The [(Cp)M(CO)3] (M=Re, 99mTc) building block for imaging agents and bioinorganic probes: Perspectives and limitations

Article abstract of DOI:10.1002/cbdv.201200076

Starting from asymmetric Thiele's acid derivatives, two different imaging probes [^99mTc(CO)₃(CpR)] (R=potential targeting vector) are generated simultaneously in one-pot and from one substrate. This extends the previously introduced

Full text of DOI:10.1002/cbdv.201200076

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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The [(Cp)M(CO)3] (M¼Re, 99mTc) Building Block for Imaging Agents and
Bioinorganic Probes: Perspectives and Limitations
by Daniel Cana), Harmel W. Peindy NꢀDongoa), Bernhard Spinglera), Paul Schmutza),
Paula Raposinhob), Isabel Santosb), and Roger Alberto*a)
a) Institute of Inorganic Chemistry, University of Zꢀrich, Winterthurerstrasse 190, CH-8057 Zꢀrich
(phone: þ41446354631; e-mail: ariel@aci.uzh.ch)
b) IST/ITN, Instituto Superior Tꢁcnico, Universidade Tꢁcnica de Lisbao, Estrada Nacional 10,
´
PT-2686-953 Sacavem
Starting from asymmetric Thieleꢂs acid derivatives, two different imaging probes [99mTc(CO)3(CpR)]
(R¼potential targeting vector) are generated simultaneously in one-pot and from one substrate. This
extends the previously introduced labeling strategy of metal-mediated retro-DielsꢀAlder reaction with
HCp-R dimers. We demonstrate that chemically active functionalities such as hydroxamic acids are not
following this labeling strategy. Adopting the principle of replacing phenyl rings by [Re(CO)3(Cp)]
entities, potent histone deacetylase (HDAC)-inhibiting Re analogs of suberoylanlilide hydroxamic acid
(SAHA; N-hydroxy-N’-phenyloctanediamide) were synthesized and characterized. Cytotoxic evaluation
on different tumor cell lines revealed low IC50 values [mm] for these compounds, comparable to their
purely organic congeners.
Introduction. – Organometallic complexes with bioactive ligands are nowadays
essential for both, the noninvasive visualization of biological features and the
therapeutic treatment of diseases. While several transition metals across the periodic
table are frequently used for therapy, 99mTc is the most prominent nuclide in nuclear
medicine [1–4]. Clinically applied imaging agents frequently consist of a [99mTcVO]3þ
or [99mTcVO2]þ core, thermodynamically stabilized by tetradentate ligands with mixed
N-, S-, and O-donors [1]. Examples are diethylenetriaminepentaacetic acid (DTPA),
mercaptoacetyl-glycine-glycine-glycine (MAG3), or oxime-bridged hexamethylpropy-
lenamine oxime (HMPAO). Currently extensively investigated 99mTc fragments are the
fac-{99mTcI(CO)3}þ, {99mTcVN(PNP)}2þ, {99mTcIII(NS3)}, or fac-{99mTcVIIO3}þ cores [5–
7]. In contrast to the aforementioned perfusion agents, molecules labeled with these
fragments follow the concept of targeted imaging with the bifunctional chelator
approach. Such complexes are often generated from a robust core fraction with a few
coordination sites occupied by weakly bound substitution-labile ligands. These ligands
correspond, for example, to the H2O molecules in [99mTc(H2O)3(CO)3]þ [8][9] or to
the halides in [99mTcN(PNP)Cl2][5] respectively. [99mTc(NS3)L]-Containing targeting
molecules are formed in a 4þ1 approach from [99mTc(edta)]ꢀ in one pot together with
the NS3 chelator and an additional bifunctional ligand L [10–14]. The fac-{99mTcO3}þ
core can be used as a derivatized [99mTcO3(TACN)]þ moiety, where the biologically
active functionality is either introduced with the 1,4,7-triazacyclononane (TACN) or,
via 3þ2 cycloaddition, to two O-atoms [15][16].
ꢃ 2012 Verlag Helvetica Chimica Acta AG, Zꢀrich

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Tridentate s- or p-donors, coupled to a targeting vector are popular ligand systems
for fac-{99mTcI(CO)3}þ [3][17–19]. Cyclopentadiene (Cp) is for imaging a conceptually
and structurally uncommon ligand. It does not contain any heteroatoms, rendering the
molecule hydrophobic and, therefore, in fact unsuitable for aqueous syntheses.
Moreover, Cp and many derivatives are unstable as a monomer and undergo
DielsꢀAlder polymerization. However, Jaouen and co-workers developed a variety of
highly potent selective estrogen receptor inhibitors by replacing a phenyl (Ph) ring in
Tamoxifen by [Re(CO)3(Cp-R)]. Bioorganometallic complexes containing this moiety
exhibit potential as therapeutic agents. The resulting molecules retain a relatively high
binding affinity for the estrogen receptor [20–26].
We developed a synthetic route to [99mTc(CO)3(Cp-R)] which allowed a fully
aqueous preparation of a variety of complexes bearing a bioactive vector R at the Cp
ligand [27][28]. The metal-mediated retro-DielsꢀAlder reaction of the (HCp-R)2 dimer
with [99mTc(H2O)3(CO)3]þ or directly with [99mTcO4]ꢀ permitted to transfer the
principle of replacing Ph rings in bioactive substances to 99mTc-labeled compounds, as
demonstrated with melanin-targeting agents or with high-affinity carbonic anhydrase
inhibitors [29][30].
Since Re and Tc belong to the same triade, it is possible to use identical compounds
for combined therapy and imaging in a theragnostics sense [30–32]. While Re-based
compounds can be used for therapy, the homologous compounds with 99mTc can serve
as imaging agents for single-photon emission computed tomography (SPECT)
[1][4][33].
Histone deacetylases (HDACs) catalyze the removal of Ac groups of e-lysine tails
on histone proteins, causing condensed chromatin and, therefore, transcriptional
silencing. Hence, cancer cells escape apoptosis. As HDAC inhibitors can counter cancer
progression, they contribute to apoptosis and the formation of growth-arrest proteins
[34][35]. Since HDACs are highly overexpressed in cancer tissues, they are targets for
both, cancer diagnosis and therapy. Suberoylanilide hydroxamic acid (SAHA; N-
hydroxy-N’-phenyloctanediamide) is a clinically approved HDAC inhibitor (HDACi).
It is administered for the treatment of a number of hematological and solid tumors
[36]. While different organometallic SAHA analogs containing ferrocene or cisplatin,
respectively, have been investigated for therapeutic application [37–39], such
bioinorganic SAHA analogs for radiopharmaceutical application are still unknown.
In this communication, we report the extensions but also the limitations of the
metal-mediated retro-DielsꢀAlder reaction of (HCp-R)2: the adaption of the afore-
mentioned synthetic pathway to asymmetric Thieleꢂs acid derivatives allows the
generation of two different bioactive imaging probes in one pot and from one substrate.
Furthermore, we demonstrate with the example of new organometallic analogs of
SAHA that the principle of replacing Ph rings by [Re(Cp)(CO)3] is consistent to a
large extent. However, chemically active functionalities like unprotected hydroxamic
acids can oxidize [99mTc(H2O)3(CO)3]þ to form [99mTcO4]ꢀ and are, therefore,
unsuitable for this labeling route.
Results and Discussion. – The Rhenium Complexes. Meggers et al. showed with
protein kinase inhibitors that organometallic complexes, as compared to organic
compounds, can have the property of enhanced three-dimensional population of

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chemically relevant biological space [40–42]. Such molecules can achieve better
selectivity for specific targets. We recently demonstrated with organometallic carbonic
anhydrase inhibitors (CAi) that compounds containing [Re(CO)3(Cp)] truly follow
this concept [30]. [Re(CO)3(Cp)] Complexes containing a bioactive targeting vector
are, therefore, very important pharmaceutical building blocks. Moreover, [Re(-
CO)3(Cp)] can be introduced into bioactive substances by replacing Ph rings.
Combining these two concepts, organometallic SAHA analogs 1–3 were synthesized
(Scheme 1). To tune interactions with the binding pocket, the investigated compounds
differ in the position of the amide linker adjacent to Cp and in the length of the
aliphatic spacer between the amide group and the hydroxamic acid portion.
[Re(CO)3(Cp-COOH)] (23) was activated with pentafluorophenyl trifluoroacetate
and coupled to the appropriate amine to form complexes 14 and 15. [M(CO)3(Cp-
NHCO(CH2)6COOMe)] (16a/b, M¼Re/Mn) was synthesized by activating suberic
acid monomethyl ester with ClCOOiBn and coupling with [M(Cp-NH2)(CO)3]. The
obtained ester 16a was hydrolyzed with aqueous LiOH. The Mn complex 16b was
synthesized exclusively for crystallographic purposes and was not exploited further.
Crystals were grown by dissolving 16b in a minimum of CH2Cl2 and slow addition of
hexane as a precipitant (Fig. 1). Crystallographic details are given in the Exper. Part.
Activation of the terminal carboxylic acids with ClCOOEt and treatment with NH2OH
gave the final products 1–3 (Scheme 1). All compounds were purified by silica-gel
chromatography and analyzed by NMR, ESI-MS, and IR.
Fig. 1. ORTEP Representation (50% probability) of [Mn(CO)3(Cp-NHCO(CH2)6COOMe)] (16b)
The 1H-NMR spectrum of 1 in MeOD shows the two aromatic Cp signals as pseudo-
triplets at 6.15 and at 5.56 ppm, respectively. The amide H-atom exhibits a triplet at
8.19 ppm. The CH2 signals were detected as multiplets between 3.26 and 1.35 ppm.
Characteristic strong nCO bonds of the fac-[Re(CO)3] were observed in the IR spectrum
at 2020 and at 1905 cmꢀ1 (KBr). ESI-MS (MeOH) indicated two adducts: m/z 559.1
([MþNa]þ ) and 537.2 ([MþH]þ ).
1
The H-NMR in (D6)DMSO for 2 showed broad singlets for OH and NH of the
hydroxamic acid at 10.32 and at 8.63, respectively. The amide H-atom signal was
observed at 8.20 as a triplet. The Cp signals appeared as pseudo-triplets at 6.27 and at
5.70 ppm, respectively. The methylene multiplets appeared between 3.13 and 1.25 ppm.
The IR nCO bands of the fac-[Re(CO)3] were observed at 2020 and at 1907 cmꢀ1 (KBr).
ESI-MS (MeOH) exhibited a peak at 545.1, corresponding to the [MþNa]þ adduct
(m/z).

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Scheme 1
i) Pentafluorophenyl trifluoroacetate, 8-aminocaprylic acid, DMF; 88%. ii) Pentafluorophenyl
trifluoroacetate, 7-aminoheptanoic acid, DMF, 86%. iii) Ethyl chloroformate (ClCOOEt), NH2OH,
THF; 80–86%. iv) Suberic acid monomethyl ester activated with isobutyl chloroformate, THF; 98%.
v) LiOH, H2O, MeOH, THF; 98%

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1H-NMR for 3 in (D6)DMSO displayed broad singlest at 10.30 and 8.63 ppm for OH
and NH of the hydroxamic acid, respectively. At 8.63 ppm, the singlet for the amide H-
atom was observed, and the Cp pseudo-triplets were found at 5.78 and at 5.48 ppm.
Multiplets between 2.14 to 1.22 ppm indicated presence of the CH2 groups. Character-
istic IR nCO bands were observed at 2019 and at 1913 cmꢀ1 (KBr). A peak at m/z 545.1
indicated the [MþNa]þ adduct in the ESI-MS (MeOH). 1H-NMR Cp Signals are due
to couplings within a high-order AA’BB’ spin system and, therefore, assigned as
multiplets in the Exper. Part.
The Organic SAHA Analogs. To support the concept of replacing Ph rings by
[Re(CO)3(Cp)] under retention of bioactivity holds also true for organometallic
HDAC inhibitors, the organic congeners of compounds 1 and 2, 4, and 5 were
synthesized. Compound 3 corresponds to native SAHA. Syntheses were performed
analogously to those of the organometallic inhibitors (Scheme 2). The compounds were
purified by silica-gel chromatography and characterized by NMR, ESI-MS, and
elemental analysis.
Scheme 2
i) Pentafluorophenyl trifluoroacetate, 8-aminocaprylic acid, DMF; 90%. ii) Pentafluorophenyl
trifluoroacetate, 7-aminoheptanoic acid, DMF; 89%. iii) ClCOOEt, NH2OH, THF; 90–92%.
Biological Studies. To analyze the antitumor activity of organometallic HDAC
inhibitors 1–3, their cytotoxicities towards five carcinoma cell lines were evaluated. For
a direct comparison, the corresponding organic congeners SAHA, 4 and 5 were treated
the same way. The cancer cell lines were exposed to increasing concentrations of the
different compounds for 72 h, and the cellular viability was determined by MTT (¼ 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The viability
of cells in the presence of the tested compounds was compared to that observed in
control cultures, and the inhibition of growth [%] was calculated. The doseꢀresponse
curves demonstrate the antiproliferative effects of all compounds, exemplified in Fig. 2

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for human cervical carcinoma HeLa and vulva epidermal carcinoma A431 cells. The
IC50 (i.e., the concentration producing 50% inhibition of growth) values (in mm)
determined for five cells lines are compiled in the Table.
Fig. 2. Cytotoxicity on human cervical carcinoma HeLa cells (a) and vulva epidermal carcinoma A431
cells (b)
Table. Cytotoxicity Profile (IC50 [mm]) of Compounds 1–5 and of the Reference HDAC Inhibitor SAHA
for 72 h (378, 5% CO2) Continuous Treatment of Five Different Tumor Cell Linesa)
Compound
MCF-7
A431
HeLa
A375
B16F1
MeanꢁSD
1
2
3
4
9.47
15.2
11.4
1.71
7.19
3.74
13.7
17.1
17.3
2.51
5.22
4.44
14.4
13.3
8.34
1.65
5.83
4.45
14.7
23.3
12.5
2.63
4.85
4.58
9.82
12.6
15.2
3.10
8.88
3.67
12.4ꢁ2.6
16.3ꢁ4.3
12.9ꢁ3.4
2.32ꢁ0.62
6.39ꢁ1.65
4.18ꢁ0.43
5
SAHA
a) MCF-7, Human breast carcinoma; A431, human vulva epidermal carcinoma; HeLa, human cervical
carcinoma; A375, human melanoma; B16F1, murine melanoma.
The analysis of the data presented in the Table allows us to understand how the
position of the amide linker and the length of the alkyl chain, attached to the
[Re(CO)3(Cp)] framework, may affect the antitumoral capacity of organometallic
HDCA inhibitors. Moreover, it allows the possibility of finding evidence whether the
replacement of Ph rings by [Re(CO)3(Cp)] is tolerated in the investigated examples.
The Table reveals that, after a drug-administration period of 72 h, SAHA displayed
a broad cytotoxicity across cell lines from different tumor entities with a mean IC50
value of 4.2ꢁ0.4 mm. This value, obtained from the MTTassay, is in agreement with the
antiproliferative activity of SAHA previously reported by using the Alamar Blue cell
viability assay (IC50 3.3 mm, a mean value for several tumor cell lines) [43].
Among the new compounds reported here, the organic SAHA analog 4 showed the
lowest IC50 values for all cell lines used. This indicates a superior potency for HDCA
inhibition in vitro, even higher than the one described for the native SAHA. In fact, the
mean IC50 value for compound 4 (2.32ꢁ0.62 mm; 4 contains a longer aliphatic chain and
inverted amide linker as compared to SAHA) was about two or three times lower than

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that obtained for SAHA or compound 5, respectively (4.2ꢁ0.4 and 6.39ꢁ1.65 mm,
resp.), both containing the shorter hexyl alkyl chain. These results suggest that
cytotoxicity of HDAC inhibitors of the class of benzamide derivatives may depend on
the length of the CH2 chain attached to the benzamide framework.
The organometallic HDACis, 1, 2, and 3, are less active in each case as compared to
the corresponding organic congeners SAHA, 4, and 5. The mean IC50 value for 1 was
12.4 mm, compared to 2.3 mm for 4. Complex 2 and the corresponding congener 5
displayed IC50 values of 16.3 and 6.4 mm, respectively, and while SAHA presented a
value of 4.2 mm, complex 3 displayed a higher IC50 value of 12.9 mm. This slight decrease
of antiproliferative capacity of the Re complexes indicates that the bulkier [Re(-
CO)3(Cp)] moiety is disfavored over a simple planar Ph ring and might be the cause of
reduced ability of cellular penetration. Nevertheless, the Re complexes still retain a
high cytotoxic activity, comparable to their organic congeners. No significant effect on
cytotoxicity was observed by altering the position of the amide linker at the Cp. Similar
mean values of IC50 were obtained for 2 and 3 (16.3ꢁ4.3 vs. 12.9ꢁ3.4 mm). All
biological experiments were performed by using MeOH as co-solvent (to a final
concentration of ꢂ0.1%). Although this solvent is known to be toxic to cells, here, due
to its low concentration, the cell viability in the control experiments was higher than
90%.
The Ligands. [99mTc(CO)3(Cp-R)] complexes are accessible via metal-mediated
retro-DielsꢀAlder reaction from amide coupled Thieleꢂs acid derivatives and
[
99mTcO4]ꢀ in H2O [28]. So far, (HCp-R)2 dimers contained two identical substituents
R which led to the generation of one imaging agent [99mTc(CO)3(Cp-R)]. Demon-
strating the versatile possibilities of [M(CO)3(Cp)]-type complexes in medicinal
inorganic chemistry and extending the labeling strategy of metal-mediated retro-
DielsꢀAlder reaction, we synthesized the asymmetric derivatives of Thieleꢂs acid, 7–9
(cf. Scheme 3). If none of the Cp fragments in (HCp-R)2 dimers is favored for the
formation of [99mTc(CO)3(Cp-R)], this would allow for the synthesis of two different
imaging agents with one single substrate. According to the theragnostic concept, the
potent Re-HDACis 1–3 could be applied in combination with the corresponding 99mTc-
labelled compounds; macroscopic amounts of Re for therapy and microscopic amounts
of the 99mTc analogs for diagnosis. We synthesized 6, a dimeric species of the ligand of
compound 2 evaluated in vitro for its cytotoxicity.
Compounds 6–9 were prepared from one single isomer of Thieleꢂs acid (Scheme 3).
Products were obtained after activation of both carboxylic acids with pentafluoro-
phenyl trifluoroacetate and coupling to the appropriate amines. Depending on the
amount of amine used and the presence of a base, mono- or disubstituted products were
obtained. Singly substituted derivatives were mainly observed as their pentafluoro-
phenyl esters at the bridged side of the (HCp)2 dimer which could be isolated.
Subsequent addition of the second substituent along with Et3N led to the final products
7–9.
Two equiv. of 7-aminoheptanoic acid together with an excess of Et3N led to the
formation of the doubly substituted compound 20. Activation of the terminal carboxy
groups with O-(7-aza-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluoro-
phosphate (HATU) and treatment with O-[(tert-butyl)dimethylsilyl]hydroxylamine
gave product 6.

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Scheme 3
i) Pentafluorophenyl trifluoroacetate. ii) 7-Aminoheptanoic acid, DMF; 34%. iii) 8-Aminocaprylic acid,
Et3N, DMF; 32%. iv) BnOH, Et3N, DMF; 9%. v) 4-(Aminomethyl)benzensulfonamide, Et3N, DMF;
80%. vi) 7-Aminoheptanoic acid, Et3N, DMF; 54%. vii) 2-(7-Aza-1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU), O-[(tert-butyl)dimethylsilyl]hydroxylamine, H2O,
DMF; 70%.
The 99mTc Complexes. Labeling of bioactive functionalized ligands with the fac-
{
99mTcI(CO)3}þ core was significantly simplified with the introduction of the Isolinkꢀ
Kit enabling the fast formation of the [99mTc(H2O)3(CO)3]þ precursor [8][9].
99mTc(CO)3(Cp-R)]-type complexes are accessible from aqueous solutions [27][28].
The metal-mediated retro-DielsꢀAlder reaction of the (HCp-R)2 with
[
[
99mTc(H2O)3(CO)3]þ or with [99mTcO4]ꢀ allowed transfer of the principle of replacing
Ph rings in bioinorganic substances to 99mTc-labelled compounds. Introducing suitable
biologically active residues R, this labeling approach can be regarded as an applicable
easy-to-use protocol for routine use in nuclear medicine [29][30][44].

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To complement the Re-HDACis 1–3 with the corresponding 99mTc-labelled
compounds, attempts were made to label 6 with [99mTc(H2O)3(CO)3]þ . However,
heating 1 mm solution of 6 with [99mTc(H2O)3(CO)3]þ at 908 resulted only in the
quantitative formation of [99mTcO4]ꢀ . Obviously, hydroxamic acids are oxidants
towards [99mTc(H2O)3(CO)3]þ . Thus, the desired compound was not obtained. On the
other hand, the labeling of 20 with [99mTcO4]ꢀ afforded after 80 min at 908 the expected
product 10 in 87% yield and high radiochemical purity (Scheme 4). The nature of the
obtained 99mTc compound was confirmed by co-injection with the corresponding Re
complex 15.
Scheme 4. Bottom-Up Trace: g-Trace of the One-Pot Labeling of 20 with [99mTcO4]ꢀ and Isolink Kit.
Top-Down Trace: UV Trace of Re Compound 15.
In extension to the labeling strategy of symmetrical (HCp-R)2, the asymmetric
derivatives of Thieleꢂs acid, 7–9, were treated accordingly. Compounds 7, 8, and 9 all
exhibited two main peaks in the HPLC trace when reacted with [99mTc(H2O)3(CO)3]þ .
Ligands again were present in mm concentrations, and reactions were performed at 908
in aqueous saline. Beside the two main products 10 and 13, the labeling of 7 furnished a
small portion (7%) of [99mTcO4]ꢀ (Scheme 5). The product ratio of 45 :55 for 13/10
indicated no clear preference of complex formation concerning localization of the
derivatives at the unbridged or at the bridged side of the (HCp) dimer. Co-injection of
the Re congeners [Re(CO)3(Cp-CONHCH2C6H4SO2NH2)] (22) [30] and 15, respec-
tively, clearly confirmed the nature of the products.
A very similar result was obtained with 8, having two different aliphatic chain
lengths but the same conjugation to Cp (Scheme 6). Two products, 10/11, in a similar
ratio of 45 :55 were formed as before, confirming the lack of preference for one of the
regions in the Thieleꢂs acid dimers.
Compound 9 contains an amide-bound substituent on one side and an ester group
on the other. Even though the labeling of 9, similar to 7 and 8, gave two products, the
benzyl ester [99mTc(CO)3(Cp-COOCH2C6H5)] was not obtained but hydrolyzed during
the process. Product 12 was identified as the free acid [99mTc(CO)3(CpCOOH)], the
99mTc-form of [Re(CO)3(Cp-COOH)] (23) [45]. The second peak again corresponded
to 10, the 99mTc analog 15. The product ratio was nearly 50 :50 (Scheme 7).

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme 5. Bottom-Up Trace: g-Trace of the Two-Step Labeling of 7 with [99mTc(H2O)3(CO)3]þ . Top-
Down Trace: UV Trace of Re Compounds 22 and 15.
Scheme 6. Bottom-Up Trace: g-Trace of the One-Pot Labeling of 8 with [99mTcO4]ꢀ . Top-Down Trace:
UV Trace of Re Compounds 15 and 14.
Conclusions. – A series of new organometallic HDAC inhibitors as well as their
organic congeners were synthesized and characterized. Cytotoxic evaluation revealed
low mm IC50 values for these compounds against different tumor cell lines. These values
are in the same range as for SAHA and establish the concept of replacing Ph rings by
[Re(CO)3(CpR)] for organometallic HDAC inhibitors. Slight alterations in the
aliphatic spacer as compared to the hexyl chain in SAHA do not affect activity of
HDACis. Increasing the number by one CH2 group even showed increased cytotoxic
capacity. No significant effect was observed by altering the position of the amide linker
at the Cp ring.
Using asymmetrically derivatized Thieleꢂs acid, two different imaging agents,
[
99mTc(CO)3(Cp-R)] (R¼potential targeting vector), were generated in one pot and
from one substrate. Introducing appropriate vectors, combined imaging of two targets
from one substrate is now possible.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Scheme 7. Bottom-Up Trace: g-Trace of the One-Pot Labeling of 9 with [99mTcO4]ꢀ . Top-Down Trace:
UV Trace of Re Compounds 23 and 15.
Ester linkers are generally hydrolyzed during labeling under the applied conditions.
Furthermore, oxidation of [99mTc(H2O)3(CO)3]þ to [99mTcO4]ꢀ prior to complex
formation occurred, when hydroxamic acids were used. Chemically labile function-
alities are, therefore, limitations to this labeling strategy. [99mTc(CO)3(CpR)] Com-
plexes, where R is a HDAC-targeting vector with hydroxamic acid, were not accessible
via metal-mediated retro-DielsꢀAlder reaction.
Experimental Part
General. Reactions were carried out in dried glassware under N2. Solvents were dried using standard
techniques and stored over molecular sieves. [Re(CO)3(Cp-COOH)] [45] (23), [Re(CO)3(Cp-NH2)]
[46], and [Re(CO)3(Cp-CONHCH2C6H4SO2NH2)] [30] (22) were prepared according to literature
procedures. RP-HPLC was performed on a Merck Hitachi LaChrom L7200 tunable UV detector and a
radiodetector, separated by a Teflon tube, which causes a ca. 0.4–0.7-min delay compared to UV/VIS
detection. UV/VIS Detection was performed at 250 nm. Anal. columns (Macherey-Nagel Nucleosil 100-5
C18) were eluted with a flow rate of 0.5 ml minꢀ1 using 0.1% TFA in H2O (solvent A) and MeOH
(solvent B) as eluents with a variable gradient (0–3 min, 100% A; 3–3.1 min, 0–25% B; 3.1–9 min, 25%
B; 9–9.1 min, 25–34% B; 9.1–20 min, 34%–100% B; 20–25 min, 100% B; 25–25.1 min 100% B to
100% A; 25.1–30 min 100% A). IR Spectra were recorded as KBr pellets on a Perkin-Elmer BX II IR
spectrometer; in cmꢀ1 1H- and 13C-NMR spectra were recorded on a Bruker Advance 400 MHz or
.
500 MHz spectrometer; d in ppm, J in Hz. Mass spectra were recorded on a Bruker Esquire HCT (ESI)
instrument; in m/z. The detection of radioactive 99mTc complexes was performed with a Berthold LB 507
radiodetector equipped with a NaI(Tl) scintillation detector.
For biological studies, Dulbeccoꢂs Modified Eagle Medium (DMEM) with GlutaMax-I, fetal bovine
serum (FBS), penicillin/streptomycin antibiotic soln., phosphate-buffered saline (PBS), and trypsin
were purchased from Gibco Invitrogen Co. (Alfagene, Portugal). MeOH, DMSO, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and Trypan Blue were bought from SigmaChemical
Co. (Sigma, Portugal). Suberoylanilide hydroxamic acid (SAHA) was purchased from Cayman
Corporation. For the determination of the IC50 values, absorbance at 570 nm was measured using a
microplate spectrophotometer (PowerWave Xs, Bio-Tek Instruments, Winooski, VT, USA).
Crystallographic data was collected at 183(2) K with MoKa radiation (l¼0.7107 ꢄ) that was
graphite-monochromated on an Oxford Diffraction CCD Xcalibur system with a Ruby detector. Suitable

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crystals were covered with oil (Infineum V8512, formerly known as Paratone N), mounted on top of a
CryoLoopTM (Hampton Research) and immediately transferred to the diffractometer. The program suite
CrysAlisPro was used for data collection, multi-scan absorption correction, and data reduction [47]. The
structure was solved with direct methods using SIR97 [48] and were refined by full-matrix least-squares
methods on F2 with SHELXL-97 [49]. The H-atoms were placed on calculated positions. CCDC
No. 868628 contains the supplementary crystallographic data for this paper. These data can be obtained
free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/
cif.
Tricarbonyl(8-{[(h5-cyclopentadienyl)carbonyl]amino}-N-hydroxyoctanamide)rhenium (1). To a
soln. of 14 (318 mg, 0.61 mmol) in 5 ml of dry THF, Et3N (123 ml, 0.88 mmol) and ClCOOEt (78 ml,
1.0 mmol) were added. After stirring the mixture for 1 h at r.t., a soln. of NH2OH·HCl (141 mg,
2.03 mmol) and MeONa (30% in MeOH, 381 ml, 2.03 mmol) in 2 ml of MeOH was added, and the
mixture was stirred at r.t. for 18 h. Solvents were evaporated at reduced pressure, and the residue was
redissolved in THF, filtered, and evaporated to dryness. Silica-gel chromatography with a gradient from
CH2Cl2/AcOH 100 :5 to CH2Cl2/MeOH 100 :5 afforded 1 (80%). Calc. for Mr (C16H13N2O6ReS) 535.5.
1
IR: 2020, 1905, 1633, 1548, 1367, 1303, 1036. H-NMR (500 MHz, MeOD): 8.19 (t, J ¼ 5.6, 2 NHCH2);
6.15 (m, 2 H, Cp); 5.56 (m, 2 H, Cp); 3.26 (m, CH2); 2.08 (m, CH2); 1.60 (m, 2 CH2); 1.35 (m, 3 CH2).
13C-NMR (125 MHz, MeOD): 194.3 (CO); 173.2 (CONHOH); 164.9 (CONH); 96.2 (Cp1); 87.9 (Cp2);
86.6 (Cp3); 40.7, 33.9, 30.5, 30.2, 30.1, 27.9, 26.8 (CH2). ESI-MS (MeOH): 559.1 ([MþNa]þ ), 537.2 ([Mþ
H]þ ).
Tricarbonyl(7-{[(h5-cyclopentadienyl)carbonyl]amino}-N-hydroxyheptanamide)rhenium (2). Com-
pound 2 was prepared as described for 1 using 15. Silica-gel chromatography with a gradient from
CH2Cl2/AcOH 100 :5 to CH2Cl2/MeOH 100 :5 afforded 2 (83%). Calc. for Mr (C16H19N2O6Re) 521.5. IR:
2020, 1907, 1633, 1549, 1368, 1303, 1036. 1H-NMR (400 MHz, (D6)DMSO): 10.32 (s, OH); 8.63 (s,
NHOH); 8.20 (t, J ¼ 5.6, 2 NHCH2); 6.27 (m, 2 H, Cp); 5.70 (m, 2 H, Cp); 3.13 (m, CH2); 1.92 (m, CH2);
1.45 (m, 2 CH2); 1.25 (m, 2 CH2). 13C-NMR (125 MHz, (D6)DMSO): 194.2 (CO); 169.1 (CONHOH);
161.2 (CONH); 96.1 (Cp1); 87.3 (Cp2); 86.2 (Cp3); 38.7, 32.3, 29.0, 28.4, 26.1, 25.1 (CH2). ESI-MS
(MeOH): 545.1 ([MþNa]þ ).
Tricarbonyl[N1-(h5-cyclopentadienyl)-N8-hydroxyoctanediamide]rhenium (3). Compound 3 was
prepared as described for 1 using 17. Silica-gel chromatography with a gradient from CH2Cl2/AcOH
100 :5 to CH2Cl2/MeOH 100 :5 afforded 3 (86%). Calc. for Mr (C16H19N2O6Re) 521.5. IR: 2019, 1913,
1640, 1558, 1487, 1385. 1H-NMR (400 MHz, (D6)DMSO): 10.30 (s, OH); 10.02 (s, NH); 8.63 (s, NHOH);
5.78 (m, 2 H, Cp); 5.48 (m, 2 H, Cp); 2.14 (m, CH2); 1.92 (m, CH2); 1.47 (m, 2 CH2); 1.22 (m, 2 CH2).
13C-NMR (125 MHz, (D6)DMSO): 195.5 (CO); 171.3 (NHCO); 169.0 (CONHOH); 118.7 (Cp1); 81.4
(Cp2); 72.5 (Cp3); 35.7, 32.2, 28.3, 28.2, 25.0, 24.8 (CH2). ESI-MS (MeOH): 545.1 ([MþNa]þ ). Anal.
calc.: C 39.02, H 4.09, N 2.84; found: C 38.91, H 3.95, N 2.76.
N-[8-(Hydroxyamino)-8-oxooctyl]benzamide (4). Compound 4 was prepared as described for 1
using 18. Silica gel chromatography with a gradient from CH2Cl2/AcOH 100 :5 to CH2Cl2/MeOH 100 :5
1
afforded 4 (90%). Calc. for Mr (C15H22N2O3) 278.4. H-NMR (400 MHz, (D6)DMSO): 10.33 (s, OH);
8.62 (s, NHOH); 8.41 (t, J ¼ 5.5, NH); 7.81 (m, 2 arom. H); 7.46 (m, 3 arom. H); 3.24 (m, CH2); 1.93 (m,
CH2); 1.50 (m, 2 CH2); 1.15 (m, 3 CH2). 13C-NMR (125 MHz, MeOD): 173.2 (CONHOH); 170.4
(CONH); 136.0 (Ar1); 132.7 (Ar2); 129.7 (Ar3); 128.4 (Ar4); 48.0, 41.1, 33.9, 30.6, 30.2, 28.0, 26.8 (CH2).
ESI-MS (MeOH): 279.1 ([MþH]þ ), 301.1 ([MþNa]þ ), 277.1 ([MꢀH]ꢀ ). Anal. calc.: C 64.73, H 7.97,
N 10.06; found: C 64.32, H 7.88, N 9.91.
N-[7-(Hydroxyamino)-7-oxoheptyl]benzamide (5). Compound 5 was prepared as described for 1
using 19. Silica-gel chromatography with a gradient from CH2Cl2/AcOH 100 :5 to CH2Cl2/MeOH 100 :5
1
afforded 5 (92%). Calc. for Mr (C14H20N2O3) 264.3. H-NMR (400 MHz, (D6)DMSO): 10.33 (s, OH);
8.64 (s, NHOH); 8.42 (t, J ¼ 5.5, NH); 7.83 (m, 2 arom. H); 7.46 (m, 3 arom. H); 3.24 (m, CH2); 1.93 (m,
CH2); 1.50 (m, 2 CH2); 1.15 (m, 2 CH2). ESI-MS (MeOH): 265.2 ([MþH]þ ), 263.2 ([MꢀH]ꢀ ). Anal.
calc. for 5þ0.5 H2O: C 61.52, H 7.74, N 10.25; found: C 61.14, H 7.67, N 9.89.
3a,4,7,7a-Tetrahydro-N2,N6-bis[7-(hydroxyamino)-7-oxoheptyl]-4,7-methano-1H-indene-2,6-dicar-
boxamide (6). Compound 20 (50 mg, 0.11 mmol) was dissolved in 1 ml of DMFand N-methylmorpholine
(3 equiv., 35.1 ml, 0.32 mmol) and O-(7-aza-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexa-

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1861
fluorophosphate (NATU; 3 equiv., 121.1 mg, 0.32 mmol) was added. The soln. was stirred for 30 min at
r.t. O-[(tert-Butyl)dimethylsilyl]hydroxylamine (3 equiv., 47 mg, 0.32 mmol) dissolved in 1 ml of DMF
was added, and the mixture was stirred for 5 h at r.t. The solvent was evaporated in vacuo. The residue
was suspended in dist. H2O and extracted three times with 20 ml of AcOEt. The aq. phase was
evaporated, and the residue was purified by silica-gel chromatography with CH2Cl2/MeOH/AcOH
10 :1:0.1 (70%). Calc. for Mr (C26H40N4O6) 504.6. 1H-NMR (400 MHz, MeOD): 7.86 (t, J ¼ 5.7, NH); 7.59
(t, J ¼ 5.6, NH); 6.68 (m, CH); 6.33 (m, CH); 3.53 (m, CH); 3.38 (m, CH); 3.21 (m, 5 H, CH2
superimposed with CH); 3.02 (m, CH); 2.49 (m, CH); 2.12 (dt, J ¼ 7.4, 4 CH); 2.06 (m, CH); 1.65 (m,
5 H, CH2 superimposed with CH); 1.51 (m, 5 H, CH2 superimposed with CH); 1.36 (m, 4 CH2). ESI-MS
(MeOH): 527.3 ([MþNa]þ ).
7-[({6-[({[4-(Aminosulfonyl)phenyl]methyl}amino)carbonyl]-3a,4,7,7a-tetrahydro-4,7-methano-1H-
inden-2-yl}carbonyl)amino]heptanoic acid (7). 4-(Aminomethyl)benzensulfonamide hydrochloride
(1.2 equiv., 92.4 mg, 0.42 mmol) was dissolved in 1 ml of DMF. Et3N (5 equiv., 235.6 ml, 1.7 mmol) and
21 (174.6 mg, 0.34 mmol) were added. The reaction progress was monitored by HPLC. When the starting
compound was consumed, the solvent was evaporated in vacuo, and the residue was purified by silica-gel
chromatography with CH2Cl2/MeOH/AcOH 100 :5 :5 (80%). Calc. for Mr (C26H33N3O6S) 515.6.
1H-NMR (400 MHz, MeOD): 7.86 (m, 2 arom. H); 7.61 (t, J ¼ 5.9, NH); 7.41 (m, 2 arom. H); 6.77 (m,
CH); 6.35 (m, CH); 3.56 (m, CH); 4.52 (m, CH2) 3.41 (m, CH); 3.22 (m, 3 H, CH2 superimposed with
CH); 3.03 (m, CH); 2.51 (m, CH); 2.27 (t, J ¼ 7.4, CH2); 2.11 (m, CH); 1.69 (m, CH); 1.56 (m, 5 H, CH2
superimposed with CH); 1.35 (m, 2 CH2). ESI-MS (MeOH): 514.1 ([MꢀH]ꢀ ), 538.2 ([MþNa]þ ).
8-{[(2-{[(6-Carboxyhexyl)amino]carbonyl}-3a,4,7,7a-tetrahydro-4,7-methano-1H-inden-6-yl)carbo-
nyl]amino}octanoic acid (8). Compound 8 was synthesized as described for 7 using 8-aminocaprylic acid.
The crude product was purified by flash chromatography (FC) on silica gel (with gradient from CH2Cl2 to
CH2Cl2/MeOH/AcOH 20 :1:1) to give pure 8 (32%). Calc. for Mr (C27H40N2O6) 488.3. 1H-NMR
(500 MHz, MeOD): 7.83 (t, J ¼ 5.7, NH); 7.54 (t, J ¼ 5.7, NH); 6.66 (m, CH); 6.33 (m, CH); 3.52 (m,
CH); 3.37 (m, CH); 3.17 (m, 2 CH2); 3.13 (m, CH); 3.01 (m, CH); 2.48 (m, CH); 2.30 (dt, J ¼ 7.3, 2 CH2);
2.06 (m, CH); 1.65 (m, 5 H, CH2 superimposed with CH); 1.51 (m, 5 H, CH2 superimposed with CH);
1.35 (m, 5 CH2). ESI-MS (MeOH): 527.3 ([MþK]þ ), 511.3 ([MþNa]þ ), 489.3 ([MþH]þ ).
6-Benzyl 2-{[(6-Carboxyhexyl)amino]carbonyl}-3a,4,7,7a-tetrahydro-4,7-methano-1H-indene-6-car-
boxylate (9). To a soln. of 21 (934.5 mg, 1.82 mmol) in 3 ml of DMF, BnOH (4 equiv., 754 ml, 7.28 mmol)
and Et3N (4 equiv., 1.01 ml, 7.28 mmol) were added, and the mixture was stirred at r.t. under N2. Reaction
control by NMR still showed incomplete coupling after 7 d. Hence, more BnOH (4 equiv., 754 ml,
7.28 mmol) and Et3N (2 equiv., 500 ml, 3.64 mmol) were added, and the mixture was stirred during
additional 24 h at 508. The solvent was evaporated at reduced pressure. The residue was suspended in
4 ml of sat. NaHCO3 soln. diluted 20-fold with H2O and extracted with 100 ml of AcOEt. The aq. phase
was then acidified with 1m HCl to pH 1, and extracted three times with 100 ml of AcOEt. The combined
org. phases were dried (Na2SO4), and the solvent was removed in vacuo to yield 700 mg of crude product.
The crude product was purified by FC on silica gel (with gradient from CH2Cl2 to CH2Cl2/MeOH 9 :1) to
1
give pure 9 (70 mg, 9%). Calc. for Mr (C26H31NO5) 473.2. H-NMR (500 MHz, MeOD): 7.64 (t, J ¼ 5.5,
NH) 7.35 (m, 5 arom. H); 6.95 (m, CH); 6.32 (m, CH); 3.55 (m, CH); 3.36 (m, CH); 3.20 (m, 3 H, CH2
superimposed with CH); 3.01 (m, CH); 2.49 (m, CH); 2.28 (t, J ¼ 7.4, CH2); 2.02 (m, CH); 1.67 (m, CH);
1.60 (m, CH); 1.48 (m, 2 CH2); 1.33 (m, 2 CH2). ESI-MS (MeOH): 460.1 ([MþNa]þ ), 438.1 ([MþH]þ ),
436.1 ([MꢀH]ꢀ ).
Tricarbonyl(7-{[(h5-cyclopentadienyl)carbonyl]amino}heptanoic Acid)-99m-technetium (10). A vial
was charged with [H3BCOOH] (4 mg), Na2[C4H4O6]·2 H2O (7 mg), Na2B4O7 ·10 H2O (7 mg), and 20
(5 mg). The vial was sealed and flushed with N2 for 5 min. Freshly eluted Na[99mTcO4] (1 ml) was
injected, and the mixture was heated at 85–908. After cooling to r.t. and filtering, the products were
analyzed by HPLC. After 80 min, 87% conversion of [99mTcO4]ꢀ was observed. Co-injection of the
corresponding Re complex confirmed the nature of the product.
Tricarbonyl(7-{[(h5-cyclopentadienyl)carbonyl]amino}heptanoic Acid)-99m-technetium (10) and
Tricarbonyl(8-{[(h5-cyclopentadienyl)carbonyl]amino}octanoic Acid)-99m-technetium (11). A vial was
charged with [H3BCOOH] (4 mg), Na2[C4H4O6]·2 H2O (7 mg), Na2B4O7 ·10 H2O (7 mg), and 8 (8 mg).
The vial was sealed and flushed with N2 for 5 min. Freshly eluted Na[99mTcO4] (1 ml) was injected, and

1862
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
the mixture was heated at 85–908. After cooling to r.t. and filtering, the products were analyzed by
HPLC. After 150 min, 80% conversion of [99mTcO4]ꢀ was observed. The radio 10/11 was 45 :55. The
nature of the products was confirmed by co-injection of the corresponding Re complex.
Tricarbonyl(7-{[(h5-cyclopentadienyl)carbonyl]amino}heptanoic Acid)-99m-technetium (10) and
Tricarbonyl[(h5-cyclopentadienyl)carboxylic Acid]-99m-technetium (12). A vial was charged with
[H3BCOOH] (4 mg), Na2[C4H4O6]·2 H2O (7 mg), Na2B4O7 ·10 H2O (7 mg), and 9 (3–4 mg). The vial
was sealed and flushed with N2 for 5 min. Freshly eluted Na[99mTcO4] (1 ml) was injected, and the
mixture was heated at 85–908. After cooling to r.t. and filtering, the products were analyzed by HPLC.
After 200 min, 98% conversion of [99mTcO4]ꢀ was observed. The radio 12/10 was nearly 50 :50. The
nature of the products was confirmed by co-injection of the corresponding Re complex.
Tricarbonyl(7-{[(h5-cyclopentadienyl)carbonyl]amino}heptanoic Acid)-99m-technetium (10) and
Tricarbonyl(N-{[4-(aminosulfonyl)phenyl]methyl}(h5-cyclopentadienyl)carboxamide)-99m-technetium
(13). To a commercially available Isolink-Kit, freshly eluted Na[99mTcO4] (1 ml) was injected, and the
mixture was heated at 85–908. The full conversion of [99mTcO4]ꢀ to [99mTc(H2O)3(CO)3]þ was confirmed
by HPLC after 30 min. A new vial was charged with 7 (3–5 mg), sealed, and flushed with N2 for 5 min. A
[
99mTc(H2O)3(CO)3]þ soln. (1 ml) was injected into the vial, and the mixture was heated at 85–908. After
cooling to r.t. and filtering, the products were analyzed by HPLC. After 150 min, 81% conversion of
99mTc(H2O)3(CO)3]þ to the products was observed; 7% of [99mTcO4]ꢀ was formed as a side product. The
[
radio 13/10 was 45 :55. The nature of the products was confirmed by co-injection of the corresponding Re
complexes.
Tricarbonyl(8-{[(h5-cyclopentadienyl)carbonyl]amino}octanoic Acid)rhenium (14). [(CpꢀCOOH)-
Re(CO)3] was activated with pentafluorophenyl trifluoroacetate according to the procedure described in
[28]. PFP-Activated acid (360.4 mg, 0.53 mmol) was dissolved in 5 ml of DMFand cooled to 08. A soln. of
8-aminocaprylic acid (100 mg, 0.63 mmol) and NaHCO3 (53 mg, 0.63 mmol) in 5 ml of H2O was slowly
added. A thick cream-colored precipitate formed instantly. The mixture was allowed to reach r.t. and was
stirred for 18 h, whereupon the precipitate was consumed. Removal of the solvent under reduced
pressure and silica-gel chromatography with CH2Cl2/MeOH/AcOH 100 :1:1 afforded 14 (88%). Calc.
for Mr (C16H13N2O6ReS) 547.6. IR: 2022, 1907, 1706, 1621, 1551. 1H-NMR (400 MHz, (D6)DMSO): 8.16
(t, J ¼ 5.7, NH); 6.26 (m, 2 H, Cp); 5.70 (m, 2 H, Cp); 3.13 (m, CH2); 2.17 (m, CH2); 1.45 (m, 2 CH2); 1.25
(m, 3 CH2). 13C-NMR (125 MHz, (D6)DMSO): 194.2 (CO); 174.5 (COOH); 161.2 (NHCO); 96.0 (Cp1);
87.2 (Cp2); 86.2 (Cp3); 38.7, 33.7, 29.0, 28.6, 28.5, 26.2, 24.5 (CH2). ESI-MS (MeOH): 519.8 ([MꢀH]ꢀ ).
Anal. calc.: C 39.07, H 4.24, N 2.68; found: C 38.19, H 3.97, N 2.86.
Tricarbonyl(7-{[(h5-cyclopentadienyl)carbonyl]amino}heptanoic Acid)rhenium (15). Compound 15
was prepared as described for 14 using 7-aminoheptanoic acid. Silica-gel chromatography with CH2Cl2/
MeOH/AcOH 100 :1:1 afforded 15 (86%). Calc. for Mr (C16H18NO6Re) 506.5. IR: 2022, 1909, 1705,
1623, 1551. 1H-NMR (400 MHz, (D6)DMSO): 8.17 (t, J ¼ 5.7, NH); 6.26 (m, 2 H, Cp); 5.70 (m, 2 H, Cp);
3.13 (m, CH2); 2.18 (m, CH2); 1.30 (m, 2 CH2); 1.26 (m, 2 CH2). 13C-NMR (125 MHz, (D6)DMSO):
194.2 (CO); 174.5 (COOH); 161.2 (CONH); 96.0 (Cp1); 87.2 (Cp2); 86.2 (Cp3); 38.6, 33.6, 28.9, 28.3,
26.1, 24.5 (CH2). ESI-MS (MeOH): 508.0 ([MþH]þ ), 530.0 ([MþNa]þ ), 505.7 ([MꢀH]ꢀ ). Anal.
calc.: C 37.94, H 3.58, N 2.77; found: C 37.74, H 3.61, N 2.60.
Tricarbonyl{methyl 8-[(h5-Cyclopentadienyl)amino]-8-oxooctanoate}rhenium (16a) and Tricarbo-
nyl{methyl 8-[(h5-Cyclopentadienyl)amino]-8-oxooctanoate}manganese (16b). Suberic acid monomethyl
ester (309 ml, 1.72 mmol) was dissolved in 10 ml of dry THF. Addition of N-methylmorpholine (190 ml,
1.72 mmol) and ClCOOiBu of (225.5 ml, 1.72 mmol) resulted in formation of a colorless precipitate. After
stirring the suspension for 10 min, [M(CO)3(Cp-NH2)] (16a: M¼Re, 16b: M¼Mn) [46] (0.86 mmol)
was added, and the mixture was stirred for further 17 h at r.t. The solvent was removed at reduced
pressure. H2O (30 ml) was added, and the residue was extracted three times with 50 ml of AcOEt. The
combined org. fractions were dried (Na2SO4) and evaporated under reduced pressure. Silica-gel
chromatography with hexane/AcOEt 2 :1 afforded 16a/16b in quant. yield.
Data of 16a. Calc. for Mr (C17H20NO6Re) 520.6. IR: 2024, 1910, 1719, 1703, 1541, 1487, 1235, 1163.
1H-NMR (400 MHz, (D6)DMSO): 10.02 (s, NH); 5.78 (m, 2 H, Cp); 5.48 (m, 2 H, Cp); 3.57 (s, CH3); 2.27
(m, CH2); 2.14 (m, CH2); 1.25 (m, 2 CH2); 1.23 (m, 2 CH2). 13C-NMR (125 MHz, (D6)DMSO): 195.5
(CO); 173.3 (COOMe); 171.3 (NHCO); 118.7 (Cp1); 81.4 (Cp2); 72.5 (Cp3); 51.2 (COOMe); 35.6, 33.2,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1863
28.1, 28.0, 24.7, 24.2 (CH2). ESI-MS (MeOH): 544.0 ([MþNa]þ ). Anal. calc.: C 39.02, H 4.09, N 2.84;
found: C 38.91, H 3.95, N 2.76.
Data of 16b. ESI-MS (MeOH): 412.0 ([MþNa]þ ). Crystals of 16b were grown from CH2Cl2/hexane.
Crystallographic parameters: empirical formula, C17H20 MnNO6; Mr, 398.28 g/mol; crystal system,
monoclinic; space group, P21/c; unit cell parameters: a¼12.2182(4), b¼19.1058(7), c¼7.5939(3) ꢄ, a¼
90, b¼90.240(3), g¼908; V, 1772.69(11) ꢄ3; density (calc.), 1.459 Mg/m3; absorption coefficient,
0.777 mmꢀ1; Z¼4; crystal size, 0.35ꢃ0.23ꢃ0.17 mm3; crystal description, colorless block; reflections
collected, 12863; independent reflections (R(int)), 5402 (0.0334); reflections observed (I>2s(I)), 4080;
number of parameters, 228; completeness to V, 99.7% to 30.518; goodness-of-fit on F2, 1.066; final R
indices (I>2s(I)), R1 ¼0.0460 and wR2 ¼0.1119; R indices (all data), R1 ¼0.0650 and wR2 ¼0.1217; diff.
peak and hole, 0.400 and ꢀ0.914 e/ꢄ3. Relevant bond lengths [ꢄ] and angles [8]: Mn(1)ꢀC(1) 1.784(3),
Mn(1)ꢀC(2) 1.784(3), Mn(1)ꢀC(3) 1.776(3), Mn(1)ꢀC(4) 2.194(2), Mn(1)ꢀC(5) 2.146(2), Mn(1)ꢀC(6)
2.102(3), Mn(1)ꢀC(7) 2.105(3), Mn(1)ꢀC(8) 2.133(3), C(1)ꢀO(1) 1.147(4), C(2)ꢀO(2) 1.138(4),
C(3)ꢀO(3) 1.143(4), C(4)ꢀN(1) 1.400(3), C(9)ꢀO(4) 1.212(3), C(16)ꢀO(5) 1.192(3), C(16)ꢀO(6)
1.325(3), C(3)ꢀMn(1)ꢀC(2) 93.72(15), C(3)ꢀMn(1)ꢀC(1) 92.19(15), C(2)ꢀMn(1)ꢀC(1) 90.54(14),
O(4)ꢀC(9)ꢀN(1)
121.7(3),
O(4)ꢀC(9)ꢀC(10)
123.6(3),
O(5)ꢀC(16)ꢀO(6)
122.9(3),
O(5)ꢀC(16)ꢀC(15) 124.8(3), C(16)ꢀO(6)ꢀC(17) 116.0(2).
Tricarbonyl{8-[(h5-Cyclopentadienyl)amino]-8-oxooctanoic Acid}rhenium (17). Compound 16a
(342 mg, 0.66 mmol) was dissolved in a mixture of 2 ml of THF and 4 ml of MeOH. LiOH (138 mg,
3.28 mmol) in 2 ml of H2O was added, and the mixture was stirred at r.t. for 2 h. Solvents were evaporated
at reduced pressure to ca. 2 ml, and the mixture was acidified with HCl (conc.) to pH 1. Extraction of the
aq. layer with AcOEt afforded 17 (310 mg, 92%). Calc. for Mr (C16H18NO6Re) 506.5. 1H-NMR
(400 MHz, (D6)DMSO): 10.02 (s, NH); 5.78 (m, 2 H, Cp); 5.48 (m, 2 H, Cp); 2.17 (m, 2 CH2); 1.47 (m,
2 CH2); 1.24 (m, 2 CH2). ESI-MS (MeOH): 530.0 ([MþNa]þ ), 506.0 ([MꢀH]ꢀ ).
8-(Benzoylamino)octanoic Acid (18). Benzoic acid (250 mg, 2.05 mmol) was dissolved in 5 ml of dry
DMF. Pyridine (180.6 ml, 2.24 mmol) and pentafluorophenyl trifluoracetate (386.8 ml, 2.24 mmol) were
added, and the soln. was stirred for 3 h at r.t. DMF was evaporated at reduced pressure. AcOEt was
added, and the residue was extracted three times with 0.1m HCl and three times with 5% NaHCO3. The
org. fraction was washed with fresh H2O, dried (Na2SO4), and evaporated to dryness. The activated acid
(151.3 mg, 0.53 mmol) was dissolved in 5 ml of DMF and cooled to 08. A soln. of 8-aminocaprylic acid
(100 mg, 0.63 mmol) and NaHCO3 (53 mg, 0.63 mmol) in 1 ml of H2O was slowly added. A thick cream-
colored precipitate formed instantly. The mixture was allowed to reach r.t. and was stirred for 18 h. Silica-
gel chromatography with AcOEt/hexane 1:1 afforded 18 (90%). Calc. for Mr (C15H21NO3) 263.3.
1H-NMR (400 MHz, (D6)DMSO): 8.41 (t, J ¼ 5.4, NH); 7.81 (m, 2 arom. H); 7.46 (m, 3 arom. H); 3.24
(m, CH2); 2.19 (m, CH2); 1.50 (m, 2 CH2); 1.29 (m, 3 CH2). 13C-NMR (125 MHz, MeOD): 177.9
(COOH); 170.4 (CONH); 136.0 (Ar1); 132.7 (Ar2); 129.7 (Ar3); 128.4 (Ar4); 41.1, 35.1, 30.6, 30.3, 30.2,
28.1, 26.2 (CH2). ESI-MS (MeOH): 286.1 ([MþNa]þ ), 264.0 ([MþH]þ ), 262.0 ([MꢀH]ꢀ ). Anal.
calc.: C 68.42, H 8.04, N 5.32; found: C 67.99, H 7.76, N 5.26.
7-(Benzoylamino)heptanoic Acid (19). Compound 19 was prepared as described for 18 using 7-
aminoheptanoic acid. Silica-gel chromatography with AcOEt/hexane 1:1 afforded 19 (89%). Calc. for
Mr (C15H19NO3) 249.3. 1H-NMR (400 MHz, (D6)DMSO): 8.41 (t, J ¼ 5.3, NH); 7.81 (m, 2 arom. H); 7.46
(m, 3 arom. H); 3.24 (m, CH2); 2.19 (m, CH2); 1.50 (m, 2 CH2); 1.30 (m, 2 CH2). 13C-NMR (125 MHz,
MeOD): 177.9 (COOH); 170.5 (CONH); 136.1 (Ar1); 132.7 (Ar2); 129.7 (Ar3); 128.4 (Ar4); 41.1, 35.1,
30.5, 30.1, 27.9, 26.2 (CH2). ESI-MS (MeOH): 250.0 ([MþH]þ ), 272.0 ([MþNa]þ ), 247.9 ([MꢀH]ꢀ ).
Anal. calc.: C 67.45, H 7.68, N 5.62; found: C 67.25, H 7.48, N 5.46.
7,7’-[(3a,4,7,7a-Tetrahydro-4,7-methano-1H-indene-2,6-diyl)bis(carbonylimino)]bisheptanoic acid
(20). Thieleꢂs acid was activated with pentafluorophenyl trifluoroacetate as described in [28]. 7-
Aminoheptanoic acid (2.2 equiv., 115.7 mg, 0.80 mmol) was dissolved in 1 ml of DMF, and Et3N
(4.4 equiv., 220.8 ml, 1.59 mmol) was added. The activated acid (200 mg, 0.36 mmol) was added in one
portion, and the mixture was stirred for 20 h at r.t. The solvent was evaporated in vacuo, and the residue
was purified by silica-gel chromatography with CH2Cl2/MeOH/AcOH 100 :0.5 :0.1 to give 20 (54%).
Calc. for Mr (C26H38N2O6) 474.6. 1H-NMR (400 MHz, MeOD): 7.81 (t, J ¼ 5.7, NH); 7.52 (t, J ¼ 5.7, NH);
6.63 (m, CH); 6.30 (m, CH); 3.48 (m, CH); 3.34 (m, CH); 3.18 (m, 2 CH2); 3.10 (m, CH); 2.97 (m, CH);

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2.45 (m, CH); 2.27 (dt, J ¼ 7.3,2 CH2); 2.00 (m, CH); 1.60 (m, 5 H, CH2 superimposed with CH); 1.47 (m,
5 H, CH2). ESI-MS (MeOH): 497.2 ([MþNa]þ ), 475.3 ([MþH]þ ).
6-(2,3,4,5,6-Pentafluorophenyl) 2-[[(6-Carboxyhexyl)amino]carbonyl]-3a,4,7,7a-tetrahydro-4,7-
methano-1H-indene-6-carboxylic acid (21). Thieleꢂs acid was activated with pentafluorophenyl trifluoro-
acetate as described in [28]. 7-Aminoheptanoic acid (1.2 equiv., 60.3 mg, 0.42 mmol) was dissolved in
1 ml of DMF. The activated acid (191 mg, 0.34 mmol) was added in one portion, and the mixture was
stirred for 20 h at r.t. The solvent was evaporated in vacuo, and the residue was purified by silica-gel
chromatography (CH2Cl2/MeOH/AcOH 100 :1:1) to afford 21 (34%). Calc. for Mr (C25H24F5NO5) 513.5.
1H-NMR (400 MHz, MeOD): 7.67 (t, J ¼ 5.5, NH); 7.35 (m, CH); 6.38 (m, CH); 3.67 (m, CH); 3.47 (m,
CH); 3.22 (m, 3 H, CH2 superimposed with CH); 3.11 (m, CH); 2.58 (m, CH); 2.29 (t, J ¼ 7.4, CH2); 2.08
(m, CH); 1.81 (m, CH); 1.57 (m, 5 H, CH2 superimposed with CH); 1.35 (m, 2 CH2). ESI-MS (MeOH):
536.1 ([MþNa]þ ).
Tumor Cell Lines. The human breast carcinoma MCF-7, prostate carcinoma PC3, cervical carcinoma
HeLa, vulva epidermal carcinoma A43, and melanoma A375 cell lines as well as the murine melanoma
B16F1cell line used in this study were grown in Dulbeccoꢂs Modified Eagle Medium (DMEM)
containing GlutaMax-I supplemented with 10% heat-inactivated foetal bovine serum (FBS) and 1%
penicillin/streptomycin antibiotic soln. (all from Gibco, Alfagene, Lisbon). Cells were cultured in a
humidified atmosphere of 95% air and 5% CO2 at 378 (Heraeus, Germany), with the medium changed
every other day. The cells were adherent in monolayers and, when confluent, were harvested from the cell
culture flasks with trypsin EDTA (Gibco, Alfagene, Lisbon) and seeded farther apart.
Cytotoxic Activity. The antiproliferative activity on tumor cells was evaluated using a colorimetric
method based on MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide), which is
reduced in viable cells to yield purple formazan crystals. The optimal plating density of each cell line,
which ensures exponential growth throughout all of the exper. period, was first optimized. MCF-7 (15ꢃ
103 cells), HeLa (9ꢃ103 cells), A431 (7.5ꢃ103 cells), A375 (7ꢃ103 cells), PC3 (6ꢃ103 cells), and B16F1
(6ꢃ103 cells) cells were plated in 96-well sterile plates in 150 ml of culture medium per well and incubated
for 24 h at 378/5% CO2 for seeding. Then, the cells were incubated with various concentrations of the
compounds 1–5 for 72 h at 378/5% CO2, dissolved in MeOH and diluted in the culture medium (final
concentration in MeOH 0.025% and 0.1%, resp.). The effect of the vehicle solvent (MeOH) on the
growth of these cell lines was evaluated in all experiments by exposing untreated control cells to both
concentrations of MeOH used (0.025% and 0.1%).
At the end of the incubation period, the compounds were removed and cells were washed with 200 ml
of PBS. Cell viability was then determined by incubating cells with 200 ml of MTT soln. (0.5 mg mlꢀ1 in
PBS). After 3 h at 378/5% CO2, the soln. was removed and the purple formazan crystals formed inside
the cells were dissolved in 200 ml of DMSO by thorough shaking. For each test compound and for each
cell line, a doseꢀresponse curve was obtained. The cytotoxic effects of ligands and Re complexes were
quantified by calculating the compound concentration inhibiting tumor cell growth by 50% (IC50), based
on non-linear regression analysis of doseꢀresponse data (GraphPad Prisma 5 software). For comparison,
suberoylanilide hydroxamic acid (SAHA) was evaluated under the same experimental conditions. All
compounds were tested in at least two independent studies with eight replicates for each concentration.
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Received March 2, 2012