Aqueous syntheses of [(Cp-R)M(CO)3] type complexes (Cp = cyclopentadienyl, M = Mn, 99mTc, Re) with bioactive functionalities

Article abstract of DOI:10.1016/j.jorganchem.2008.12.006

We describe reactions of [^99mTc(H₂O)₃(CO)₃)]⁺ (1) with Diels-Alder products of cyclopentadiene such as "Thiele's acid" (HCp-COOH)₂ (2) and derivatives thereof in which the corresponding [(C

Full text of DOI:10.1016/j.jorganchem.2008.12.006

Journal of Organometallic Chemistry 694 (2009) 981–987
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Journal of Organometallic Chemistry
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Aqueous syntheses of [(Cp-R)M(CO)3] type complexes (Cp = cyclopentadienyl,
M = Mn, 99mTc, Re) with bioactive functionalities
*
H.W. Peindy N’Dongo, Y. Liu, D. Can, P. Schmutz, B. Spingler, R. Alberto
Institute of Inorganic Chemistry, University of Zürich, 190, CH-8057, Switzerland
a r t i c l e i n f o
a b s t r a c t
We describe reactions of [99mTc(H2O)3(CO)3)]+ (1) with Diels–Alder products of cyclopentadiene such as
‘‘Thiele’s acid” (HCp-COOH)2 (2) and derivatives thereof in which the corresponding [(Cp-
COOH)99mTc(CO)3)] (3) complex did form in water. We propose a metal mediated Diels–Alder reaction
mechanism. To show that this reaction was not limited to carboxylate groups, we synthesized conjugates
Article history:
Received 13 October 2008
Received in revised form 28 November 2008
Accepted 1 December 2008
Available online 10 December 2008
of 2 (HCp-CONHR)2 (4a–c) (4a, R = benzyl amine; 4b, R = N -Boc-L-2,3-diaminopropionic acid and 4c,
a
R = glycine). The corresponding 99mTc complexes [(4a)99mTc(CO)3)] 6a, [(4b)99mTc(CO)3)] 6b and
[(4c)99mTc(CO)3)] 6c have been prepared along the same route as for Thiele’s acid in aqueous media dem-
onstrating the general applicability of this synthetic strategy. The authenticity of the 99mTc complexes on
the no carrier added level have been confirmed by chromatographic comparison with the structurally
characterized manganese or rhenium complexes.
Keywords:
Bioorganometallic chemistry
Technetium
Radiopharmaceuticals
Labelling
Studies of the reaction of 1 with Thiele’s acid bound to a solid phase resin demonstrated the formation
of [(Cp-COOH)99mTc(CO)3)] 3 in a heterogeneous reaction. This is the first evidence for the formation of no
carrier added 99mTc radiopharmaceuticals containing cyclopentadienyl ligands via solid phase syntheses.
Macroscopically, the manganese analogue 5a and the rhenium complexes 5b–c have been prepared and
characterized by IR, NMR, ESI-MS and X-ray crystallography for 5a (monoclinic, P21/c, a = 9.8696(2) Å,
b = 25.8533(4) Å, c = 11.8414(2) Å, b = 98.7322(17)°) in order to unambiguously assign the authenticity
of the corresponding 99mTc complexes.
Retro Diels–Alder
Solid phase synthesis
Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction
Cyclopentadienyl (Cpꢀ) is one of the most basic ligand in orga-
nometallic chemistry. Cyclopentadienyl is relevant since it has a
The group 7 transition metals Tc and Re are in the main focus
for novel radiopharmaceuticals in diagnostic imaging (99mTc) or
for therapeutic (186Re, 188Re) purposes [1–4]. Organometallic com-
pounds are generally considered to require water and air free syn-
thetic conditions and to be therefore not compatible with
biomedical applications. However, the preparation of the complex
low molecular weight, it blocks three coordination sites and in-
cludes the possibility of conjugating targeting vectors. Keeping
the principle of using inert complexes for radiopharmaceutical
in vivo applications in mind, Cpꢀ forms robust organometallic
[(Cp-R)M(CO)3] (M = Re, Tc) complexes with Tc and Re being in
the oxidations state +1. In fact, complexes [(Cp-R)M(CO)3] have
been attached both to antibodies [9,10] and to steroidal hormones
[11,12] without substantial loss of receptor recognition and affin-
ity. A more prominent and well explored example is in the context
of tamoxifen/ferrocifen where [CpRe(CO)3] was shown to surrogate
ferrocene under retention of biological activity [13,14]. Cyclopen-
tadienyl on other hand has severe disadvantages for applications
in water. It is essentially insoluble and unstable in water, tends
to di- or polymerize and can hardly be deprotonated (pKa ꢁ 15).
For radiopharmaceutical applications on a routine base, it is desir-
able to prepare e.g. [(Cp-R)99mTc(CO)3] directly in water. Some ap-
proaches to [Cp-R99mTc(CO)3] have been reported but most of them
require harsh condition and organic solvents [15–18]. We reported
a fully aqueous synthesis of [(Cp-R)99mTc(CO)3] at <100 °C [19].
Still, the drawback of this approach was the sensitivity of Cp group
pendent to the biomolecule. To circumvent this issue, protection of
[
99mTc(CN-R)6]+ directly from [99mTcO4]ꢀ in water clearly elimi-
nated this prejudice [5]. This complex contained monodentate li-
gands only but is still perfectly stable under in vivo conditions
due to the kinetic stability of the d6 electronic configuration. This
isocyanide 99mTc complex is the most widely used radiopharma-
ceutical in myocardial imaging [6]. Chemically, its high stability to-
wards substitution reactions makes it not suitable for the so called
third generation radiopharmaceuticals, labeled targeting biomole-
cules. On the other hand, the need for innovative and efficient syn-
thetic methods of organometallic compounds from water is crucial
for application in the bio-organometallic field [7,8].
* Corresponding author.
E-mail address: ariel@aci.uzh.ch (R. Alberto).
0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2008.12.006

982
H.W. Peindy N’Dongo et al. / Journal of Organometallic Chemistry 694 (2009) 981–987
the Cpꢀ ligand would be helpful. The Diels–Alder product of dimer-
ized [HCp-R] would be an ideal precursor provided that it could be
concertedly cleaved and coordinated. We recently showed that the
reaction of [99mTc(H2O)3(CO)3)]+ (1) with Diels–Alder products
such as Thiele’s acid (HCp-COOH)2 (2) gave the corresponding
[(Cp-COOH) 99mTc(CO)3)] (3) complex. The labelling conditions
were such that 2 did not thermally cleave [20]. Hence, we proposed
that the retro Diels–Alder reaction with concerted coordination to
the [99mTc(CO)3]+ core was metal mediated. A first step might in-
volve the coordination of the carboxylate followed by cleavage
and coordination. Dimerized cyclopentadiene compounds can
therefore be considered as ‘‘pre-cyclopentadiene” able to form
to react with benzyl amine to yield 4a in 43% yield as white solid.
Using the same protocol, N -Boc- -2,3-diaminopropionic acid
L
a
(Boc = tert-butoxycarbonyl) was reacted with 4 and the reaction
was monitored by HPLC. Deprotection with a trifluoroacetic acid
(TFA)/CH2Cl2 mixture 1:1 gave 4b in 95% yield as an off-white so-
lid. The analytical data (see Section 4) confirmed the authenticity
of the compounds. The synthesis of 4c (HCp-gly)2 was previously
reported by us [20]. We would like to emphasize at this point, that
this strategy can be followed by principally any biomolecule. Along
this route, targeting vectors such as peptides or CNS receptor li-
gands can be introduced. Taking the clean labelling with 99mTc into
account (vide infra) it became clear that new labelled biomolecules
can be introduced in which the amide functionality acts as an
anchoring and activation group for the formation of [(Cp-
R)99mTc(CO)3] complexes.
the {(g
5-Cp)Tc} core.
As an important extension to the previous synthetic ap-
proaches, we describe inhere a more general approach to [(Cp-
R)99mTc(CO)3] complexes which also allows the introduction of
additional functionalities R on the Cp ring. Thiele’s acid was cou-
pled with amines to give amides. We show that amides can also
act as anchoring group and the corresponding 99mTc complexes
be synthesized along a retro Diels–Alder reaction. The scope of this
finding is the principal possibility of coupling different families of
biomolecules, including peptides or other small amine containing
vectors to the cyclopentadiene ring. Furthermore, we will show
that the retro Diels–Alder can also be performed directly on a solid
phase support. Thereby, the synthesis of no carrier added [(Cp-
R)99mTc(CO)3] type complexes is enabled.
2.2. Synthesis of manganese and rhenium complexes
A comparison of the HPLC retention times of the fully character-
ized manganese, rhenium or technetium complexes with the cor-
responding 99mTc compounds is an accepted method to assess
the authenticity of the 99mTc complexes. Accordingly, the manga-
nese complex [(Cp-ba)Mn(CO)3] (5a) and the rhenium complexes
[(Cp-dap)Re(CO)3] (5b) and [(Cp-gly)Re(CO)3] (5c) were synthe-
sized by different methods and fully characterized with spectro-
scopic methods including X-ray crystallography for 5a (Scheme
2). As described above for the free ligands, the complexes 5a–c
were synthesized by activation of cymantrene-carboxylic acid
[(Cp-COOH)Mn(CO)3] or its rhenium analogue with pentafluor-
ophenyl-trifluoroacetate [22] in the presence of pyridine. These
activated complexes were then allowed to react with benzylamine
to give 5a as a yellow powder in 65% yield. Compound 5a could be
recrystallised from CH2Cl2/hexane to afford X-ray quality crystals.
The proton NMR of 5a in CDCl3 showed the aromatic protons at
7.25 ppm as multiplet and the protons of the cyclopentadienyl ring
were observed at 5.23 and 4.70 ppm, respectively, as two distinct
multiplets. The NH amide proton and the methylene proton are ob-
served at 5.91 and 4.31 ppm as two broad signals. The infrared
spectrum in KBr showed the two characteristic strong bands for
mCO at 2028 and 1927 cmꢀ1, respectively, which confirmed the
presence of the fac-[Mn(CO)3]+ moiety. The amide C@O bond
stretch showed a strong absorption at 1638 cmꢀ1. Furthermore,
the ESI-MS in both, positive and negative mode showed single
peaks at 360 and 336 corresponding to M+Na, respectively, MꢀH.
5b was prepared using the same protocol with [(Cp-COOH)-
2. Results and discussion
2.1. Synthesis of ligands
Thiele’s acid is a very convenient precursor for the preparation
of cyclopentadiene derivatives. Starting from [HCp-COOH], Diels–
Alder dimerisation gave different stereoisomers [21]. One of the
isomers could be separated by fractioned crystallization and its
structure being elucidated. An ORTEP together with important
bond lengths and angles is given in Fig. 1. All subsequent function-
alisations were then performed with this single isomer (Scheme 1).
The preparation of ligand 4a (HCp-ba)2 and 4b (HCp-dap)2 was
performed in a two step reaction. Thiele’s acid was easily activated
with pentafluorophenyl-trifluoroacetate in an N,N-dimethylform-
amide (DMF)/pyridine mixture at room temperature to yield 4 in
quantitative yield in solution [22]. Precursor 4 was then allowed
Re(CO)3] and N -Boc-L-2,3-diaminopropionic acid followed by
a
TFA deprotection. After workup 5b was isolated as TFA salt giving
a yield of 70% with respect to the starting material [(Cp-COOH)-
Re(CO)3]. The ESI-MS positive mode measurements realized in
methanol, indicated a peak at 467 as a main peak corresponding
to M+H. As expected, the IR spectrum displays two strong vibration
mCO at 2026 and 1926 cmꢀ1 associated to fac-[Re(CO)3]+ fragment
and one amide C@O bond stretch at 1639 cmꢀ1. The proton NMR
spectrum in CD3OD confirmed the proposed structure with the
presence of two triplets associated to Cp ring at 6.16 and
5.59 ppm while the
a proton to amino acid function and the CH2
appear as two multiplets at 3.75 and 3.68 ppm.
Crystals suitable for an X-ray structure analysis were obtained
for cymantrene functionalized with benzyl amine 5a by recrystal-
lization from CH2Cl2/hexane. An ORTEP is given in Fig. 2. The com-
pound crystallizes in the monoclinic crystal system (Table 1). The
manganese central atom is
g
5-coordinated to the C5H4–CO–NH–
Fig. 1. ORTEP presentation of Thiele’s acid dimethyl ester. Important bond lengths
(Å) and angles (°) are: C(2)–C(6) = 1.3374(19); C(12)–C(16) = 1.5009(18); C(12)–
C(13) = 1.3343(17); C(13)–C(14) = 1.4947(17); C(6)–C(2)–C(1) = 124.76(12); C(6)–
C(2)–C(3) = 107.74(11); C(13)–C(12)–C(11) = 126.00(12); C(13)–C(12)–C(16) =
113.05(11); C(12)–C(13)–C(14) = 112.40(11).
CH2–C6H5 ligand and the three CO groups complete the coordina-
tion sphere. The geometry around manganese is pseudo octahedral
and, accordingly, C–M–C between CO are close to 90°. The amide
group O4–C9–N1 has the usual geometrical parameters with a

H.W. Peindy N’Dongo et al. / Journal of Organometallic Chemistry 694 (2009) 981–987
983
H
N
H
N
O
4a
O
O
C6F5-O2C2F3
F
F
F
HOOC
H2N
HOOC
O
H
N
HO
H
O
F
F
F
Pyridine / DMF
OH
F
N
NH2
O
O
4
O
2
O
F
O
4b
4c
F
F
H
H
N
N
HOOC
COOH
O
O
Scheme 1. Syntheses of the activated form of Thiele’s acid 4, and amide derivatives 4a–4c containing a benzyl group, an amino acid and glycine.
F
F
F
F
O
O
O
OH
F
C6F5-O2C2F3
M
M
CO
CO
OC
OC
Pyridine / DMF
CO
CO
3 / 3´
M = Mn, Re
M = Mn, Re
O
O
O
NH2
N
H
COOH
N
N
H
H
COOH
Re
5c
Re
Mn
CO
CO
CO
CO
CO
OC
OC
OC
CO
5b
5a
Scheme 2. Syntheses of the manganese and rhenium complexes 5a–c.
Table 1
Crystallography parameters for complex 5a and Thiele’s dimethyl ester
Formula
C16H12MnNO4
337.21
C14H16O4
248.27
M (g molꢀ1
)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
Monoclinic
P21/c
Monoclinic
P21/n
9.8696(2)
25.8533(4)
11.8414(2)
98.7322(17)
2986.45(9)
8
1.500
0.900
Multi-scan
0.88553/1.00000
38865
8.0642(2)
6.0850(1)
24.9771(5)
94.453(2)
1221.94(4)
4
1.350
0.099
Multi-scan
0.91246/1.00000
13181
b (°)
V (Å3)
Fig. 2. ORTEP presentation of [(Cp-ba)Mn(CO)3] (5a). Important bond lengths (Å)
and angles (°) are: Mn(1)–C(1) = 1.7905(16); Mn(1)–C(3) = 1.796(2); Mn(1)–
C(2) = 1.7996(17); Mn(1)–C(4) = 2.1210(15); Mn(1)–C(5) = 2.1363(16); C(9)–
N(1) = 1.339(2); C(10)–N(1) = 1.452(2); C(1)–O(1) = 1.1414(19); C(2)–O(2) =
Z
Dcalc. (g cmꢀ3
)
Linear absorption coefficient (mmꢀ1
Absorption correction
)
1.1423(19);
C(4)–C(8) = 1.428(2);
C(4)–C(5) = 1.430(2);
C(1)–Mn(1)–C(3) =
Relative transmin/transmax
Measured reflections
92.11(8); C(1)–Mn(1)–C(2) = 92.47(7); C(2)–Mn(1)–C(3) = 92.26(8); C(1)–Mn(1)–
C(4) = 99.81(7); O(1)–C(1)–Mn(1) = 178.07(15); O(2)–C(2)–Mn(1) = 178.72(15).
Unique reflections [R(int)
]
9105/0.0387
397
0.0347/0.0835
0.953
3734/0.0242
165
0.0480/0.1230
1.057
Refined parameters
R1(F)/wR2 (F2) (I > 2
r
(I))a
GOF
C–N bond length of 1.339(2) Å and is essentially in the same plane
with the neighboring cyclopentadienyl ring (1.2° torsion angle be-
tween the amide group and the Cp plane). This lead to orthogonal
orientation between the Cp and the phenyl ring. All the geometri-
cal parameters agreed well with the data reported for
(C5H4COCH3)Mn(CO)3 in the literature [23].
R1 ¼ jFo ꢀ Fcj=jFoj; wR2 ¼ ½wðF2o ꢀ F2c Þ =ðwFo2Þꢂꢀ1=2
.
2
a
Diels–Alder reactions with building blocks different from 2. We
were especially interested in other functionalities, particularly
amide groups, bound to the cyclopentadiene ring. Although this
has principally been shown with ligand 4c, the presence of the car-
boxylate group in this ligand might still be the anchoring group re-
quired for cyclopentadienyl complex formation. In ligand 4a, this
was not the case anymore. Thus, if 4a could be labelled, a clear hint
was given that a carboxylate group is not an absolute requirement
for retro Diels–Alder reaction. This should lead to a more general
approach towards [(Cp-R)99mTc(CO)3] complexes conjugated to
2.3. Syntheses of 99mTc complexes
We recently reported the metal mediated retro Diels–Alder
reaction of (HCp-COOH)2 with [99mTc(OH2)3(CO)3]+ or [99mTcO4]ꢀ
under formation of the corresponding cyclopentadienyl complex
[(Cp-COOH)99mTc(CO)3] (3) [20,24]. On the basis of these results,
we investigated the possibility of extending this method to retro

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H.W. Peindy N’Dongo et al. / Journal of Organometallic Chemistry 694 (2009) 981–987
targeting molecules. Complexes 5a–c are the models based on
which the identity of the corresponding 99mTc complexes can be
confirmed.
We used three different procedures to synthesize the 99mTc
complexes 6a–6c. In particular, pH, reaction time and temperature
were varied in order to find optimal conditions for the metal med-
iated retro Diels–Alder reaction. Method A involved the classical
‘‘one pot” reaction which was the direct synthesis from [99mTcO4]ꢀ
in an IsolinkÒ Kit and in the presence of ligands 4a–c. Method B
was a two step procedure under alkaline conditions, thereby
[
99mTc(OH2)3(CO)3]+ was synthesized first and then reacted with
the corresponding ligand. Method C finally consisted also in a
two step reaction, initial synthesis of 1, neutralizing the solution
with phosphate buffer to pH 7.4 and subsequent addition and reac-
tion with 4a–c. The reactions and the corresponding products are
depicted in Scheme 3.
Fig. 3. HPLC trace of the cold manganese complex 5a and the corresponding 99mTc
complex 6a complex as prepared directly from [99mTcO4]ꢀ. The time difference is
due to detector separation.
The compounds (HCp-CONHR)2 (4a-b) were reacted directly
with [99mTcO4]ꢀ in the presence of boranocarbonate [H3BCOOH]ꢀ
to give 6a–b after 30–180 min at 95 °C in quantitative yield. In
the reaction solutions, we could not detect measurable amounts
of the respective monomers of 4a or 4b by HPLC. Still, for all reac-
tions, one HCp-R per 99mTc must be released. Since the concentra-
tion of [99mTcO4]ꢀ is very low (10ꢀ8–10ꢀ7 M) we did not expect to
find the monomer provided that no thermal retro Diels–Alder reac-
tion occurred, which was obviously not [25] the case. Decreasing
the temperature to 70 °C reduced the rate of the reaction and we
started to observe the formation of side products especially for
4b. Since no corresponding model compounds could be used to
identify the composition of these side products, we assume that
under more moderate conditions coordination might also take
place at the amino acid part in 4b. Though not a very favorable
bidentate ligand, we showed previously that coordination takes
place under formation of well defined amino acid complexes
[20]. Unlike of basic direct synthesis of 6a and 6c, the preparation
of 6b is best realized with the buffered method which gave quan-
titative conversion. The pH of the reaction seemed of to be crucial
for the metal mediated retro Diels–Alder reaction especially for the
zwitterions of amino acid derivatives. The authenticity of the 99mTc
compounds could be assessed by comparison with the cold manga-
nese and rhenium analogues synthesized before. An example for an
HPLC comparison is given in Fig. 3. The inverted trace on top shows
the radiochromatogram of 6a whereas the lower one gives the UV–
Vis absorption trace of the manganese surrogate 5a.
crucial in application when working with receptor targeting
agents. A too high concentration of e.g. unlabelled peptide would
avoid good images since the cold peptides preferentially bind to
the receptors. Thus, reasonable images could not be received due
to insufficient target to background ratio. The solid phase polymer
was a polystyrene matrix functionalized with polyethyleneglycol
and bearing terminal NH2 groups. These polymers had good swell-
ing properties in water. The polymer was then linked via an amide
bond to 2 previously activated with the O-(7-Azabenzotriazol-1-
yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HATU)/
N,N-diisopropylethylamine procedure. The efficiency of the cou-
pling reaction was performed according to literature procedure
with a 2,4,6-trinitrobenzylsulfonic acid (TNBS) test. This allowed
a semi-quantitative analysis of the uncoupled terminal amino
groups [26]. Essentially no free NH2 groups were detected by this
method, thus, confirming the complete derivatization of the resin
by Thiele’s acid. The procedure and the products are shown in
Scheme 4. Additional IR spectroscopy analysis in KBr pellets
showed the presence of two news peaks at 1535 and 1712 cmꢀ1
,
respectively. Since these bands were not present in the unmodified
resin, we attributed them to the amide bond and the terminal car-
boxylic group of coupled Thiele’s acid.
The resulting polymer 7, Thiele’s acid PEG-NH-CO-(HCp)2COOH
derivative was first swelled in water at 4 °C for 3 h and then al-
lowed to react in one pot reaction in presence of [99mTcO4]ꢀ and
[H3BCOOH]ꢀ at 95 °C for 3 h (Scheme 4). The reaction mixture
was then cooled and the solid phase removed by filtration. The
HPLC analysis of the remaining solution revealed one single peak
at 21.35 min and residual [99mTcO4]ꢀ. The formation of [(Cp-
COOH)99mTc(CO)3] (3) at 21.35 min in 50% yield in the reaction
mixture. This is to our knowledge, the first evidence of aqua metal
mediated retro Diels–Alder on solid support. Even though the reac-
tion occurred in basic condition with [99mTc(H2O)3(CO)3] (1), the
modulation of other parameters such reaction time and tempera-
ture did not improved the yield but we observed the rising of addi-
tional peaks. It seems that [99mTcO4]ꢀ or 1 could not reach
efficiently the polymer bound Thiele’s acid in heterogeneous con-
ditions since it is reasonable to assume that the distribution of
polymer is not homogeneous in the vial. However, 3 could be easily
2.4. Retro Diels–Alder reaction on a solid phase
The Diels–Alder product of Thiele’s acid (HCp-COOH)2 (2) was
chosen as a model substrate to investigate if the corresponding
complexes [(Cp-COOH)99mTc(CO)3] could be synthesized directly
on solid phase support. This would give access to no carrier added
99mTc complexes. A low concentration of radiopharmaceutical is
OH2
H2O
OC
OH2
CO
R
R
+
Tc
or [99mTcO4]-
O
4a-c
O
CO
O
O
O
NH2
COOH
N
H
N
H
N
H
COOH
2
Tc
6b
Tc
OC
Tc
6a
CO
CO
CO
OC
OC
CO
CO
CO
6c
Scheme 3. Preparation of the 99mTc complexes 6a–c under aqueous conditions.
Scheme 4. Solid phase synthesis and labeling of Thiele’s acid.

H.W. Peindy N’Dongo et al. / Journal of Organometallic Chemistry 694 (2009) 981–987
985
assigned by direct synthesis from Thiele’s acid and 1 in water buf-
fer with almost quantitative conversion as previously described
[20].
4.3. N,N0-Dibenzyl-3a,4,7,7a-tetrahydro-1H-4,7-methanoindene-2,6-
dicarboxamide (4a)
Compound 4 (811 mg, 1.47 mmol) was dissolved in 15 ml
(DMF) and stirred under N2 at 0 °C. A solution of benzyl amine
(376 mg, 3.52 mmol) and NaHCO3 (296 mg, 3.52 mmol) in 15 ml
water was added drop-wise via syringe through a rubber seal
cap. The reaction mixture was stirred under N2at 0 °C for 10 h.
The solvent was then removed under reduced pressure and the res-
idue dissolved in 150 ml H2O and acidified with 1 N HCl to pH 3.
The solution was extracted three times with 150 ml of CH2Cl2.
After each extraction step the pH of the aqueous phase was re-ad-
justed to 2–3. The combined organic phases were dried over
Na2SO4. The solvent was removed in vacuo and the crude product
purified by flash chromatography with a gradient from pure CH2Cl2
to CH2Cl2/MeOH 1:1. Compound 4a was isolated as white powder
(266 mg, 43% yield). Calcd. for Mr (C26H26N2O2) 398.5; ESI-
MS(CH3OH, pos. Mode): 421.3 [M+Na]+. Anal. Calc. for
C26H26N2O2: C, 78.36; H, 6.58; N, 7.03. Found: C, 78.30; H, 6.74;
N, 6.92%. 1H NMR (200 MHz, CDCl3), d 7.29–7.32 (m, 10H, Ph),
6.43 (d, 1H, J = 3.2 Hz, CH), 6.15 (d, 1H, J = 2.8 Hz, CH), 5.75–5.86
(m, 2H, NH), 4.35–4.52 (m, 4H, CH2), 3.65 (m, 1H, CH), 3.43 (m,
1H, CH), 3.06 (m, 1H, CH), 2.96 (m, 1H, CH), 2.01–2.43 (m, 2H,
CH2), 1.45–1.67 (2H, CH2).
3. Conclusion
Thiele’s acid has been conjugated to different functional groups.
These dimers ligands were found to be compatible to generated pia-
no stool-like 99mTc complexes in water. Metal mediated retro Diels–
Alder seems to be a new general approach to produce biomolecules
[(Cp-R)99mTc(CO)3] with more complex functionalities bound to
cyclopentadienyl. Furthermore, promising preliminary studies
shown that the scope of this retro Diels–Alder reaction can be ex-
tend to the solid phase synthesis in aqueous media. This water solid
phase synthesis of 99mTc complexes is to our knowledge, the first
evidence of metal mediated retro Diels–Alder on solid support.
4. Experimental
4.1. General remarks
Reactions were carried out in oven-dried Schlenk glassware un-
der an atmosphere of pure nitrogen when necessary. Solvents were
dried over molecular sieves and degassed prior to use. All chemi-
cals were obtained from commercial sources and used without fur-
ther purification. The acid [(Cp-COOH)M(CO)3] (M = Mn, Re) and 5c
were prepared according to literature procedure [20,21]. NMR
spectra were recorded on Bruker Advance 500, 400 and Varian
200 spectrometers. Chemical shifts d in ppm relative to tetrameth-
ylsilane (TMS) and coupling constants J are given in Hz. Mass spec-
tra were measured on Bruker Esquire HCT (ESI) instrument, only
characteristic fragments are given. The solvent flow rate for ESI
4.4. 3,30-[Tricyclo[5.2.1.02,6]deca-3,8-diene-4,8-
diylbis(carbonylimino)]bis(2-aminopropanoic acid) (4b)
Compound 4 (1.620 g, 2.93 mmol) was dissolved in 33 ml DMF
and stirred under N2 at 0 °C. A solution of N -Boc-L-2,3-diamino-
a
propionic acid (Boc-Dap-OH) (1.438 g, 7.04 mmol) and NaHCO3
(591 mg, 7.04 mmol) in 33 ml H2O was added drop-wise via syr-
inge. The reaction mixture was stirred under N2 at 0 °C during
10 h. The solvent was removed and the residue dissolved in
300 ml H2O and acidified with 1 N HCl to pH 3. The solution
was extracted 3ꢃ with 300 ml CH2Cl2. After each extraction step
the pH of the aqueous phase was re-adjusted to 2–3. The com-
bined organic phases were dried over Na2SO4. The crude product
was purified by flash chromatography as for 4a. The product
(1.020 g, 59% yield) was obtained as an off-white powder. For
deprotection, 103 mg (0.17 mmol) was dissolved in 2 ml of a 1:1
(v/v) mixture of F3CCOOH and CH2Cl2. After stirring for 4 h at
r.t. under N2, the solution was cooled to 0 °C and 2 ml of H3CCN
were added, followed by solvent removal in vacuo. This procedure
was repeated twice. The dry residue was dissolved in 8 ml 1 N HCl
and lyophilized, resulting in 100 mg (95% yield) 4b as a slightly
brown solid. Calcd. for Mr (C18H24N4O6) 392.41; ESI-MS(CH3OH,
neg. Mode): 391.1 [MꢀH]ꢀ; 1H NMR (500 MHz, D2O): d 6.84 (m,
1H, CH), 6.48 (m, 1H, CH), 4.28–4.16 (m, 2H, CH2), 3.90–3.72
(m, 4H, CH2), 3.68–3.59 (m, 1H, CH), 3.38–3.31 (m, 1H, CH),
3.29–3.22 (m, 1H, CH), 3.12–3.02 (m, 1H, CH), 2.58–2.47 (m, 1H,
CH), 2.01–1.83 (m, 1H, CH), 1.73–1.66 (m, 1H, CH), 1.53–1.46
(m, 1H, CH).
measurements was 5 l
l minꢀ1 with a nebulizer pressure of 15 psi
and a dry gas flow rate of 5 l minꢀ1 at a dry gas temperature of
300 °C. IR spectra were recorded as KBr pellets on a Perkin Elmer
BX II IR spectrometer.
HPLC solvents consisted of 0.1% CF3COOH in H2O (solvent A)
and methanol (solvent B) with variable gradient (0–3 min, 100%
A; 3–3.1 min, 0–25% B; 3.1–9 min, 25% B; 9–9.1 min, 25% B to
34% B; 9.1–20 min, 34% B to 100% B; 20–25 min, 100% B; 25–
25.1 min 100% B to 100% A; 25.1–30 min 100% A).
4.2. Bis(pentafluorophenyl) 3a,4,7,7a-tetrahydro-1H-4,7-
methanoindene-2,6-dicarboxylate (4)
3a,4,7,7a-Tetrahydro-1H-4,7-methanoindene-2,6-dicarboxylic
acid (Thiele’s acid) 2 (881 mg, 4.0 mmol) was dissolved in 9 ml of
DMF. Pyridine (708 ll, 8.8 mmol) and pentafluorophenyl-trifluoro-
acetate (1.6 ml, 9.3 mmol) were added, and the solution stirred at
r.t. under N2 for 5 h. 600 ml ethyl acetate were added, and the solu-
tion washed three times with 0.1 N HCl and once with 5% (wt./vol.)
aqueous NaHCO3. The organic phase was dried over Na2SO4 and
the solvent removed in vacuo to produce 2.346 g of crude product
which contained approximately 20% of mono-activated product.
The solid was then dissolved in 200 ml of CH2Cl2 and extracted
with 400 ml of 5% Na2HCO3. The organic phase was dried under re-
duced pressure and 1.639 g (74% yield) of 4 was isolated as a
brownish oil. The product was used for the next synthesis step
without further purification.
4.5. Synthesis of [(CpCONHCH2C6H5)Mn(CO)3] (5a)
Cymantrene-carboxylic acid (248 mg, 1 mmol) was dissolved in
1 ml of dry DMF. Pyridine (81
ll, 0.853 mmol) and pentafluorophe-
nyl-trifluoroacetate (PFT, 174
ll, 1.0 mmol) were added and the
solution was stirred for 3 h under N2 at r.t. The reaction mixture
was diluted with 30 ml ethyl acetate and washed 3ꢃ with 30 ml
of 0.1 M HCl and once with 30 ml of 5% NaHCO3. The organic phase
was dried over Na2SO4 the solvent removed under reduced pres-
sure and the yellow solid 3 (350 mg, 85% yield) was used without
further purification.
1H NMR (400 MHz, CDCl3): d 7.28 (d, 1H, J = 3.2 Hz, CH), 6.88 (d,
1H, J = 2.1 Hz, CH), 3.71 (m, 1H, CH), 3.53 (m, 1H, CH), 3.35 (m, 1H),
3.12 (m, 1H, CH), 2.68 (m, 1H, CH), 2.25 (m, 1H, CH), 1.86 (m, 1H,
CH), 1.55 (m, 1H, CH). 19F{1H} NMR (CDCl3): d ꢀ154.6, ꢀ159.8,
ꢀ164.0.

986
H.W. Peindy N’Dongo et al. / Journal of Organometallic Chemistry 694 (2009) 981–987
Activated cymantrene-carboxylic acid (3) (207 mg, 0.5 mmol)
5. Technetium complexes synthesis
was dissolved in 8.5 ml of DMF and stirred under N2 at 0 °C. A solu-
tion of benzyl amine (53 mg, 0.5 mmol) and NaHCO3 (66 mg,
0.78 mmol) in 8.5 ml of H2O was added via syringe. The reaction
mixture was stirred under N2 at 0 °C for 4 h. After solvent removal,
the residue was dissolved in 50 ml of CH2Cl2 and washed with
50 ml of 1 mM HCl. A small amount of 0.1 M HCl was added during
extraction to ensure a final pH of 3 in the aqueous phase which was
then extracted 3ꢃ with 150 ml of CH2Cl2. The combined organic
phases were dried over Na2SO4. The crude product was purified
5.1. Solid phase synthesis
NovaSynÒ TG amino resin (400 mg, 0.116 mmol –NH2 groups)
was left to swell in DMF during 1 h and washed several times with
DMF. The supernatant was removed. Thiele’s acid (64 mg,
0.29 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyl-
uronium hexafluorophosphate (HATU) (220 mg, 0.58 mmol) were
dissolved in 1 ml of DMF and added to the resin. N,N-diisopropyl-
by flash chromatography, using
a gradient from CH2Cl2 to
ethylamine (200 ll, 1.16 mmol) was added, and the suspension
CH2Cl2/MeOH 5:1. The product (396 mg, 65% yield) was obtained
as a slightly yellow powder which could be recrystallised from
CH2Cl2/hexane mixture. Calcd. for Mr (C16H12MnNO4) 337.21;
ESI-MS (CH3OH, pos. Mode): 360.0 [M+Na]+, ESI-MS (CH3OH, neg.
Mode): 336.0 [MꢀH]ꢀ. Anal. Calc. for C16H12MnNO4: C, 56.99; H,
3.59; N, 4.15. Found: C, 56.81; H, 3.63; N, 4.16%. 1H NMR
(500 MHz, CDCl3): d 7.25 (m, 5H, Ph), 5.91 (m, 1H, NH), 5.23 (m,
2H, Cp), 4.70 (m, 2H; Cp), 4.31 (m, 2H, CH2); 13C{1H} NMR (125
MHz, CDCl3): d 223.90, 138.25 (1C, Ph), 129.34 (1C, Ph), 128.38
(1C, Ph), 128.26 (1C, Ph), 91.13 (1C, Cp), 85.12 (1C, Cp), 83.41
(1C, Cp), 44.36 (1C, CH2); IR (KBr): 3300, 2028, 1927, 1638,
was stirred very gently for 18 h under N2 at r.t. The resin was fil-
tered and washed 5ꢃ each with 5 ml of DMF, CH2Cl2, and finally
2-propanol (to de-swell the resin). The resin was dried in vacuo
overnight and stored at 4 °C prior to use.
To check the completeness of the coupling a TNBS test was per-
formed according to literature procedure [26]. No free –NH2 groups
were detected, thus confirming the complete derivatization of the
resin by Thiele’s acid. IR (KBr pellet) showed peaks at 1535 and
1712 cmꢀ1 not present in the unmodified resin, which were attrib-
uted to the amide bond and the terminal carboxylic group of Thi-
ele’s acid, respectively.
1560 cmꢀ1
.
5.2. Labelling method A (one-pot labeling)
4.6. Synthesis of [(CpCOOC6F5)Re(CO)3] (30)
A vial was charged with [H3BCOOH]– (4 mg) and Na2[tar-
trate]2H2O (7 mg), Na2B4O7 ꢄ 10H2O (7 mg) and the ligands 4a–c.
The vial was then sealed and flushed with N2. The [99mTcO4]ꢀ elute
(1 mL) was injected into the vial and the resulting mixture was
heated at 95 °C for 30 min, after which time the reaction products
were analyzed with HPLC coupled with gamma detector.
[(Cp-COOH)Re(CO)3] (274 mg, 0.775 mmol) was dissolved in
1 ml of dry DMF. Pyridine (69 ll, 0.853 mmol) and PFT (155 ll,
0.900 mmol) were added and the solution was stirred for 3 h un-
der N2 at r.t. The reaction mixture was diluted with 75 ml ethyl
acetate and washed three times with 75 ml of 0.1 M HCl and then
with 75 ml of 5% NaHCO3. The organic phase was dried with
Na2SO4 and the solvent was removed in vacuo to yield 344 mg
(81%) of 30.
5.3. Method B: 99mTc-complexes in alkaline conditions
1H NMR (200 MHz; acetone-d6): d 6.55 (t, 2H, J = 2.4 Hz, Cp),
5.89 (t, 2H, J = 2.4 Hz, Cp); 19F{1H} NMR (acetone-d6): d ꢀ155.0,
ꢀ159, 164.5.
To a solution of fac-[99mTc(H2O)3(CO)3]+ (1) (900
ll) 100 ll of
10ꢀ2 or 10ꢀ3 M stock solution of the ligands 4a–c in MeOH under
N2, were added. The vials was then brought to 95 °C, and the final
solution analyzed by HPLC coupled with gamma detector after
cooling to r.t.
4.7. Synthesis of [Re(CO)3(CpCONHCH2CH(NH3)COOH)](CF3COO) (5b)
5.4. Method C: 99mTc-complexes in buffered conditions
Compound 30 (711 mg, 1.30 mmol) was dissolved in 8.5 ml of
DMF and stirred under N2 at 0 °C. A solution of N -Boc-L-2,3-diami-
a
The pH of a solution containing 1 was adjusted (pH 7.4) by a
nopropionic acid (Boc-Dap-OH) (319 mg, 1.56 mmol) and NaHCO3
(132 mg, 1.56 mmol) in 8.5 ml of water was added drop-wise via
syringe. The reaction mixture was stirred under N2 at 0 °C for
4 h, the solvent removed in vacuo and the residue dissolved in
150 ml of CH2Cl2 and washed with 150 ml of 1 mM HCl. A small
amount of 0.1 M HCl was added during extraction to ensure a final
pH of 3 in the aqueous phase which was extracted 3ꢃ with 150 ml
of CH2Cl2. The combined organic phases were dried with Na2SO4
and the solvent was removed under reduced pressure. The crude
product was purified by flash chromatography, using a gradient
from CH2Cl2 to CH2Cl2/MeOH 5:1. The product (396 mg, 65% yield)
was obtained as a slightly brownish powder which was used for
the next step by dissolving 104 mg (0.175 mmol) in mixture of
1 ml CH2Cl2 and 1 ml TFA. The removal of Boc group reaction
was complete after stirring at r.t. for 2 h under N2 as indicated by
HPLC. Twenty five milliliters of CH3CN were then added, followed
by solvent removal. This procedure was repeated four times to pro-
duce 436 mg (72%) of 5b as a brownish solid. Calcd. for Mr
(C12H11N2O6Re) 466.43; ESI-MS(CH3OH, pos. Mode): 467.0
[M+H]+. 1H NMR (500 MHz, CD3OD): d 6.16 (m, 2H, Cp), 5.59 (t,
2H, J = 2.9 Hz, Cp), 3.75 (m, 1H, CH), 3.68 (m, 2H, CH2); 13C{1H}
NMR (125 MHz, CD3OD): d 194.0, 169.9 166.9, 94.5, 88.7, 41.0. IR
solution of 1 N HCl (respectively 1 N NaOH) and phosphate buffer.
Nine hundred microliters of 1 was then added to 100 l
l of 10ꢀ2 or
10ꢀ3 M stock solution of ligands 4a–c were mixed together and
protected with N2. The mixture was allowed to heat at 95 °C, and
the labeling products were analyzed with HPLC coupled with gam-
ma detector.
5.5. Solid phase labeling of Thiele’s acid
Polymer 7 (4 mg) was previously swelled in water at 4 °C for 3 h
and then the solid was filtrated off. The vial was charged with
Na[H3BCO2H] (4 mg), Na2B4O7 ꢄ 10H2O (7 mg) and Na2[tar-
trate]2H2O (7 mg). The vial was then sealed and flushed with N2.
The [99mTcO4]ꢀ elute (1 mL) was injected into the vial and the result-
ing mixture was heated at 95 °C for 3 h, after which time the reaction
products were analyzed with HPLC coupled with gamma detector.
5.6. X-ray crystallographic data collection and refinement of the
structures of dimethyl ester of Thiele’s acid and 5a
Crystallographic data were collected at 183(2) K on an Oxford
Diffraction Xcalibur system with a Ruby detector using Mo Ka radi-
(KBr) 3420, 2026, 1926, 1639 cmꢀ1
.

H.W. Peindy N’Dongo et al. / Journal of Organometallic Chemistry 694 (2009) 981–987
987
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Supplementary material
CCDC 704728 contains the supplementary crystallographic data
for 5a. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Acknowledgements
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This work was supported by the Swiss Federal Secretariat for
Research and Education under Contract No. SBF C06.0109 and the
University of Zurich.
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