Syntheses of bifunctional 2,3-diamino propionic acid-based chelators as small and strong tripod ligands for the labelling of biomolecules with 99mTc

Article abstract of DOI:10.1039/c002796k

The labelling of targeting biomolecules requires small and hydrophilic complexes in order to not affect the binding properties of the vectors. 2,3-Diamino propionic acid (dap) is a small and strong, albeit scarcely used, tripod ligand for the fac-[^99mTc(CO)₃]⁺ moiety. We have introduced at the α-carbon atom in the basic dap structure various second functionalities such as carboxylato, amino and α-amino acid groups via various spacers in order to yield bifunctional chelators. These dap derivatives can be coupled to targeting molecules for application in molecular imaging. Full characterizations of the bifunctional chelators, X-ray structures of intermediates and of one rhenium complex, as well as labelling studies with ^99mTc, are presented.

Full text of DOI:10.1039/c002796k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Syntheses of bifunctional 2,3-diamino propionic acid-based chelators as small
and strong tripod ligands for the labelling of biomolecules with 99mTc†
Yu Liu,‡a Bruno L. Oliveira,b Joa˜o D. G. Correia,b Isabel C. Santos,b Isabel Santos,b Bernhard Spinglera and
Roger Alberto*a
Received 12th February 2010, Accepted 6th April 2010
First published as an Advance Article on the web 6th May 2010
DOI: 10.1039/c002796k
The labelling of targeting biomolecules requires small and hydrophilic complexes in order to not affect
the binding properties of the vectors. 2,3-Diamino propionic acid (dap) is a small and strong, albeit
scarcely used, tripod ligand for the fac-[99mTc(CO)3]+ moiety. We have introduced at the a-carbon atom
in the basic dap structure various second functionalities such as carboxylato, amino and a-amino acid
groups via various spacers in order to yield bifunctional chelators. These dap derivatives can be coupled
to targeting molecules for application in molecular imaging. Full characterizations of the bifunctional
chelators, X-ray structures of intermediates and of one rhenium complex, as well as labelling studies
with 99mTc, are presented.
radionuclide but combination with targeting molecules is not
Introduction
routine. Labelling approaches are based on only a few fragments,
3+
2+
namely [Tc O] , [Tc N] and fac-[Tc(CO)3]+. Our ongoing
attempts to optimize ligands for the fac-[Tc(CO)3]+ core with
respect to size, hydrophilicity and labelling efficiency brought
2,3-diamino propionic acid 1 (dap) into our interest. Compound
1 (Scheme 1) is ubiquitous in the natural world and has been
found in the Bombyx larva12 on earth as well as on the Murchison
meteorite from the sky.13 Consequently, 1 and its derivatives have
stimulated both chemists and biochemists through the years to
find the biological significance of its unique structural roles from
chemistry to peptide synthesis14 and the origin of life.15 From an
inorganic point of view, 1 is a classical tripod ligand of very low
molecular weight; still, its coordination chemistry has not been
explored in great detail. In the context of radiolabelling, our group
has demonstrated that 1 is an efficient tripod ligand for the fac-
[Tc(CO)3]+ moiety.10,16 The resulting complex fac-[Tc(1)(CO)3] is
small, forms at low concentration and is highly hydrophilic. We
have also shown that 1 conjugated by the a-C to a spacer and an
a-amino acid group was recognized and transported by the L-type
amino acid transporter LAT1 (Scheme 1).10
Molecular imaginghas become amajor diagnostic toolinmedicine
which relies on labelled compounds targeting specific biological
events on a molecular level.1 Gene expression, receptors up-
regulation, messenger molecules or enzymes such as tyrosine
kinase are prominent molecular examples in current research.2–4
Chemists and biologists aim to find and explore labelled molecules
for imaging with highest specificity and sensitivity. Among other
examples of imaging modalities are Magnetic Resonance Imaging
(MRI), Single Photon Emission Computer Tomography (SPECT)
or Positron Emission Tomography (PET) and combined methods.
Probably the most developed example is the molecular targeting
of hexokinase with 18FDG visualizing increased cell proliferation
related to oncology.5 Active transport of 18FDG into cells with
high glucose (energy) demand and intracellular trapping results
in substantial signal amplification for imaging. All molecular
imaging modalities finally rely on affinity ligands which confer
molecular or cellular specificity for the target of interest.
A basic chemical challenge to targeting vectors for imaging
is the combination of the signaling compound with the affinity
ligand since the latter should not interfere with the former.
The combination of a signaling PET or SPECT metal with a
targeting vector is particularly challenging since metals must
be stably bound to chelators. Numerous examples of successful
compounds have been described but few have entered clinical
application so far.6–11 The radionuclide 99mTc is a very favorable
=
≡
aUniversity of Zu¨rich, Institute of Inorganic Chemistry, Winterthurerstr. 190,
8057 Zu¨rich, Switzerland. E-mail: ariel@aci.uzh.ch
Scheme 1 Basic building blocks for bifunctional chelators containing the
dap ligand (1).
bUnidade de Cieˆncias Qu´ımicas e Radiofarmaceˆuticas, ITN, Estrada Na-
cional 10, 2686-953 Sacave´m, Portugal
Due to the very favorable properties of the dap ligand, it would
be desirable to derivatize it at the a-carbon for coupling to different
targeting biomolecules with specific receptor binding properties
for going beyond perfusion agents.17,18 We present in this synthetic
and labelling study the preparation of a-carbon derivatives of 1,
carrying pendant amino acid groups for integration as artificial
amino acids into peptide chains, and with a simple carboxylate or
† Electronic supplementary information (ESI) available: 13C NMR data
for all new compounds, labelling yields for 20 and 25, and crystallographic
data for 4. CCDC reference numbers 761460 (4), 761459 (5), 761461(6),
751390 (19), 751391 (26) and 761462 (28). For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c002796k
‡ Current address: Key Laboratory of Nuclear Analysis Techniques, Institute
of High Energy Physics, Chinese Academy of Sciences, Beijing 100049,
China
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amine group for coupling to the corresponding functionalities in
other biomolecules (Scheme 1). The unique tripod structure of dap
and the properties of its 99mTc complexes renders it a basic building
block in future development of vectors for molecular imaging.
von hochgesellt function, the conjugation group to the targeting
molecule or an amino acid. The mild reducing agent NaBH4 does
not reduce –CN groups alone at ambient temperature. However, it
has been reported that a combination of NaBH4 and NiCl2,20
21
22
CoCl2 or I2 does reduce –CN to –CH2NH2 efficiently. The
respective metal cations coordinate to nitriles, thereby activating
them for reduction. This method was applied to 2 alone to test if
simple dap can be received. The combination of NaBH4 and NiCl2
catalytically reduced the –CN group in 2 and in situ protection with
Boc2O gave the protected dap derivative 3 in 80% yield (Scheme 3).
It is interesting to note that both the –NHAc and –COOEt did
not interfere with the reduction and no trans-esterification or
reduction was observed.
Thus, the reductive conversion of 2 to 3 provides a straight-
forward and convenient method for the synthesis of protected
dap. Some syntheses of dap as reported are multi-step or use
rather expensive reagents/catalyst.23 The Hoffman rearrangement
of asparagines, for example, is probably the most convenient
approach to dap reported so far, requires costly and not rou-
tine trivalent iodine from bis(trifluoroacetoxy)iodobenzene.24 The
conjugation of an a-amino acid via an alkyl spacer of various
lengths to 2 was performed as shown in Scheme 3. Compound
2 was deprotonated with NaOEt and then alkylated with 4, a
typical precursor in the Sorensen method of amino acid synthesis,
to give 5 in 79% yield. The same –CN to –CH2NH2 reduction
as before was employed for compound 5, and 6 was isolated in
73% yield. Despite some steric crowd around the –CN group, the
smooth conversion of 5→6 demonstrated that the alkylation of 2
had little effect on its reduction. This implied that the procedures
illustrated in Scheme 3 provide a general and efficient way for
the synthesis of dap derivatives functionalized at the a-carbon
position. The same syntheses in comparable yields were applied
for pentyl and hexyl spacers, underscoring the general synthetic
principle. The X-ray structures of 5 and 6 could be elucidated and
ORTEP presentations are given in Fig. 1.
Results and discussion
The syntheses of the building blocks shown in Scheme 1 requires
a-carbon derivatization. To be general, the synthetic approach
should be flexible with respect to the spacer between the two
functions. A retro-synthetic analysis revealed that derivatization
at the a-carbon of the latter dap ligand is best achieved by starting
from commercially available N-acetyl-3-nitriloalanine (2), a useful
starting material for a-amino acids (Scheme 2). Alkylation of 2
with e.g. diethyl 2-acetamido-2-(4-bromobutyl)malonate (4), pre-
pared by alkylation of diethyl malonate with 1,4-dibromobutane19
will ultimately lead to dap-based compounds (Scheme 3). The
key step in the retro-synthetic analysis is the functional group
interconversion step, involving the reduction of the nitrile (–CN) to
the amine (–CH2NH2). While there are many catalysts for –CN to
–CH2NH2 conversion, it was important to find a selective one for
this transfer in the presence of other functional groups. It should
be emphasized that the retro-synthetic analysis as presented in
Scheme 2 will not be stereospecific at the a-carbon. After labeling,
two diastereomeric complexes will be received. However, since the
label is distant and does not interact with the target, enantiopure
metal complexes are not crucial at this point.
Scheme 2 Retrosynthetic analysis of the target compound with (CH2)4
spacer (protection groups are omitted for clarity during analysis).
Both 5 and 6 crystallized in racemic form in the space groups
Pbca and P21/n, respectively. Data collection and structure
solution can be found in Table 1. The bond length 1.138(2) A of
Syntheses
˚
C(10)–N(3) in 5 is typical for a triple bond. The two AcNH– groups
in compound 5 are not equal. The bond length of C(19)–N(2)
The crucial step in the preparation of the bifunctional chelators
is the conjugation of the latter tripod building block 2 to the 2nd
Scheme 3 (a) 1) MeOH, NaBH4, NiCl2, Boc2O, r.t. 2) diethylenetriamine, r.t., 80.0%; (b) EtOH, NaOEt, diethyl 2-acetamido-2-(4-bromobutyl)malonate
(4), reflux, 79.0%; (c) 1) MeOH, NaBH4, NiCl2, Boc2O, r.t. 2) diethylenetriamine, r.t., 73.0%; (d–f) in situ, 1 M NaOH 80 ◦C, conc. HCl, 8 h (35%).
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Table 1 Crystallographic data for 5, 6, 19, 26 and 28
Compound
5
6
19
26
28
Empirical formula
Formula weight
Diffractometer
C20H31N3O8
441.48 g mol-1
IPDS Stoe
C25H43N3O10
545.62 g mol-1
IPDS 2T Stoe
C13H25N3O5
C7H13ClN2O3
C10H13N2O7Re
459.42 g mol-1
303.36 g mol-1
Bruker-AXS APEX
208.64 g mol-1
Bruker-AXS APEX
Oxford Diffraction
Xcalibur Ruby
˚
˚
˚
˚
˚
Wavelength
0.7107 A
1.5418 A
0.7107 A
0.7107 A
0.7107 A
Crystal system
Space group
Unit cell dimensions
Orthorhombic
Pbca
Monoclinic
P21/n
Orthorhombic
Pca21
Triclinic
P1
Triclinic
P1
¯
¯
˚
˚
˚
˚
˚
˚
˚
a = 17.2196(7) A
a = 8.8810(11) A
a = 10.1904(8) A
a = 6.4348(2) A
a = 6.7581(10) A
˚
˚
˚
˚
b = 27.2811(13) A
b = 20.9682(19) A
b = 17.6150(12) A
b = 8.0509(2) A
b = 8.7934(2) A
˚
˚
˚
˚
c = 10.2859(4) A
c = 16.241(2) A
c = 9.4373(5) A
c = 9.6331(3) A
c = 11.4890(2) A
a = 74.2580(10)◦
b = 101.327(10)◦
a = 86.1958(16)◦
b = 79.7708(16)◦
b = 101.327(10)◦
g = 78.232(2)◦
g = 83.8740(17)◦
3
3
3
3
3
˚
˚
˚
˚
˚
Volume
4832.0(4) A
2965.5(6) A
1694.0(2) A
469.81(2) A
667.28(2) A
Z
8
4
4
2
2
Density (calculated)
Absorption coefficient
F(000)
Crystal size
q range
Index ranges
1.214 Mg m-3
0.094 mm-1
1888
1.222 Mg m-3
0.787 mm-1
1176
1.189 Mg m-3
0.091 mm-1
656
1.475 Mg m-3
0.385 mm-1
220
2.287 Mg m-3
0.913 mm-1
436
0.57 ¥ 0.21 ¥ 0.20 mm3 0.2 ¥ 0.18 ¥ 0.08 mm3 0.25 ¥ 0.10 ¥ 0.06 mm3 0.50 ¥ 0.10 ¥ 0.04 mm3 0.2 ¥ 0.2 ¥ 0.1 mm3
2.42–27.10◦
3.49–58.99◦
3.16–25.02◦
2.99–25.68◦
2.88–27.99◦
-8 ≤ h ≤ 8, -11 ≤ k
≤ 11, -15 ≤ l ≤ 15
16418
-22 ≤ h ≤ 22, -34 ≤ k -9 ≤ h ≤ 9, -21 ≤ k
-12 ≤ h ≤ 8, -15 ≤ k -7 ≤ h ≤ 7, -9 ≤ k
≤ 34, -13 ≤ l ≤ 13
≤ 23, -16 ≤ l ≤ 16
≤ 19, -11 ≤ l ≤ 11
≤ 9, -11 ≤ l ≤ 11
Reflections collected
Independent reflections
Completeness to q
Max. and min. transmission 0.9843 and 0.9618
(rel. for 28)
43838
30674
4537
3354
5312 [R(int) = 0.0577] 4152 [R(int) = 0.0957] 2321 [R(int) = 0.0545] 1781 [R(int) = 0.0171] 3211 [R(int) = 0.0385]
99.7% (27.10◦)
97.4% (58.99◦)
0.9372 and 0.8557
96.1% (25.02◦)
0.9945 and 0.9775
99.7% (25.68◦)
0.9848 and 0.8310
99.9% (27.99◦)
1.0000 and 0.2585
Data/restraints/parameters 5312/0/304
Goodness-of-fit on F2
4152/1/362
1.107
2321/1/215
0.985
1781/0/134
1.057
3211/0/182
1.064
1.102
Final R indices [I > 2s(I)]
R1 = 0.0604,
wR2 = 0.1629
R1 = 0.0720,
wR2 = 0.1686
R1 = 0.0660,
wR2 = 0.1751
R1 = 0.0984,
wR2 = 0.2198
0.0049(9)
R1 = 0.0552,
wR2 = 0.0931
R1 = 0.0870,
wR2 = 0.1011
R1 = 0.0282,
wR2 = 0.0708
R1 = 0.0324,
wR2 = 0.0729
R1 = 0.0177,
wR2 = 0.0469
R1 = 0.0192,
wR2 = 0.0474
R indices (all data)
Extinction coefficient
Largest diff. peak and hole
-3
-3
-3
-3
-3
˚
˚
˚
˚ ˚
0.305 and -0.177 e A 0.899 and -0.623 e A
0.327 and -0.194 e A
0.305 and -0.292 e A
0.206 and -0.208 e A
Fig. 1 ORTEP drawing of compounds 5 and 6 (H atoms and minor conformations of disordered ethyl (5) and methyl groups (6) were omitted for clarity)
in 50% probability.
˚
˚
important to have the L-form only. This could be achieved by
starting directly from amino acids such as lysine (Scheme 4).
The e-NH2 group was oxidized to the –OH group (10) as
reported25 and activated with mesitylchloride to give compound
11. Proceeding as described above finally gave compound 14
bearing an enantiomerically pure L-form amino acid.
Besides the direct conjugation of bioactive fragments such
as amino acids to dap as described above, the bifunctional
chelator (BFC) concept is very important in radiopharmaceutical
chemistry. To prepare bifunctional chelators comprising the dap
(1.355(2) A) is significantly longer than C(15)–N(1) (1.338(2) A).
˚
This bond length difference of 0.017 A was also indirectly reflected
by the difference of 1H-NMR shift of AcNH– groups (d 6.80 and
6.26).
In situ hydrolysis of the ethyl ester groups in 6 gave the tri-
carboxylic acid 7 and subsequent decarboxylation gave compound
8 which, after deprotection, afforded the final a-amino acid 9
conjugated to the dap ligand in D- and L-forms.
With respect to later incorporation of artificial amino acids in
peptide syntheses or to use them as labelled amino acids, it is
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Scheme 4 (a) MesCl, Et3N, CH2Cl2, -78 ◦C, 96%; (b) EtOH, NaOEt, 2, reflux, (c) 1) MeOH, NaBH4, NiCl2, Boc2O, r.t. 2) diethylenetriamine, r.t.;
(d) 1 M NaOH 80 ◦C, then conc. HCl, 8 h.
Scheme 5 Reagents and conditions: A: (a) EtOH, NaOEt, benzyl-N-(3-bromopropyl)carbamate (15), reflux, 41.2%; (b) 1) MeOH, NaBH4, NiCl2, Boc2O,
r.t. 2) Diethylenetriamine, r.t., 40.3%; (c) EtOH, H2, Pd/c, r.t., 89.9%; (d) HCl 4 M, reflux, 92.6%. B: (e) DMF, NaH, ethyl-4-bromobutyrate, reflux,
51.1%; (f) 1) MeOH, NaBH4, NiCl2, Boc2O, r.t. 2) Diethylenetriamine, r.t., 97.0%; (g) MeOH/NaOH 2 M, r.t., 67.9%; (h) HCl 4 M, reflux, 52.0%.
(numbering scheme for NMR assignments is displayed for 25 as an example).
coordination motif together with a single functionality for con-
jugation to biomolecules of interest, we proceeded to dap-based
compounds derivatised with a primary amine or a carboxylic acid.
With the scope of subjecting these compounds to derivatization
of biomolecules, the conjugating functions should be unprotected
but the dap unit be protected. The synthetic strategy is similar as
before and the reaction sequences are shown in Scheme 5.
The dap derivatives 20 and 25 have been prepared starting
from 2 or tert-butyl acetamidocyanoacetate 21, respectively. The
precursor 21 was synthesized following a similar procedure
as described for 2 but starting from tert-butyl cyanoacetate.
Deprotonation of the a-C–H in 2 with NaOEt and subsequent
reaction with benzyl-N-(3-bromopropyl)carbamate (15), prepared
by bromination of benzyl-N-(3-hydroxypropyl)carbamate with
CBr4 and PPh3, gave 16 in moderate yield (Path A in Scheme 5).
The reduction of the –CN group in 16 and in situ protection with
Boc2O led to 17. Deprotection of the Cbz group in 17 to give 18 was
accomplished almost quantitatively by catalytic hydrogenation,
using an optimized amount of catalyst (Pd/C, Pd content 10%).
When a large excess of Pd/C catalyst was used, compound 18 was
always obtained in a lower yield (51.4%) due to the formation of a
side product, which was formulated as 19 (18.7% yield) based on
multinuclear NMR spectroscopy, ESI-MS and X-ray diffraction
analysis (see experimental section and Fig. 3). The formation of
19 was due to in situ cyclisation from the terminal amine with
the ethyl ester. The goal compound 20 finally was obtained in
good yield by full deprotection of 18 under acidic conditions. The
preparation of 22 by alkylation of 21 with ethyl-4-bromobutyrate
in the presence of NaOEt/EtOH was unsuccessful due to a trans-
esterification reaction (path B in Scheme 5). Compound 22 could
only be obtained using NaH as a base in DMF. Compound 23 was
obtained in almost quantitative yield starting with 22 and using
the same conditions as described for 17. Sequential removal of the
ethyl ester and protecting groups of the dap unit in 23 gave 24, and
then the desired product 25, respectively. As for 18, the product
of this reaction was always a mixture of the goal compound 25
and a second species formulated as a lactam (26). Compounds
25 and 26 were obtained in 60 and 40% yield, respectively, based
on 1H-NMR data. All the attempts to minimize the formation of
the undesired lactam 26 remained unsuccessful. At higher pH, 25
even converted almost quantitatively to 26. The identification of
25 and 26 in the reaction mixture was based on NMR. In fact,
1D and 2D 1H- and 13C-NMR experiments at different pH values
allowed a complete assignment of the resonances for each species
1
(see experimental section). For illustration, Fig. 2 shows the H-
NMR spectra of a mixture of 25 and 26 immediately after BOC
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Synthesis and characterization of the Re(I) complexes
fac-[Re(k3-L)(CO)3] 27 and 28
In order to characterize the 99mTc complexes at very high dilution,
it is common to prepare and fully characterize the macroscopic
rhenium homologues and to compare their HPLC retention time
with those of the corresponding 99mTc complex.
Reaction of 20 with the precursor fac-[Re(H2O)3(CO)3]+ in
refluxing water afforded the rhenium complex fac-[Re(20)(CO)3]
(27) in 79% yield (Scheme 6). The analogue reaction of fac-
[ReBr3(CO)3]2- with a mixture of 25 + 26 overnight at pH ~ 4
gave a white precipitate. After washing with water and warm
CH2Cl2, the solid obtained was dried under vacuum. Based on
multinuclear NMR and IR spectroscopy, and ESI-MS the complex
was formulated as fac-[Re(25)(CO)3] (28). X-ray structure analysis
confirmed this composition (Fig. 4).
Fig. 2 1H-NMR spectra (D2O) of the mixture 25 + 26 at pH 1.4 (A) and
4.3 (B).
deprotection, at pH 1.4 (Fig. 2, A). The same mixture at pH 4.3
(Fig. 2, B) exhibits almost complete lactamisation of 25 → 26.
Scheme 6 Reaction pathways to Re(I) complexes with the tripod ligands
20 and 25. X = Br, n = -2; X = H2O, n = +1.
1
The main differences in the H-NMR spectra of 25 and 26
are related to the splitting and/or chemical shifts of Ha and Hd
protons. In 25, the 2Ha protons appear as a sharp singlet at d 3.17,
while in the lactam 26 these protons appear as a quartet centered at
d 3.03. The two CH2d protons in 26 become diastereotopic due to
the rigidity imposed by cyclisation. Accordingly, two resonances
at d 2.00 and d 1.60 appeared, each integrating for one proton. The
assumed structure of 26 could be confirmed by recrystallization of
a CH2Cl2–EtOH solution containing mainly 26. Single crystals
suitable for X-ray diffraction analysis were obtained. Fig. 3
(right) shows an ORTEP diagram of 26, confirming its lactam
authenticity. The ESI-MS of the mixture 25 + 26 presented two
main peaks at m/z values which agree with the expected values for
25 ([M + H]+, 191.0) and 26 ([M + H]+, 172.9).
After precipitation of 28 from the reaction mixture, HPLC
analysis of the solution still revealed small amounts of dissolved
28 (Rt = 9.9 min), unreacted fac-[ReBr3(CO)3]2- precursor (Rt =
6.5 min) and the lactam 26 (Rt = 5.5 min). To force the reaction
to completion, the pH of the solution was increased to 8 and
the mixture refluxed overnight. After this time, HPLC analysis of
the solution indicated no residual fac-[ReBr3(CO)3]2-, the presence
of a small amount of 28 (Rt = 9.9 min), and the formation
of a defined new species (Rt = 13.2 min), which could be
isolated by chromatography. Based on multinuclear NMR and IR
spectroscopy, and ESI-MS we tentatively formulated this complex
as “fac-[Re(26)(CO)3]”.
˚
Fig. 3 ORTEP drawing of compounds 19 (left) and 26 (right) in 50% probability (H atoms were omitted for clarity). Selected bond lengths (A)
◦
◦
˚
˚
˚
˚
and angles ( ): For 19: C(5)–C(1), 1.528(4) A; C(5)–C(4), 1.545(4) A; C(5)–C(8), 1.550(5) A; C(5)–N(3), 1.453(4) A; C(1)–C(5)–C(4), 112.9(3) ;
N(3)–C(5)–C(8), 107.5(3) . For 26: C(5)–N(1), 1.4656(19) A; C(5)–C(4), 1.536(2) A; C(5)–C(7), 1.544(2) A; C(5)–C(6), 1.553(2) A; N(1)–C(5)–C(4),
◦
˚
˚
˚
˚
110.10(12)◦; C(7)–C(5)–C(6), 107.83(12)◦.
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such as peptides, ligands with higher labelling rates might be more
suitable for proteins. The radiochemical purity of the complexes
has been determined by RP-HPLC analysis, and the chemical
identity ascertained by comparing their HPLC traces with the
corresponding HPLC traces of the Re congeners 27 and 28. All
complexes could be kept in solution for at least 18 h at 37 ◦C
without any noticeable decomposition (RP-HPLC analysis). A
table with the radiochemical labelling yields with 20 and 25 at
75 and at 100◦ C and at different concentrations is given in the
supplementary information.†
It is worth mentioning that the reaction of a mixture of 25 + 26
with fac-[99mTc(H2O)3(CO)3]+ gave only fac-[99mTc(25)(CO)3], most
probably for kinetic reasons. Incubation of◦fac-[99mTc(25)(CO)3]
with a large excess (100¥) of 26 (2 h at 100 C) revealed its high
stability since no trans-chelation could be observed. Interestingly,
if the pure complex “fac-[99mTc(26)(CO)3]” was incubated (2 h
at 100 ◦C) with a large excess (100¥) of pure unsubstituted dap
amino acid, trans-chelation occurred with formation of “fac-
Fig. 4 ORTEP view of complex 28; thermal ellipsoids are drawn ◦at
˚
the 40% probability level. Selected bond lengths (A) and angles ( ):
˚
˚
˚
Re1–C1, 1.915(3) A; Re1–C2, 1.906(3) A; Re1–C3, 1.914(3) A; Re1–N1,
˚
˚
˚
2.218(2) A; Re1–N2, 2.206(2) A; Re1–O4: 2.154(2) A; C1–Re1–N1,
168.48(10)◦; C3–Re1–N2, 172.90(11)◦; C2–Re1–N1, 100.11(12)◦;
C1–Re1–O4, 96.25(11)◦; C3–Re1–C1, 89.67(14)◦; C3–Re1–C2, 87.28(14)◦;
O4–Re1–N1, 74.32(8)◦; N2–Re1–N1, 76.49(9)◦; O4–Re1–N2, 77.96(8)◦.
[
99mTc(dap)(CO)3]” in ca. 70% radiochemical yield.
The complex fac-[99mTc(20)(CO)3] is very hydrophilic and eluted
in the standard gradient 0.1% CF3COOH–MeOH with the
same retention time as the precursor fac-[99mTc(H2O)3(CO)3]+. A
different gradient was, thus, required in order to differentiate
between the two complexes. This was achieved by replacing
the 0.1% CF3COOH aqueous solution with Et3N–CH3COOH
[2.1 : 2.8 (v/v)] which might be of useful help in characterizing
such hydrophilic radiocomplexes (Fig. 5).
The infrared spectra of 27 and 28 showed strong CO stretching
bands in the 2028–1906 cm-1 range, which is in coincidence with
a fac-[Re(CO)3]+ motif for both complexes. The ESI-MS spectra
showed dominant single peaks with the expected isotopic pattern
and correct m/z values (27, [M + H]+ = 431.8 m/z; 28, [M + Na]+ =
482.9 m/z). The 1H-NMR spectra of 27 and 28 (D2O) were very
similar. The most interesting features are four broad multiplets
in the range d 5.32–4.31, integrating for 1 H each (assigned to
the diastereotopic protons of the coordinated NH2 groups), and
a
two other resonances, integrating for 1 H each due to the CH2
protons, which became diastereotopic upon coordination of the
dap derivatives to the metal (27, d 2.79 and 2.51; 28, d 2.76
and 2.59). Together with the 13C-NMR spectra, these data are
consistent with a tridentate coordination of the chelators in 27 and
28. The structure was confirmed by an X-ray structure analysis of
28 (Fig. 4).
Labelling studies: syntheses of fac-[99mTc(20)(CO)3] and
fac-[99mTc(25)(CO)3]
For the labelling of targeting biomolecules, quantitative labelling
at the lowest possible concentrations is crucial. A high amount of
unlabelled biomolecules entails purification which is not feasible
in clinical routine. High hydrophilicity is desirable since it prevents
accumulation of the radiosensor in non-targeted organs, such as
the liver or the lungs. Our labelling studies focused on these two
aspects to reveal the advantages of the dap ligand and its deriva-
tives. The synthesis of the precursor complex [99mTc(OH2)3(CO)3]+
was done as previously reported.26 The 99mTc(I)-complexes fac-
Fig.
5
RP-HPLC radioactive traces of fac-[99mTc(20)(CO)3] and
fac-[99mTc(H2O)3(CO)3]+ obtained using a gradient with Et3N–CH3COOH
[2.1 : 2.8 (v/v)] and MeOH.
Conclusions
[
99mTc(20)(CO)3] and fac-[99mTc(25)(CO)3] were prepared in high ra-
diochemical yield (>95%) by reaction of fac-[99mTc(H2O)3(CO)3]+
with 20 or a mixture of 25 + 26, respectively. The reaction with 20
at 8 ¥ 10-5 M and with 25 + 26 at 1 ¥ 10-4 M was completed after
30 min at 100 ◦C and pH = 7.4. The lowest concentration at which
quantitative labelling after ª 60 min was achieved was 10 mM. At
lower temperature, higher concentrations are required. However,
since this ligand is very useful for small hydrophilic biomolecules
It has been shown that NaBH4/NiCl2 reagent system can be
adopted as a good catalyst for the facile synthesis of dap-
containing compounds starting from ethyl or tert-butyl acetami-
docyanoacetate. The synthetic strategy reportedhereinprovides an
efficient method for the preparation of a-positioned dap deriva-
tives which involves carbon–carbon bond formation. Therefore,
it has been possible to prepare in a straightforward way an
2834 | Org. Biomol. Chem., 2010, 8, 2829–2839
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artificial amino acid (9) combining a pendant amino acid group
and a dap chelating unit, which can be incorporated into peptide
chains for (radio)metallation after introduction of orthogonal
protecting groups in the amino acid and chelating moieties.
It has also been possible to prepare new versatile bifunctional
chelators for radiopharmaceutical applications comprising a dap
coordinating unit and a pendant amine (20) or carboxylate (25)
group for conjugation to relevant biomolecules. Such chelators
react efficiently with the organometallic moiety “M(CO)3” (M =
Re, 99mTc), yielding well-defined complexes of the type fac-[M(k3-
L)(CO)3] (L = 20 and 25). In these complexes the dap unit
acts as tridentate chelator through the N,N,O donor atom set,
without any interference of the amine or carboxylate functional
groups.
(25 mg, 0.1 mmol) to afford a green solution. To this solution was
added NaBH4 (0.230 g, 6 mmol) in small portions with stirring.
The purple mixture was allowed to warm to 20 ◦C slowly and
stirred overnight and then diethylenetriamine (0.2 mL) was added.
The reaction was stirred for 1 h before the volatile part of the
mixture was removed by vacuum. The residue was partitioned
between EtOAc and saturated NaHCO3 solution. The organic
phase was dried with MgSO4. Removal of organic solvent gave
a colorless residue, which was purified by silica chromatography
1
(hexane–EtOAc) to give a colourless oil of 3 (0.220 g, 80%). H-
NMR (200 MHz, CDCl3, ppm) dH 6.71 (1H, s, AcNH-), 4.96
(1H, s, BocNH-), 4.58 (1H, m, CH), 4.20 (2H, q, -OCH2CH3),
3.69 (1H, br m, BocNHCH2-), 3.31 (1H, br m, BocNHCH2-), 2.02
(3H, s, -COCH3), 1.44 (9H, s, -C(CH3)3), 1.21 (3H, t, -OCH2CH3).
Anal. Calcd. for C12H22N2O5: C, 52.54; H, 8.08; N, 10.21. Found:
C, 52.83; H, 8.02; N, 10.27%.
Experimental section
General
Triethyl-1,6-diacetamido-6-cyanohexane-1,1,6-tricarboxylate 5
Chemicals and solvents of reagent grade were purchased from
Aldrich and used without further purification. Ethyl-N-acetyl-3-
nitriloalaninate (2), (Et4N)2[ReBr3(CO)3] and [Re(H2O)3(CO)3]Br
were prepared according to published methods.27–29 All reactions
were carried out under N2. NMR spectra were recorded on
Varian Mercury 200 MHz, Varian Gemini 300 MHz or Bruker
500 MHz instrument. 1H and 13C chemical shifts were referenced
with the residual solvent resonances relative to TMS. The spectra
were fully assigned with the help of 2D experiments (1H–1H
correlation spectroscopy, gCOSY and 1H–13C heteronuclear single
quantum coherence, HSQC). Assignments of the 1H and 13C
NMR resonances are given in accordance with the identification
system displayed for compounds 25 and 28. Electron impact mass
spectra were taken using Merck Hitachi M-8000 LCMS. HPLC
analyses were performed on a Perkin Elmer LC pump 200 coupled
to a Shimadzu SPD 10AV UV/Vis and to a Berthold-LB 509
radiometric detector, using an analytic Macherey-Nagel C18
reversed-phase column (Nucleosil 100-10, 250 ¥ 4 mm) with a flow
rate of 1 mL min-1. Purification of the rhenium compounds was
achieved on a semi-preparative Macherey-Nagel C18 reversed-
phase column (Nucleosil 100-7, 250 ¥ 8 mm) or on a preparative
Waters m Bondapak C18 (150 ¥ 19 mm) with a flow rate of 2.0 and
5.5 mL min-1, respectively. UV detection: 254 or 220 nm. Method
1—Eluent A was aqueous 0.1% CF3COOH and eluent B was
MeOH. For complex fac-[99mTc(20)(CO)3], an alternative method
was used: Method 2—Eluent A was aqueous Et3N–CH3COOH
[2.1 : 2.8 (v/v)] solution and eluent B was MeOH. The HPLC
gradient was the same for both methods: t = 0–3 min, 0% B;
3–3.1 min, 0→25% B; 3.1–9 min, 25% B; 9–9.1 min, 25→34%
B; 9.1–20 min, 34→100% B; 20–25 min, 100% B; 25–25.1 min,
100→0% B; 25.1–30 min, 0% B.
To a flask containing absolute ethanol (20 mL) was added sodium
(0.023 g, 1 mmol) with stirring. After complete dissolution of
sodium, 2 (0.170 mg, 1 mmol) was added and warmed up to
◦
60 C for 30 min. After cooling down to room temperature, 410
(0.352 mg, 1 mmol) was added to the solution in one portion.
The reaction mixture was refluxed overnight and then the solvent
was removed under reduced pressure. The resulting residue was
treated with H2O (20 mL) and was extracted with EtOAc (2 ¥
50 mL). The organic phase was washed with brine and dried over
MgSO4. After filtration, the organic phase was evaporated to give a
colorless gel, which was recrystallised from EtOAc–hexane to yield
1
colorless crystals (0.350 g, 79%). H-NMR (200 MHz, CDCl3,
ppm) dH 6.80 (1H, br s, NH), 6.26 (1H, br s, NH), 4.31 (6H, m,
-OCH2CH3), 2.09 (3H, s, -COCH3), 2.06 (3H, s, -COCH3), 2.02 -
2.39 (4H, m), 1.20 - 1.51 (13H, m). Anal. Calcd. for C20H31N3O8:
C, 54.41; H, 7.08; N, 9.52. Found: C, 54.57; H, 7.02; N,
9.44%.
Triethyl-1,6-diacetamido-7-(tert-butoxycarbonylamino)heptane-
1,1,6-tricarboxylate 6
To the MeOH (10 mL) solution of 5 (0.220 g, 0.5 mmol) cooled
with an ice bath, was added (Boc)2O (0.218 g, 1 mmol) and
NiCl2·6H2O (0.012 g, 0.05 mmol) to give a green solution. To
this solution was added NaBH4 (0.152 g, 4 mmol) in portions
◦
with stirring. The purple mixture was stirred overnight at 20 C
and then diethylenetriamine (0.06 mL) was added. The reaction
was stirred for 1 h before the volatile part of the mixture was
removed by vacuum. The residue was partitioned between EtOAc
and saturated NaHCO3 solution. The organic phase was dried
with MgSO4. Removal of organic solvent gave a colorless residue,
which was re-crystallized with EtOAc–hexane to yield colorless
Na[99mTcO4] was eluted from a 99Mo/99mTc generator, using
0.9% saline. The radioactive precursor fac-[99mTc(H2O)3(CO)3]+
R
1
IsoLinkꢀ kit (Malinckrodt,
crystals of 6 (0.200 g, 73%). H-NMR (500 MHz, CDCl3, ppm)
was prepared using
Med B.V.).
a
d
H 6.74 (1H, s, NH), 6.48 (1H, s, NH), 4.83 (1H, s, BocNH-), 4.24
(m, 6H, -OCH2CH3), 3.85 (1H, br m, BocNHCH2-), 3.62 (1H, br
m, BocNHCH2-), 2.05 (3H, s, -COCH3), 2.03 (3H, s, -COCH3),
1.75 - 2.29 (4H, m), 1.42 (9H, s, -C(CH3)3), 1.03 - 1.32 (13H, m).
Anal. Calcd. for C25H43N3O10: C, 54.02; H, 7.95; N, 7.70. Found:
C, 55.21; H, 7.76; N, 7.99%.
Ethyl-2-acetamido-3-(tert-butoxycarbonylamine) propanoate 3
To a MeOH (10 mL) solution of 2 (0.170 g, 1 mmol) cooled with
an ice bath, was added (Boc)2O (0.440 g, 2 mmol) and NiCl2·6H2O
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2,7-Diamino-2-(aminomethyl)octanedioic acid 9
2-Amino-2-(aminomethyl)-7-
(benzyloxycarbonylamino)octanedioic acid 14
This compound was directly prepared from 6 without isolation
of the intermediates 7 and 8. Compound 6 (430 mg, 0.81 mmol)
was dissolved in a mixture of 4 ml 1 M NaOH and MeOH. The
mixture was heated to 80◦ C overnight causing de-esterification
and decarboxylation. After neutralization with HCl, the solution
was brought to dryness and the residue re-dissolved in 2 ml conc.
HCl. Heating to 90◦ C for 8 h resulted in complete removal of
the amine protecting groups. Slow reduction of the volume to
about 1/3 caused precipitation of a white solid. (Yield 0.090 g,
35%). The residual solution contains more product but could not
be separated from NaCl. Anal. Calcd. for C9H17N3O4·3HCl: C,
31.85; H, 5.95; N, 12.39. Found: C, 30.72; H, 6.17; N, 11.69%.
Compound 14 was obtained as described for 9, alkaline de-
esterification followed by conc. HCl for Boc and acetate protecting
group removal.
Benzyl-N-(3-bromopropyl)carbamate 15
A solution of triphenylphosphine (9.4 g, 28.60 mmol) and carbon
tetrabromide (7.5 g, 28.60 mmol) in dry THF was added dropwise
(30 mL) to a solution of benzyl-N-(3-hydroxypropyl)carbamate
(3.0 g, 14.30 mmol) in the same solvent (30 mL). After 48 h
of stirring at room temperature, the solution was filtrated to
remove an insoluble solid. After evaporation of filtrate, the
residue was dissolved in CH2Cl2 and the solution washed with
H2O. The organic layer was dried over MgSO4. Removal of the
solvent yielded an oily residue, which was purified by column
chromatography (CH2Cl2 to MeOH). After evaporation of the
solvents, benzyl-N-(3-bromopropyl)carbamate was obtained as an
(S)-2-Benzyloxycarbonylamino-6-methanesulfonyloxy-hexanoic
acid ethyl ester 11
Compound 10 (0.804 g, 2.6 mmol) and methanesulfonylchloride
(0.22 mL, 2.86 mmol) were dissolved in 5 mL CH2Cl2 and cooled
to -78 ◦C under nitrogen. Triethylamine (0.38 mL, 2.86 mmol)
was added dropwise to the stirred solution at -78 ◦C. The solution
was then allowed to warm up to room temperature and stirred for
2 h. The reaction mixture was then diluted by 5 mL CH2Cl2, and
washed with water and brine. The organic phase was dried with
Na2SO4 and evaporated under reduced pressure. The product was
obtained as a yellow oil (0.962 g, 96%) and was used without
further purification. 1H NMR (500 MHz, CDCl3, ppm): dH 7.36
(5H, m, Cbz), 5.45 (1H, br m, CbzNH), 5.11 (2H, s, Cbz), 4.37
(1H, m), 4.20 (4H, m), 2.99 (3H, s, CH3), 2.10 - 1.40 (6H, m), 1.29
(3H, t, -OCH2CH3).
1
orange oil (3.2 g, 86.4%). Rf (silica gel, CHCl3) = 0.5. H-NMR
(300 MHz, CDCl3, ppm) dH 7.32 (5H, m, Cbz), 5.15 (1H, br s,
CBzNH-), 5.06 (2H, s, Cbz), 3.39 (2H, t), 3.30 (2H, q), 2.06–1.96
(2H, m).
Ethyl-2-acetamido-5-(benzyloxycarbonylamino)-2-
cyanopentanoate 16
Metallic sodium (0.141 g, 5.88 mmol) was added to dry EtOH
under stirring. After complete dissolution of sodium, ethyl ac-
etamidocyanoacetate (2) (1.0 g, 5.88 mmol) was added and the
◦
solution warmed up to 60 C for 30 min. After cooling down to
room temperature, 15 (1.6 g, 5.88 mmol) was added to the solution
in one portion. The reaction mixture was refluxed overnight and,
after this time, turned deep brown from the initially orange colour.
Evaporation of the solvent gave a dark residue which was treated
with H2O and extracted three times with EtOAc. The organic
phases were collected and washed with brine, and dried over
MgSO4. After filtration, the organic phase was evaporated to give
an orange oil (0.841 g, 41.2%), which was purified by column
chromatography (EtOAc to MeOH). Rf (silica gel, hexane–EtOAc
Diethyl-2-acetamido-7-(benzyloxycarbonylamino)-2-
cyanooctanedioate 12
Compound 12 was obtained by the same coupling conditions as
described for 5 but with different purification. After evaporation of
the solvent, the reaction mixture was loaded on a silica gel column
and eluted with an EtOAc–hexane gradient. The product 12 was
obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3, ppm):
dH 7.38 (5H, m, Cbz), 6.30 (1H, m, AcNH-), 5.40 (1H, CbzNH)
5.14 (s, 2H, Cbz), 4.20 - 4.37 (5H, m), 2.10 (3H, s, -COCH3),
2.30 - 1.40 (8H, m), 1.30 (6H, m, -OCH2CH3). Anal. Calcd. for
C23H31N3O7: C, 59.86; H, 6.77; N, 9.10. Found: C, 60.03; H, 6.84;
N, 9.16%.
1
80%) = 0.45. H-NMR (300 MHz, CDCl3, ppm) dH 7.98 (1H, s,
AcNH-), 7.34 (5H, m, Cbz), 5.42 (1H, t, CbzNH-), 5.09 (2H, s,
Cbz), 4.28 (2H, q, -OCH2CH3), 3.26 (2H, m, -CHf-), 2.09 (2H, m,
-CHd-), 2.04 (3H, s, -COCH3), 1.81 (1H, m, -CHe-), 1.68 (1H, m,
-CHe¢-), 1.28 (3H, t, -OCH2CH3). Anal. Calcd. for C18H23N3O5:
C, 59.82; H, 6.41; N, 11.63. Found: C, 59.60; H, 6.40; N, 11.63%.
Retention time (analytic RP-HPLC, 220 nm): 23.4 min.
Diethyl-2-acetamido-7-(benzyloxycarbonylamino)-2-
((tert-butoxycarbonylamino)methyl) octanedioate 13
Ethyl-2-acetamido-5-(benzyloxycarbonylamino)-2-
((tert-butoxycarbonylamino)-methyl)pentanoate 17
Compound 13 was obtained by the same reduction conditions
as described for 6. Instead of recrystallization, the residue
was loaded on silica gel and purified with EtOAc–hexane
gradient. The product of 13 was obtained as a colourless oil.
1H NMR (300 MHz, CDCl3): dH 7.38 (5H, m, Cbz), 6.30 (1H,
br s, AcNH-), 5.40 (1H, CbzNH-) 5.14 (2H, s, Cbz), 4.87 (1H,
m, BocNH-), 4.20 - 4.37 (5H, m), 3.86 (1H, m, BocNHCH2-),
3.62 (1H, m, BocNHCH2-), 2.07 (3H, s, -COCH3), 1.42 (9H, s,
-C(CH3)3), 1.50 - 1.03 (14H, m). Anal. Calcd. for C28H43N3O9: C,
59.45; H, 7.66; N, 7.43. Found: C, 59.63; H, 7.72; N, 7.50%.
Compound 17 was prepared by using the same reduction con-
ditions described above for 6. An excess of (Boc)2O (1.015 g,
4.65 mmol), NiCl2·6H2O (0.055 g, 0.23 mmol), NaBH4 (0.700 g,
18.61 mmol) and diethylenetriamine (0.239 g, 2.32 mmol) was
added to 16 (0.841 g, 2.32 mmol). Compound 17 was purified
by column chromatography (EtOAc to MeOH) and obtained as
a yellow pale oil (0.437 g, 40.3%). Rf (silica gel, hexane–EtOAc
80%) = 0.50. Isomer a: 1H-NMR (300 MHz, CDCl3, ppm) dH 7.35
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(5H, m, Cbz), 6.53 (1H, s, AcNH-), 5.09 (2H, s, Cbz), 4.88 (2H,
m, CbzNH- + BocNH-), 4.24 (2H, q, -OCH2CH3), 3.85 (1H, m,
BocNHCHa-), 3.61 (1H, m, BocNHCHa¢-), 3.17 (2H, m, -CHf-),
2.23 (2H, m, -CHd-), 2.01 (3H, s, -COCH3), 1.82 (1H, m, -CHe-),
1.72 (1H, m, -CHe¢-), 1.41 (9H, s), 1.27 (3H, t, -OCH2CH3). Isomer
162.0 [M+H]+; calcd for C6H15N3O2 = 161.2. Anal. Calcd. for
C6H15N3O2.3Cl: C, 26.90; H, 5.65; N, 15.70. Found: C, 27.20; H,
6.00; N, 16.00%. Retention time (analytic RP-HPLC, 220 nm):
2.4 min.
1
b: H-NMR (300 MHz, CDCl3, ppm) dH 7.58 (1H, s, AcNH-),
tert-Butyl acetamidocyanoacetate 21
7.37 (5H, m, Cbz), 5.12 (2H, s, Cbz), 4.88 (2H, m, CbzNH- +
BocNH-), 4.36 (2H, q, -OCH2CH3), 3.85 (1H, m, BocNHCHa-),
3.61 (1H, m, BocNHCHa¢-), 3.30 (2H, m, -CHf-), 2.23 (2H, m,
-CHd-), 2.08 (3H, s, -COCH3), 1.82 (1H, m, -CHe-), 1.72 (1H,
m, -CHe¢-), 1.41 (9H, s), 1.31 (3H, t, -OCH2CH3). ESI-MS (+)
(m/z): 488.1 [M+Na]+; calcd for C23H35N3O7Na = 488.2. Anal.
Calcd. for C23H35N3O7: C, 59.34; H, 7.58; N, 9.03. Found: C, 59.00;
H, 7.64; N, 8.99%. Retention time (analytic RP-HPLC, 220 nm):
27.1 min.
To a stirred solution of tert-butyl cyanoacetate (5.6 g, 40.0 mmol)
and 45% aq HOAc (50 mL) at 0 ◦C was added portion-wise
NaNO2 (8.3 g, 120.0 mmol) over 1.5 h. After the addition was
completed, the stirring was continued at room temperature for
3 h. The reaction mixture was extracted with Et2O (3¥). The
ethereal solution containing tert-butyl isonitrosocyanoacetate was
immediately mixed with Ac2O (10 mL, 105.0 mmol) and HOAc
(28 mL, 500.0 mmol). With vigorous stirring, zinc powder (8.0 g,
125.0 mmol) was added in small portions and the stirring was then
continued for 6 h. After filtration, the solvent was evaporated at
reduced pressure to give a pale yellow oil, which was purified by
column chromatography (hexane–EtOAc 25–100%). Compound
21 was recovered as yellowish oil (0.761 g, 35.0%). Rf (silica gel,
hexane–EtOAc 50%) = 0.30. 1H-NMR (300 MHz, CDCl3, ppm)
dH 6.39 (1H, s, AcNH-), 5.40 (1H, d, -CH-), 2.11 (3H, s, -COCH3),
1.55 (9H, s);
Ethyl-2-acetamido-5-amino-2-((tert-
butoxycarbonylamino)methyl)pentanoate 18
Compound 17 (0.437 g, 0.94 mmol) was dissolved in dry EtOH
(15 mL) and Pd/C (Pd content 10%, 0.215 g) was added. The
solution was bubbled with H2 at room temperature for 8 h and
left overnight under H2 atmosphere. The catalyst was filtered
through Celite, the filtrate was evaporated and the obtained residue
purified by column chromatography (CH2Cl2 to EtOH–NH4OH
5%). Compound 18 was obtained as yellow viscous oil (0.280 g,
89.9%). Rf (silica gel, CH2Cl2–EtOH 20%) = 0.20. 1H-NMR
(300 MHz, CD3OD, ppm) dH 4.15 (2H, q, -OCH2CH3), 3.57
1-tert-Butyl-6-ethyl-2-acetamido-2-cyanohexanedioate 22
Compound 22 was obtained by reaction of 21 (0.740 g, 3.73 mmol)
with NaH (0.150 g, 3.73 mmol) in DMF, followed by addition of
ethyl-4-bromobutyrate (539 mL, 3.73 mmol), using a procedure
similar to that described for 5. Purification by column chro-
matography (hexane–EtOAc 25–100%) gave 22 as a colourless oil
(2H, s, BocNHCH2 ), 2.87 (2H, t, -CHf-), 1.94 (3H, s, -COCH3),
a
1.82 (2H, m, -CHd-), 1.63 (2H, m, -CHe-), 1.42 (9H, s), 1.24
(3H, t, -OCH2CH3). ESI-MS (+) (m/z): 331.4 [M+H]+; calcd for
C15H29N3O5 = 331.2. Retention time (analyticRP-HPLC, 220 nm):
15.2 min.
1
(0.630 g, 54.1%). Rf (silica gel, hexane–EtOAc 50%) = 0.40. H-
NMR (300 MHz, CDCl3, ppm) dH 6.94 (1H, s, AcNH-), 4.15 (2H,
q, -OCH2CH3), 2.37 (2H, m), 2.26 (1H, m), 2.08 (3H, s, -COCH3),
2.01 (1H, m), 1.82 (2H, m), 1.54 (9H, s), 1.27 (3H, t, -OCH2CH3);
ESI-MS (+) (m/z): 335.2 [M+Na]+; calcd for C15H24N2O5Na =
335.2. Anal. Calcd. for C15H24N2O5: C, 57.68; H, 7.74; N, 8.97.
Found: C, 57.50; H, 7.80; N, 8.81%. Retention time (analytic RP-
HPLC, 220 nm): 20.9 min.
When the reaction is performed using a large excess of
Pd/C catalyst (1 : 1 ratio of Pd/C), two compounds, formu-
lated as 18 and 19, were obtained in 51.4 and 18.7% yield,
after column chromatography. 19: Rf (silica-gel, CH2Cl2–EtOH
20%) = 0.70. 1H-NMR (300 MHz, CDCl3, ppm) dH 7.16
(1H, s, AcNH-), 6.32 (1H, br s, NH-amide), 5.64 (1H, br m,
BocNH-), 3.64 (1H, m, BocNHCHa-), 3.50 (2H, m, -CH2 -), 3.31
f
(1H, m, BocNHCHa¢-), 2.38 (1H, m, -CHd-), 2.09 (1H, m, -CHd¢-),
2.01 (3H, s, -COCH3), 1.89 (2H, m, -CHe-), 1.50 (9H, s). ESI-
MS (+) (m/z): 308.1 [M+Na]+; calcd for C13H23N3O4Na =
308.1. Single crystals suitable for X-Ray diffraction analysis were
grown by slow evaporation of a EtOH–CH2Cl2 solution at room
temperature.
1-tert-Butyl-6-ethyl-2-acetamido-2-((tert-
butoxycarbonylamino)methyl)hexanedioate 23
Compound 23 was prepared by using the same reduction con-
ditions described above for 6. An excess of (Boc)2O (1.762 g,
8.08 mmol), NiCl2·6H2O (0.040 g, 0.40 mmol), NaBH4 (1.216 g,
32.32 mmol) and diethylenetriamine (0.416 g, 4.04 mmol) was
added to 22 (0.630 g, 2.02 mmol) to force reaction to completion.
Compound 23 was purified by column chromatography (hexane
to EtOAc) and obtained as a yellow pale oil (0.817 g, 97.0%).
2,5-Diamino-2-(aminomethyl)pentanoic acid 20
Compound 20 was obtained directly by hydrolysis of the protecting
groups of 18 (0.047 g, 0.14 mmol) with a 4 M HCl solution (5
mL). The reaction mixture was refluxed overnight, cooled down
to room temperature and washed with CH2Cl2. Compound 20
was recovered as a colorless oil, after drying the aqueous phase
under vacuum (0.035 g, 92.6%, calcd. for C6H15N3O2Cl3). IR
1
Rf (silica-gel, hexane–EtOAc 50%) = 0.45. H-NMR (300 MHz,
CDCl3, ppm) dH 6.60 (1H, s, AcNH-), 4.85 (1H, m, BocNH-),
4.13 (2H, q, -OCH2CH3), 3.85 (1H, br m, BocNHCHa-), 3.70
(1H, br m, BocNHCHa¢-), 2.29 (m, 2H), 2.08 (3H, s, -COCH3),
1.74 (1H, m), 1.60 (1H, m), 1.49 (9H, s), 1.44 (2H, m), 1.41
(9H, s), 1.22 (3H, t, -OCH2CH3); Anal. Calcd. for C20H36N2O7:
C, 57.67; H, 8.71; N, 6.73. Found: C, 57.50; H, 8.60; N,
7.00%.
1
(KBr, cm-1): 1787 m, 1619 s and 1606 s. H-NMR (300 MHz,
D2O, ppm) dH 3.32 (2H, s, NH2CH2 -), 2.88 (2H, t, -CHf-),
a
2.00–1.84 (1H, m, -CHd-), 1.82–1.78 (1H, m, -CHd¢-), 1.78–1.66
(1H, m, -CHe-), 1.66–1.47 (1H, m, -CHe¢-). ESI-MS (+) (m/z):
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5-Acetamido-6-tert-butoxy-5-((tert-
butoxycarbonylamino)methyl)-6-oxohexanoic acid 24
431.8 [M+H]+; calcd for C9H15N3O5Re = 432.0. Anal. Calcd. for
C9H14N3O5Re·3TFA: C, 23.32; H, 2.21; N, 5.44. Found: C, 23.00;
H, 2.39; N, 5.80%. Retention time (analytic RP-HPLC, 220 nm):
5.5 min.
The protected intermediate 23 (0.600 g, 1.440 mmol) was dissolved
in H2O–MeOH, and an excess of NaOH (0.144 g, 3.6 mmol) was
added. The obtained solution was refluxed overnight. The solution
was neutralized with 1 M HCl at 0 ◦C and extracted with CH2Cl2.
The organic phases were collected, dried over MgSO4, filtered
and the solvent evaporated. The crude product was purified by
column chromatography (CH2Cl2–EtOH 0–30%). Compound 24
was obtained as a colorless oil (0.380 g, 67.9%), which crystallizes
Synthesis of fac-[Re(k3-25)(CO)3] (28)
Precursor (Et4N)2[ReBr3(CO)3] (0.050 g, 0.194 mmol) reacted with
a mixture of 25 + 26 (0.146 g, 0.194 mmol) in refluxing water for
18 h (pH 4). After cooling to room temperature and concentration
of the solvent a white insoluble solid was obtained. This precipitate
was isolated by centrifugation and washed several times with water
and warm CH2Cl2 to remove excess [NEt4]Br. The spectral data of
this complex were in accordance with the formulation proposed
for 28. Analytic RP-HPLC analysis of the supernatant obtained
after precipitation of 28 revealed still the presence of 26, Re
precursor and a small amount of 28. By refluxing overnight at
pH 8, the rhenium precursor was consumed and a mixture of 28
and a second new species, formulated as “fac-[Re(26)(CO)3]”, was
obtained. Complexes 28 and “fac-[Re(26) (CO)3]” were isolated by
RP-HPLC purification. fac-[Re(k3-25)(CO)3] (28): Yield: 0.049 g,
1
upon standing. Rf (silica-gel, CH2Cl2–EtOH 10%) = 0.40. H-
NMR (300 MHz, CDCl3, ppm) dH 6.79 (1H, s, AcNH-), 4.94 (1H,
m, BocNH-), 3.83–3.65 (2H, m, BocNHCHa- and BocNHCHa¢-),
2.33 (2H, m), 2.06 (3H, s, COCH3), 1.74 (1H, m), 1.55 (3H, m),
1.48 (9H, s), 1.41 (9H, s). ESI-MS (-) (m/z): 387.1 [M - H]-,
423.1 [M+Cl]-; calcd for C18H32N2O7= 388.2. Anal. Calcd. for
C18H32N2O7: C, 55.66; H, 8.30; N, 7.21. Found: C, 55.03; H, 8.10;
N, 6.96%. Retention time (analytic RP-HPLC, 220 nm): 18.9 min.
2-Amino-2-(aminomethyl)hexanedioic acid 25
54.9% (calcd for C10H13N2O7Re). IR (KBr, cm-1): 2021 s, 1907 s,
Compound 25 was obtained directly by hydrolysis of the protecting
groups of the dap unit of 24 (0.230 g, 0.592 mmol) with a 4 M
HCl solution (5 mL). After refluxing for 18 h, the solvent was
evaporated to dryness. The oily residue was thoroughly washed
with CH2Cl2 and dried. The deprotection reaction afforded
compound 25 (60% by1H-NMR) together with a side product
which was formulated as the lactam 26 (40% by 1H-NMR).
After appropriate work-up, 0.080 g of the mixture (25 + 26) was
obtained. Mixture of 25 + 26: IR (KBr, cm-1): 1746 vs, 1605 vs,
1
=
=
1884 s, 1693 m (C O) and 1647 m (C O). H-NMR (300 MHz,
CD3OD, ppm) dH 5.32 (1H, m, NH2Cb-), 4.95 (1H, m, NH2Ca-),
b
a
4.75 (1H, m, NH2CH2 -), 4.64 (1H, m, NH2CH2 -), 2.76 (1H,
m, -CH2 -), 2.59 (1H, m, -CH2a¢-), 2.33 (2H, m, -CH2 -), 1.78
a
f
(2H, m, -CH2 -) 1.67 (2H, m, CHe). ESI-MS (+) (m/z): 482.9
d
[M+Na]+; calcd for C10H13N2O7ReNa = 483.0. Anal. Calcd. for
C10H13N2O7Re: C, 26.14; H, 2.85; N, 6.10. Found: C, 26.07; H,
2.93; N, 6.25%. Retention time (analytic RP-HPLC, 220 nm):
9.9 min. Single crystals suitable for X-ray diffraction analysis were
grown by slow evaporation of a EtOH–CH2Cl2 solution at room
1
1211 m, 1179 m and 1144 m. H-NMR (300 MHz, D2O, ppm)
a
a
dH 3.18 (2H, s, NH2CH2 -, 25), 3.03 (2H, q, NH2CH2 -, 26), 2.09
temperature. fac-[Re(26)(CO)3]: Yield: 0.025 g, 29.1% (calcd for
f
f
(2H, t, -CH2 -, 25), 1.98 (2H, t, -CH2 -, 26), 1.89–1.20 (8H, m,
-CHd- + -CHe-, 25 + 26). ESI-MS (+) (m/z): 172.9 [M+H]+ and
191.0 [M+H]+; calcd for C7H12N2O3 = 172.0 (m/z for 26) and
for C7H14N2O4 = 190.0 (m/z for 25). Retention time (analytic
RP-HPLC, 220 nm): 3.9 min. At pH > 4, 25 converted almost
-1
≡
C10H11N2O6Re). IR (KBr, cm ): 2022 s, ~1918 s (C O), 1678 m,
1
1630, 1585, and 1416 m. H-NMR (300 MHz, CD3OD, ppm)
dH 4.78 (1H, m, NH2CH2 -, overlapped with H2O peak; assigned
a
a
from gCOSY spectrum), 4.63 (1H, m, NH2CH2 -), 2.86 (1H, m,
-CH2 -), 2.55 (1H, m, -CH2a¢-), 2.45 (2H, m, -CH2 -), 2.20 (1H,
a
f
1
quantitatively to 26: H-NMR (300 MHz, D2O, ppm) dH 3.17
m, -CH2 -), 1.76 (2H, m, -CH2 -) 1.58 (1H, m, CHd¢). H-NMR
d
e
1
f
(2H, q, NH2CHa-), 2.24 (2H, m, -CH2 -), 2.00 (1H, m, -CHd-),
a
(300 MHz, DMSO, ppm) dH 4.93 (1H, m, NH2CH2 -), 4.80 (1H,
1.71 (2H, m, -CHe-), 1.60 (1H, m, -CHd¢-). ESI-MS (+) (m/z):
172.9 [M+H]+; calcd for C7H12N2O3 = 172.0. Anal. Calcd. for
C7H13N2O3·HCl: C, 40.30; H, 6.28; N, 13.43. Found: C, 39.60;
H, 6.68; N, 12.85%. Single crystals suitable for X-ray diffraction
analysis were grown by slow evaporation of a EtOH–CH2Cl2
solution at room temperature.
m, NH2CH2 -), 2.87 (1H, br s, -CH2 -), 2.71 (1H, br s, -CH2a¢-),
a
a
f
d
2.36 (2H, br m, -CH2 -), 1.98 (1H, m, -CH2 -), 1.70–1.40 (3H, m,
e
-CH2 - + -CHd¢-). ESI-MS (+) (m/z): 464.9 [M+Na]+; calcd for
C10H11N2O6ReNa = 465.0. ESI-MS (-) (m/z): 440.9 [M - H]-;
calcd for C10H11N2O6Re = 442.0. Retention time (analytic RP-
HPLC, 220 nm): 13.2 min.
Syntheses of the Re complexes 27 and 28
Synthesis of the 99mTc(I)complexes (fac-[99mTc(20)(CO)3]and
fac-[99mTc(25)(CO)3])
Precursor fac-[Re(H2O)3(CO)3]Br (0.026 g, 0.065 mmol) reacted
with 20 (0.017 g, 0.065 mmol) in refluxing water for 18 h (pH 7).
After evaporation of the solvent the resulting residue was purified
by preparative RP-HPLC. Compound 27 was obtained as a
General method. In a nitrogen-purged glass vial, 100 mL
of an aqueous solution of the compounds (20 and 25; the
concentrations are discussed in the text and ESI† [ ] = 10-3–10-4
M) were added to 900 mL of a solution of the organometallic
precursor fac-[99mTc(H2O)3(CO)3]+ (1–2 mCi) in saline (pH 7.4).
colourless oil (0.028 g, 79.2%, calcd for C9H14N3O5Re·TFA). IR
-1
≡
(KBr, cm ): 2028 s, ~1911 s (C O), ~1682 m, 1207 m and 1135
m. H-NMR (300 MHz, D2O, ppm) dH 5.08 (1H, m, NH2Cb-),
1
◦
4.74 (2H, m, NH2Cb- + NH2Ca-, overlapped with H2O peak;
The reaction mixture was then heated to 100 C for 30–60 min,
a
assigned from gCOSY spectrum), 4.31 (1H, m, NH2CH2 -), 2.88
cooled on an H2O bath and the final solution analyzed by
(2H, m, -CH2 -), 2.79 (1H, m, -CH2 -), 2.51 (1H, m, -CH2a¢-),
RP-HPLC, yielding the complexes fac-[99mTc(20)(CO)3] and fac-
f
a
d
1.69 (2H, m, -CH2 -), 1.51 (2H, m, CHe). ESI-MS (+) (m/z):
[
99mTc(25)(CO)3]). Retention times: 4.29 min (fac-[99mTc(20)(CO)3],
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method 2) and 10.0 min (fac-[99mTc(25)(CO)3], method 1). Reaction
of the pure lactam 26 wit◦h fac-[99mTc(H2O)3(CO)3]+ under the same
conditions (30 min, 100 C, 1 ¥ 10-4 M, pH 7.4) gave the complex
“fac-[99mTc(CO)3(26)])”. Retention time: 13.7 min.
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X-Ray crystallography
Single crystals were grown by slow evaporation of n-hexane–
EtOAc (5 and 6) and EtOH–CH2Cl2 (19, 26 and 28) solutions
of the compounds at room temperature. Crystallographic data
of 5 were collected at 183 K on an IPDS Stoe diffractometer.
A maximum of eight thousand reflections distributed over the
whole limiting sphere were selected by the program SELECT and
used for unit cell parameter refinement with the program CELL.30
Crystallographic data of 4 and 6 were collected at 183 K on a
Stoe IPDS 2T diffractometer with Cu-Ka radiation. Reflection
data was processed with the help of the program X-Area.31 Data
were corrected for Lorentz and polarisation effects as well as
for absorption (numerical). Crystallographic data of 28 were
collected on an Oxford Diffraction Xcalibur system with a Ruby
detector. The program suite CrysAlisPro was used for data collec-
tion, semiempirical absorption correction, and data reduction.32
Crystallographic data of 19 and 26 were collected at 150 K on a
Bruker-AXS APEX-CCD area-detector diffractometer. Empirical
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collection and data reduction were done with the SMART and
SAINT programs.34 The structures were solved by direct methods
with SIR9735 and refined by full matrix least-squares analysis
with SHELXL9736 using the WINGX suite of programs. All non-
hydrogen atoms were refined anisotropically. The hydrogen atoms
were placed in calculated positions. Molecular graphics were
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Acknowledgements
27 G. Yu, S. Z. Wang, K. Wang, Y. F. Hu and H. W. Hu, Synthesis, 2004,
This work has been supported by Mallinckrodt Med. B. V., an
Eureka project and by the Fundac¸a˜o para a Cieˆncia e Tecnologia
(FCT), Portugal, (POCI/SAU-FCF/58855/2004). B. L. Oliveira
would like to thank FCT for a PhD grant. COST Action
D39 is also acknowledged for a STSM grant. Dr Joaquim
Marc¸alo is acknowledged for performing the ESI-MS analyses.
The QITMS instrument was acquired with the support of
the Programa Nacional de Reequipamento Cient´ıfico (Contract
REDE/1503/REM/2005 - ITN) of FCT and is part of RNEM—
Rede Nacional de Espectrometria de Massa.
1021–1028.
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30 STOE-IPDS Software package, STOE & Cie, GmbH, Darmstadt,
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31 X-Area, 1.38b, STOE & Cie, GmbH, Darmstadt, Germany, 2006.
32 CrysAlisPro Software system, Oxford Diffraction Ltd., 171.32, Oxford,
UK, 2007.
33 SADABS, Area-Detector Absorption Correction, Bruker AXS Inc.,
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34 SAINT, Area-Detector Integration Software, 7.23, Bruker AXS Inc.,
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35 A. Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C.
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©