both of the carboxyl groups (a- and c-carboxyl) of fo-
lic acid are accessible for a potential modification and
thus reaction with an amino-group of any functional lig-
and (Fig. 1). The necessity of a free a-carboxyl group for
retained binding to the FR is controversially discussed in
the literature [10,41,42]. We therefore reasoned the syn-
thesis of clearly defined c- or a-folate derivatives. Since
both carboxyl groups of the glutamate moiety have sim-
ilar reactivities unspecific coupling reactions result in a
mixture of c- and a-derivatives and require a subsequent
cumbersome separation step [10,43]. To avoid this disad-
vantage the synthesis of folate derivatives via solid phase
chemistry has been described [28,41]. We developed and
applied a straight forward method for synthesizing uni-
form c- and a-folate derivatives via a converging syn-
thetic strategy.
The bifunctional, tridentate metal chelating system
was synthesized according to a previously described
procedure, starting from a mono-Boc protected diami-
nohexane 1 which was reacted with pyridine-2-carboxal-
dehyde to form a Schiff base, followed by an in situ
reduction [40]. The alkylation of the secondary amine
by bromo methylacetate and the following Boc-depro-
tection resulted in the PAMA-ligand 4 with a primary
amino group for coupling with any carboxyl group of
a biomolecule. Compound 4 was attached either to the
ml, streptomycin 100 lg/ml, fungizone 0.25 lg/ml).
Eighteen to twenty hours prior to each experiment, the
cells were seeded in 12-well plates (8 · 105 cells per well)
to form confluent monolayers over night.
2.16. Determination of IC50-values
Cell binding experiments to calculate IC50 values were
performed according to the following general procedure:
The monolayers were rinsed with ice-cold PBS pH 7.4.
Eight concentrations (0–10 lM) of cold folic acid, the
PAMA-c-folate derivative 8 and the corresponding rhe-
nium complex 9a were prepared in PBS pH 7.4. Pure spe-
cial RPMI medium (without FCS/L-glutamine/
antibiotics) (475 ll) and the corresponding solutions of
the compounds (500 ll) were added and the well plates
were pre-incubated at 37 ꢁC for 40 min. The 3H-folic acid
solution (25 ll, 0.8 lM; ꢁ0.02 lCi/ll) was added to each
well and the well plates incubated again at 37 ꢁC for 1 h
and then rinsed three times with ice-cold PBS pH 7.4.
The monolayers were dissolved in 1 N NaOH (1000 ll)
and after transfer in 4 ml-tubes homogenized by vortex.
The concentration of the proteins for each sample was
determined. Scintillation solution (4 ml) was added to
the cell suspensions, homogenized and transferred in a

4718
C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
i. / ii.
H
N
BocHN
2
BocHN
1
NH2
4
4
N
iii.
O
O
OCH3
OCH3
N
iv.
H2N
4
N
BocHN
3
N
4
4
N
v. / vi.
v. / vi.
O
O
O
O
O
O
γ
OCH3
N
OCH3
N
H
N
H
N
N
N
α
RHN
γ
RHN
α
4
4
O
10
O
5
5: R = Boc
10: R = Boc
11: R = H
vii.
vii.
6: R = H
Scheme 1. (i) Pyridine-2-carboxaldehyde, toluol, reflux 3 h; (ii) NaBH4, MeOH, rt, over night; (iii) methyl bromoacetate, Et3N, THF, reflux, 5 h;
(iv) HCl 6 N, rt, 3 h; (v) Boc–Glu–OMe or Boc–Glu(OMe)–OH, HOBt, EDC, CH2Cl2, rt, 4 h; (vi) N-ethyldiisopropylamine, DMF, 50 ꢁC, over
night; (vii) TFA/CH2Cl2 (9:1), rt, over night.
activated c-carboxyl group of the Boc-protected gluta-
mate a-methylester or the a-carboxyl group of the
Boc-protected glutamate c-methylester (Scheme 1). This
coupling reactions monitored by TLC resulted in the
formation of the PAMA-c-glutamate 5 or a PAMA-a-
glutamate derivative 10, respectively. The reactions were
relatively fast and the products 5 and 10 received in
good yields of 75% after chromatographic purification.
The following deprotection reaction performed under
acidic conditions ensured intact ester protecting groups.
The NMR spectra proved selectivity of the Boc-depro-
tection at the a-amino-group of these compounds (5,
10) by disappearance of the singlet at 1.5 ppm represent-
ing nine protons of the tert-butyl group. Compounds 6
and 11 where then coupled via the a-amino group to
N2,N10-protected pteroic acid, activated by carbonyldi-
imidazole (CDI) as previously described (Scheme 2) [44].
These reactions were followed by HPLC analyses whereby
after 12 h the HPLC trace showed a peak with a reten-
tion time of ꢂ30 min, clearly indicating the formation of
a novel product (starting material: Rt = 25 min and 28
min, respectively). The deprotection reactions were per-
formed under alkaline conditions. The final product
showed a significantly shifted peak detected after ꢂ18
min. The PAMA-c- (8) or PAMA-a-folate (12) deriva-
tives, respectively, were unambiguously identified by
means of NMR- and mass spectroscopy. The coupling
of the ligand 4 directly to the activated N2,N10-protect-
ed pteroic acid as well as the deprotection (yielding in
compound 14) were performed in the same way as de-
scribed for the folate derivatives. Based on the signifi-
cantly longer retention time of compound 14 (ꢂ33 min
before and 20 min after deprotection) it can be con-
cluded that the pteroate derivative was more lipophilic
than the folate derivatives 8 and 12. The yields of the fi-
nal coupling reaction to obtain 8, 12 and 14 were gener-
ally low (10% (8), 7% (12) and 15% (14), respectively).
Incomplete coupling as well as the purification and iso-
lation of the products by extraction and precipitation
are reasons for these low yields. However our method
exhibits the important advantage of well defined deriva-
tives accessible without the necessity of arduous separat-
ing steps.
Starting from the compounds 8, 12 and 14 the Re-
complexes 9a, 13a, 15a have been synthesized on the
macroscopic level using the organometallic precursor
(NEt4)2[ReBr3(CO)3] (Scheme 3). Due to the fact that
the folate (8, 12) and especially the pteroate (14) deriva-

C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
4719
O
OH
O
N
OH
N
O
H
N
O
N
N
H
γ
α
i. / ii./ iii.
i. / ii./ iii.
i. / ii./ iii.
O
N
N
HN
H2N
N
H
8
O
O
OH
γ
O
N
OH
N
H
N
O
O
N
N
H
α
O
N
OH
O
N
N
HN
H2N
N
H
12
N
N
HN
R'HN
N
R''
O
7
O
OH
N
N
O
N
N
H
N
N
14
HN
H2N
N
H
Scheme 2. R0 = N2-N,N-dimethylamino-group, R00 = formyl-group: (i) CDI, rt, 3h; (ii) 6, 11 or 4 in CH2Cl2, rt, over night; (iii) NaOH, rt, 4–5 h.
O
O
O
M
OH2
M
COOH
OH
N
COOH
CO
OC
N
H
N
H2O
OC
OH2
CO
H
N
R
N
O
i.
i.
i.
R
N
N
H
α
γ
γ
N
α
4
H
4
O
OC
O
CO
8
9a,b
COOH
O
COOH
γ
O
M
OH
γ
H
N
CO
OC
N
H
N
N
R
N
R
α
N
N
H
α
4
O
H
4
OC
N
O
12
13a,b
O
O
O
M
OH
N
H
CO
OC
N
H
N
N
N
R
N
R
4
4
OC
14
15a,b
Scheme 3. R = pteroic acid; (i) 9a, 13a, 15a, M = Re: MeOH/H2O (2/1), rt, 3 h; 9b, 13b, 15b: M = 99mTc: PBS pH 7.4, 75 ꢁC, 30 min.
tives were poorly soluble in neutral or acidic aqueous
solutions, but reasonably soluble under alkaline condi-
tions, the formation of the desired rhenium complexes
competed with the formation of the cluster [Re(OH)-
(CO)3]4 formed under likewise conditions [45]. Therefore
a slight excess of (NEt4)2[ReBr3(CO)3] (1.1 or 1.5 eq.,
respectively) was necessary in particular for the forma-
tion of the pteroate complex 15a. After completion of
the reaction the complexes could be isolated from the
acidified reaction solutions. The precipitates were cen-
trifugated and washed several times with water. An
additional washing step with ether was necessary in or-
der to eliminate the rhenium clusters. The IR spectra
of the rhenium complexes (9a, 13a, 15a) revealed the
characteristic fac-[M(CO)3] pattern (2024–2025 cmꢀ1
and 1897–1899 cmꢀ1, respectively) with significantly
blueshifted CO stretch frequencies compared to the
starting material (NEt4)2[ReBr3(CO)3] (2000 and 1868

4720
C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
cmꢀ1). In the H NMR spectra the typical patterns of
AB spin systems (J = 15–17 Hz) of the methylene groups
of the PAMA chelator were observed. This proved the
desired tridentate coordination of the metal-tricarbonyl
core via the PAMA chelator as previously described by
our group [40]. Compared to the unlabeled derivatives
(8, 12: Rt = 18.9 min; 14: Rt = 20.0 min) the UV-
HPLC-traces of the rhenium-complexes 9a, 13a and
15a showed a significant shift to a longer retention time
(9a, 13a: Rt = 27.2 min; 15a: Rt = 26.3 min). Similar
retention times were observed for the corresponding
99mTc-radiolabeled complexes (9b, 13b, 15b) (Fig. 2).
The radiolabeling with 99mTc-tricarbonyl under mild
and aqueous conditions was achieved (>95%) for all of
the derivatives 8, 12 and 14 in a concentration of 10ꢀ4
M and a reaction time of 30 min at 75 ꢁC to obtain
the 99mTc-complexes 9b, 13b, 15b. No ‘‘unspecific’’ radi-
olabeling via functional groups of the pterin-moiety
could be observed, indicating the proposed characteris-
tics of the PAMA-system as an effective chelator.
1
Fig. 3. In vitro experiment of the 99mTc-complexes 9b, 13b, 15b with
KB cells (human nasopharyngeal carcinoma cell line, overexpressing
the FR); cell binding, cell internalization and displacement by excess
folic acid (<1% bound of total activity).
3.2. In vitro evaluation
To investigate specific FR-binding of the derivatives
we performed in vitro experiments with KB cells, a hu-
man nasopharyngeal carcinoma cell line overexpressing
the FR. The cells were cultured in folate deficient med-
ium with a concentration similar to that of human
plasma to provoke up-regulation of the FR expression.
The 99mTc-radiolabeled folate (9b, 13b) and pteroate
(15b) derivatives, separated from unlabeled substance
(8, 12, 14) by HPLC, evidenced an unexpected high cell
binding of 48 7% (9b), 57 1% (13b), 60 2% (15b) of
total activity (Fig. 3). The internalized fractions were
approximately one third of total binding (15 2%
(9b), 16 1% (13b), 21 2% (15b) of total activity). If
the cells were pre-incubated with excess folic acid almost
complete displacement of the activity could be observed
(0.05 0.01% (9b), 0.02 0.01% (13b), 0.27 0.01%
(15b)), indicating that the binding of all of the tested
derivatives was highly specific. The amount of cell bind-
ing and internalization were in the same range for the c-,
a-folate and pteroate derivatives which clearly disproved
the necessity of a free carboxyl group or the glutamate
moiety for FR binding. This could be important consid-
ering the future development of potential folate or pter-
oate (radio)pharmaceuticals.
Fig. 4. Determination of the IC50-values, using KB cells (human
nasopharyngeal carcinoma cell line overexpressing the FR): Inhibition
of 3H-folic acid with cold folic acid (IC50 = 50 nM), compound 8
(IC50 = 100 nM) and the corresponding rhenium complex 9a
(IC50 = 110 nM).
carbonyl complex. Due to equal cell binding properties
of the c- and a-folate derivative as well as of the pteroate
derivative as earlier described it can be assumed that the
IC50-values of the a-folate and the pteroate derivative
are in the same range as for the c-folate derivative.
99mTc-radiolabeld complexes (9b, 13b, 15b) were
tested for plasma stability by means of TLC analyses.
Up to 4 h incubation in plasma at 37 ꢁC no decomposi-
tion of the complexes could be observed and after 24 h
<10% complexes were decomposed to pertechnetate.
The derivatives (9b, 13b, 15b) were tested for their
unspecific binding to plasma proteins (Rt = 12–18 min)
over 24 at 37 ꢁC. After 24 h a small amount (ꢂ1%) of
the activity of both folate derivatives (9b, 13b) and
10% for the pteroate derivative 15b were bound to
plasma proteins (Rt = 15.5 min). Slightly higher and
Inhibition experiments with KB cells (Fig. 4), per-
formed with 3H-folic acid in order to determine the
IC50-values of the unlabeled PAMA-c-folate derivative
8 and the rhenium–tricarbonyl complex 9a resulted in
100 nM (8) and 110 nM (9a), respectively, and were in
the same range as found for other folate derivatives pub-
lished in the literature [28]. These experiments demon-
strated full retention of receptor binding of the
c-folate derivative and the corresponding rhenium–tri-

C. Mu¨ller et al. / Journal of Organometallic Chemistry 689 (2004) 4712–4721
4721
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derivatives have been successfully labeled with
[M(OH2)3(CO)3]+ (M = 99mTc, Re). IC50-values of the
PAMA-c-folate derivative and the corresponding rhe-
nium complex were similar to folic acid which proves
their retained receptor binding properties. The cell bind-
ing and internalization studies showed a high and spe-
cific binding to the FR for all three 99mTc-complexes.
Therefore the glutamate moiety of folic acid seems not
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Acknowledgements
We thank Merck Eprova AG, for providing us with
the pteroic acid precursor and Judith Stahel and Chris-
tine De Pasquale for assisting the in vitro experiments.
This work was financially supported by Mallinckrodt-
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