Synthesis, characterization and biological evaluation of tricarbonyl M(i) (M = Re, 99mTc) complexes functionalized with melanin-binding pharmacophores

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www.rsc.org/njc | New Journal of Chemistry

Synthesis, characterization and biological evaluation of tricarbonyl

M(^I) (M = Re, ^99mTc) complexes functionalized with melanin-binding

pharmacophores

Carolina Moura, Teresa Esteves, Lurdes Gano, Paula D. Raposinho,

´

Antonio Paulo* and Isabel Santos

Received (in Gainesville, FL, USA) 5th April 2010, Accepted 3rd June 2010

DOI: 10.1039/c0nj00256a

Aiming to evaluate their potential as radioactive probes for in vivo targeting of melanotic

melanoma and its metastases, we have synthesized ^99mTc(I) tricarbonyl complexes (Tc1–Tc8)

anchored by pyrazolyl-containing chelators with (N₃) or (N₂O) donor atom sets and

functionalized with 2-aminoethyldiethylamine and 4-amino-N-(2-diethylaminoethyl)benzamide

groups as melanin-binding pharmacophores. The chemical identiﬁcation of the several ^99mTc

complexes has been accomplished by HPLC comparison with the Re congeners (Re1–Re8),

which were synthesized at the macroscopic level and fully characterized by common analytical

techniques. The biological evaluation of the ^99mTc(I) complexes comprised the determination of

their in vitro binding to synthetic melanin, measurement of cellular uptake in B16F1 murine

melanoma cells, as well as biodistribution studies in B16F1 melanoma-bearing mice. All the tested

complexes have shown a moderate to high in vitro aﬃnity to melanin, with percentages of binding

spanning between 60 and 94%. In agreement with the poor cellular uptake measured in vitro, the

in vivo tumor uptake of the complexes was in general relatively low, ranging between 0.12 and

1.69% ID g^ꢀ1at 4 h p.i. However, some complexes have shown favorable tumor-to-organ ratios

with values as high as 28 and 5.3 for tumor–muscle and tumor–blood ratios, respectively. This

seems to indicate that some selectivity towards melanoma tissue was conserved, and encourages

further optimization of the in vitro/in vivo biological properties of this type of complexes aimed

at ﬁnding novel radioactive probes for non-invasive imaging of melanoma.

derivatives. So far, the best results have been found for

Introduction

radioiodinated benzamide derivatives that, in some cases, hold

potential for SPECT imaging of malignant melanoma.^6–13In

Melanoma is a serious form of skin cancer that develops in the

pigment-producing skin cells – melanocytes. Around 160 000

new cases of melanoma are diagnosed worldwide each year.

Melanoma often starts as a single tumor or lesion that can

spread to nearby lymph nodes and distant sites throughout the

body – metastatic melanoma.^1,2The early detection of

melanoma and its metastases can considerably improve

management and prognosis of the disease. The nuclear

imaging techniques Positron Emission Tomography (PET)

and Single Photon Emission Computed Tomography

(SPECT) are expected to provide an early and non-invasive

detection of melanoma, given that radioactive probes suitable

for a selective in vivo targeting of melanoma tissues are

available. In the past few years, several approaches have been

explored in the design of speciﬁc radioactive probes for in vivo

targeting of melanoma tissue, based on monoclonal antibodies

(MAbs),³peptide analogs of a-melanocyte stimulating

particular,

N-(2-diethylaminoethyl)-4-[¹²³I]iodobenzamide

([¹²³I]BZA) and its ortho analog ([¹²³I]BZA₂) allowed the

detection of melanoma and its metastases with high sensitivity

and selectivity in phase II clinical trials involving several

melanoma patients.^9,11

Due to several reasons, the cyclotron-produced ¹²³I is

less convenient for SPECT imaging compared with the

inexpensive and readily available ^99mTc, which is the most

used radionuclide for diagnostic applications in nuclear

medicine. Taking these advantages into consideration, various

research groups have pursued the ﬁnding of ^99mTc-labelled

benzamide derivatives as alternatives to radioiodinated

benzamides. Until now, several ^99mTc(V) complexes

functionalized with benzamide derivatives, or their fragments,

have been prepared and evaluated as potential SPECT probes

for in vivo targeting of melanoma. This included complexes

with the [^99mTcN]²⁺and [^99mTcO]³⁺cores anchored by N₂S₂

tetradentate bifunctional chelators, as well as [3+1] mixed-

ligand Tc(^V) oxocomplexes.^14–21However, the tested

complexes have shown in most cases only low to moderate

tumor uptake, and did not emerge as an alternative to

radioiodinated benzamides.

hormone (a-MSH)⁴and small organic molecules such as

5

precursors of melanin

or melanin-binders.^6–13Melanin-

binders constitute a heterogeneous drug family that comprises

polycyclic aromatic compounds, polyamines and benzamide

´

ˆuticas,

Unidade de Cieˆncias Quımicas e Radiofarmace

Estrada Nacional 10, 2686-953 Sacavem Codex, Portugal.

ITN,

In the past few years, the so-called tricarbonyl approach has

gained considerable attention, following the introduction by

´

E-mail: apaulo@itn.pt

c

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Alberto and co-workers of a convenient and fully aqueous-

based kit preparation of the organometallic precursor

fac-[^99mTc(OH₂)₃(CO)₃]⁺.²²The fac-[^99mTc(CO)₃]⁺core can

be stabilized by a large variety of ligands and/or donor sets,

without compromising the kinetic inertness of the resulting

complexes. Such versatility allows ﬁne-tuning of the physico-

chemical properties of the complexes, and their adjustment

to those of the carrier biomolecules to be labelled. These

favorable features justify the exploration of this approach

for labelling of a large variety of biomolecules. To the best

of our knowledge, the fac-[^99mTc(CO)₃]⁺core has only

recently been explored in the ^99mTc-labelling of melanin-

binders, using cyclopentadienyl ligands as bifunctional

chelators.²³The reported results were encouraging in terms

of tumor uptake in B16F1 melanoma-bearing mice, but the

pharmacokinetics and biological proﬁle of the evaluated

organometallic complexes were rather unfavourable.

melanoma detection, should also freely diﬀuse through the cell

membrane to interact with melanin in the cytosol. The

capability of the complexes to cross the cell membrane will

be dependent on their physico-chemical properties, such as

size, topology, charge and lipophilicity. As mentioned in the

introductory section, we have focused on pyrazole-diamine or

pyrazole-aminocarboxylic bifunctional chelators to stabilize

the Re(^I)/^99mTc(I) tricarbonyl complexes functionalized with

melanin-binding pharmacophores. By exploring these N,N,N-

and N,N,O-tridentate chelators, we have taken into considera-

tion their excellent coordination capability towards the

fac-[M(CO)₃]⁺(M = Re, ^99mTc) moiety, which enables the

formation of complexes with excellent in vitro and in vivo

stability.^29–31Moreover, these compounds coordinate to the

metal as neutral and monoanionic chelators, respectively,

aﬀording complexes with a diﬀerent overall charge that might

inﬂuence their ability to cross the cell membrane. Proﬁting

from the versatility of pyrazole-diamine and pyrazole-

aminocarboxylic ligands, in terms of possibilities of

functionalization, we have prepared an enlarged family of

bifunctional chelators containing 2-aminoethyldiethylamine

and 4-amino-N-(2-diethylaminoethyl)benzamide (procainamide)

as melanin-binding groups, which were introduced at the

4-position of the pyrazole ring or at the central amine.

The functionalization of the pyrazolyl-diamine ligands

through the central amine started with a previously reported

BOC-protected derivative (compound 1) of 4-{(2-aminoethyl)-

[2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl]amino}butanoic acid.²⁹

As shown in Scheme 1, the derivative contains a butyric

pendant arm that has been used for the attachment of the

melanin-binding pharmacophores by amide coupling reactions,

using HBTU (O-benzotriazole-N,N,N⁰,N⁰-tetramethyluronium-

hexaﬂuorophosphate) as an activating reagent in the presence

of triethylamine. The resulting compounds, 2 and 3, were then

treated with triﬂuoroacetic acid (TFA) to remove the BOC

protecting group. The deprotection reaction aﬀorded, almost

quantitatively, the ﬁnal ligands L¹and L², which were

recovered as yellow oils.

Using the tricarbonyl approach, we have embarked on the

synthesis and biological evaluation of ^99mTc(I) tricarbonyl

complexes anchored by pyrazole-diamine (N,N,N-donors) or

pyrazole-aminocarboxylic

(N,N,O-donors)

bifunctional

chelators bearing 2-aminoethyldiethylamine and 4-amino-N-

(2-diethylaminoethyl)benzamide (procainamide) groups as

melanin-binding pharmacophores, aiming to evaluate their

potential interest for the design of novel radioactive probes

for the detection of malignant melanoma. Herein, we report

on the synthesis and characterization of new pyrazole-

diamine (L¹, L², L⁵, L⁶) or pyrazole-aminocarboxylic ligands

(L³, L⁴, L⁷, L⁸) that carry the aforementioned melanin-binding

pharmacophores at diﬀerent positions of the chelator frame-

work, i.e. at the 4-position of the pyrazol ring or at the central

amine. These new ligands were used to prepare ^99mTc(I)/Re(I)

tricarbonyl complexes (Tc1–Tc8/Re1–Re8), which is expected

to be useful for the design of melanoma-seeking radioactive

probes. The Re complexes were fully characterized by

common analytical techniques, and were used as surrogates

for the ^99mTc congeners. Reported herein is an evaluation of

the potential of the ^99mTc complexes for SPECT imaging of

melanoma, achieved by evaluation of their in vitro binding to

synthetic melanin, measurement of cellular uptake in B16F1

murine melanoma cells and biodistribution studies in B16F1

melanoma-bearing mice.

A similar strategy has been explored for coupling of the

2-aminoethyldiethylamine and 4-amino-N-(2-diethylaminoethyl)-

benzamide pharmacophores to a butyric arm attached at the

central amine of the pyrazole-aminocarboxylic ligands. As can

be seen in Scheme 2, this involved the synthesis of a mixed

ethyl/benzyl diester, 6, which was converted to 7 by selective

deprotection of the benzyl ester by hydrogenation over Pd/C.

Compound 7 was then linked to the melanin-binding groups,

as described above for L¹and L². The ethyl ester function of

the resulting amide derivatives (8 and 9) was hydrolyzed with

NaOH in reﬂuxing THF–H₂O, yielding L³and L⁴. L³and L⁴

were recovered as yellow oils, in moderate to high yield

(60–98%), after neutralization of the respective reaction

mixtures and extraction with dichloromethane.

Results and discussion

Chemical synthesis: ligands and rhenium complexes

The mechanisms involved in the uptake of benzamide deriva-

tives by melanoma tumor cells are not fully understood, but it

has been shown that the compounds co-localize with melanin

in the melanocytes of pigmented cells. Their uptake has been

correlated to melanin binding, suggesting a non-receptor

binding uptake mechanism. Moreover, for radioiodinated

N-(2-dialkylaminoethyl)benzamides passive diﬀusion appears

to be the dominant uptake mechanism on melanoma cells,

it being reported in some cases that the tumor uptake is

positively correlated with the lipophilicity of the compounds.^24–28

Therefore, ^99mTc(I) tricarbonyl complexes bearing melanin-

binding pharmacophores, to be explored as SPECT probes for

The incorporation of the melanin-binding groups at the

pyrazolyl rings of the pyrazole-diamine and pyrazoleamine-

carboxylic chelators involved the synthesis of derivatives

having a methylacetate substituent at the 4-position of the

rings (Schemes 3 and 4).

For the pyrazole-diamine ligands, the precursor ethyl

2-(1-(2-bromoethyl)-3,5-dimethylpyrazolyl)acetate (11) was

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Scheme 1 Synthesis of L¹and L². Reagents and conditions: (i) N,N-Diethylethylenediamine or procainamide, HBTU, NEt₃, CH₃CN, 48 h,

reﬂux; (ii) TFA, CH₂Cl₂, 2.5 h, r.t.

Scheme

2 3 days; (ii)

Synthesis of L³and L⁴. Reagents and conditions: (i) H₂NCH₂COOEt, CH₃CN, K₂CO₃, KI, reﬂux,

Br(CH₂)₃COOCH₂C₆H₅, CH₃CN, K₂CO₃, KI, reﬂux, 3 days; (iii) H₂, Pd/C, dry EtOH, 20 h; (iv) N,N-diethylethylenediamine or procainamide,

HBTU, NEt₃, CH₃CN, 72 h, reﬂux; (v) NaOH, THF–H₂O, reﬂux, overnight.

readily obtained by bromination of the corresponding alcohol

groups from 17 and 18 led to the ﬁnal pyrazolyl-diamine

ligands, L⁵and L⁶, containing melanin-binding pharmaco-

phores at the 4-position of the pyrazolyl ring.

(Scheme 3). Treatment of 11 with BOCHNCH₂CH₂NH₂

aﬀorded

a pyrazolyl-diamine derivative (12) bearing a

(tert-butoxy)carbonyl protecting group at the terminal amine.

Compound 12 reacted with di-tert-butyl-dicarbonate anhydride

giving a bis-protected derivative (13) that has been converted

to an NHS-activated ester (15), after deprotection of the ethyl

ester group of 14. Unlike 4-amino-N-(2-diethylaminoethyl)-

benzamide, 2-aminoethyldiethylamine reacted smoothly with

15 leading to the formation of the amide derivative 17. This

diﬀerence in reactivity reﬂects the aromatic nature of the

reacting amine group in the former case. To introduce

the 4-amino-N-(2-diethylaminoethyl)benzamide group at the

4-position of the pyrazolyl ring, we have functionalized this

pharmacophore with a 2-aminoethylcarboxy linker. The

resulting derivative (16) was reacted with 15 to aﬀord the

desired compound (18). Removal of the BOC protecting

As shown in Scheme 4, the introduction of the melanin-

binding groups at the 4-position of the pyrazole-

aminocarboxylic ligands followed a similar strategy, starting

from compound 24 which has a terminal ethyl ester function

and a methylacetic group at the 4-position of the azolyl ring.

Compound 24 was coupled to the two pharmacophores,

upon activation with HBTU under basic conditions.

Treatment of the resulting compounds, 25 and 26, with NaOH

gave L⁷and L⁸, respectively. All the ligands bearing the

pharmacophores at the 4-position of the pyrazolyl ring,

L⁵–L⁸, are yellow oils that have been obtained in moderate

to high yield (42–98%).

All the new ligands, L¹–L⁸, were characterized by common

spectroscopic techniques (IR, ¹H and ¹³C NMR) and by

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Scheme 3 Synthesis of L⁵and L⁶. Reagents and conditions: (i) PBr₃, toluene, reﬂux, overnight; (ii) NH₂(CH₂)₂NHBOC, CH₃CN, NEt₃, NaI,

reﬂux, 24 h; (iii) (BOC)₂O, THF, r.t., overnight; (iv) NaOH, THF–H₂O, reﬂux, overnight; (v) NHS, EDC, CH₂Cl₂, r.t., 24 h;

(vi) 2-aminoethyldiethylamine or 16, dry DMF, DIPEA, r.t., 48 h; (vii) TFA, CH₂Cl₂, r.t., 2.5 h.

Scheme 4 Synthesis of L⁷and L⁸. Reagents and conditions: (i) benzyl 2-bromobutyrate, NaH, THF, 0 1C, 3 h; (ii) hydrazine monohydrate,

ethanol, r.t., overnight; (iii) CBr₄, PPh₃, THF, r.t., overnight; (iv) H₂N(CH₂)₂COOEt, CH₃CN, NaI, NEt₃, reﬂux, 24 h; (v) H₂, Pd/C, dry CH₂Cl₂,

r.t., 20 h; (vi) 2-aminoethyldiethylamine or 16, HBTU, NEt₃, dry CH₃CN, reﬂux, 24 h; (vii) NaOH, THF–H₂O, reﬂux, overnight.

HR-ESI-MS; such characterization conﬁrmed the proposed

formulations, namely the presence of the 2-aminoethyl-

diethylamine and 4-amino-N-(2-diethylaminoethyl)benzamide

pharmacophores, and showed that all compounds were

suﬃciently pure to be used in the synthesis of the respective

Re(^I) and ^99mTc(I) tricarbonyl complexes.

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The synthesis of the rhenium complexes was performed by

ligand exchange reactions of fac-[Re(H₂O)₃(CO)₃)]Br with

L¹–L⁸in reﬂuxing methanol. These reactions led to cationic

complexes (Re1, Re2, Re5 and Re6) and neutral complexes

(Re3, Re4, Re7 and Re8) anchored by bifunctional pyrazolyl-

diamine and pyrazolyl-aminocarboxylic ligands, respectively

(Scheme 5). Complexes Re1–Re8 were analyzed by spectro-

pyrazolyl-aminocarboxylic ligands, which was consistent with

the diastereotopic character of such protons. These multiplets

exhibited chemical shifts signiﬁcantly diﬀerent from those of

the corresponding protons in the respective free ligands. In

contrast, the signals due to the protons of the 2-aminoethyl-

diethylamine and 4-amino-N-(2-diethylaminoethyl)benzamide

pharmacophores, as well as those due to the methylenic linkers

attaching the pharmacophores to the ligand backbone, have

shown splitting patterns and chemical shifts very similar for

the free ligands and respective Re complexes. These ﬁndings

conﬁrmed that the pharmacophores were not interacting with

the metal. In the positive mode, the HR-ESI-MS spectra of

Re1–Re8 have shown prominent peaks corresponding to the

expected molecular-ions, with the correct isotope distribution

pattern and without signiﬁcant fragmentation.

1

scopic techniques (IR and H/¹³C NMR) and mass spectro-

metry, which allowed for the unambiguous identiﬁcation of

their chemical structures. The IR spectra of Re1–Re8 showed

intense absorption bands between 1878 and 2032 cm^ꢀ1

,

easily assigned to the n(CRO) stretching modes of the

fac-[Re(CO)₃]⁺unit. These frequencies compare well with

those that we have previously reported for other Re(^I)

tricarbonyl complexes anchored by pyrazolyl-diamine or

pyrazolyl-aminocarboxylic ligands.^29,30The ¹H NMR data

obtained for Re1–Re8 corroborated a facial coordination of

the ligands through the pyrazolyl ring, the central nitrogen

atom and the terminal amine (Re1, Re2, Re5 and Re6) or

carboxylate (Re3, Re4, Re7 and Re8) groups, since all the

spectra have shown a set of multiplets for the methylenic

protons of the framework of the pyrazolyl-diamine or

Synthesis, characterization and in vitro evaluation of the ^99mTc

complexes

The synthesis of the ^99mTc complexes (Tc1–Tc8) has

been performed in aqueous solution by reaction of

fac-[^99mTc(H₂O)₃(CO)₃]⁺with the appropriate ligand (L¹–L⁸)

Scheme 5 Synthesis of the Re and ^99mTc complexes.

c

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at 100 1C for 30 min (Scheme 5). The reactions were almost

quantitative (radiochemical yield 495%) for concentrations

of the ligands as low as 10^ꢀ4M. In the case of the pyrazolyl-

aminocarboxylic ligands functionalized through the central

amine, the pH of the reaction mixture strongly inﬂuenced

the radiochemical purity of the respective complexes

(Tc3 and Tc4). Tc3 and Tc4 could be obtained with a radio-

chemical purity higher than 95% only when their synthesis

was performed at relatively acidic pH, i.e. at pH = 5.5.

At neutral pH, the same reactions were accompanied by the

formation of another radioactive species, which was identiﬁed

as [^99mTc(CO)₃(3,5-Me₂pzCH₂CH₂NHCH₂COO)]. The forma-

tion of the latter complex resulted from the hydrolytic cleavage

of the butyric pendant arm used to couple the melanin-binding

groups to the chelator. This behavior has been previously

found in labeling with fac-[^99mTc(CO)₃]⁺of pyrazolyl-

aminocarboxylic ligands functionalized with other biologically

active fragments.³¹Hydrolytic cleavage of a C–N bond in a

tridentate diethylenetriamine chelator coordinated to

fac-[^99mTc(CO)₃]⁺has been also reported by Alberto et al.³²

Unlike Tc3 and Tc4, all the other ^99mTc complexes could be

obtained with high radiochemical purity (495%) at pH 7. The

chemical identity of Tc1–Tc8 was ascertained by comparison

of their HPLC proﬁles with those of the corresponding

rhenium complexes (Re1–Re8). The respective retention times

are presented in Table 1.

pharmacophore at the pendant arm of the central amine. This

trend reﬂects the presence, or not, of a central NH group,

which contributes to the increases hydrophilicity of the

complexes.

The aﬃnity for melanin of Tc1–Tc8 was assessed in vitro by

co-incubation of the complexes with synthetic melanin in

distilled water. The experiments were optimized in terms of

incubation time and melanin concentration. Relatively fast

binding kinetics were observed, since almost identical percen-

tages of binding were found after 1 h and 24 h of incubation.

As shown in Table 2, the percentages of bound complexes

spanned between 60 and 94%, for a melanin concentration of

0.5 mg per 10 mL after 1 h of incubation. These ﬁndings show

that the complexes have a moderate to high aﬃnity for

melanin, which indicates that the metalation of the 2-amino-

ethyldiethylamine and 4-amino-N-(2-diethylaminoethyl)-

benzamide pharmacophores with the fac-[^99mTc(CO)₃]⁺unit

did not compromise their ability to interact with this pigment.

Apparently, the charge of the complexes has some inﬂuence on

their interaction with melanin, the percentage of binding of the

cationic complexes being generally higher than the values

found for the neutral counterparts. In contrast, we have

veriﬁed that the lipophilicity of the complexes did not have a

well-deﬁned eﬀect on their aﬃnity to melanin. As proposed by

other authors, the binding of benzamide derivatives to melanin

is a reversible process that involves two classes of binding sites,

one ionic and another one hydrophobic.³³The behavior that

has been observed for Tc1–Tc8 indicates that for these

complexes electrostatic interactions with melanin seem to be

more determinant than the hydrophobic ones, which reﬂects

the polyanionic nature of melanin.

The in vitro evaluation of complexes Tc1–Tc8 involved the

study of their lipophilicity and their binding to synthetic

melanin. As discussed above, the lipophilicity could be a

decisive factor in the ability of the complexes to cross the

membrane of melanoma cells, while binding to melanin was

expected to promote their intracellular retention.

The lipophilicity of complexes Tc1–Tc8 was assessed by

measurement of the respective log P_o/wvalues (n-octanol/0.1 M

PBS, pH 7.4) using the multiple back-extraction method. The

measured log P_o/wvalues are shown in Table 1. Unlike Tc4

(log P_o/w= 1.02 ꢁ 0.02), all the other complexes exhibited

a hydrophilic character with log P_o/wvalues ranging from

ꢀ1.64 to ꢀ0.16. For the same pharmacophore, the neutral

complexes are less hydrophilic than the cationic congeners, as

would be expected. The complexes functionalized with the

pharmacophores at the 4-position of the pyrazolyl ring

are also more hydrophilic than those having the same

Biological evaluation of the ^99mTc complexes: in vitro cellular

uptake and biodistribution studies

To evaluate the ability of the complexes to accumulate in

melanoma tumor cells, in vitro cell uptake studies were

performed for Tc1–Tc8 in murine B16F1 melanoma cells,

which were also used for the in vivo studies with tumor-bearing

mice. As can be seen in Fig. 1, the measured cell uptake was

almost independent of the incubation time (15–240 min), and

the maximum cell uptake values ranged between 0.07 and

1.11%, when expressed as a percentage of total activity. These

low uptake values, corresponding to internalized activity, have

shown that all the complexes have a poor ability to accumulate

into the cells.

Table 1 HPLC retention times, log P values and in vitro binding to

synthetic melanin for complexes Tc1–Tc8

We have pursued with the in vivo evaluation of the

complexes in B16F1 melanoma-bearing mice, aiming to assess

their biodistribution proﬁle and pharmacokinetics, in terms of

tumor uptake, excretory pathways, metabolic stability and

target/non-target ratio. The biodistribution data obtained

for Tc1–Tc8 at 1 h and 4 h p.i., expressed in percent injected

dose per gram tissue (% ID g^ꢀ1), are presented in Table 2.

With the exception of Tc3, all the complexes have shown a

relatively fast blood clearance. Unlike other non-target tissues

like the muscle, some retention of radioactivity was observed

in the excretory organs, i.e. kidney and liver, which reﬂects the

urinary and hepatobiliary excretion of Tc1–Tc8. A more

pronounced kidney and liver uptake has been found for the

Retention

time/min^ab

% Bound to

synthetic melanin

Compound

log P_o/P_w

Tc1

Tc2

Tc3

Tc4

Tc5

Tc6

Tc7

Tc8

17.8 (17.1)

18.5 (18.5)

19.4 (19.0)

20.3 (20.1)

17.1 (16.5)

18.5 (18.1)

18.5 (18.2)

19.5 (19.2)

ꢀ0.38 ꢁ 0.04

ꢀ0.29 ꢁ 0.08

ꢀ0.16 ꢁ 0.03

1.02 ꢁ 0.02

79 ꢁ 5

94 ꢁ 2

77 ꢁ 4

71 ꢁ 3

76 ꢁ 2

67 ꢁ 5

60 ꢁ 5

87 ꢁ 7

ꢀ1.64 ꢁ 0.01

ꢀ1.24 ꢁ 0.05

ꢀ0.93 ꢁ 0.01

ꢀ0.31 ꢁ 0.01

a

Using a gradient of aqueous 0.1% CF₃COOH and methanol as the

b

solvent. The values in parentheses are for the Re complexes

Re1–Re8. Concentration of melanin 0.05 mg mL^ꢀ1, 1 h incubation.

c

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Table 2 Relevant biodistribution data (% ID g^ꢀ1) of Tc1–Tc8 at 1 and 4 h post-injection (p.i.) in C57/B16 mice with palpable hind limb B16

melanoma nodules

Mean tissue concentrations (% ID g^ꢀ1organ)

Time

(h, p.i.)

^99mTc complex

Muscle

Blood

Eyes

Tumor

Kidneys

Liver

Intestine

Tc1

1

4

1

4

1

4

1

4

1

4

1

4

1

4

1

4

0.42 ꢁ 0.01

0.28 ꢁ 0.04

0.12 ꢁ 0.04

0.07 ꢁ 0.02

0.17 ꢁ 0.04

0.24 ꢁ 0.15

0.06 ꢁ 0.00

0.08 ꢁ 0.01

0.15 ꢁ 0.06

0.10 ꢁ 0.02

0.16 ꢁ 0.02

0.06 ꢁ 0.00

0.13 ꢁ 0.09

0.04 ꢁ 0.02

0.05 ꢁ 0.02

0.03 ꢁ 0.01

0.70 ꢁ 0.06

0.29 ꢁ 0.03

0.53 ꢁ 0.22

0.26 ꢁ 0.19

1.02 ꢁ 0.08

0.86 ꢁ 0.68

0.33 ꢁ 0.06

0.35 ꢁ 0.05

0.26 ꢁ 0.03

0.20 ꢁ 0.20

1.54 ꢁ 0.28

0.32 ꢁ 0.08

0.25 ꢁ 0.05

0.18 ꢁ 0.07

0.35 ꢁ 0.02

0.20 ꢁ 0.02

1.09 ꢁ 1.00

0.45 ꢁ 0.19

—

1.20 ꢁ 0.08

0.87 ꢁ 0.18

1.24 ꢁ 0.27

0.60 ꢁ 0.29

0.44 ꢁ 0.16

0.45 ꢁ 0.24

0.44 ꢁ 0.22

0.54 ꢁ 0.04

1.00 ꢁ 0.22

0.68 ꢁ 0.23

1.21 ꢁ 0.02

1.69 ꢁ 0.39

0.24 ꢁ 0.04

0.12 ꢁ 0.03

0.26 ꢁ 0.04

0.17 ꢁ 0.03

18.52 ꢁ 3.07

12.61 ꢁ 1.04

6.93 ꢁ 1.06

4.28 ꢁ 0.33

2.4 ꢁ 0.4

18.83 ꢁ 6.48

10.24 ꢁ 0.88

46.15 ꢁ 5.98

34.09 ꢁ 3.29

2.44 ꢁ 0.05

2.07 ꢁ 0.82

5.67 ꢁ 1.01

1.37 ꢁ 0.46

26.24 ꢁ 2.62

24.81 ꢁ 0.68

26.94 ꢁ 0.84

18.28 ꢁ 1.24

3.36 ꢁ 1.20

1.54 ꢁ 0.17

4.70 ꢁ 2.64

2.93 ꢁ 2.79

15.35 ꢁ 4.85

15.19 ꢁ 2.82

16.14 ꢁ 3.15

26.24 ꢁ 5.81

12.94 ꢁ 3.93

15.71 ꢁ 2.39

38.86 ꢁ 3.14

20.81 ꢁ 14.29

6.18 ꢁ 0.97

9.28 ꢁ 0.79

8.45 ꢁ 1.41

16.87 ꢁ 0.85

12.14 ꢁ 1.37

16.03 ꢁ 1.96

29.97 ꢁ 4.88

34.87 ꢁ 7.26

Tc2

Tc3

Tc4

Tc5

Tc6

Tc7

Tc8

—

0.33 ꢁ 0.09

0.39 ꢁ 0.07

0.17 ꢁ 0.04

0.25 ꢁ 0.04

0.62 ꢁ 0.45

0.23 ꢁ 0.03

0.70 ꢁ 0.21

0.31 ꢁ 0.09

0.14 ꢁ 0.02

0.14 ꢁ 0.05

0.39 ꢁ 0.10

0.28 ꢁ 0.05

1.89 ꢁ 0.62

0.93 ꢁ 0.09

1.55 ꢁ 0.35

40.21 ꢁ 4.95

34.42 ꢁ 24.26

57.14 ꢁ 4.79

16.65 ꢁ 1.58

1.01 ꢁ 0.18

0.67 ꢁ 0.08

1.15 ꢁ 0.06

0.62 ꢁ 0.08

for melanin binding, which exhibited a considerable tumor

uptake of 7.62% ID g^ꢀ1at 1 h p.i. in a murine melanoma

model.²⁰The melanoma uptake of ^99mTc complexes bearing

melanin-avid pharmacophores might depend on various

factors, such as the nature of the Tc core and chelator, size,

topology and lipophilicity of the complex, structure of the

pharmacophore and position used for its attachment to the

chelator framework. In particular, the lipophilicity should

strongly inﬂuence their ability to freely diﬀuse across the cell

membrane. In the published studies, no simple correlation has

been found between melanoma uptake and lipophilicity, but

all the evaluated ^99mTc complexes having a hydrophilic

character presented a low tumoral accumulation.^15–17With

the exception of Tc4, all the other complexes reported in this

work are hydrophilic, which explains their relatively poor

melanoma uptake.

Fig. 1 In vitro cell uptake of complexes Tc1–Tc8 in murine B16F1

melanoma cells.

cationic complexes (Tc1, Tc2, Tc5 and Tc6) compared with the

neutral congeners (Tc3, Tc4, Tc7 and Tc8).

The in vivo stability of Tc1–Tc8 has been studied by HPLC

analysis of urine and blood samples taken from mice injected

with these complexes. Unlike Tc6 and Tc8, all the other

complexes have shown a remarkable resistance to metabolic

transformation. In the latter case, the intact compounds were

the unique radioactive species detected in the blood and urine.

By contrast, signiﬁcant amounts of metabolites were detected

in the blood and/or urine of mice injected with Tc6 and Tc8, as

can be seen in the HPLC radiochromatograms presented in

Fig. 2. For Tc6, the HPLC analysis of serum at 1 h p.i. has

shown the presence of the intact complex (t_R= 18.90 min)

together with a more lipophilic radioactive species (t_R= 22.38 min)

in higher percentage (Fig. 2A). In the blood, only the original

Tc8 was found (see Fig. 2B). However, HPLC analysis

of the urine of mice injected with Tc6 and Tc8 has shown

that both complexes are transformed into more hydrophilic

metabolites appearing at t_R= 17.87 min and t_R= 18.87 min,

respectively. The common feature of Tc6 and Tc8 is the

All the complexes have shown a negligible or modest tumor

uptake, both at 1 h p.i. (0.24–1.21% ID g^ꢀ1) or at 4 h p.i.

(0.17–1.69% ID g^ꢀ1). Nevertheless, the neutral complexes

showed a reduced tumor uptake compared to the cationic ones,

with uptake values at 4 h p.i. in the ranges 0.17–0.54% ID g^ꢀ1

and 0.60–1.69% ID g^ꢀ1, respectively. Apparently, the increasing

lipophilicity of the complexes did not improve their tumor

uptake, as the neutral and lipophilic Tc4 (log P = 1.02)

has shown one of the lowest values of tumor uptake

(0.54% ID g^ꢀ1at 4 h p.i.). In spite of their relatively poor

tumor uptake, some of the complexes (e.g. Tc2 and Tc6) have

shown encouraging tumor-to-organ ratios, with tumor–muscle

and tumor–blood ratios as high as 28 and 5.3, respectively, in

the case of Tc6. Compared to radioiodinated benzamides,

^99mTc(V) complexes functionalized with the same type of

pharmacophore have commonly shown modest tumor uptake

values,^14–22as reported herein for the organometallic

complexes Tc1–Tc8. So far, the best results have been reported

by Eisenhut et al. for a [^99mTcO]³⁺complex anchored by a

N₂S₂chelator and bearing a 2-aminoethyldiethylamine group

presence of

a 4-amino-N-(2-diethylaminoethyl)benzamide

pharmacophore that has been attached at the pyrazolyl ring

through formation of an amide bond. As discussed above,

the attachment of this pharmacophore required, in the case of

c

2570 New J. Chem., 2010, 34, 2564–2578 This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientiﬁque 2010

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Fig. 2 HPLC analysis (radiometric detection) of blood and urine from mice injected with Tc6 (A) and Tc8 (B), at 60 min p.i.

these complexes, the use of a propylenic spacer and the

formation of an aliphatic amide. Eventually, the more hydro-

philic metabolites detected in the urine may result from an

enzymatic cleavage of such amide bonds. However, no further

eﬀorts were made to identify chemically the hydrophilic

metabolites detected in the urine for Tc6 and Tc8, or the more

lipophilic species found in the blood for Tc6.

4-(2-aminoethyl)[2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl]amino-

butanoic acid (1),²⁹ethyl 2-(1-(2-hydroxyethyl)-3,5-dimethyl-

pyrazol)acetate³⁴were prepared according to published

methods. The starting material fac-[Re(H₂O)₃(CO)₃]Br was

synthesized by the literature method.³⁵Na[^99mTcO₄] was

eluted from a commercial ⁹⁹Mo/^99mTc generator, using 0.9%

1

saline. H and ¹³C NMR spectra were recorded on a Varian

1

Unity 300 MHz spectrometer; H and ¹³C chemical shifts are

given in ppm and were referenced with the residual solvent

resonances relative to SiMe₄. IR spectra were recorded as KBr

pellets on a Bruker, Tensor 27 spectrometer. Electrospray

ionisation mass spectrometry (ESI-MS) was performed

at ITN on a QITMS instrument in positive ion mode.

High-resolution mass spectra (HR-MS) were performed at

the Faculty of Science of the University of Lisbon in a ApexQe

FTICR Mass Spectrometer from Bruker Daltonics (Billerica,

MA, USA) equipped with an electrospray ion source (ESI)

and a 7 T actively shielded superconducting magnet.

Conclusion

We have successfully synthesised an enlarged family of

Re(^I)/^99mTc (I) tricarbonyl complexes anchored by bifunctional

pyrazolyl-containing chelators of the N₃- or N₂O-types, bearing

2-aminoethyldiethylamine or 4-amino-N-(2-diethylaminoethyl)-

benzamide groups as melanin-binders. Proﬁting from the

versatility of these systems the melanin-avid fragments were

introduced either at the 4-position of the azolyl ring or at the

central amine of the chelator backbone. The in vitro evaluation

of the ^99mTc complexes in B16F1 murine melanoma cell lines

has shown a poor cellular uptake. In line with this result, the

in vivo tumor uptake of these complexes in B16F1 melanoma-

bearing mice was rather disappointing. Nevertheless, the

complexes anchored by the N₃-donors have shown, in general,

an increased tumor uptake compared with the congeners

anchored by the N₂O-donors. This trend reﬂects the highest

binding aﬃnity of the former for melanin, which is due most

probably to the polyanionic nature of melanin and to the

cationic character of the complexes with the N₃-donors. It is

worthwhile to mention that some of these cationic compounds

have shown favorable tumor–non-target ratios, which seems to

indicate that they have some selectivity towards melanoma

tissue. Such selectivity encourages the further optimization of

the in vitro/in vivo biological properties of these types of

complexes, particularly concerning their ability to cross the cell

membrane and target intracellular melanin. To achieve this goal

we are currently evaluating the possibility of synthesizing

related, but more compact, complexes.

Thin-layer chromatography (TLC) was performed on

Merck silica gel 60 F254 plates. Column chromatography

was performed with silica gel 60 (Merck). HPLC analysis of

the Re and ^99mTc complexes was performed on a Perkin–

Elmer LC pump 200 coupled to a LC 290 tunable UV-Vis

detector and to a Berthold LB-507A radiometric detector,

using an analytic Macherey-Nagel C18 reversed-phase column

(Nucleosil 100–10, 250 ꢂ 3 mm) with a ﬂow rate of

1 mL min^ꢀ1. HPLC puriﬁcations were performed using a

preparative Waters m Bondapak C18 column (150 ꢂ 19 mm)

at a ﬂow rate of 5.0 mL min^ꢀ1; UV detection, 254 nm; eluents,

A – aqueous 0.1% CF₃COOH solution, B – acetonitrile; method:

0–3 min, 100% A; 3–3.1 min, 100%–75% A; 3.1–9 min, 75% A;

9–9.1 min 75%–66% A; 9.1–20 min, 66%–0% A; 20–25 min,

0% A; 25–25.1 min, 0%–100% A; 25.1–30 min, 100% A.

Synthesis of pyrazolyl-diamine ligands (L¹and L²) functionalized

with melanin-avid pharmacophores at the central amine group

tert-Butyl 2-((4-(2-(diethylamino)ethylamino)-4-oxobutyl)-

(2-(3,5-dimethylpyrazol-1-yl)ethyl)amino)ethylcarbamate (2).

To a solution of 4-(2-aminoethyl)[2-(3,5-dimethyl-1H-pyrazol-

1-yl)ethyl]amino}butanoic acid (1) (200 mg, 0.543 mmol) in

dry CH₃CN was added N,N-diethylethylenediamine (76 mL,

0.543 mmol), HBTU (206 mg, 0.543 mmol) and NEt₃(228 mL;

1.62 mmol). The reaction mixture was reﬂuxed for 72 h;

after cooling to room temperature, the solvent was removed

under vacuum. The residue was puriﬁed by silica-gel column

Experimental section

Chemistry

Unless otherwise stated, the synthesis of the ligands and

complexes was carried out under a nitrogen atmosphere, using

standard Schlenk techniques and dry solvents; the work-up

procedures were performed under air. The compounds

c

This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientiﬁque 2010 New J. Chem., 2010, 34, 2564–2578 2571

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chromatography using a gradient from 100% MeOH to

MeOH/NH₄OH (98 : 2). Compound 2 (98%, 312 mg) was

recovered from the collected fractions as a yellow oil,

after removal of the solvent under reduced pressure.

R_f(SiO₂and MeOH) = 0.28; ¹H NMR (CDCl₃): d 0.97

(CH₂), 47.4 (CH₂), 52.1 (CH₂), 52.8 (CH₂), 54.1 (CH₂), 54.2

(CH₂), 60.3 (CH₂), 106.3 (C(H₄-pz), 112.4 (C_aromatic), 115.97

(C_TFA), 116.3 (C_aromatic), 120.2 (C_aromatic), 129.4 (C_3/5-pz)),

141.1 (C_aromatic), 143.7 (C_3/5-pz), 165.33 (C_TFA), 170.6 (CQO),

174.6 (CQO). HR ESI-MS (m/z): calcd for [M+H]⁺,

486.36922; found, 486.35510.

(6H, t, (NCH₂CH₃), 1.39 (9H, s, CH₃(BOC)), 1.66

ꢀ

(2H, t, CH₂), 2.07 (2H, t, CH₂), 2.17 (3H, s, CH₃-pz), 2.19

(3H, s, CH₃-pz), 2.47 (8H, m, CH₂), 2.67 (2H, t, CH₂), 2.95

(4H, q, CH₂), 3.25 (2H, t, CH₂), 3.95 (2H, t, CH₂), 5.17 (1H, b,

NH-BOC), 5.75 (1H, s, H(4)-pz), 6.96 (1H, b, NH).

Synthesis of pyrazolyl-aminocarboxylic ligands (L³and L⁴)

functionalized with melanin-avid pharmacophores at the

central amine group

Ethyl 2-(2-(3,5-dimethylpyrazol-1-yl)ethylamino)acetate (5).

To a solution of 1-(2-bromoethyl)-3.5-dimethylpyrazole (4)

(2.80 g, 13.7 mmol) in dry CH₃CN (35 mL) was added glycine

ethyl ester (2.88 g, 20.6 mmol), K₂CO₃(2.85 g, 20.6 mmol) and

KI (171 mg, 1.03 mmol). The reaction mixture was reﬂuxed for

3 days. After cooling to room temperature, the solvent was

removed under vacuum. The residue obtained was applied on

the top of a silica-gel column, which was eluted with a gradient

of CH₂Cl₂–MeOH (95 : 5 to 85 : 15). Removal of the solvent

from the collected fractions gave 5 (1.480 g, 48%) as a brown

tert-Butyl 2-((4-(4-(2-(diethylamino)ethylcarbamoyl))phenyl-

amino)-4-oxobutyl)(2-(3,5-dimethylpyrazol-1-yl)ethyl)amino)-

ethylcarbamate (3). Compound 3 was synthesized and recovered

as described above for 2, starting from compound 1 (120 mg,

0.543 mmol) and procainamide (89 mg, 0.326 mmol) and

with reﬂux of the reaction mixture for 24 h. Compound 3

(41 mg, 13%) was obtained as a yellow oil after puriﬁcation by

silica-gel column chromatography with a gradient from

CH₂Cl₂–MeOH (10 : 90) to 100% MeOH. R_f(SiO₂and

CH₂Cl₂–MeOH–NH₄OH (95 : 4 : 1)) = 0,4;¹H NMR (CD₃OD):

1

oil. R_f(SiO₂and CH₂Cl₂–MeOH (90 : 10)) = 0.44; H NMR

d 1.34 (15H, m, NCH₂CH₃+ BOC); 2.08 (2H, t, CH₂); 2.13

ꢀ

(CDCl₃): d 1.20 (3H, t, CH₃(Et)), 2.15 (3H, s, CH₃-pz), 2.18

(3H, s, CH₃-pz), 2.96 (2H, t, CH₂), 3.31 (2H, s, CH₂), 4.00

(2H, t, CH₂), 4.10 (2H, q, CH₂), 5.72 (1H, s, H(4-pz)).

(3H, s, CH₃-pz); 2.28 (3H, s, CH₃-pz); 2.62 (2H, t, CH₂); 3.33

(12H, m, 6 CH₂); 3.76 (4H, t, 2CH₂); 4.41 (2H, t, CH₂); 5.89

(1H, s, H(4)-pz); 7.67 (2H, d, Ph); 7.83 (2H, d, Ph).

Benzyl 4-((2-(3,5-dimethylpyrazol-1-yl)ethyl)(2-ethoxy-2-

oxoethyl)amino)butanoate (6). To a solution of ethyl 2-(2-

(3,5-dimethylpyrazol)ethylamino)acetate (5) (700 mg, 3.11 mmol)

in dry CH₃CN (20 mL) was added NEt₃(871 mL, 6.22 mmol)

and KI (47 mg, 0.31 mmol). Then, a solution of benzyl

4-bromobutanoate (800 mg, 3.11 mmol) in dry CH₃CN

(5 mL) was added dropwise. The reaction mixture was reﬂuxed

for 3 days. The residue obtained was applied on the top of a

silica-gel column which was eluted with a gradient of

CH₂Cl₂–MeOH (95 : 5 to 80 : 20). The removal of the solvent

from the collected fractions gave 6 (422 mg, 34%) as a brown

4-((2-Aminoethyl)(2-(3,5-dimethylpyrazol-1-yl)ethyl)amino)-

N-(2-diethylamino)ethyl)butanamideꢃCF₃COOH (L¹). To

a

solution of 2 (156 mg, 0.267 mmol) in CH₂Cl₂(5 mL) was

added triﬂuoroacetic acid (600 mL, 6.68 mmol), and the

mixture was stirred for 4 h at room temperature. The solvent

was removed under vacuum and the crude product was

puriﬁed by RP-HPLC on a preparative Waters m Bondapak

C18 (150 ꢂ 19 mm) with a ﬂow rate of 5.0 mL min^ꢀ1and using

the method described above. Compound L¹(98 mg, 77%) was

recovered from the collected fractions as a transparent oil,

after removal of the solvent under vacuum. IR u_max(KBr)/cm^ꢀ1

1702 (CQO); ¹H NMR (CD₃OD): d 1.36 (6H, t, 2 CH₃

1

oil. R_f(SiO₂and CH₂Cl₂–MeOH (90 : 10)) = 0.96; H NMR

(CDCl₃): d 1.24 (3H, t, CH₃), 1.70 (2H, m, CH₂), 2.17

(3H, s, CH₃-pz), 2.18 (3H, s, CH₃-pz), 2.28 (2H, t, CH₂),

2.62 (2H, t, CH₂), 2.99 (2H, t, CH₂), 3.32 (2H, s, CH₂), 3.97

(2H, t, CH₂), 4.13 (2H, q, CH₂), 5.10 (2H, s, CH₂), 5.72 (1H, s,

H₄-pz), 7.34 (5H, s, C₆H₅).

(NCH₂CH₃), 1.99 (2H, m, CH₂), 2.25 (3H, s, CH₃-pz), 2.35

ꢀ

(3H, s, CH₃-pz), 2.41 (2H, t, CH₂), 3.21 (2H, t, CH₂), 3.32

(10H, m, CH₂), 3.46 (2H, m, CH₂), 3.57 (2H, t, CH₂), 4.43

(2H, t, CH₂), 6.00 (1H, s, H(4)-pz); ¹³C NMR (CD₃OD):

d 8.97 (CH₃), 10.72 (CH₃-pz), 13.08 (CH₃-pz), 21.24 (CH₂),

33.20 (CH₂), 35.57 (CH₂), 36.32 (CH₂), 44.37 (CH₂), 49.58

(CH₂), 51.36 (CH₂), 52.16 (CH₂), 53.95 (CH₂), 54.84 (CH₂),

107.01 (C₄-pz), 119.72 (C_TFA), 142.45 (C(H_3/5-pz)), 149.45

(C_3/5-pz), 162.61 (C_TFA), 176.22 (CQO). HR ESI-MS (m/z):

calcd for [M+H]⁺, 367.31799; found, 367.32606.

4-((2-(3,5-Dimethylpyrazol-1-yl)ethyl)(2-ethoxy-2-oxoethyl)-

amino)butanoic acid (7). To a solution of benzyl 4-((2-(3,5-

dimethylpyrazol)ethyl)(2-ethoxy-2 oxoethyl)amino)butanoate

(6) (422 mg, 1.05 mmol) in dry ethanol (40 mL) was added

palladium on charcoal, 10% (500 mg). The reaction mixture

was saturated with H₂and stirred at room temperature for

20 h. The catalyst was removed by vacuum ﬁltration through a

pad of celite; the supernatant was evaporated under vacuum to

aﬀord 7 (257 mg, 79%) as a transparent oil. ¹H NMR

(CDCl₃): d 1.22 (3H, t, CH₃), 1.72 (2H, m, CH₂), 2.17

(3H, s, CH₃-pz), 2.20 (3H, s, CH₃-pz), 2.35 (2H, t, CH₂),

2.68 (2H, t, CH₂), 2.99 (2H, t, CH₂), 3.24 (2H, s, CH₂), 3.73

(2H, t, CH₂), 4.10 (2H, q, CH₂), 5.75 (1H, s, H₄-pz).

4-(4-((2-Aminoethyl)(2-(3,5-dimethylpyrazol-1-yl)ethyl)amino)-

butanamido)-N-(2-(diethylamino)ethyl)benzamide (L²). L²was

obtained as described above for L¹, starting from 41 mg

(0.070 mmol) of compound 3. L²(31 mg, 73%), was recovered

as a yellow oil after removal of the solvent. IR u_max(KBr)/cm^ꢀ1

1635, 1688 (CQO); ¹H NMR (CD₃OD): d 1.34 (6H, t, 2 CH₃),

2.02 (2H, t, CH₂), 2.15 (3H, s, CH₃-pz), 2.29 (3H, s, CH₃-pz),

2.56 (2H, t, CH₂), 3.33 (12H, m, 6 CH₂), 3.74 (4H, t, 2CH₂),

4.39 (2H, t, CH₂), 5.91 (1H, s, H(4)-pz), 7.67 (2H, d, aromatic),

7.83 (2H, d, aromatic);¹³C NMR (CD₃OD): 9.1 (CH₃), 10.9

(CH₃), 13.1 (CH₃), 23.9 (CH₂), 34.8 (CH₂), 36.3 (CH₂), 38.8

Ethyl 2-((4-(2-(Diethylamino)ethylamino)-4-oxobutyl)(2-(3,5-

dimethypyrazol-1-yl)ethyl)amino)acetate (8). Compound

8

was obtained as above described for 2, starting from 128 mg

c

2572 New J. Chem., 2010, 34, 2564–2578 This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientiﬁque 2010

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(0.41 mmol) of 7 and reﬂux of the reaction mixture for 48 h.

The puriﬁcation of 8 (70 mg, 42%) was performed by silica-gel

column chromatography using a gradient from 100% MeOH

to MeOH–NH₄OH (98 : 2); R_f(SiO₂and MeOH) = 0.29;

¹H NMR (CDCl₃): d 1.06 (6H, t, CH₃), 1.18 (3H, t, CH₃), 1.65

(2H, m, CH₂), 2.11 (3H, s, CH₃-pz), 2.13 (2H, m, CH₂), 2.17

(3H, s, CH₃-pz), 2.53 (2H, t, CH₂), 2.69 (6H, m, CH₂), 2.93

(2H, t, CH₂), 3.23 (2H, s, CH₂), 3.35 (2H, m, CH₂), 3.96

(2H, t, CH₂), 4.06 (2H, q, CH₂), 5.68 (1H, s, H₄-pz), 7.26

(1H, b, NH).

13.19 (CH₃-pz), 21.25 (CH₂), 34.35 (CH₂), 36.15 (CH₂), 43.88

(CH₂), 48.94 (CH₂), 52.65 (CH₂), 54.81 (CH₂), 55.96 (CH₂),

57.54 (CH₂), 106.49 (C₄-pz), 120.17 (C_aromatic), 129.29

(C_aromatic), 129.33 (C_aromatic), 141.73 (C_3/5-pz)), 143.42

(C_aromatic), 149.69 (C_3/5-pz)), 170.24 (CQO), 170.93 (CQO),

173.14 (CQO). HR ESI-MS (m/z): calcd for [M + H]⁺,

501.31565; found, 501.23801.

Synthesis of pyrazolyl-diamine ligands (L⁵and L6) functionalized

with melanin-avid pharmacophores at the 4-position of the azolyl

ring

Ethyl 2-((4-(4-(2-(diethylamino)ethylcarbamoyl)phenylamino)-

4-oxobutyl)(2-(3,5-dimethypyrazol-1-yl)ethyl)amino)acetate (9).

Compound 9 was obtained as above described for 3, starting

from 450 mg (14.5 mmol) of 7 and with reﬂux of the reaction

mixture for 72 h. The puriﬁcation of 9 (107 mg, 14%) was

performed by silica-gel column chromatography with

CHCl₃–MeOH–NH₄OH (80 : 18 : 2) as eluent; R_f(SiO₂and

Ethyl 2-(1-(2-bromoethyl)-3,5-dimethylpyrazol-1-yl)acetate (11).

To a solution of ethyl 2-(1-(2-hydroxyethyl)-3,5-dimethyl-

pyrazol-1-yl)acetate, 10 (7.500 g, 35 mmol) in toluene

(150 mL) was added PBr₃(6.58 mL, 70 mmol), and the

reaction mixture was reﬂuxed overnight. After cooling to

room temperature, the solvent was removed under vacuum,

the residue dissolved in CHCl₃and the resulting solution

washed with distilled water. The organic phase was dried over

MgSO₄, ﬁltered and evaporated under vacuum, giving a

yellow oil that was formulated as compound 11. Yield: 55%

CHCl₃–MeOH–NH₄OH (70 : 28 : 2))

=

0.80; ¹H NMR

(CDCl₃): d 1.00 (6H, t, CH₃), 1.20 (3H, t, CH₃), 1.77

(2H, m, CH₂), 2.15 (3H, s, CH₃-pz), 2.19 (3H, s, CH₃-pz),

2.31 (2H, m, CH₂), 2.57 (8H, m, 4 CH₂), 2.93 (2H, t, CH₂),

3.21 (2H, s, CH₂), 3.43 (2H, m, CH₂), 3.97 (2H, t, CH₂), 4.09

(2H, q, CH₂), 5.78 (1H, s, H_4ꢀpz), 6.93 (1H, bs, NH), 7.69

(4H, s, CH), 9.84 (1H, b, NH).

1

(5.570 g, 19.2 mmol). H NMR (CDCl₃): d 1.21 (3H, t, CH₃);

2.17 (3H, s, CH₃-pz); 2.21 (3H, s, CH₃-pz); 3.30 (2H, s, CH₂);

3.67 (2H, q, CH₂); 4.10 (2H, m, CH₂); 4.33 (2H, m, CH₂).

Ethyl 2-(1-(2-(2-(tert-butoxycarbonylamino)ethylamino)ethyl)-

3,5-dimethylpyrazol-1-yl)acetate (12). To a solution of ethyl

2-(1-(2-bromoethyl)-3,5-dimethylpyrazol-1-yl)acetate (11) (2.770 g,

9.6 mmol) in dry CH₃CN (35 mL) was added tert-butyl

2-aminoethylcarbamate (2.31 g; 14.4 mmol), K₂CO₃(1.990 g;

14.4 mmol) and KI (80 mg; 0.48 mmol). The reaction mixture

was reﬂuxed overnight, and after cooling to room temperature

the solvent was removed under vacuum. The product was

puriﬁed by silica-gel column chromatography using

CH₂Cl₂–MeOH (90/10) as the eluent. Removal of the solvent

from the collected fractions gave 12 (650 mg, 12%) as a yellow

[(3-{[2-(Diethylamino)ethyl]carbamoyl}propyl)[2-(3,5-dimethyl-

pyrazol-1-yl)ethyl]amino]acetic acid (L³). To a solution of 8

(70 mg, 0.17 mmol) in THF were added 7 mL of an aqueous

0.02 M NaOH solution (molar ratio 8/NaOH = 1 : 10), and

the mixture was reﬂuxed overnight. After cooling to room

temperature, the solution was neutralized to pH 7 and the

solvent removed under vacuum. The residue was extracted

with CH₂Cl₂and the resulting solution was dried under

vacuum giving

a yellow oil that was formulated as

L³(64 mg, 98%); IR u_max(KBr)/cm^ꢀ11581, 1659 (CQO);

¹H NMR (CD₃OD): d 1.17 (6H, t, 2CH₃), 1.66 (2H, m, CH₂),

2.06 (3H, s, CH₃-pz), 2.13 (2H, m, CH₂), 2.19 (3H, s, CH₃-pz),

2.58 (2H, t, CH₂), 2.93 (2H, t, CH₂), 2.98 (6H, m, 3 CH₂), 3.14

(2H, s, CH₂), 3.48 (2H, t, CH₂), 4.04 (2H, t, CH₂), 5.73 (1H, s,

H₄-pz); ¹³C NMR (CD₃OD): d 8.22 (2 CH₃), 9.78 (CH₃-pz),

12.07 (CH₃-pz), 22.62 (CH₂), 32.58 (CH₂), 34.90 (CH₂), 45.60

(CH₂), 47.27 (CH₂), 51.30 (CH₂), 53.62 (CH₂), 53.96 (CH₂),

61.65 (CH₂), 104.85 (C₄-pz), 140.51 (C_3/5-pz), 147.66 (C_3/5-pz);

175.69 (CQO); 176.52 (CQO). HR ESI-MS (m/z): calcd for

[M + H]+, 382.24994; found, 382.28188.

1

oil. R_f(SiO₂and CH₂Cl₂–MeOH (90 : 10)) = 0.38; H NMR

(CDCl₃): d 1.20 (3H, t, CH₃), 1.39 (9H, s, CH₃(BOC)), 2.14

(3H, s, CH₃), 2.16 (3H, s, CH₃), 2.36 (2H, t, CH₂), 2.70 (2H, t,

CH₂), 2.98 (2H, t, CH₂), 3.16 (2H, s, CH₂), 4.00 (2H, t, CH₂),

4.05 (2H, q, CH₂), 5.17 (1H, b, NH).

Ethyl 2-(1-(2-(tert-butoxycarbonyl)(2-(tert-butoxycarbonyl-

amino)ethyl)amino)ethyl)-3,5-dimethylpyrazol-1-yl)acetate (13).

To a solution of 12 (650 mg; 1.75 mmol) in dry THF (25 mL)

was added dropwise, at 0 1C, a solution of di-tert-butyl

dicarbonate (382 mg, 1.75 mmol) in dry THF (25 mL). The

reaction mixture was stirred overnight, at room temperature.

The solvent was removed under vacuum, and the crude

product extracted with a mixture of aqueous Na₂CO₃/CHCl₃.

The organic phase was separated, dried over MgSO₄, ﬁltered

and the solvent was removed under vacuum, giving 13

(820 mg, 99%) as a yellow oil. ¹H NMR (CDCl₃): d 1.21

(3H, t, CH₃), 1.39 (18H, m, CH₃(BOC)), 2.16 (3H, m,

CH₃-pz), 3.00 (4H, m, CH₂), 3.29 (2H, s, CH₂), 3.50 (2H, m,

CH₂), 4.07 (4H, m, 2 CH₂), 4.80 (1H, b, NH).

Ethyl 2-((4-(4-(2-(diethylamino)ethylcarbamoyl)phenylamino)-

4-oxobutyl)(2-(3,5-dimethypyrazol-1-yl)ethyl)amino)acetic acid

(L⁴). L⁴(60 mg, 60%) was prepared as above described for L³,

starting from 107 mg (0.27 mmol) of 9. Compound 9 was

isolated as a yellow oil after puriﬁcation by silica-gel column

chromatography with MeOH–NH₄OH (99 : 1) as eluent. R_f

(SiO₂and MeOH–NH₄OH (99 : 1)) = 0.56; IR u_max(KBr)/cm^ꢀ1

1

1544, 1632, 1685 (CQO); H NMR (CD₃OD): d 1.33 (6H, t,

CH₃); 1.97 (2H, m, CH₂), 2.14 (3H, s, CH₃-pz), 2.25 (3H, s,

CH₃-pz), 2.53 (2H, t, CH₂), 3.24 (10H, m, CH₂), 3.55 (2H, s,

CH₂), 3.74 (2H, t, CH₂), 4.28 (2H, t, CH₂), 5.83 (1H, s, H₄-pz),

6.93 (1H, b, NH), 7.69 (2H, d, CH-aromatic), 7.83 (2H, d, CH-

aromatic). ¹³C NMR (CDCl₃): d 9.06 (2 CH₃), 10.64 (CH₃-pz),

2-(1-(2-tert-Butoxucarbonyl(2-(tert-butoxycarbonylamino)-

ethyl)amino)ethyl)-3,5-dimethylpyrazol-1-yl)acetic acid (14). To

a solution of 13 (830 mg, 1.75 mmol) in dry THF (10 mL) was

c

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added a solution of NaOH (700 mg, 17.5 mmol) in H₂O

(10 mL). The reaction mixture was reﬂuxed overnight, after

cooling to room temperature, the mixture was neutralized to

pH 7 with aqueous 1 M HCl. The solvent was evaporated

under vacuum and the residue extracted with CH₂Cl₂. After

ﬁltration, dichloromethane was removed under vacuum,

tert-Butyl N-(2-{[(tert-butoxy)carbonyl](2-{4-[({3-[(4-{[2-(diethyl-

amino)ethyl]carbamoyl}phenyl)carbamoyl]propyl}carbamoyl)-

methyl]-3,5-dimethylpyrazol-1-yl}ethyl)amino}ethyl)carbamate (18).

Compound 18 was synthesized as described above for 17,

starting from 15 (333 mg, 0.58 mmol) and 16 (186 mg,

0.58 mmol). 18 (70 mg, 16%) was puriﬁed by silica-gel column

chromatography using CHCl₃–MeOH–NH₄OH (80 : 18 : 2) as

the eluent. R_f(SiO₂and CHCl₃–MeOH–NH₄OH (80 : 18 : 2)) =

0.64; ¹H NMR (CDCl₃): d 0.97 (6H, t, 2CH₃), 1.25 (9H, s, CH₃

(BOC)), 1.39 (9H, s, CH₃(BOC)), 1.78 (2H, m, CH₂), 2.05

(3H, s, CH₃-pz), 2.11 (3H, s, CH₃-pz), 2.29 (2H, m, CH₂), 2.51

(4H, q, CH₂), 2.58 (2H, t, CH₂), 3.39 (12H, m, 6-CH₂), 4.09

(2H, m, CH₂), 6.61 (1H, b, NH), 6.83 (1H, b, NH), 6.97

(1H, b, NH), 7.69 (4H, s, H_aromatic), 9.87 (1H, b, NH-BOC).

1

giving 14 (749 mg, 97%) as a yellow oil. H NMR (CDCl₃):

d 1.37 (18H, m, CH₃(BOC)), 2.11 (3H, m, CH₃-pz), 2.97

(4H, m, CH₂), 3.29 (2H, m, CH₂), 3.47 (2H, m, CH₂), 4.09

(2H, m, CH₂).

tert-Butyl N-(2-{[(tert-butoxy)carbonyl](2-{4-[2-(2,5-dioxo-

pyrrolidin-1-yl)-2-oxoethyl]-3,5-dimethylpyrazol-1-yl}ethyl)amino}-

ethyl)carbamate (15). To a solution of 14 (400 mg, 0.90 mmol)

in dry CH₂Cl₂(15 mL) was added, at 0 1C, N-hydroxy-

succinimide (NHS) (115 mg, 0.99 mmol) and 1-ethyl-3-

[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)

(190 mg, 0.99 mmol). The resulting mixture was stirred at

room temperature for 72 h and, after this time, was washed

with distilled water. The organic phase was separated and

dried over MgSO₄; after ﬁltration, the solvent was removed

2-(1-(2-(2-Aminoethylamino)ethyl)-3,5-dimethylpyrazol-1-yl)-

N-(2-(diethylamino)ethyl)acetamideꢃCF₃COOH (L⁵). L⁵(107 mg,

73%) was synthesized and recovered as described above for

L², starting from 177 mg (0.33 mmol) of 17. IR u_max(KBr)/cm^ꢀ1

1

1710 (CQO); H NMR (CD₃OD): d 1.30 (6H, t, CH₃), 2.15

(3H, s, CH₃-pz), 2.23 (3H, s, CH₃-pz), 3.23 (6H, m, CH₂), 3.43

(6H, m, CH₂), 3.55 (4H, m, CH₂), 4.38 (2H, t, CH₂); ¹³C NMR

(CD₃OD): d 9.12 (CH₃); 9.39 (CH₃-pz); 11.83 (CH₃-pz); 31.09

(CH₂); 35.87 (CH₂); 36.79 (CH₂); 44.95 (CH₂); 45.73 (CH₂);

48.72 (CH₂); 49.85 (CH₂); 52.34 (CH₂); 115.59 (C₄-pz); 140.48

(C_3/5-pz); 149.29 (C_3/5-pz), 162.92 (C_TFA), 175.23 (CQO). HR

ESI-MS (m/z): calcd for [M + H]⁺, 339.28669; found,

339.29361.

1

under vacuum, giving 15 (478 mg, 93%) as a yellow oil. H

NMR (CDCl₃): d 1.37 (18H, m, CH₃(BOC)), 2.13 (3H, m,

CH₃-pz), 2.77 (4H, s, CH₂), 2.85 (4H, m, CH₂), 3.48 (2H, m,

CH₂), 3.60 (2H, m, CH₂), 4.09 (2H, m, CH₂).

4-(4-Aminobutanoyloxyamino)-N-(2-(diethylamino)ethyl)-

benzamide (16). To a solution of 4-(tert-butoxycarbonylamino)-

butanoic acid (2.00g, 7.06 mmol) in dry CH₃CN (30 mL) was

added procainamaide (1.44 g, 5.30 mmol), HBTU (2.678 g,

7.06 mmol) and NEt₃(2.96 mL, 21.2 mmol). The reaction

mixture was reﬂuxed for 48 h and, after cooling to room

temperature, the solvent was removed under vacuum. The

resulting amide derivative tert-butyl 4-(4-(2-(diethylamino)-

ethylcarbamoyl)phenylaminooxy)-4-oxobutylcarbamate was

puriﬁed by silica-gel column chromatography using methanol

as eluent. The product obtained from the collected fractions

was dissolved in CH₂Cl₂(5 mL) and stirred for 4 h in the

presence of TFA (5 mL). Removal of the solvent under

vacuum gave 16 (773 mg, 90%) as a yellow oil. R_f(SiO₂and

4-(4-(2-(1-(2-Aminoethylamino)ethyl)3,5-dimethylpyrazol-1-yl)-

acetamido)butanamido)-N-(2-(diethylamino)ethyl)benzamideꢃ

CF₃COOH (L⁶). L⁶(230 mg, 80%) was synthesized and

recovered as described above for L², starting from 234 mg

(0.44 mmol) of 18. IR u_max(KBr)/cm^ꢀ11646, 1699 (CQO);

¹H NMR (CDCl₃): d 1.27 (6H, t, CH₃); 1.81 (2H, m, CH₂),

2.09 (3H, s, 2CH₃-pz), 2.16 (3H, s, 2CH₃-pz), 2.36 (2H, m, CH₂),

3.25 (14H, m, 7 CH₂), 3.49 (2H, m, CH₂), 3.68 (2H, m, CH₂),

4.34 (2H, m, CH₂), 7.63 (2H, d, H_aromatic), 7.75 (2H, d,

H

_aromatic); ¹³C NMR (CDCl₃): d 7.92 (CH₃); 8.21 (CH₃-pz),

10.49 (CH₃-pz), 25.15 (CH₂), 29.95 (CH₂), 34.06 (CH₂), 35.20

(CH₂), 35.62 (CH₂), 38.93 (CH₂), 43.83 (CH₂), 44.58 (CH₂),

46.97 (CH₂), 51.80 (CH₂), 53.92 (CH₂), 109.16 (C_4ꢀpz), 114.37

(C_TFA), 119.14 (C_aromatic), 120.48 (C_aromatic), 128.14 (C_aromatic),

141.87 (C_3/5-pz), 142.51 (C_aromatic), 148.02 (C_3/5-pz)), 160.35

(C_TFA), 169.53 (CQO), 171.25 (CQO), 172.89 (CQO). HR

ESI-MS (m/z): calcd for [M + H]⁺, 543.37656; found,

543.39389.

CHCl₃–MeOH–NH₄OH (80 : 18 : 2))

=

0.42; ¹H NMR

(CD₃OD): d 1.26 (3H, t, 2CH₃), 1.95 (2H, m, CH₂), 2.50

(2H, t, CH₂), 2.96 (2H, t, CH₂), 3.24 (4H, q, 2 CH₂), 3.32

(2H, m, CH₂), 3.68 (2H, m, CH₂), 7.62 (2H, m, H_aromatic), 7.75

(2H, m, H_aromatic).

tert-Butyl N-(2-{[(tert-butoxy)carbonyl]({2-[4-({[2-(diethyl-

amino)ethyl]carbamoyl}methyl)-3,5-dimethylpyrazol-1-yl]ethyl})-

amino}ethyl)carbamate (17). To a solution of 15 (400 mg,

0.67 mmol) in dry DMF was added N,N-diethylethylene-

diamine (103 mL; 0.73 mmol) and N,N-diisopropylethylamine

(DIPEA) (183 mL; 1.05 mmol). The reaction mixture was

stirred at room temperature for 72 h. After removal of the

solvent, 17 (340 mg, 91%) was puriﬁed by silica-gel column

chromatography with CHCl₃–MeOH–NH₄OH (88/10 : 2) as

the eluent. R_f(SiO₂and CHCl₃–MeOH–NH₄OH (88/10 : 2)) =

Synthesis of pyrazolyl-amino carboxylic ligands (L⁷and L⁸)

functionalized with melanin-avid pharmacophores at the

4-position of the azolyl ring

Benzyl 3-acetyl-4-oxopentanoate (20). To a suspension of

NaH (3.16 g, 79 mmol) in dry THF (85 mL) was added

dropwise, at

0 1C, freshly distilled pentane-2,4-dione

(7.50 mL, 72 mmol). The resulting suspension was stirred at

0 1C for 1 h; then, benzyl bromoacetate (1.36 mL, 72 mmol)

was added dropwise at the same temperature. The reaction

mixture was stirred for 3 h at 0 1C, and overnight at room

temperature. After this time, the mixture was treated with

1

0.59; H NMR (CDCl₃): d 0.86 (6H, m, CH₃), 1.34 (18H, m,

(CH₃)), 2.10 (6H, m, 2CH₃-pz), 2.51 (8H, m, 4 CH₂), 3.24

(6H, m, 3 CH₂), 3.49 (2H, m, CH₂), 4.09 (2H, m, CH₂).

c

2574 New J. Chem., 2010, 34, 2564–2578 This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientiﬁque 2010

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aqueous 2 M HCl (383 mL) and then extracted with diethyl

ether. The organic phase was separated, dried over MgSO₄,

ﬁltered and the solvent removed under vacuum, giving 20

(16.0 g, 90%) as a yellow oil. ¹H NMR (CDCl₃): d 2.11 (3H, s,

CH₃–CQO enol), 2.14 (3H, s, CH₃–CQO, enol), 2.23 (6 H, s,

2 CH₃–CQO keto), 2.91 (2H, d, CH₂COOEt, enol), 3.28 (2H,

d, CH₂COOEt, keto), 5.09 (4H, m, COOCH_2,enol + keto),

7.32 (10H, m, H_aromatic, keto + enol).

for 24 h. The puriﬁcation of 25 (108 mg, 34%) was performed

using silica-gel column chromatography using a gradient

elution from CHCl₃–MeOH (80 : 18) to CHCl₃–MeOH–

NH₄OH (80 : 18 : 2). R_f(SiO₂and CHCl₃–MeOH–NH₄OH

1

(85 : 13 : 2)) = 0.53; H NMR (CDCl₃): d 0.79 (6H, t, CH₃),

1.16 (3H, t, CH₃), 2.06 (3H, s, CH₃), 2.09 (3H, s, CH₃), 2.35

(6H, m, CH₂), 2.92 (2H, t, CH₂), 3.12 (2H, t, CH₂), 3.19 (2H, s,

CH₂), 3.29 (2H, s, CH₂), 4.01 (2H, t, CH₂); 4.08 (2H, q, CH₂);

6.16 (1H, s, NH).

Benzyl 2-(1-(2-hydroxyethyl)-3,5-dimethylpyrazol-1-yl)acetate

(21). To a solution of 20 (9.00 g, 36 mmol) in ethanol (70 mL)

was added dropwise, at 0 1C, a solution of 2-hydroxyethyl-

hydrazine (2.46 mL, 36 mmol) in ethanol (30 mL). The

reaction mixture was stirred overnight at room temperature

and, after this time, the solvent was removed under vacuum

aﬀording 21 (5.0 g, 98%) as an orange solid. ¹H NMR

(CDCl₃): d 2.17 (6H, s, 2 CH₃-pz), 3.37 (2H, s, CH₂), 3.92

(2H, t, CH₂), 4.01 (2H, m, CH₂), 5.09 (2H, s, CH₂), 7.31

(5H, m, H_aromatic).

Ethyl 2-(2-(4-(2-(4-(4-(2-(diethylamino)ethylcarbamoyl)phenyl-

amino)-4-oxobutylamino)-2-(oxoethyl)-3,5-dimethylpyrazol-1-yl)-

ethylamino)acetate (26). Compound 26 was prepared as above

described for 2, starting from 24 (450 mg, 1.60 mmol) and 16

(509 mg, 1.60 mmol) and by reﬂuxing the reaction mixture for

48 h. The puriﬁcation of 25 (100 mg, 10%) was performed

using silica-gel column chromatography using a gradient

elution from CHCl₃–MeOH–NH₄OH (90 : 8 : 2) to MeOH–

NH₄OH (98 : 2)). R_f(SiO₂and CHCl₃–MeOH–NH₄OH

1

(85 : 13 : 2)) = 0.63; H NMR (CDCl₃): d 1.16 (9H, t, CH₃),

Benzyl 2-(1-(2-bromoethyl)-3,5-dimethylpyrazol-1-yl)acetate

(22). To a solution of 21 (9.527 g, 36 mmol) in dry THF

(70 mL) was added, at 0 1C, carbon tetrabromide (21.89 g,

66 mmol) and a solution of triphenylphosphine (19.04 g,

73 mmol) in dry THF (70 mL). The temperature was slowly

raised and the reaction mixture was stirred overnight at room

temperature. The solvent was evaporated to dryness and the

residue was puriﬁed by silica-gel column chromatography with

CH₂Cl₂–MeOH (95 : 5) as eluent. Compound 22 (3.235 g,

30%) was obtained as a transparent oil, after removal of the

solvent from the collected fractions. ¹H NMR (CDCl₃): d 2.15

(3H, s, CH₃-pz), 2.18 (3H, s, CH₃-pz), 3.37 (2H, s, CH₂), 3.63

(2H, t, CH₂), 4.29 (2H, m, CH₂), 5.08 (2H, s, CH₂), 7.31

(5H, m, H_aromatic).

1.78 (2H, m, CH₂), 2.11 (3H, s, CH₃-pz), 2.14 (3H, s, CH₃-pz),

2.32 (2H, m, CH₂), 2.94 (8H, m, CH₂), 3.31 (4H, m, CH₂), 3.61

(4H, m, CH₂), 4.09 (4H, m, CH₂), 7.71 (4H, m, H_aromatic).

2-(2-(4-(2-(2-(diethylamino)ethylamino)-2-oxoethyl)-3,5-dimethyl-

pyrazol-1-yl)ethylamino)acetic acid (L⁷). L⁷(100 mg, 98%) was

prepared and recovered in the form of a yellow oil as above

described for L³, starting from 108 mg (0.28 mmol) of 25. IR

u

_max(KBr)/cm^ꢀ11590, 1664 (CQO); ¹H NMR (CD₃OD):

d 1.31 (6H, t, CH₃), 2.13 (3H, s, CH₃), 2.24 (3H, s, CH₃),

3.22 (6H, m, CH₂), 3.38 (2H, s, CH₂), 3.47 (2H, t, CH₂), 3.55

(2H, s, CH₂), 3.59 (2H, s, CH₂), 4.37 (2H, t, CH₂); ¹³C NMR

(CD₃OD): d 8.11 (CH₃), 8.51 (CH₃-pz), 10.80 (CH₃-pz), 30.32

(CH₂), 34.79 (CH₂), 44.37 (CH₂), 47.04 (CH₂), 48.01 (CH₂),

49.38 (CH₂), 51.40 (CH₂), 110.27 (C₄-pz), 139.20 (C_3/5-pz),

148.10 (C_3/5-pz), 169.85 (CQO), 173.66 (CQO). HR ESI-MS

(m/z): calcd for [M + H]⁺, 354.24884; found, 354.24885.

Benzyl 2-(1-(2-(2-ethoxy-2-oxoethylamino)ethyl)-3,5-dimethyl-

pyrazol-1-yl)acetate (23). To a solution of 22 (1.632 g,

5.25 mmol) in dry CH₃CN (35 mL) was added glycine ethyl

ester (1.100 g, 7.88 mmol), NEt₃(1.47 mL, 10.50 mmol) and

KI. The resulting mixture was stirred overnight. After removal

of the solvent, compound 23 (900 mg, 46%) was recovered as

an yellow oil upon puriﬁcation by silica-gel column chromato-

graphy with CH₂Cl₂–MeOH (95 : 5) as eluent. R_f(SiO₂and

CH₂Cl₂–MeOH (95 : 5)) = 0.38; ¹H NMR (CDCl₃): d 1.26

(3H, t, CH₃), 2.18 (3H, s, CH₃), 2.19 (3H, s, CH₃), 3.02 (2H, t,

CH₂), 3.38 (2H, t, CH₂), 3.39 (2H, t, CH₂), 4.07 (2H, t, CH₂),

4.16 (2H, q, CH₂), 5.12 (2H, s, CH₂), 7.34 (5H, m, H_aromatic).

Ethyl

2-(2-(4-(2-(4-(4-(2-(diethylamino)ethylamino)ethyl)-

phenylamino)-4-oxobutylamino)(2-oxoethyl)3,5-dimethypyrazol-

1-yl)ethyl)amino)acetic acid (L⁸). L⁸was synthesized as

described above for L³, starting from 100 mg (0.17 mmol) of

26. L⁸(40 mg, 42%) was recovered as a yellow oil after

puriﬁcation by silica-gel column chromatography with

gradient elution from CHCl₃–MeOH–NH₄OH (75 : 13 : 2) to

MeOH–NH₄OH (98 : 2). R_f(SiO₂and CHCl₃–MeOH–

NH₄OH (75 : 13 : 2)) = 0.42; IR u_max(KBr)/cm^ꢀ11586, 1652,

1687 (CQO); ESI/MS (+) (m/z): 558.2 [M + H]⁺; ¹H NMR

(CD₃OD): d 1.35 (6H, t, CH₃), 1.88 (2H, m, CH₂), 2.13 (3H, s,

CH₃-pz), 2.22 (3H, s, CH₃-pz), 2.44 (2H, m, CH₂), 2.42

(2H, m, CH₂), 3.32 (12H, m, 6 CH₂), 3.64 (2H, m, CH₂),

3.78 (2H, m, CH₂), 4.33 (2H, m, CH₂), 7.68 (2H, d, H_aromatic),

7.84 (2H, d, H_aromatic); ¹³C NMR (CDCl₃): d 8.02 (CH₃), 8.33

(CH₃-pz), 10.76 (CH₃-pz), 25.04 (CH₂), 30.27 (CH₂), 34.16

(CH₂), 35.23 (CH₂), 38.99 (CH₂), 44.01 (CH₂), 47.08 (CH₂),

47.90 (CH₂), 48.41 (CH₂), 51.73 (CH₂), 110.74 (C₄-pz), 119.21

(C_aromatic), 128.31 (C_aromatic), 139.00 (C_aromatic), 139.47

(C_3/5-pz), 142.45 (C_aromatic), 147.98 (C_3/5-pz), 169.37 (CQO),

169.67 (CQO), 172.80 (CQO), 173.00 (CQO). HR ESI-MS

(m/z): calcd for [M + H]⁺, 354.24884; found, 354.24885.

2-(1-(2-(2-Ethoxy-2-oxoethylamino)ethyl)-3,5-dimethylpyrazol-

1-yl)acetic acid (24). Compound 24 was synthesized and

recovered as above described for 7, starting from 900 mg

1

(2.42 mmol) of 23 (630 mg, 92%). H NMR (CDCl₃): d 1.24

(3H, t, CH₃), 2.03 (3H, s, CH₃), 2.13 (3H, s, CH₃), 3.24 (2H, t,

CH₂), 3.28 (2H, t, CH₂), 3.79 (2H, s, CH₂), 4.14 (2H, q, CH₂),

4.22 (2H, m, CH₂).

Ethyl 2-(2-(4-(2-(2-(diethylamino)ethylamino)-2-oxoethyl)-

3,5-dimethylpyrazol-1-yl)ethylamino)acetate (25). Compound

25 was prepared as above described for 2, starting from 24

(238 mg; 0.84 mmol) and N,N-diethylethylenediamine

(130 mL, 0.92 mmol) and by reﬂuxing the reaction mixture

c

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General procedure for the synthesis of the Re complexes (Re1–Re8)

(CRO), 196.97 (CRO). HR ESI-MS (m/z): calcd for

[M + H]⁺, 652.21637; found, 652.21636.

[Re(H₂O)₃(CO)₃]Br was reacted with equimolar amounts of

L¹–L⁸in reﬂuxing methanol for 18 h. After this time, the

solvent was removed under vacuum and the desired complexes

were puriﬁed by washing with organic solvents or water, by

column chromatography, recrystallization or by RP-HPLC. In

the case of RP-HPLC, the puriﬁcation was achieved using a

preparative Waters m Bondapak C18 (150 ꢂ 19 mm) with a

ﬂow rate of 5.0 mL min^ꢀ1and the method described above.

fac-[Re(CO)₃(k³-L⁴)] (Re4). Re4 (60 mg, 71%) was puriﬁed

by silica-gel column chromatography using CHCl₃–MeOH–

NH₄OH (80 : 18 : 2) as eluent. R_f(SiO₂and CHCl₃–MeOH–

NH₄OH (80 : 18 : 2)) = 0.73; IR u_max(KBr)/cm^ꢀ11653, 1687,

1865 (CQO), 1909, 2024 (CRO); ¹H NMR (CD₃OD): d 1.30

(6H, t, CH₃); 2.23 (2H, m, CH₂), 2.33 (3H, s, CH₃-pz), 2.46

(3H, s, CH₃-pz), 2.53 (3H, m, CH₂), 3.24 (2H, m, CH₂), 3.34

(2H, m, CH₂), 3.37–3.75 (9H, m, CH₂), 4.27 (1H, m, CH₂);

4.49 (1H, m, CH₂), 6.13 (1H, s, H₄-pz); 7.72 (2H, d, C_aromatic),

7.85 (2H, d, C_aromatic); ¹³C NMR (CD₃OD): d 9.23 (CH₃),

11.38 (CH₃-pz), 15.84 (CH₃-pz), 20.44 (CH₂), 34.27 (CH₂),

36.46 (CH₂), 46.76 (CH₂), 49.11 (CH₂), 53.06 (CH₂), 56.56

(CH₂), 63.80 (CH₂), 66.29 (CH₂), 108.79 (C₄-pz), 120.36

(C_aromatic), 129.33 (C_aromatic), 129.48 (C_aromatic), 143.6

(C_aromatic), 143.60 (C_3/5-pz), 145.12 (C_3/5-pz), 170.59 (CQO),

173.05 (CQO), 181.69 (CQO), 195.23 (CRO), 196.27

(CRO), 196.94 (CRO). HR ESI-MS (m/z): calcd for

[M + H]⁺, 771.25397; found, 771.25395.

fac-[Re(CO)₃(k³-L¹)](CF₃COO) (Re1). Re1 (36 mg, 45%)

was puriﬁed by recrystallization from MeOH–diethyl ether at

0 1C. IR u_max(KBr)/cm^ꢀ11735 (CQO), 1931, 2028 (CRO);

¹H NMR (CD₃OD): d 1.34 (6H, t, CH₃), 2.05 (1H, m, CH₂),

2.20 (1H, m, CH₂), 2.37 (3H, s, CH₃-pz), 2.41 (2H, t, CH₂);

2.44 (3H, s, CH₃-pz), 2.58 (1H, m, CH₂), 2.72 (1H, m, CH),

2.90 (2H, m, CH₂), 3.17 (1H, m, CH₂), 3.31 (8H, m, CH₂), 3.41

(1H, m, CH₂), 3.60 (3H, m, CH₂), 3.72 (1H, m, CH₂); 4.10

(1H, b, NH₂); 4.21 (1H, m, CH₂), 4.56 (1H, m, CH₂), 5.54

(1H, b, NH), 6.19 (1H, s, H₄-pz); ¹³C NMR (CDCl₃): 9.23

(2CH₃), 11.62 (CH₃-pz), 16.10 (CH₃-pz), 21.01 (CH₂), 33.33

(CH₂), 35.77 (CH₂), 43.72 (CH₂), 48.97 (CH₂), 49.83 (CH₂),

52.57 (CH₂), 53.88 (CH₂), 62.55 (CH₂), 67.30 (CH₂), 109.20

(C₄-pz), 119.87 (C_TFA), 145.36 (C_3/5-pz), 155.10 (C_3/5-pz),

165.35 (C_TFA), 175.90 (CQO), 193.72 (CRO), 194.90

(CRO), 195.27 (CRO). HR ESI-MS (m/z): calcd for M⁺,

637.25072; found, 637.27338.

fac-[Re(CO)₃(k³-L⁵)](CF₃COO) (Re5). Re5 (12 mg, 20%)

was puriﬁed by RP-HPLC, as described above. IR u_max(KBr)/

cm^ꢀ1: 1696 (CQO), 1923, 2026 (CRO); ¹H NMR (CD₃OD):

d 1.28 (6H, t, 2 CH₃); 2.31 (3H, s, CH₃); 2.34 (1H, m, CH);

2.39 (3H, s, CH₃); 2.55 (4H, m, 2 CH₂); 2.87 (2H, s, CH₂); 3.24

(2H, m, CH₂); 3.46 (2H, s, CH₂); 3.58 (4H, q, 2CH₂); 3.89

(1H, m, CH); 4.11 (1H, m, CH); 4.51 (1H, m, CH); ¹³C NMR

(CDCl₃): d 7.92 (CH₃), 9.13 (CH₃-pz), 13.27 (CH₃-pz), 29.82

(CH₂), 34.69 (CH₂); 41.97 (CH₂), 46.98 (CH₂), 47.55 (CH₂),

47.83 (CH₂), 48.80 (CH₂), 51.00 (CH₂), 54.69 (CH₂), 112.39

(C₄-pz), 142.68 (C_3/5-pz), 152.44 (C_3/5-pz), 173.0 (CQO),

195.77 (CRO), 197.00 (CRO), 197.80 (CRO). HR ESI-MS

(m/z): calcd for M⁺, 609.21941; found, 609.24015.

fac-[Re(CO)₃(k³-L²)](CF₃COO) (Re2). Re2 (249 mg, 67%)

was puriﬁed by washing of the crude with CH₂Cl₂and with a

mixture of n-hexane–diethyl ether. IR u_max(KBr)/cm^ꢀ11667,

1701 (CQO), 1942, 2017 (CRO); ¹H NMR (CD₃OD): d 1.34

(6H, t, CH₃), 2.12 (2H, m, CH₂), 2.25 (2H, m, CH₂), 2.37

(3H, s, CH₃), 2.53 (3H, s, CH₃), 2.67 (3H, m, CH₂), 2.76

(1H, m, CH₂), 2.89 (2H, m, CH₂), 3.19 (1H, m, CH), 3.39

(4H, q, CH₂), 3.54 (2H, m, CH₂), 3.67–3.75 (3H, m, CH₂), 3.92

(1H, m, NH), 4.22 (1H, m, CH), 4.54 (1H, m, CH), 5.53

(1H, m, NH), 6.20 (1H, s, H₄-pz), 7.71–7.87 (4H, m, Ar);

¹³C NMR (CD₃OD): 9.1 (CH₃), 11.5 (CH₃), 13.1 (CH₃), 16.0

(CH₂), 20.8 (CH₂), 34.2 (CH₂), 36.4 (CH₂), 43.7 (CH₂), 53.0

(CH₂), 53.8 (CH₂), 62.4 (CH₂), 67.2 (CH₂), 109.2 (C₄-pz),

116.3 (C_aromatic), 120.3 (C_aromatic), 120.67 (C_TFA), 129.4

(C_aromatic), 143.6 (C_3/5-pz), 145.3 (C_aromatic), 155.1 (C_3/5-pz),

165.78 (C_TFA), 170.6 (CQO), 174.6 (CQO), 190.72 (CRO),

fac-[Re(CO)₃(k³-L⁶)](CF₃COO) (Re6). Re6 (150 mg, 98%)

was puriﬁed by washing of the crude with CH₂Cl₂and with a

mixture of n-hexane–diethyl ether. IR u_max(KBr)/cm^ꢀ11677,

1

1694 (CQO) 1923, 2032 (CRO); H NMR (CD₃OD): d 1.21

(6H, t, CH₃), 1.81 (2H, m, CH₂), 2.16 (3H, s, CH₃-pz), 2.25

(3H, s, CH₃-pz), 2.32 (2H, m, CH₂), 2.42 (CH₂), 2.74 (2H, m,

CH₂), 3.26 (10H, m, 5 CH₂), 3.64 (2H, m, CH₂), 3.98 (2H, m,

CH₂), 3.97 (1H, m, CH₂), 4.41 (1H, m, CH₂), 7.59 (2H, d,

H

_aromatic), 7.71 (2H, d, H_aromatic); ¹³C NMR (CDCl₃): d 8.03

193.67 (CRO), 195.32 (CRO). ESI/MS (+) (m/z): 378.5 [M]²⁺

.

(CH₃), 9.22 (CH₃-pz), 13.33 (CH₃-pz), 25.16 (CH₂), 30.13

(CH₂), 34.15 (CH₂), 35.19 (CH₂), 38.95 (CH₂), 42.09 (CH₂),

47.05 (CH₂), 47.90 (CH₂), 48.80 (CH₂), 51.85 (CH₂), 54.70

(CH₂), 112.85 (C₄-pz), 119.11 (C_aromatic), 128.11 (C_aromatic),

128.29 (C_aromatic), 142.52 (C_3/5-pz), 142.57 (C_aromatic), 152.32

(C_3/5-pz), 169.46 (CQO), 171.85 (CQO), 172.88 (CQO),

192.97 (CRO), 193.44 (CRO), 194.17 (CRO). HR ESI-MS

(m/z): calcd for M⁺, 813.30936; found, 813.34603.

fac-[Re(CO)₃(k³-L³)] (Re3). Re3 (60 mg, 71%) was puriﬁed

by washing of the crude with distilled water. IR u_max(KBr)/cm^ꢀ1

1629, 1845 (CQO), 1971, 2020 (CRO); ESI/MS (+) (m/z):

1

651.9 [M + H]⁺; H NMR (CD₃OD): d 1.37 (6H, t, 2CH₃);

2.12 (2H, m, CH₂); 2.36 (3H, s, CH₃-pz); 2.43 (2H, m, CH₂);

2.48 (3H, s, CH₃-pz); 2.56 (1H, m, CH₂); 3.43 (4H, m, CH₂);

3.53 (7H, m, CH₂); 3.71 (1H, s, CH₂); 3.73 (1H, s, CH₂); 4.30

(1H, m, CH₂); 4.48 (1H, m, CH₂); 6.16 (1H, s, H₄-pz);

¹³C NMR (CD₃OD): d 9.27 (CH₃), 11.46 (CH₃-pz), 15.86

(CH₃-pz), 20.51 (CH₂), 33.19 (CH₂), 35.80 (CH₂), 46.80 (CH₂),

48.15 (CH₂), 52.66 (CH₂), 56.64 (CH₂), 63.80 (CH₂), 66.31

(CH₂), 108.79 (C(H₄-pz), 145.13 (C_3/5-pz), 155.17 (C_3/5-pz)),

175.89 (CQO), 181.65 (CQO), 195.24 (CRO), 196.40

fac-[Re(CO)₃(k³-L⁷)] (Re7). Re7 (18 mg, 24%) was puriﬁed

by silica-gel column chromatography using CHCl₃–MeOH–

NH₄OH (75 : 23 : 2) as eluent. R_f(SiO₂and CHCl₃–MeOH–

NH₄OH (80 : 18 : 2)) = 0.65; IR u_max(KBr)/cm^ꢀ11665, 1870

(CQO), 1919, 2023 (CRO); ¹H NMR (CD₃OD): d 1.28

(6H, t, 2 CH₃), 2.32 (3H, s, CH₃), 2.39 (1H, m, CH), 2.45

c

2576 New J. Chem., 2010, 34, 2564–2578 This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientiﬁque 2010

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(3H, s, CH₃), 3.20 (6H, m, CH₂), 3.35 (2H, s, CH₂), 3.45

(2H, s, CH₂), 3.57 (2H, t, CH₂), 3.66 (1H, m, CH₂); 4.17

(1H, m, CH₂); 4.47 (1H, m, CH₂); ¹³C NMR (CDCl₃): d 10.18

(CH₃), 11.11 (CH₃-pz), 13.19 (CH₃-pz), 31.24 (CH₂), 36.30

(CH₂), 47.47 (CH₂), 49.40 (CH₂), 50.45 (CH₂), 52.61 (CH₂),

56.24 (CH₂), 113.24 (C₄-pz), 143.62 (C_3/5-pz), 153.70 (C_3/5-pz),

174.18 (CQO), 182.82 (CQO), 195.33 (CRO), 195.88

(CRO), 196.35 (CRO). HR ESI-MS (m/z): calcd for

[M + H]⁺, 624.18311; found, 624.18313.

Cell uptake studies

Cell culture. B16F1 murine melanoma cells (ECACC, UK)

were grown in DMEM containing GlutaMax I supplemented

with 10% heat-inactivated fetal bovine serum and 1%

penicillin–streptomycin antibitiotic solution (all from Gibco,

Alfagene, Lisbon). Cells were cultured in a humidiﬁed

atmosphere of 95% air and 5% CO₂at 37 1C (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.

fac-[Re(CO)₃(k³-L⁸)] (Re8). Re8 (26 mg, 56%) was puriﬁed

by silica-gel column chromatography using CHCl₃–MeOH–

NH₄OH (80 : 18 : 2) as eluent. R_f(SiO₂and CHCl₃–MeOH–

NH₄OH (80 : 18 : 2)) = 0.73; IR u_max(KBr)/cm^ꢀ11648, 1692,

1877 (CQO), 1945, 2019 (CRO); ¹H NMR (CD₃OD): d 1.17

(6H, t, CH₃), 1.87 (2H, m, CH₂), 2.26 (3H, s, CH₃-pz), 2.35

(2H, m, CH₂), 2.41 (3H, s, CH₃-pz), 2.89 (6H, m, CH₂), 3.33

(6H, m, CH₂); 3.61 (4H, m, CH₂), 4.11 (1H, m, CH₂), 4.48

(1H, m, CH₂), 7.67 (2H, d, H_aromatic), 7.80 (2H, d, H_aromatic);

¹³C NMR (CDCl₃): d 8.97 (CH₃), 9.42 (CH₃-pz), 12.76

(CH₃-pz), 25.13 (CH₂), 30.17 (CH₂), 34.03 (CH₂), 36.32

(CH₂), 38.93 (CH₂), 47.37 (CH₂), 49.09 (CH₂), 50.93 (CH₂),

51.57 (CH₂), 55.08 (CH₂), 112.48 (C₄-pz), 119.11 (C_aromatic),

128.09 (C_aromatic), 128.81 (C_aromatic), 142.30 (C_3/5-pz), 142.40

(C_aromatic), 152.46 (C_3/5-pz), 168.83 (CQO), 171.83 (CQO),

172.80 (CQO), 181.65 (CQO), 194.20 (CRO), 195.18

Cellular uptake studies. Cellular uptake assays of the ^99mTc

complexes (Tc1–Tc8) were performed in B16F1 murine

melanoma cells seeded at a density of 0.2 million per 0.5 mL

culture medium per well in 24-well tissue culture plates and

allowed to attach overnight. After that period, the medium

was removed and replaced by fresh medium containing

approximately 2 ꢂ 10⁵cpm per 0.5 mL of each ^99mTc complex.

The cells were incubated again under humidiﬁed 5% CO₂

atmosphere, at 37 1C for a period of 15 min to 4 h. After 0.25,

0.5, 1, 2, 3 and 4 h incubation period the cells were washed

twice with cold PBS, lysed with 0.1 M NaOH and the cellular

extracts were counted for radioactivity. Each experiment was

performed in quadruplicate. Cellular uptake data were

expressed as an average value plus the standard deviation.

(CRO). HR ESI-MS (m/z): calcd for [M

827.27686; found, 827.27782.

+

H]⁺,

In vivo evaluation of the ^99mTc complexes. All animal

experiments were performed in compliance with Portuguese

regulations for animal treatment. The animals were housed in

a temperature- and humidity-controlled room with a 12 h

light–12 h dark schedule.

Synthesis and in vitro evaluation of the ^99mTc(I) complexes

Synthesis of Tc1–Tc8. General method. In a nitrogen-purged

glass vial, 40 mL of a 4.4 ꢂ 10^ꢀ5M aqueous solution of L¹–L⁸

Biodistribution studies. Biodistribution of the radioconjugates

was evaluated in melanoma-bearing C57BL/6 female mice

(8–10 weeks old). Mice were previously implanted subcuta-

neously with 1 ꢂ 10⁶B16F1 cells to generate a primary skin

melanoma. Ten to 12 days after the inoculation, tumors

reached a weight of 0.2–1 g. Animals were intravenously

injected into the retro-orbital sinus with complexes Tc1–Tc8

(3–10 MBq) diluted in 100 mL of PBS pH 7.2. Mice were killed

by cervical dislocation at 1, 4, and 24 h after injection. The

dose administered and the radioactivity in the killed animals

was measured using a dose calibrator (Curiemeter IGC-3,

Aloka, Tokyo, Japan or Carpintec CRC-15W, Ramsey,

USA). The diﬀerence between the radioactivity in the injected

and sacriﬁced animals was assumed to be due to excretion.

Tumors and normal tissues of interest were dissected, rinsed to

remove excess blood, weighed, and their radioactivity was

measured using a g-counter (LB2111, Berthold, Germany).

The uptake in the tumor and healthy tissues was calculated

and expressed as a percentage of the injected radioactivity dose

per gram of tissue. For blood, bone, muscle, and skin, total

activity was estimated assuming that these organs constitute 6,

10, 40, and 15% of the total body weight, respectively. Urine

was also collected and pooled together at the time the animals

were killed.

was added to 400 mL of

a solution of the organo-

metallic precursor fac-[^99mTc(CO)₃(H₂O)₃]⁺(1–2 mCi) in

saline at pH 7.4 (Tc1, Tc2, Tc5–Tc8) or at pH 5.5

(Tc3, Tc4). The reaction mixture was then heated to 100 1C

for 30 min, cooled to room temperature and the ﬁnal solution

analyzed by RP-HPLC.

Partition coeﬃcient measurements. The log P_o/wvalues of

complexes Tc1–Tc8 (Table 1) were determined by the

‘‘shake ﬂask’’ method under physiological conditions

(n-octanol–0.1 M PBS, pH 7.4).³⁶

In vitro binding to melanin. The binding aﬃnity to melanin

of Tc1–Tc8 was assessed using synthetic tyrosine–melanin

(Sigma). The general procedure used was as follows: A

100 mL aliquot of the radioactive preparations of Tc1–Tc8

was added to a melanin suspension (0.5 mg/10 ml) in distilled

water. The reaction mixture was incubated at room tempera-

ture for 1 h with stirring. After incubation, the tubes

were centrifuged at 30 000 g for 10 min, and aliquots of the

supernatant were counted in the gamma counter. Control

tubes were also used containing the radioactive preparation

without melanin. The diﬀerence between the activity of

aliquots from the supernatants of the test tubes (with melanin)

and the control tubes (without melanin) allowed the

calculation of the percentage of unbound complexes.

Metabolism studies. The in vivo stability of Tc1–Tc8 was

evaluated by urine and murine serum HPLC analysis, using an

c

This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientiﬁque 2010 New J. Chem., 2010, 34, 2564–2578 2577

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analytic Macherey-Nagel C18 reversed-phase column

(Nucleosil 100–10, 250 ꢂ 3 mm) with a ﬂow rate of

1 mL min^ꢀ1and the method described above. The urine was

collected at the time of sacriﬁce and ﬁltered through a Millex

GV ﬁlter (0.22 mm) before RP-HPLC analysis. Blood collected

from mice was immediately centrifuged for 15 min at 3000 rpm

at 4 1C, and the serum was separated. Aliquots of 100 mL of

serum were treated with 200 mL of ethanol to precipitate the

proteins. Samples were centrifuged at 4000 rpm for 15 min,

at 4 1C. Supernatant was collected and passed through a

Millex GV ﬁlter (0.22 mm) prior to RP-HPLC analysis.

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Acknowledgements

The authors would like to acknowledge the Fundac¸ ao para a

Ciencia e Tecnologia (FCT) for ﬁnancial support (PPCDT/

QUI/59588/2004). Carolina Moura thanks the FCT for a

Doctoral research grant (SFRH/BD/38469/2007). J. Marc¸ alo

is acknowledged for the ESI-MS analyses which were run on a

QITMS instrument acquired with the support of the Programa

Nacional de Reequipamento Cientıﬁco (Contract REDE/

´

1503/REM/2005 - ITN) of FCT and as part of RNEM – Rede

Nacional de Espectrometria de Massa. We also acknowledge

Pedro Vaz e Paulo Madeira for the HR-ESI-MS which

were run on a ApexQe FTICR acquired with the support of

22 R. Alberto, R. Schibli, A. Egli, A. P. Schubiger, U. Abram and

T. A. Kaden, J. Am. Chem. Soc., 1998, 120, 7987–7988.

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D. Can, P. Schmutz, B. Spingler and R. Alberto, Nucl. Med. Biol.,

2010, 37, 255–264.

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2578 New J. Chem., 2010, 34, 2564–2578 This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientiﬁque 2010

Article Doi

Synthesis, characterization and biological evaluation of tricarbonyl M(i) (M = Re, 99mTc) complexes functionalized with melanin-binding pharmacophores

DOI: 10.1039/c0nj00256a

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