Synthesis of a novel 'smart' bifunctional chelating agent 1-(2-[β,d-galactopyranosyloxy]ethyl)-7-(1-carboxy-3-[4-aminophenyl]propyl)-4,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (Gal-PA-DO3A-NH2) and its Gd(III) complex

Article abstract of DOI:10.1016/j.bmc.2007.05.004

A new synthetic pathway to 1-(2-[β,d-galactopyranosyloxy]ethyl)-7-(1-carboxy-3-[4-aminophenyl]propyl)-4,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (Gal-PA-DO3A-NH₂) and 1-(2-[β,d-galactopyranosyloxy]ethyl)-4,7,10-tris(carboxymethyl)-1, 4,7,10-tetraazacyclododecane (Gal-DO3A) chelating agents was developed involving full hydroxyl- and carboxyl-group protection in precursors to product. Two sequences of cyclen-N-functionalisation were subsequently investigated, one successfully, towards synthesis of the novel 'smart' bifunctional Gal-PA-DO3A-NH₂ chelate. The longitudinal proton relaxivities of the neutral [Gd-(Gal-PA-DO3A-NH₂)] and [Gd-(Gal-DO3A)] complexes were increased by 28% and 37% in the presence of β-galactosidase, respectively.

Full text of DOI:10.1016/j.bmc.2007.05.004

Bioorganic & Medicinal Chemistry 15 (2007) 4714–4721
Synthesis of a novel ‘smart’ bifunctional chelating agent
1-(2-[b,D-galactopyranosyloxy]ethyl)-7-(1-carboxy-3-[4-
aminophenyl]propyl)-4,10-bis(carboxymethyl)-1,4,7,10-
tetraazacyclododecane (Gal-PA-DO3A-NH2) and its
Gd(III) complex
Nick J. Wardle,a Amy H. Herlihy,b Po-Wah So,b Jimmy D. Bellb and S. W. Annie Bligha,*
aInstitute for Health Research and Policy, Tower Building, London Metropolitan University, 166-220 Holloway Rd.,
London N7 8DB, UK
bMolecular Imaging Group, Imaging Sciences Department, MRC Clinical Sciences Centre, Imperial College London,
Hammersmith Hospital Campus, London, UK
Received 2 December 2006; revised 20 April 2007; accepted 2 May 2007
Available online 6 May 2007
Abstract—A new synthetic pathway to 1-(2-[b,D-galactopyranosyloxy]ethyl)-7-(1-carboxy-3-[4-aminophenyl]propyl)-4,10-bis(carb-
oxymethyl)-1,4,7,10-tetraazacyclododecane (Gal-PA-DO3A-NH2) and 1-(2-[b,D-galactopyranosyloxy]ethyl)-4,7,10-tris(carboxy-
methyl)-1, 4,7,10-tetraazacyclododecane (Gal-DO3A) chelating agents was developed involving full hydroxyl- and carboxyl-
group protection in precursors to product. Two sequences of cyclen-N-functionalisation were subsequently investigated, one suc-
cessfully, towards synthesis of the novel ‘smart’ bifunctional Gal-PA-DO3A-NH2 chelate. The longitudinal proton relaxivities of
the neutral [Gd-(Gal-PA-DO3A-NH2)] and [Gd-(Gal-DO3A)] complexes were increased by 28% and 37% in the presence of b-galac-
tosidase, respectively.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
isothiocyanato)phenyl]propyl)-4,7,10-tris(carboxymeth-
yl)-1,4,7,10-tetraazacyclododecane (PA-DOTA, an
N-functionalised DOTA derivative) have been conju-
gated with monoclonal antibody CC49 and complexed
Since the emergence of paramagnetic contrast agents
(PCA) as clinical aids in magnetic resonance imaging
(MRI) protocols,1–4 approaches to introducing selectiv-
ity of action have fallen into two broad categories; those
generating selective tissue distribution of the PCA by
conjugation to physiologically relevant macromolecules,
and those relying on metabolic PCA-activation to
achieve localisation of differential water relaxation char-
acteristics. The former approaches are exemplified in the
development of bifunctional chelating agents capable of
conjugating macromolecular systems such as human ser-
um albumin5,6 and the membrane translocation signal
peptide ‘Tat’.7,8 Related strategies have also been ap-
plied in the fields of radioimmunoimaging and therapy,
whereby ligands such as 1-(1-carboxy-3-[4-(nitro/amino/
177
with radioisotopes such as Lu.9–12
Meanwhile gadolinium(III) complexes of 1-([b,D-galacto-
pyranosyloxy]alkyl)-4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododecane ([b,D-galactopyranosyloxy]alkyl-
DO3A conjugates, e.g., EGad, EGadMe) have been
developed as substrates to b-galactosidase, an expres-
sion-product of the reporter gene Lac-Z commonly
co-administered intracellularly with the subject-gene of
gene-therapy studies.13–15 Metabolic cleavage of the
galactopyranosyl moiety of EGad/EGadMe unveils a free
coordinate site on the central Gd3+ ion, allowing binding
of water with an increase in relaxivity. While significant
differentials in water-relaxation-time have been obtained
by this method, introduction to the intracellular domain
has thus far been achieved by microinjection alone.
Since intracellular delivery is a prerequisite for the
practical success of this strategy, it is reasoned that
Keywords: DO3A; MRI; Gd(III) complex; Contrast agents.
*
Corresponding author. Tel.: +44 207 133 2142; fax: +44 207 133
2096; e-mail: a.bligh@londonmet.ac.uk
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.004

N. J. Wardle et al. / Bioorg. Med. Chem. 15 (2007) 4714–4721
4715
additional functionalisation of the chelate moiety, facil-
itating appendage to macromolecular carrier systems,
could enhance efficacy of action in vivo, representing
a confluence of the two broad approaches to PCA-de-
sign described above. In this context, investigation
of appropriate synthetic strategies to the bifunctional
conjugate 1-[b,D-galactopyranosyloxy]ethyl)-7-(1-carboxy-
3-[4-aminophenyl]propyl)-4,10-bis(carboxymethyl)-1,4,
7,10-tetraazacyclododecane (Gal-PA-DO3A-NH2), and
the associated Gd(III) complex, was carried out, report-
ing characterisations of species isolated. The
longitudinal proton relaxivities of both Gd(III) com-
plexes of Gal-PA-DO3A-NH2 and Gal-DO3A were
determined in aqueous medium and in the presence of
b-galactosidase.
tem in order to maintain nucleophilic N-function during
these alkylation procedures, characterising also the
tenacity of solvent retention in free amine products. This
latter observation necessitated isolation of protected
conjugate 3 from chromatographed solutes as hydro-
chloride salt (35%) for analytical purpose; achieved
through product-precipitation from strictly anhydrous
Et2O/CH2Cl2 solution (50:4 v/v) via application of dry
HCl gas. Appropriate chromatographic fractions (elu-
ent—CH2Cl2/MeOH 89:11 v/v) of free amine were easily
1
identified for this purpose via H NMR analysis on the
basis of the integral ratios of resonances associated with
tert-butyl and acetyl CH3 groups, respectively. N-(tetra-
acetyl-b,D-galactopyranosyloxy)ethyl-DO3A-tert-butyl
esters 3 were subsequently deprotected to give (b,D-
galactopyranosyloxy)ethyl-DO3A (Gal-DO3A ꢀ56%)
as a hygroscopic solid, through consecutive treatments
with trifluoroacetic acid reagent (TFA, cleaving tert-
Bu groups)24 and NaOMe/MeOH solution under Zem-
plen conditions (cleaving Ac groups).25 The glycosyl
linkage remained intact and no loss of anomeric integ-
rity at C-1 was detected throughout this coupling/depro-
2. Results and discussion
2.1. Ligand synthesis and characterisation
Solubility considerations arising from the presence of
pendant-group aromatic functionality in the targeted
Gal-PA-DO3A-NH2 conjugate, and its precursors,
encouraged investigation of synthetic pathways retain-
ing fully protected DO3A-carboxyl and pyranosyl-hy-
droxyl groups in the developing molecular framework
up to the point of product generation. Such a methodol-
ogy was investigated in the first instance towards a sim-
ple conjugate b,D-galactopyranosyloxy)ethyl-DO3A
(Gal-DO3A, Scheme 1) coupling chelate and sugar moi-
eties as the DO3A-tert butyl ester and galactopyrano-
3
tection process, as indicated by JHH values associated
with anomeric protons; J = 7.7 Hz (3) and J = 7.4 Hz
(Gal-DO3A).
As with approaches reported in pursuit of the bifunc-
tional chelating agent PA-DOTA,12,23,26 mono-alkyl-
ation of free cyclen with the N-functionalisation group
(PA) was envisaged as the first in the sequence of
cyclen-derivatisation steps to the proposed Gal-PA-
DO3A-NH2 conjugate. To this end, the alkyl bromide
of the N-functionalisation pendant group was con-
structed from 4-(4-nitrophenyl)butyric acid (4), through
a-halogenation of the acyl chloride by treatment with
N-bromosuccinimide, and HBr (aq 48%) catalyst.27
Consistent with the formulations of carboxymethyl
groups to be appended subsequently, the pendant-group
carboxyl function was protected as a tert-butyl ester
using tert-butanol, employing the poor nucleophile
N,N-dimethylaniline as the base of choice, generating
side tetraacetate, respectively, prior to
a
final,
deprotection step to product. This constitutes a signifi-
cant departure from the approach adopted during pio-
neering work to generate the latter conjugate,13,15 in
which hydroxyl sites were deprotected on the cyclen-
galactopyranoside adduct, and an aqueous medium
was employed for subsequent cyclen-N-alkylation with
unprotected carboxymethyl groups. The presence of
additional functionality on one carboxymethyl-arm in
the target Gal-PA-DO3A-NH2 subsequently necessi-
tated investigation of various sequences of N-alkylation,
directed primarily by steric factors, and successful mod-
ifications to the approach ascertained in the preliminary
studies concerning the Gal-DO3A are also outlined in
Scheme 1.
tert-butyl
(R,S)-2-bromo-4-(4-nitrophenyl)butanoate
(5) in 42% yield from 4. Mono-alkylation of cyclen
was subsequently achieved in a gently refluxing acetoni-
trile/K2CO3 heterologous system, employing cyclen in
2:1 molar excess of the organobromide, giving 6 in
66% yield.
Working from 2,3,4,6-tetra-O-acetyl-a,D-galactopyrano-
syl bromide (1), bromoethyl-b,D-galactopyranoside tet-
raacetate (2) was isolated anomerically pure in 52%
yield from AgOTf/pyranosyl bromide procedure,
employing the non-nucleophilic base 2,6-di-tert-butyl-
4-methylpyridine16–19 in anhydrous diethyl ether. Orga-
nobromide 2 was subsequently used to functionalise the
4th N-site of DO3A tert-butyl ester20,21 in the initial
investigations, employing K2CO3 (2.1 equiv) base in
DMF solvent at room temperature under strictly anhy-
drous conditions to furnish the corresponding fully pro-
tected (b,D-galactopyranosyloxy)ethyl-DO3A chelate.
The strong propensity for intramolecular hydrogen-
bonding associated with the cyclen ring system22,23
necessitated complete deprotonation of the alicyclic sys-
From this point, both possible sequences of cyclen-N-
derivatisation to Gal-PA-DO3A-NH2 were investigated;
one involving alkylation of two N-sites with carboxym-
ethyl functions and subsequent appendage of the galac-
topyranosyl moiety, and another more felicitous
pathway involving initial linkage of the sugar, followed
by appendage of carboxymethyl functions. In the former
approach, chromatographically inseparable isomeric
mixtures of 1-(PA)-4,7/10-bis(DO3A-tert-butyl ester)-
1,4,7,10-tetraazacyclododecane (7a/b, Scheme 1) were
generated from 6 in exhaustive procedures (i.e., 21–23
days, rt) employing strictly stoichiometric quantities of
tert-butyl bromoacetate in a N,N-dimethylacetamide/
NaOAc (solvent/base) system. Consistent with the
observations of Chappell et al.,12 as structures

4716
N. J. Wardle et al. / Bioorg. Med. Chem. 15 (2007) 4714–4721
Scheme 1. Reagents and conditions: (i) AgOTf/Et2O, ꢁ78 ꢁC; then HO(CH2)nBr; (ii) DO3A-tBu ester/K2CO3 (2.1 equiv)/dry DMF, rt; (iii) TFA, rt;
(iv) NaOMe/MeOH, rt; then Amberlite IR-120 (H+); (v) SOCl2/CCl4, D; (vi) NBS (1.2 equiv)/HBr (aq 48%, cat), D; (vii) PhN(Me)2/tBuOH,
t
0 ꢁC ! rt; (viii) cyclen (2 equiv)/K2CO3 (1.4 equiv)/MeCN, D; (ix) BrCH2CO2 Bu (2 equiv)/NaOAc (2 equiv)/dry DMA, rt, 21–23 days; (x) K2CO3
t
(1.1 equiv)/dry DMF, rt; (xi) BrCH2CO2 Bu (excess)/K2CO3 (excess)/dry DMF, rt; (xii) Pd/C (10%, cat)/aq/H2, 1 atm.
containing one chiral centre, the isomeric mixtures of 7a/
b were readily identifiable in H NMR spectra as multi-
ple (exceeding two) downfield doublets associated with
aromatic protons. In addition, steric congestion
1

N. J. Wardle et al. / Bioorg. Med. Chem. 15 (2007) 4714–4721
4717
precluded subsequent alkylation at the fourth N-site
with bromoethyl-b,D-galactopyranoside tetraacetate 2
(available in minor stoichiometric excess only).
(H2O)2] are 3.59 and 4.82 mMꢁ1 sꢁ1, respectively, in
water which are higher than those of [Gd-(Gal-PA-
DO3A-NH2)] and [Gd-(Gal-DO3A)] indicating that
the number of water molecule coordinating to the
Gd(III) is likely less than one for the latter complexes.
The number of water molecules that are in fast exchange
(q) with the complex [Tb-(Gal-DO3A)] was reported to
be 0.7 by fluorescence study, and the enzymatically
cleaved terbium complex was 1.2.13 These data have
supported the q value of [Gd-(Gal-PA-DO3A-NH2)] is
probably similar to that of [Gd-(Gal-DO3A)].
Meanwhile, alkylation of 6 with bromoethyl-b,D-galac-
topyranoside tetraacetate 2 in minor stoichiometric ex-
cess, using the DMF/K2CO3 system, achieved greater
site-selectivity of alkylation (presumably due to steric
factors), giving chelate 8 which was isolated as the
hydrochloride salt from chromatographic fractions of
free amine (eluent—CH2Cl2/MeOH 9:1 v/v, analysed
1
for tert-Bu and Ac-CH3 integral ratios H NMR spec-
tra). While the presence of two chiral centres in 8 inevi-
2.3. Conclusion
1
tably complicates the H NMR spectrum, broadening
multiplets associated with aromatic protons therein, res-
onance profiles associated with methyl protons of the
tert-butyl (dH = 1.51, 1.55 [1.54sh]) and acetyl groups
(dH = 1.99, 2.05, ꢀ2.11 br m., 2.17; 4· CH3 [Ac], also
CH2Ca [PA]) are consistent with the identification of
hydrochloride 8 as the 1,7-disubstituted cyclen formula-
tion to good isomeric purity. Treatment with large ex-
cess of tert-butyl bromoacetate in an anhydrous DMF/
K2CO3 system over an exhaustive period (15 days, rt)
subsequently yielded 9, which was isolated as the hydro-
chloride salt. Steric congestion in this structure was evi-
denced in the broadening of resonance signatures
A novel neutral Gd(III) complex was prepared with the
capability of conjugating to a peptide and biomacromol-
ecule, and responding to the presence of a specific en-
zyme effecting a change in water relaxation time in
magnetic resonance imaging. The synthetic methodol-
ogy and the characterisation of the cyclen based ligands
with four pendant arms and their precursors were re-
ported in detail.
3. Experimental
3.1. General
1
observed in both H and 13C NMR spectra resulting
from slow conformational interconversion, however
spectral characteristics were comparable to those of 8,
displaying the appropriate modification of tert-butyl
NMR spectra were recorded at ambient temperature on
a Bruker AM-250 or a Bruker Avance-500 spectrometer.
1
1
CH3 resonance integral in the H NMR spectrum, and
All 13C spectra are H broadband decoupled. Chemical
assignments were carried out according to chemical shift
criteria. Consecutive carboxyl and hydroxyl group-
deprotections were carried out as for 3, and catalytic
(Pd/C) hydrogenation in aqueous medium at atmo-
spheric pressure effected transformation of the nitro-
phenyl function to the aniline. This was evidenced in
the upfield shift of (broad) multiplets associated with
shifts (d) are expressed in ppm and J values are given in
Hz. 1H and 13C NMR spectra were referenced internally
to Me4Si apart from those carried out in D2O, where
TSP-d4 was used. Infrared spectra were recorded on a
Bruker Vector 22 FT-IR spectrophotometer, with sam-
ples prepared as 16 mM diameter KBr discs. Mass spec-
trometry was performed using a Kratos Profile HV3
mass spectrometer in liquid secondary ionisation mass
spectrometry mode (employing glycerol as a matrix).
Melting points were obtained on an Electrothermal
Eng. Ltd digital melting point apparatus, and are uncor-
rected. A Carlo Erba 1108 elemental analyser was used
for C, H and N microanalyses. TLC was performed
on pre-coated silica plates (Whatman Al Sil G/UV,
250-lm layer) in the solvent systems stated. Column
1
aromatic protons in the H NMR spectrum of the ana-
lytically homogeneous Gal-PA-DO3A-NH2 ligand, ob-
tained in 20% overall yield for the two deprotection
steps and the catalytic hydrogenation.
2.2. Gadolinium(III) complexes and their longitudinal
proton relaxivities (R1)
Complexations of ligands Gal-PA-DO3A-NH2 and Gal-
DO3A were carried out by refluxing in aqueous suspen-
sions of Gd2(CO3)3 over 24 h, the reaction progress indi-
cated by gradual disappearance of the suspension.
Elemental analyses of the Gd(III) complexes of
Gal-PA-DO3A-NH2 and Gal-DO3A (EGad)13 were
consistent with the anticipated 1:1 Gd(III):ligand stoi-
chiometry forming neutral complexes. Both Gal-PA-
DO3A-NH2 and Gal-DO3A ligands consist of 4-N
(cyclen) and 3-O (carboxylate) donor atoms and one
ethyl-galactose pendant arm. The R1 of the neutral
[Gd-(Gal-PA-DO3A-NH2)] and [Gd-(Gal-DO3A)] com-
plexes were 1.47 and 1.52 mMꢁ1 sꢁ1 in water, respec-
tively, and, 1.89 and 2.09 mMꢁ1 sꢁ1 in the presence of
b-galactosidase, respectively. The increases in relaxivi-
ties were 28% and 37% in the presence of the enzyme.
The R1 of [Gd-(DOTA)(H2O)]ꢁ and [Gd-(DO3A)
chromatography was performed over Aldrich silica gel
˚
(Merck grade 10180, 70–230 mesh 40 A).
0
Reagents were used as purchased. Solvents (DMF, ace-
tonitrile, diethyl ether and methanol) were dried and dis-
tilled before use. All other solvents were used as
purchased. Filtrations were carried out through scin-
ter-glass, or in the case of Pd/C suspensions and fine sus-
pensions of dessicants, etc. (e.g., Na2SO4 and MgSO4),
through Celite filter-aid (521) on scinter-glass.
3.2. Syntheses
Cyclen, 1,4,7,10-tetraazacyclododecane (bis H2SO4 salt),
was prepared according to the method of Atkins et al.28
and the free amine isolated by continuous extraction
from conc. KOH solution employing chloroform.22

4718
N. J. Wardle et al. / Bioorg. Med. Chem. 15 (2007) 4714–4721
1,4,7-Tris(carboxymethyl-tert-butyl ester)-1,4,7,10-tet-
raazacyclododecane, HBr salt (DO3A-tert-butyl ester,
HBr salt) was prepared as a white solid from cyclen
according to the method of Schultze and Bulls.20
stirred under N2 at rt for 1.5 h thereafter. The mixture
was cooled to 5 ꢁC and neutralised with Amberlite IR-
120 (H+) ion-exchange resin. Immediate filtration, full
removal of solvent and subsequent addition of methanol
(2 mL), yielded a yellow solution from which crude solid
was precipitated by addition of diethyl ether (50 mL).
Two repetitions of the latter precipitation procedure
and a further precipitation from aqueous solution
(2 mL) by dropwise addition to 2-propanol (75 mL,
seeded with diethyl ether), followed by consecutive
washings of isolated material with 2-propanol and
diethyl ether, yielded a hygroscopic white solid which
was stored under N2 (320.1 mg, 56%): mp 132–135 ꢁC
(dec): (Found: C, 42.8; H, 6.7; N, 7.25. Calcd for
3.2.1.
1-O-(2-Bromoethyl)-2,3,4,6-tetra-O-acetyl-b,D-
galactopyranoside (2).29–31 A yellow oil (34%); (Found:
C 42.1; H 5.18. Calcd. for C16H23O10Br: C, 42.2; H,
5.12%): dH(CDCl3): 4.55 [1H, d, J 7.9, H-1]; lit.21 4.51
[1H, d, J 7.9, H-1]; dC(CDCl3).29–31
3.2.2. 1-(2-[2,3,4,6-Tetra-O-acetyl-b,D-galactopyranosyl-
oxy]ethyl)-4,7,10-tris(carbo-[1,1-dimethyl]ethoxymethyl)-
1,4,7,10-tetraazacyclododecane, hydrochloride salt (3).
An anhydrous DMF suspension (25 mL) of 2 (1.9 mmol
scale, 1.1 equiv), DO3A-tert-butyl ester, HBr salt (1.0
equiv) and anhydrous K2CO3 (2.1 equiv) was vigorously
stirred under anhydrous conditions at rt for 5 days. The
mixture was decanted into dichloromethane (400 mL),
washed with deionised water (6· 200 mL) and saturated
brine (3· 200 mL), and dried over MgSO4/Na2SO4.
Evaporation yielded a yellow oil which was subjected
to column chromatography (eluent—CH2Cl2/MeOH
89:11 v/v; TLC—Rf ꢀ0.42), leaving a colourless oil (3)
of a free amine product. A cooled dichloromethane solu-
tion (4 mL) of amine, diluted with anhydrous diethyl
ether (50 mL) to the point of turbidity, was bubbled
with dry HCl gas for 5 min, precipitating crude hydro-
chloride salt as a white solid. Immediate isolation of so-
lid and thorough washing with anhydrous ether,
followed by dissolution in dichloromethane and evapo-
ration of the filtered solution, generated the product as
a cream/yellow solid (1.05 g, 52%): mp 126–131 ꢁC
(dec): (Found: C, 47.8; H, 6.75; N, 4.3. Calcd for
C42H72N4O16ÆHClÆKHCO3Æ7/4MeOHÆCH2Cl2Æ1/4Et2O:
C, 47.4; H, 7.2; N, 4.7%): vmax/cmꢁ1 (KBr): 3433, 3096,
2981, 2936, 1745, 1638, 1460, 1370, 1253, 1228, 1163,
1079; dH(CDCl3): 1.21 [trace, t, CH3 (Et2O)]; 1.45
[18H, s, 6· CH3 (tBu)]; 1.49 [9H, s, 3· CH3 (tBu)];
1.98, 2.07, 2.13, 2.18 [12H, 4· s, 4· CH3 (Ac)]; 2.40–
4.80 [ꢀ38H, br m, CH2CH2 (Et linker), 11· NCH2
(DO3A ester), H-1,5,6,60 (gal), 1/2· CH2 (Et2O), 7/4·
CH3OH] in which 4.58 [1H, d, J 7.7, H-1 (gal)]; 5.02
[1H, dd, J1 10.4, J2 2.9, H-3 (gal)]; 5.13 [1H, dd, J1
10.4, J2 7.7, H-2 (gal)]; 5.31 [2H, s, CH2Cl2]; 5.39 [1H,
d, J 2.9, H-4 (gal)]; dC(CDCl3): 20.53, 20.73, 21.11
i
C22H40N4O12.5/4 PrOH.3/2TFA: C, 43.2; H, 6.5; N,
7.00%): vmax/cmꢁ1 (KBr): 3412, 2972, 2867, 1685,
1635, 1460, 1401, 1355, 1321, 1204, 1160, 1131, 1082;
dH(D2O): 1.18, 1.20 [10H, 2· s, CH3’s (iPr)]; 2.83–3.62
[18H, br m’s, NCH2 (Et linker), 8· CH2 (cyclen ring)];
3.62–3.92 [12H, br m., OCH2 (Et linker), 3· CH2CO2H,
H-3,5,6,60 (gal)]; 3.92–4.12 [13/4 H, m., H-2,4 (gal), 5/4·
Me2CHOH] in which 3.93 [1H, d, J 3.0, H-4]; 4.42 [1H,
d, J 7.4, H-1 (gal)]; also 4.14–4.35 [trace, br]; dC(D2O):
i
26.44 [CH3’s, Pr]; 52.43br., 58.10 [NCH2’s]; 63.73 [C-6
(gal), OCH2 (Et linker)]; 66.99 [CH (iPrOH)]; 71.43,
73.40, 75.50, 77.92 [C-2,3,4,5 (gal)]; 105.78 [C-1 (gal)];
117.00 [CF3, TFA]; 177.50 [C@O’s]; MS (LSIMS):
576, 575 (55, 94% resp. [MNa]+); 554, 553 (5, 8% resp.
[MH]+).
3.2.4. Gd(III) complexes of Gal-DO3A. An aqueous
suspension (20 mL) of Gal-DO3A (145.8 mg,
0.182 mmol) and Gd2(CO3)3 (0.5 equiv) was refluxed
for 24 h, then filtered. Azeotropic evaporation with 2-
propanol yielded crude material. Consecutive tritur-
ations in 2-propanol and diethyl ether generated a
cream/white powder which was stored under N2
(114.8 mg, 89%): mp 175–178 ꢁC (dec); (Found: C,
37.5; H, 5.3; N, 7.85. Calcd for C22H37N4O12.Gd: C,
37.4; H, 5.2; N, 7.9%): vmax/cmꢁ1 (KBr): 3409, 2873,
1630sh., 1601, 1384, 1326, 1244, 1158, 1085, 939; MS
(LSIMS): 709, 708 (2, 2% resp. [MH]+); 577, 576 (9,
8% resp. [LigandH]+).
3.2.5. (1,1-Dimethyl)ethyl (R,S)-2-bromo-4-(4-nitro-
phenyl)butanoate (5). The intermediate (R,S)-2-bromo-
4-(4-nitrophenyl)butanoyl chloride was obtained
by refluxing 4-(4-nitrophenyl)butyric acid (4, 11.86 g,
56.7 mmol) with thionyl chloride in CCl4 under N2, fol-
lowed by a-halogenation of the acyl chloride according
to the literature method,26,27 generating a mobile green
oil. A mixture of N,N-dimethylaniline (20 mL) and
2-methyl-2-propanol (40 mL) was cooled to 5 ꢁC under
N2, and the crude a-brominated acyl chloride was added
dropwise. Thereafter, the reaction mixture was warmed to
rt (1 h), stirred under N2 for 18 h and decanted into anhy-
drous diethyl ether (400 mL). The resulting suspension
was washed with 1N HCl solution (2· 200 mL), deionised
water (2· 200 mL), saturated NaHCO3 solution
(2· 200 mL) and saturated brine (200 mL), and the
organic phase dried over MgSO4. Evaporation gave a
brown oil which was subjected to flash chromatography
(eluent—CH2Cl2; TLC—Rf 0.83) to yield 5 as a yellow
t
[CH3’s (Ac)]; 28.08, 28.14 [CH3’s, Bu]; 42.56, 48.10,
50.78, 51.51, 52.31, 53.46, 53.99, 54.33, 54.66, 54.89
[NCH2’s]; 61.15 [C-6 (gal)]; 63.93 [OCH2 (Et linker)];
66.97, 68.56, 70.63, 71.07 [C-2,3,4,5 (gal)]; 82.44, 82.62,
t
84.65, 84.77 [CMe3’s, Bu]; 100.77 [C-1 (gal)]; 165.04,
165.39, 169.61, 169.87, 170.14, 170.38 [C@O’s]; MS
(LSIMS): 890, 889, 888 (27, 51, 18% resp. [MH]+).
3.2.3. 1-(2-[b,D-Galactopyranosyloxy]ethyl)-4,7,10-tris-
(carboxymethyl)-1,4,7,10-tetraazacyclododecane (Gal-
DO3A). An anhydrous dichloromethane solution
(5 mL) of 3 (0.85 g, 0.72 mmol) was charged with triflu-
oroacetic acid (TFA, 100 mL) under N2, and the reac-
tion mixture was stirred under N2 at rt for 3 h.
Azeotropic evaporation with chloroform (2· 50 mL)
yielded a residue which was treated with methanolic
NaOMe solution (0.25 M, 100 mL), and the mixture

N. J. Wardle et al. / Bioorg. Med. Chem. 15 (2007) 4714–4721
4719
oil (8.02 g, 41%); (Found: C, 48.95; H, 5.4; N, 4.2. Calcd
for C14H18NO4Br: C, 48.8; H, 5.2; N, 4.1%): vmax/cmꢁ1
(KBr): 2980, 2936, 1733, 1601, 1521, 1456, 1370, 1347,
1260, 1149, 1110, 853; dH(CDCl3): 1.49 [9H, s, 3· CH3
(tBu)]; 2.16–2.46 [2H, m, CH2Ca]; 2.71–3.04 [2H, m,
CH2Ar]; 4.07 (0.8H, maj.) and 4.14 (0.2H, min.) [1H, 2·
dd, J1 8.0, J2 6.3 and J1 8.2, J2 6.0 resp. CaH]; 7.38 [2H,
d, J 8.5, ArH-2,6]; 8.17 [2H, d, J 8.5, ArH-3,5];
MgSO4 and evaporated. The residual oil was then re-
fluxed in anhydrous acetonitrile/K2CO3 (large excess)
suspension for 18 h under anhydrous conditions, fil-
tered, evaporated, and the residue subjected to column
chromatography (eluent – CH2Cl2/MeOH 9:1 v/v;
TLC—Rf ꢀ0.60) to give the free-amine–isomer mix-
ture 7a/b as a dark red oil (0.42 g): vmax/cmꢁ1 (KBr):
3448, 2978, 2933, 1726, 1671, 1603, 1520, 1458, 1369,
1346, 1253, 1221, 1155, 854; dH(CDCl3): 1.42, 1.43,
1.45, 1.45, 1.48, 1.49, 1.49 [27H, 7· s, 9· CH3
(tBu)]; 1.75–2.25 [2H, br m., CH2Ca]; 2.20–4.30 [ꢀ23H,
br m’s, CH2Ar, CaH, 8· CH2 (cyclen ring), 2· CH2CO2
tBu]; 7.22, 7.37br.d, 7.44 [2H, 3· d, J 9.1, ꢀ5.4, 8.7
resp., ArH-2,6]; 8.13, 8.14, 8.16, 8.17 [2H, 4· d, J 6.6,
6.6, 6.7, 6.7, ArH-3,5]; 8.00–10.50 [ꢀ1H, br, NH]; also
2.15 [1.5H, s] and 4.50 [0.75H, s]; dC(CDCl3):
t
dC(CDCl3): 27.73 [CH3’s, Bu]; 33.15 [CH2Ca]; 35.66
[ArCH2]; 45.49 (min., DEPT-135), 46.47 (maj.) [Ca];
82.79, 83.00 [CMe3, tBu]; 123.88 [ArC-3,5]; 129.38 [ArC-
2,6]; 146.68 and 147.91 [ArC-4,1 resp.]; 168.32 [C@O];
also 57.36, 95.66 (DEPT-135); MS (LSIMS): 346, 344
(20, 24% resp. [MH]+).
3.2.6. N-(1-Carbo-[1,1-dimethyl]ethoxy-3-[4-nitrophenyl]-
propyl)-1,4,7,10-tetraazacyclododecane (6). An anhy-
drous acetonitrile suspension (30 mL) of cyclen
(0.92 g, 5.4 mmol) and fine-ground anhydrous K2CO3
(0.51 g, 3.7 mmol) was heated under N2 to the point
of reflux, and an anhydrous acetonitrile solution
(10 mL) of a-bromobutanoate 11 (0.91 g, 2.65 mmol)
was added. Thereafter, the mixture was gently refluxed
under anhydrous conditions (changing quickly from
deep blue, to pea-green colour) for 24 h, cooled to rt
and filtered. The filtrate was evaporated and the residual
brown oil subjected to column chromatography
(eluent—CH2Cl2/MeOH/0.88 aq NH3, 15:5:1 v/v/v)
and consecutive azeotropic evaporations with 2-propa-
nol and chloroform (50 mL) left residue 6 as a translu-
cent static red oil (0.80 g, 66%); (Found: C, 57.3; H,
8.3; N, 15.3. Calcd for C22H37N5O4.0.2 KBr: C, 57.5;
H, 8.1; N, 15.3%): vmax/cmꢁ1 (KBr): 3386, 2933, 2846,
1720, 1601, 1519, 1459, 1345, 1255, 1151, 853;
dH(CDCl3): 1.48 [9H, s, 3· CH3 (tBu)]; 1.86–3.09 [3H,
v.br., 3· NH]; 1.86–1.98 and 2.01–2.18 [2H, 2· m,
CH2Ca]; 2.44–2.61 [2H, m, CH2Ar]; 2.62–3.09 [16H,
m, 8· CH2 (cyclen ring)]; 3.27 [1H, dd, J1J2 7.2, CaH];
7.39 [2H, d, J 8.8, ArH-2,6]; 8.14 [2H, d, J 8.8,
ArH-3,5]; dC(CDCl3): 28.35 [CH3’s, tBu]; 31.08
[CH2Ca]; 32.99 [CH2Ar]; 45.44, 45.93, 47.34, 49.11
t
28.02, 28.12 [CH3’s, Bu]; 32.80 [DEPT-135, CH2CH2
(Pr)]; 42.00 and 42.70 (both DEPT-135), 47.00–
52.00w.br, 51.76 (DEPT-135), 55.00–57.00 br., 59.10
[NCH2’s]; 67.10 [Ca]; 81.18, 81.64, 81.94, 82.14, 82.38
t
[CMe3’s, Bu]; 123.70, 123.76 [ArC-3,5]; 129.33 [ArC-
2,6]; 146.57, 148.93 [ArC-4,1 resp.]; 166.90, 169.37,
170.28, 170.46, 171.12 [C@O’s], also 61.15; MS (LSIMS;
C34H57N5O8): 665, 664 (1, 1% resp. [MH]+).
3.2.8. 1-(2-[2,3,4,6-Tetra-O-acetyl-b,D-galactopyrano-
syloxy]ethyl)-7-(1-carbo-[1,1-dimethyl]ethoxy-3-[4-nitro-
phenyl]propyl)-1,4,7,10-tetraazacyclododecane, hydro-
chloride salt (8). A DMF solution (5 mL) of N-
functionalised cyclen derivative 6 (0.70 g, 1.5 mmol)
was added dropwise to the anhydrous DMF suspension
(5 mL) of protected bromoethyl-b,D-galactopyranoside
2 (0.83 g, 1.8 mmol) and anhydrous K2CO3 (0.28 g,
2.0 mmol) under an atmosphere of N2. Thereafter, the
mixture was vigorously stirred at rt under anhydrous
conditions for 16 days (progress monitored by TLC; elu-
ent—CH2Cl2/MeOH 9:1 v/v), decanted into dichloro-
methane (300 mL) and washed with deionised water
(6· 300 mL) and saturated brine (300 mL). The organic
phase was dried over MgSO4, evaporated and subjected
to column chromatography (eluent—CH2Cl2/MeOH 9:1
v/v; TLC—Rf 0.38–0.46) to give a red oil of free amine.
This was treated briefly in dichloromethane/diethyl ether
solution (1:5, 18 mL) with dry HCl gas and isolated as
for 3, yielding hydrochloride 8 as a red glass (0.48 g,
35%); (Found: C, 50.8; H, 7.30; N, 7.6. Calcd for
C38H59N5O14Æ2HClÆ1/4CH2Cl2Æ0Æ2Et2O: C, 51.0; H, 6.9;
N, 7.6%): vmax/cmꢁ1 (KBr): 3433, 2978, 1749, 1669,
1519, 1370, 1346, 1228, 1153, 1058, 855; dH(CDCl3):
1.21 [1.2H, t, J 7.1, CH3 (Et2O)]; 1.51, 1.55 (1.54sh)
[9H, 2· s, 3· CH3 (tBu)]; 1.99, 2.05, ꢀ2.11 br m., 2.17
[14H, 3· s and br m., 4· CH3 (Ac) and CH2Ca]; 2.40–
4.80 [ꢀ27.8H, br m’s, CaH, CH2Ar, CH2CH2 (Et lin-
ker), 8· CH2 (cyclen ring), H-1,5,6,60 (gal), 0.4· CH2
(d = 3.47, q, J 7.1, Et2O)]; 4.85–5.20 [2H, br m, H-2,3
(gal)]; 5.31 [0.5H, s, 0.25· CH2Cl2]; 5.41 [1H, br.s, H-4
(gal)]; 7.32–7.60 [2H, br m., ArH-2,6]; 7.95–8.30 [2H,
br.d, ArH-3,5]; dC(CDCl3): 20.51, 20.70 [CH3’s (Ac)];
t
[CH2’s (cyclen ring)]; 63.15 [Ca]; 81.42 [CMe3, Bu];
123.71 [ArC-3,5]; 129.32 [ArC-2,6]; 146.50 and 149.73
[ArC-4,1 resp.]; 171.57 [C@O]; MS (LSIMS): 436
(100%, [MH]+).
3.2.7. Alkylation of 6 with tert-butyl bromoacetate (2
equiv); mixture of 1-(1-[carbo-(1,1-dimethyl)ethoxy]-3-[4-
nitrophenyl]propyl)-4,7-bis(carbo-[1,1-dimethyl]ethoxym-
ethyl)-1,4,7,10-tetraazacyclododecane (7a) and 1-(1-[car-
bo-(1,1-dimethyl)ethoxy]-3-[4-nitrophenyl]propyl)-4,10-
bis(carbo-[1,1-dimethyl]ethoxymethyl)-1,4,7,10-tetraaz-
acyclododecane (7b). An anhydrous N,N-dimethylaceta-
mide solution (10 mL, DMA) of N-functionalised cyclen
derivative 6 (1.89 g, 4.0 mmol) was charged with anhy-
drous sodium acetate (0.66 g, 8.0 mmol), cooled under
N2 to 5 ꢁC, and tert-butyl bromoacetate (1.56 g,
8.0 mmol) added dropwise (residue washed in with
2 mL DMA). The reaction mixture was stirred under
anhydrous conditions for 21 days at rt, evaporated,
and a chloroform solution of the residue (200 mL) was
washed with deionised water (3· 200 mL) and saturated
brine (200 mL), before the organic phase was dried over
t
28.13, 28.35 [CH3’s, Bu]; ꢀ33.00 br, 34.40 [(CH2)2Ar];
ꢀ47.00 w.br (DEPT-135), 53.50 [CH2’s (cyclen ring),
NCH2 (Et linker)]; 61.03 [C-6 (gal), OCH2 (Et linker)];
66.90, ꢀ69.00 br., 70.46, 71.05 [C-2,3,4,5 (gal), Ca];
101.50 br [DEPT-135, C-1 (gal)]; 123.91 [ArC-3,5];

4720
N. J. Wardle et al. / Bioorg. Med. Chem. 15 (2007) 4714–4721
129.55 [ArC-2,6]; 147.00 [ArC-4,1]; 169.88, ꢀ170.13,
170.41 [C@O’s]; also 95.70 [DEPT-135]; MS (LSIMS):
810, 811 (3, 3% resp. [MH]+).
ised water (10 mL), filtered and stirred with Pd/C (10%
w/w, 50 mg) catalyst under H2 (replenished at regular
intervals) at atmospheric pressure in a gas chamber until
uptake of H2 had ceased (48 h). The reaction mixture
was filtered and the filtrate azeotropically evaporated
with absolute ethanol. Crystallisation from EtOH/H2O
(10:1 v/v, 11 mL) over 3 days yielded a pink/brown solid
(17.8 mg, 21%): mp 178–185 ꢁC (dec); (Found: C, 44.2;
H, 7.2; N, 6.7. Calcd for C30H49N5O12Æ2TFAÆ3EtOHÆ3-
H2O: C, 44.0; H, 6.9; N, 6.4%): vmax/cmꢁ1 (KBr): 3422,
2929, 1633, 1519, 1385, 1078; dH(H2O): 1.03 [9H, t, J
7.5, CH3 (EtOH)]; 1.25–1.45 [2H, br m, CH2Ca]; 1.60–
4.80 [ꢀ40H, v.br., CaH, CH2Ar, CH2CH2 (Et linker),
8· CH2 (cyclen ring), 7· gal-H’s, 2· CH2CO2H, 3· CH2
(d = 3.57, q, J 7.5, EtOH)] in which 4.42 [1H, br m., H-1
(gal)]; 6.60–7.50 [ꢀ4H, br m, ArH’s]; also 7.70–8.05 [trace,
br.]; dC(D2O): 16.73, 16.78 [CH3 (Et)]; 27.43, 31.00–
32.00 br [CH2CH2Ar]; 40.50, 42.00–56.00 br, 57.38,
57.42, 58.80–60.30 br. [CH2’s]; 61.04 [C-6 (gal)],
62.80 br [OCH2 (Et linker), Ca]; 68.63, 70.65, 72.60,
75.27 [C-2,3,4,5 (gal)]; 102.81 [C-1 (gal)]; 120.00–
123.00 br, 130.11 [CF3 (TFA), ArC’s]; (C@O’s not dis-
cernable from noise; dC = 160–180 ppm); MS (LSIMS):
673, 672 (2, 2% resp. [MH]+).
3.2.9. 1-(2-[2,3,4,6-Tetra-O-acetyl-b,D-galactopyrano-
syloxy]ethyl)-7-(1-carbo-[1,1-dimethyl]ethoxy-3-[4-nitro-
phenyl]propyl)-4,10-bis(carbo-[1,1-dimethyl]ethoxymeth-
yl)-1,4,7,10-tetraazacyclododecane, hydrochloride salt
(9) (Gal-PA-DO3A-NO2 ester conjugate). An anhy-
drous DMF suspension (5 mL) of 8 (0.35 g, 0.39 mmol)
and anhydrous K2CO3 (0.78 g, 5.6 mmol) was stirred at
rt under N2 as tert-butyl bromoacetate (0.78 g,
4.0 mmol) was added dropwise. Thereafter, the reaction
mixture was stirred under anhydrous conditions for 15
days, decanted into dichloromethane (400 mL) and
washed with deionised water (6· 200 mL) and saturated
brine (200 mL). The organic phase was dried over
MgSO4, evaporated and subjected to gradient chroma-
tography (eluent—0% ! 2% ! 4% ! 5% MeOH in
CH2Cl2 v/v). Purified free amine was treated for 2 min
in dichloromethane/diethyl ether solution (1:27,
84 mL) with dry HCl gas, and isolated as for 3, yielding
hydrochloride 9 as a brown glass (102.7 mg, 22%);
(Found: C, 50.9; H, 6.6; N, 5.8. Calcd for
C50H79N5O18Æ2HClÆKHCO3Æ0.1Et2O: C, 50.7; H, 6.8; N,
5.75%): vmax/cmꢁ1 (KBr): 3432, 2979, 2937, 1748, 1669,
1520, 1457, 1370, 1346, 1229, 1155, 1075, 845; dH(CDCl3):
1.21 [0.6H, t, J 7.0, 0.2· CH3 (Et2O)]; 1.46, 1.48, 1.49
[27H, 3· s (overlapped), 9· CH3 (tBu)]; 1.99, 2.06,
ꢀ2.11 br m., 2.16 [14H, 3· s and br m., 4· CH3 (Ac)
and CH2Ca]; 2.40–4.80 [ꢀ31.4H, br m’s, CaH, CH2Ar,
CH2CH2 (Et linker), 8· CH2 (cyclen ring), H-1,5,6,60
3.2.11. Gd(III) complex of Gal-PA-DO3A-NH2. An
aqueous suspension (2 mL) of Gal-PA-DO3A-NH2
(11.4 mg, 0.011 mmol) and Gd2(CO3)3 (2.7 mg,
0.0055 mmol) was refluxed for 24 h, then filtered and
the brown filtrate azeotropically evaporated with abso-
lute ethanol. Precipitation from EtOH over several days
yielded the Gd(III) complex as a brown solid (7.2 mg,
62%): mp 223–227 ꢁC (dec); (Found: C, 38.0; H, 4.8;
N, 5.8. Calcd for C30H46N5O12GdÆ2TFAÆ5/4EtOHÆ11/
4 H2O: C, 37.75; H, 5.1; N, 6.0%): vmax/cmꢁ1 (KBr):
3423, 2926, 1619, 1515, 1385, 1079.
t
(gal), 2· CH2CO2 Bu, 0.2· CH2 (d = 3.48, q, J 7.0,
Et2O)]; 4.85–5.20 [2H, br m, H-2,3 (gal)]; 5.41 [1H, br
m, H-4 (gal)]; 7.32–7.65 [2H, br m, ArH-2,6]; 7.95–8.30
[2H, br,m, ArH-3,5]; dC(CDCl3): 20.53, 20.71 [CH3’s
(Ac)]; 28.10 [CH3’s, tBu]; 31.20 br, 32.70, 34.70
[(CH2)2Ar]; [CH2’s not discernable from noise; dC = 40–
50 ppm]; 61.09, 65.87 [C-6 (gal), OCH2 (Et linker)];
64.20 [DEPT-135, Ca]; 66.96, 68.49, 70.70 br [C-2,3,4,5
(gal)]; 101.50 [C-1 (gal)]; 123.82 [ArC-3,5]; 129.61 [ArC-
2,6]; 146.61, ꢀ149.00 br [ArC-4,1 resp.]; 169.94, 170.43
[C@O’s]; MS (LSIMS): 1039, 1038 (3, 3% resp. [MH]+).
3.3. R1 Relaxivity measurements
Stock solutions (17.5 mM) of [Gd-(Gal-PA-DO3A-
NH2)] and [Gd-(Gal-DO3A)] complexes were prepared
in water. Both complexes (2 mM) in phosphate buffer
(pH 7.4) were incubated with b-galactosidase (5 lM
from Sigma EC 3.2.1.23) at 37 ꢁC for 20 h prior to mea-
surements. Samples of the above solutions were placed
in Eppendorf tubes and imaged on a Varian Inova (Palo
Alto, CA, USA) 9.4 T horizontal bore MRI scanner.
The tubes were placed in a holder within a 63 mm inner
diameter RF coil. A spin-echo sequence was used with
the following scan parameters: TR = 50, 100, 200, 300,
500, 700, 1200, 3000, 5000, 7000 ms, TE = 10 ms, num-
ber of signal averages = 2, 3 slices coronal slices 2 mM
thick with a 1 mM gap between the slice, FOV;
100 · 100 mm2, matrix of 256 · 128. The T1 value of
each compound was determined by fitting the data to
Eq. (1), the T1 Saturation Recovery equation.
3.2.10. 1-(2-[b,D-Galactopyranosyloxy]ethyl)-7-(1-car-
boxy-3-[4-aminophenyl]propyl)-4,10-bis(carboxymeth-
yl)-1,4,7,10-tetraazacyclododecane (Gal-PA-DO3A-
NH2). A chloroform solution (4 mL) of [b,D-galactopyr-
anosyloxy]ethyl-PA-DO3A-NO2 ester (9, 100.2 mg,
0.085 mmol) was added dropwise to trifluoroacetic acid
(TFA, 20 mL) under N2, and the reaction mixture was
stirred at rt for 3.5 h, before being evaporated alone,
and azeotropically with chloroform (2· 25 mL). The res-
idue was treated with methanolic NaOMe solution
(0.7 M, 10 mL) and the mixture was stirred under N2
for 1 h, cooled to 5 ꢁC and neutralised with Amberlite
IR-120 (H+) ion-exchange resin. The resin was immedi-
ately separated by filtration, washed with methanol, and
the filtrate/washings reduced down to 10 mL before azeo-
tropic evaporation with chloroform (20 mL) yielded a
brown oil. Consecutive precipitations from methanol
solutions (0.25 mL) by addition of diethyl ether (50 mL)
yielded a cream/pink solid. This was dissolved in deion-
Si ¼ S0ð1 ꢁ ex=T
Þ
ð1Þ
1
where x is the TR value, and Si is the measured signal
for a given TR value). To ensure reproducibility and
consistency, each measurement was carried out three
times under identical conditions.

N. J. Wardle et al. / Bioorg. Med. Chem. 15 (2007) 4714–4721
4721
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