Analogs of Eu3+ DOTAM-Gly-Phe-OH and Tm3+ DOTAM-Gly-Lys-OH: Synthesis and magnetic properties of potential PARACEST MRI contrast agents

Article abstract of DOI:10.1016/j.bmc.2008.04.038

Chelated lanthanide ions, especially gadolinium, have found wide use as contrast agents in magnetic resonance imaging. A new paradigm for generating contrast, termed PARACEST, was recently described that requires the slow exchange of water or other exchan

Full text of DOI:10.1016/j.bmc.2008.04.038

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6156–6166
Analogs of Eu3+ DOTAM-Gly-Phe-OH and Tm3+
DOTAM-Gly-Lys-OH: Synthesis and magnetic properties
of potential PARACEST MRI contrast agents
a,b
Mojmır Suchy, Alex X. Li,b Robert Barthab and Robert H. E. Hudsona,
´
*
´
aDepartment of Chemistry, The University of Western Ontario, Chemistry Building, Room 209, London, Ont., Canada N6A 5B7
bCenter for Functional and Metabolic Mapping, Robarts Research Institute, The University of Western Ontario,
London, Ont., Canada N6A 5K8
Received 25 February 2008; revised 12 April 2008; accepted 16 April 2008
Available online 24 April 2008
Abstract—Chelated lanthanide ions, especially gadolinium, have found wide use as contrast agents in magnetic resonance imaging.
A new paradigm for generating contrast, termed PARACEST, was recently described that requires the slow exchange of water or
other exchangeable protons present in the ligand framework. In previous work, we have described a synthetic method for the prep-
aration of dipeptide conjugates of DOTAM for use as PARACEST agents. Two compounds possessed interesting magnetic prop-
erties: the Eu3+ complex of DOTAM-Gly-Phe-OH and the Tm3+ complex of DOTAM-Gly-Lys-OH. To understand the relationship
between the structure of these complexes and their magnetic properties, we have expanded our synthetic methodology and prepared
several new complexes. Ligands have been prepared in which the terminal phenylalanine moieties have been replaced with trypto-
phan or tyrosine, the distance to the amino acid residue possessing an a-substituent has been changed, or phenylalanine and lysine
have been combined in the peptide sequence. The preparation of lanthanide(III) complexes of these ligands has been achieved and
their PARACEST properties have been determined.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
New ligands for PARACEST agents have been designed
and synthesized, often derived from DOTA6 or DO-
TAM,7,8 that increase MRI detection sensitivity or pro-
vide functional groups. We have recently established a
synthetic methodology that allowed the synthesis of a
large collection of different lanthanide(III) complexes
of DOTAM-derived oligopeptide conjugates.9 From
an initial examination of the PARACEST effects associ-
ated with these complexes, two compounds were identi-
fied to be of particular interest. Firstly, the Eu3+
complex of DOTAM-Gly-Phe-OH (3) (Fig. 1) has the
potential for in vivo temperature measurement as de-
scribed recently.10 Secondly, the Tm3+ complex of DO-
TAM-Gly-Lys-OH (4) (Fig. 1) was found to show a
PARACEST effect due to exchangeable amide protons
and may have applicability for in vivo pH measurement.
To understand the relationship between the structure of
the aforementioned complexes and their CEST detection
sensitivity, we have prepared several new complexes re-
lated to 3 and 4.
It is now well established that chelated lanthanide ions
can be used as contrast agents in magnetic resonance
imaging (MRI). These paramagnetic lanthanide ions
are known to induce a large hyperfine shift of the bound
water protons and other exchangeable protons that may
be present in the surrounding ligand framework.1 Selec-
tive saturation of these exchangeable spins has led to the
discovery of a new methodology for generation of MRI
contrast: chemical exchange saturation transfer
(CEST).2 In combination with paramagnetic ions
(PARA) to generate large chemical shifts, the PARA-
CEST effect has the potential to be a powerful tool for
the in vivo measurement of important physiologic
parameters, such as temperature3 and pH4 that vary be-
tween normal physiologic and disease states.5
Keywords: Cyclen; MR imaging; DOTAM; Europium.
*
Three groups of ligands have been designed as shown in
Figure 1. In comparison to compound 3, Type 1 ligands
Corresponding author. Tel.: +1 519 661 2166; fax: +1 519 661
3022; e-mail: rhhudson@uwo.ca
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.038

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M. Suchy et al. / Bioorg. Med. Chem. 16 (2008) 6156–6166
6157
HO
OH H2N
NH2
HOPheGly
GlyPheOH
HOLysGly
GlyLysOH
N
N
N
N
N
N
N
N
O
O
O
O
N
N
N
N
O
O
N
N
N
O
O
1
2
Eu
Tm
O
O
O
O
O
O
O
N
O
HO
H2N
OH
NH2
HOPheGly
HOLysGly
GlyPheOH
GlyLysOH
DOTA
DOTAM
3
4
R
R
O
R
R
R
R
N
N
N
N
N
N
N
O
N
O
O
O
N
N
O
O
O
O
R
N
O
R
O
N
O
R
R
R
R
Type 3
Type 2
Type 1
5, R = Gly-Trp-OH
6, R = Gly-Tyr-OH
9, R = Gly-Phe-Lys-OH
7, R = Phe-OH
10, R = Lys-Phe-OH
8, R = Lys-OH
Figure 1. Structure of DOTA (1), DOTAM (2), complexes 3 and 4 and ligands 5–10.
have had the terminal phenylalanine moieties replaced
with other aromatic amino acids, such as tryptophan
(5) or tyrosine (6) (Fig. 1). We also wanted to investigate
the effect the number of intervening atoms between the
cyclen ring to the amino acid residue possessing a-sub-
stitution, such as is present in ligands 3 and 4, on the
CEST sensitivity of the resultant complexes. Conse-
quently, Type 2 ligands (7 and 8, Fig. 1), which have
had the glycine deleted from the sequence in comparison
to 3 and 4 respectively, were prepared. Finally, Type 3
ligands (9 and 10, Fig. 1), combine phenylalanine and ly-
sine in their sequence.
17 which were intended to be used as electrophiles in
the synthesis of Type 1 ligands were obtained in 75%
(14) and 93% (17) yields, respectively (Scheme 1).
Chlorides 20 and 23 have been prepared by treatment of
chloroacetyl chloride (18) with H-Phe-OEtÆHCl (19)11
(78% yield) or H-Lys(Boc)-OMeÆHCl (22) (96% yield)
(Scheme 2). It is worth mentioning that halide 23 has
been recently prepared by NHS/EDC-mediated cou-
pling of chloroacetic acid with H-Lys(Boc)-OMeÆHCl.12
Finkelstein reaction of chloroderivatives 20 and 23
afforded electrophiles 21 and 24 (synthesis of Type 2 li-
gands) in high yields (ca. 80%) (Scheme 2).
A synthetic route allowing for the preparation of the no-
vel lanthanide (III) complexes of ligands 5–10 has been
developed and their PARACEST properties have been
investigated. The results of these studies are described
herein.
Preparation of iodoacetyl tripeptide 26 as a Type 3 li-
gand precursor was found to be somewhat troublesome.
The main difficulty was the removal of dicyclohexyl urea
(DCU) which formed as a side product in two consecu-
tive NHS/DCC-mediated couplings. The use of NHS/
EDCÆHCl coupling proved to give very low yields. N-
Chloroacetyl glycine (11) was coupled to H-Phe-OH
(25) (Scheme 3) and the crude dipeptide was extracted
together with large quantities of DCU after acidic aque-
ous work-up. The resulting mixture was subjected to the
second NHS/DCC-mediated coupling with H-Lys(Boc)-
OMeÆHCl (22), the corresponding chloroacetyl tripep-
tide was extracted with CHCl3 and was found to contain
approximately 20% of DCU (by 1H NMR). Purification
at this point was not attempted and the mixture was
subjected to NaI-promoted halogen exchange. Pure iod-
2. Results and discussion
2.1. Synthesis
Treatment of N-chloroacetylglycine (11) with H-Trp-
OMeÆHCl (12) or H-Tyr(t-Bu)-OMeÆHCl (15) under
the conditions of NHS/EDC or NHS/DCC-mediated
couplings9 afforded chloroacetyl dipeptides 13 and 16
in good yields (Scheme 1). Halogen exchange under
the conditions of Finkelstein reaction (NaI, acetone)
proceeded smoothly and iodoacetyl dipeptides 14 and
O
H
N
1) H-Trp-OMe.HCl (12)
1) H-Phe-OEt.HCl (19)
X
OEt
NHS, EDC.HCl,
NH
Na2CO3, DMF
O
O
H
N
Et3N, THF
OMe
2) NaI, acetone
X
N
H
2) NaI, acetone
O
O
20: X= Cl
21: X = I
Cl
Cl
O
13: X = Cl
14: X = I
H
N
O
18
Cl
OH
1) H-Lys(Boc)-OMe.HCl (22)
Na2CO3, DMF
O
H
O
N
O
X
OMe
1) H-Tyr(t
-Bu)-OMe.HCl (15)
NHS, DCC, Et3N, CH2Cl2
11
O
O
H
2) NaI, acetone
N
N
OMe
X
H
O
O
NHBoc
23: X = Cl
24: X =I
2) NaI, acetone
16: X = Cl
17: X = I
Scheme 1. Synthesis of iodoacetyl dipeptides 14 and 17.
Scheme 2. Synthesis of electrophiles 21 and 24.

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M. Suchy et al. / Bioorg. Med. Chem. 16 (2008) 6156–6166
6158
1) H-Phe-OH (25)
(NaOH in THF/H2O) and Boc or t-Bu protecting
BocHN
NHS, DCC, Et3N, THF
2) H-Lys(Boc)-OMe.HCl (22)
NHS, DCC, Et3N, THF
groups in 32 and 34–36 were removed by treatment with
TFA. Reaction mixtures containing TFA were concen-
trated and crude products obtained were saponified
(NaOH, THF, or MeOH/H2O) to afford ligands 5–10
(Scheme 5). Ligands 5, 6, 8, 9, and 10 represent new
structures, while ligand 7 and its Cd2+, Cu2+, and
Zn2+ complexes have been recently described.13 The
metalation of ligands 6–10 proceeded smoothly (Table
1) resulting in complexes 38–42 which were purified by
size exclusion chromatography (SEC) as previously de-
scribed.9 Identification of fractions containing com-
plexes 37–39 was made by UV visualization on TLC
or staining by I2 vapors (37–39). The ninhydrin test
was used to identify the fractions containing complexes
40–42. Complexes 37–42 were obtained in moderate to
very good yields (Table 1). The absence of free Eu3+
or Tm3+ after size exclusion chromatography was con-
firmed for all complexes by employing the xylenol or-
ange test.14 HPLC chromatograms (Method B, general
experimental procedures) of fractions obtained after size
exclusion chromatography (complexes 38 and 39) indi-
cated that <5% (based on peak area) of free ligands 6
and 7 were present. The complexes were characterized
by 1H NMR spectroscopy and mass spectrometry
(ESI-TOF) (Table 1, experimental procedures). The
metalation of Trp-OH containing ligand 5 was found
to be difficult and was not completed even using 5 equiv-
alents of EuCl3Æ6H2O and elevated temperature. After
SEC, the Eu3+ complex 37 obtained was found to con-
tain ca. 15% of free ligand 5 (HPLC analysis, Method
B, general experimental procedures). Since the impure
complex 37 did not possess particularly striking mag-
netic properties (Fig. 2), no studies on the optimization
of the metalation reaction were carried out. It is worth
mentioning that a similar problem occurred when at-
tempts to introduce Eu3+ to DOTAM-Gly-Arg(NO2)-
OMe were made.9
O
O
O
N
H
H
N
N
I
OMe
Cl
OH
N
O
H
H
O
O
3) NaI, acetone
11
26
Scheme 3. Synthesis of iodoacetyl tripeptide 26.
1) H-Lys(Boc)-OH (28)
O
H
N
NHS, DCC, Et3N, THF
OH
OEt
Cl
I
N
H
O
27
2) H-Phe-OEt.HCl (19)
NHS, DCC, Et3N, THF
3) NaI, acetone
O
O
29
NHBoc
Scheme 4. Synthesis of iodoacetyl dipeptide 29.
oacetyl tripeptide 26 was obtained in 52% yield, based
on N-chloroacetyl glycine (11) (Scheme 3), after chro-
matography and recrystallization.
A similar one-pot strategy has been used for the synthe-
sis of another Type 3 ligand precursor, iodoacetyl dipep-
tide 29 (Scheme 4). Chloroacetic acid (27) was subjected
to consecutive NHS/DCC-mediated couplings, first with
H-Lys(Boc)-OH (28), followed by H-Phe-OEtÆHCl (19,
Scheme 4), to yield the chloroacetyl dipeptide that was
1
contaminated with DCU (10%, by H NMR) despite
having undergone column chromatography. This mate-
rial was treated with NaI in acetone to give the pure
electrophile 29 (Scheme 4) in 40% yield, based on chlo-
roacetic acid (27).
Peralkylation of cyclen (30) with iodine containing elec-
trophiles 14, 17, 21, 24, 26 and 29 was carried out using
a recently established protocol.9 Tetraalkylated cyclens
31–35 were obtained in very good yield (ca. 90%), and
the yield of tetraalkylated cyclen 36 was somewhat lower
(64%) (Scheme 5). Compounds 31–36 were purified by
trituration in hexanes. The protecting groups were re-
moved using standard protocols: ester groups in conju-
gates 31 and 33 were removed by saponification
2.2. PARACEST properties associated with complexes
37–42
Complexes 37–42 have been designed based on the
structures of Eu3+ complex 3 and Tm3+ complex 4 and
R
R
R
R
H
H
H
H
N
N
N
N
O
O
N
N
N
N
N
N
N
N
LnCl3 . 6H2O
NaOH, H2O
O
O
14, 17, 21, 24, 26 or 29
30
Ln
DIPEA, MeCN
O
O
R
O
O
R
R
R
31, R = Gly-Trp-OMe
37, Ln = Eu3+, R = Gly-Trp-OH
38, Ln = Eu3+, R = Gly-Tyr-OH
39, Ln = Eu3+, R = Phe-OH
32, R = Gly-Tyr(
t-Bu)-OMe
33, R = Phe-OEt
34, R = Lys(Boc)-OMe
40, Ln = Tm3+, R = Lys-OH
35, R = Gly-Phe-Lys(Boc)-OMe
36, R = Lys(Boc)-Phe-OEt
41, Ln = Eu3+, R = Gly-Phe-Lys-OH
42, Ln = Eu3+, R = Lys-Phe-OH
see legend
5, R = Gly-Trp-OH
6, R = Gly-Tyr-OH
7, R = Phe-OH
8, R = Lys-OH
9, R = Gly-Phe-Lys-OH
10, R = Lys-Phe-OH
Scheme 5. Synthesis of ligands 5–10 and their lanthanide(III) complexes 37–42; 31 ! 5. Reagents: NaOH, THF/H2O; 32 ! 6: (i) TFA, CH2Cl2; (ii)
NaOH, THF/H2O; 33 ! 7: NaOH, THF/H2O; 34 ! 8: (i) TFA; (ii) NaOH, MeOH/H2O; 35 ! 9: (i) TFA; (ii) NaOH, MeOH/H2O; 36 ! 10: (i)
TFA, CH2Cl2; (ii) NaOH, THF/H2O.

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6159
Table 1. Selected data for the synthesis and characterization of complexes 37–42
Complex
Yielda (mass, percent)
HRMS (ESI-TOF) m/z
37
38
39
40
41
42
Eu3+ DOTAM-Gly-Trp-OH
Eu3+ DOTAM-Gly-Tyr-OH
Eu3+ DOTAM-Phe-OH
28 mg, 44%
28 mg, 36%
22 mg, 32%
84 mg, 77%
127 mg, 98%
34 mg, 34%
1528.5098 [Mꢀ2H]+ (1528.5072 calcd for C68H79N16O16Eu)
1433.4343 [Mꢀ2H]+ (1433.4341 calcd for C60H74N12O20Eu)
1142.3730 [Mꢀ2H]+ (1142.3764 calcd for C52H63N8O12Eu)
1083.4867 [Mꢀ2H]+ (1083.4891 calcd for C40H74N12O12Tm)
1884.8392 [Mꢀ2H]+ (1884.8435 calcd for C84H123N20O20Eu)
1653.7559 [Mꢀ2H]+ (1653.7484 calcd for C76H110N16O16Eu)
Tm3+ DOTAM-Lys-OH
Eu3+ DOTAM-Gly-Phe-Lys-OH
Eu3+ DOTAM-Lys-Phe-OH
a Complex 37 contained ca. 15% of free ligand 5.
Figure 2. PARACEST spectra of compounds 3, 4, 37–42, determined at 10 mM concentration, pH 7.0. All spectra were collected using a 9.4 T NMR
spectrometer with saturation power of 14 lT (saturation time = 10 s) at the temperature indicated. A single repetition was used at each saturation
frequency and there was no additional delay without saturation aThe PARACEST spectrum of Eu3+ DOTAM-Gly-Trp-OH (37) was obtained using
the complex containing ca.15% (HPLC analysis) of free ligand 5.

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M. Suchy et al. / Bioorg. Med. Chem. 16 (2008) 6156–6166
6160
synthesized as potentially new MRI PARACEST
agents. With the complexes 37–42 in hand, the survey
of their CEST efficiency under the conditions of near
physiological temperature and pH was performed. The
results were compared with those for Eu3+ DOTAM-
Gly-Phe-OH (3) and Tm3+ DOTAM-Gly-Lys-OH (4)
as shown in Figure 2. Weak to moderate PARACEST
effects (<20%) due to bound water protons are observed
with Eu3+ DOTAM-Gly-Trp-OH (37, ca. 45–50 ppm),
Eu3+ DOTAM-Phe-OH (39, ca. 45–50 ppm), Eu3+ DO-
TAM-Gly-Phe-Lys-OH (41, ca. 45–50 ppm), and Eu3+
DOTAM-Lys-Phe-OH (42, ca. 35 ppm) designed as ana-
logs of Eu3+ DOTAM-Gly-Phe-OH (3) (Fig. 2). The
PARACEST effect (at ca. ꢀ50 ppm) associated with
Tm3+ DOTAM-Lys-OH (40) (due to amide protons) is
slightly weaker (15%) compared to the PARACEST ef-
fect of the glycine-containing analog Tm3+ DOTAM-
Gly-Lys-OH (4) (25%, ca. ꢀ50 ppm). The PARACEST
spectrum of Eu3+ DOTAM-Gly-Tyr-OH (38) exhibits
the presence of a strong PARACEST effect due to
bound water protons (40%, ca. 50 ppm) similar to par-
ent compound 3.
indicates that analogous complexes bearing similar
PARACEST properties might be synthesized without
significant loss in the PARACEST effect associated with
amide protons.
3. Conclusions
We have shown that our recently developed synthetic
methodology for the synthesis and purification of lan-
thanide(III) complexes of cyclen oligopeptide conju-
gates9 may be easily modified such that complexes
with peptidic appendages that are shorter (glycine dele-
tion), longer (e.g., tripeptides), or vary the amino acid
(Trp, Tyr) can be prepared in reasonable yields. The
PARACEST spectra associated with new complexes
were acquired under standard conditions9 and were
compared to those associated with potentially useful
MRI contrast agents Eu3+ DOTAM-Gly-Phe-OH (3)
(for temperature measurement) and Tm3+ DOTAM-
Gly-Lys-OH (4) (for pH measurement). Most notably,
it was found that only the conservative replacement of
the terminal phenylalanine (Phe) subunit in 3 by tyro-
sine (Tyr, 38) was tolerated and resulted in a compound
with a strong PARACEST effect due to bound water
protons. Conversely, replacement of Phe by tryptophan
(Trp, 37) resulted in a compound with very poor PARA-
CEST properties under the conditions of measurement.
Other changes to the length or composition of the pep-
tide arms (39, 41, and 42) were all found to be detrimen-
tal to the PARACEST effect due to bound water
protons. To aid the rational design of subsequent con-
trast agents, insight in to the origin of the PARACEST
effect is needed, especially with respect to structural
studies on the complexes and the effect of structure on
the rate of exchange of water with the metal center.
The results show that replacement of the terminal phen-
ylalanine (Phe) subunit in 3 with other natural aromatic
amino acids has dramatically different effects. While the
structurally modest change from Phe (3) to tyrosine
(Tyr, 38) preserves the PARACEST properties, the
change to tryptophan (Trp, 37) results in significant loss
of the PARACEST effect. Similarly, a substantial loss in
the PARACEST effect (due to bound water protons)
was also observed with complexes bearing shorter
(Eu3+ DOTAM-Phe-OH, 39), longer (Eu3+ DOTAM-
Gly-Phe-Lys-OH, 41) or modified (Eu3+ DOTAM-Lys-
Phe-OH, 42) side chains. The exact reasons for the
exceptionally large PARACEST effect associated with
Eu3+ DOTAM-Gly-Phe-OH10 (3) are not fully under-
stood at the moment; however, it is reasonable to as-
cribe this effect to favorable bound-bulk water
exchange rate. The rate of exchange of the pool of
bound water with bulk water influences the magnitude
of the PARACEST effect and the exchange rate in turn
is affected by the nature of the ligand, the identity of the
metal, and environmental factors such as pH and tem-
perature. Recent studies have elucidated some elec-
tronic15,16 and steric17 factors affecting water exchange
rates within closely related series of DOTA and DO-
TAM analogs. The electronic effect is observed when
the basicity of a chelating atom, either the nitrogen of
the cyclen ring or the oxygen of the carbonyl, is modu-
lated by substituent effects. Due to the structural similar-
ity of the ligands described herein, and all other factors
(pH, temperature, and metal ion) being constant, the
changes in the observed PARACEST effect is likely
caused by changes in the water exchange rate due to
varying the steric environment around the metal.
The PARACEST effect due to amide protons was found
to be slightly less dependent on the changes in the struc-
ture of the complex, as evidenced by comparison of the
data for Tm3+ DOTAM-Lys-OH (40) and Tm3+ DO-
TAM-Gly-Lys-OH (4). This observation indicates that
more analogous complexes bearing similar PARACEST
properties could be synthesized without significant loss
in the PARACEST effect associated with amide protons.
4. Experimental
4.1. General experimental procedures
All amino acids (naturally occurring L isomers) and re-
agents were commercially available, unless otherwise sta-
ted. All solvents were of HPLC grade and used as such,
except for CH2Cl2 and THF (dried over Al2O3, in a sol-
vent purification system) and deionized water (18.2 MX/
cm). Organic extracts were dried over Na2SO4 and sol-
vents were removed under reduced pressure in a rotary
evaporator. Flash column chromatography (FCC) was
The PARACEST effect due to amide protons was also
found to be dependent on the changes in the structure
of the complex, as evidenced by comparison of the data
for Tm3+ DOTAM-Lys-OH (40) and Tm3+ DOTAM-
Gly-Lys-OH (4). Although the PARACEST effect is
diminished, it is not obliterated and this observation
˚
carried out using silica gel, mesh size 230–400 A. Size
exclusion chromatography was carried out on BIO-
GEL P2, 45–90 lm mesh resin (20 g, column size
15 · 2 cm per 0.1 mmol of compound). Ten fractions

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M. Suchy et al. / Bioorg. Med. Chem. 16 (2008) 6156–6166
6161
(10 mL each) were collected. Fractions were identified by
5% solution of ninhydrin in AcOH/EtOH (tetraacetyl-
Lys-OH, Gly-Phe-Lys-OH, and Lys-Phe-OH derivatives)
or by UV/I2 vapors (tetraacetyl-Gly-Trp-OH, Gly-Tyr-
OH, and Phe-OH derivatives). Thin-layer chromatogra-
phy (TLC) was carried out on Al backed silica gel plates,
and compounds were visualized by UV light or I2 vapors.
Melting points were obtained on Fisher–Johns apparatus
and are uncorrected. Specific rotations [a]D were deter-
mined by Atago Polax-2L polarimeter at ambient temper-
ature using a 5-mL, 10-cm path length cell; the units are
10ꢀ1 deg cm2 gꢀ1 and the concentrations are reported in
g/100 mL. HPLC analysis (Waters 600E) was carried
out using a Microsorb-CN column (particle size 5 lm;
(351.0986 calcd for C16H18ClN3O4). LRMS (EI) m/z
(rel abundance): 351 [M+H]+ (10), 201 (23), 130 (100).
4.1.2. NHS/DCC-mediated coupling of N-chloroacetyl-
glycine (11) with H-Tyr(t-Bu)-OMeÆHCl (15). To a stir-
red suspension of N-chloroacetylglycine (11, 758 mg,
5 mmol) in dry CH2Cl2 (40 mL) at 0 ꢁC were added
NHS (575 mg, 5 mmol) and DCC (1.341 g, 6.5 mmol).
The stirring was continued for 30 min at 0 ꢁC and then
H-Tyr(t-Bu)-OMeÆHCl (15, 1.275 g, 5 mmol) and Et3N
(1.4 mL, 6 mmol) were added and the resultant mixture
was stirred for 18 h at rt and then set aside for 6 h at
ꢀ20 ꢁC. The precipitate (N,N0-dicyclohexylurea (DCU)
and Et3NÆHCl) was filtered off and the filtrate was
washed with 1 M HCl, was dried, concentrated, and
then subjected to FCC on 60 g SiO2 (CH2Cl2/MeOH,
9:1). The eluate was concentrated, the residue was dis-
solved in a small amount of acetone (ca. 5 mL), and
the solution was set aside for 2 h at ꢀ20 ꢁC. An addi-
tional amount of DCU was filtered off and the filtrate
˚
100 A pore; 4.6 id · 250 mm). Mobile phase: Method A:
75% H2O/25% MeCN–25% H2O/75% MeCN over
35 min, linear gradient, at a flow rate 1 mL/min; Method
B: 25% H2O/75% MeCN–100% MeCN over 30 min, lin-
ear gradient, at a flow rate 1 mL/min. NMR spectra were
recorded on Varian-Mercury 400 MHz spectrometer at
room temperature(ꢁ21 ꢁC); for 1H (400.1 MHz), d values
were referenced as follows CDCl3 (7.26 ppm); DMSO-d6
(2.49 ppm); D2O (4.75 ppm) for 13C (100.6 MHz) CDCl3
(77.0 ppm); DMSO-d6 (39.5 ppm). Mass spectra (MS)
were obtained on mass spectrometers, using electron im-
pact (EI, Finnigan MAT 8200) and electron spray (ESI,
Micromass LCT) for ionization.
was concentrated to give N-chloroacetyl-Gly-Tyr(t-
25
D
Bu)-OMe (16) (1.18 g, 61%). Colorless oil; ½aꢂ +85 (c
1
0.47, CH2Cl2). HPLC: tR 7.2 min; H NMR (CDCl3) d
7.27 (s, D2O exch., 1H); 6.97 (d, J = 8 Hz, 2H); 6.89
(d, J = 8 Hz, 2H); 6.48 (d, D2O exch., J = 7.5 Hz, 1H);
4.82 (dd, J = 6, 6 Hz, 1H); 4.04 (s, 2H); 3.95 (d,
J = 5 Hz, 1H); 3.69 (s, 3H); 3.08 (dd, J = 14, 6.5 Hz,
1H); 3.03 (dd, J = 14, 6.5 Hz, 1H); 1.30 (s, 9H); 13C
NMR (CDCl3) d 171.8, 167.8, 166.6, 154.4, 130.3,
129.6, 124.1, 78.4, 53.4, 52.3, 42.9, 42.2, 37.1, 28.7.
HRMS (EI) m/z: found 384.1448 (384.1452 calcd for
C18H25ClN2O5). LRMS (EI) m/z (rel abundance): 384
[M+H]+ (10), 178 (100), 151 (10), 107 (48).
4.1.1. NHS/EDCÆHCl-mediated coupling of N-chloroace-
tylglycine (11) with H-Trp-OMeÆHCl (12). To a stirred
solution of N-chloroacetylglycine (11, 303 mg, 2 mmol)
in dry THF (6 mL) were added NHS (231 mg, 2 mmol)
and EDCÆHCl (767 mg, 4 mmol). The mixture was stir-
red for an additional 18 h at rt, after which it was con-
centrated. The residue was dissolved in water (30 mL)
and was extracted against EtOAc (30 mL +;3· 20 mL).
The combined organic extract was dried and concen-
trated to afford 353 mg (71%) of O-hydroxysuccinimidyl
N-chloroacetylglycinate. The crude product was dis-
solved in dry THF (12 mL), the solution was cooled to
0 ꢁC, and H-Trp-OMeÆHCl (12, 360 mg, 1.41 mmol)
and Et3N (590 lL, 4.24 mmol) were added. The result-
ing mixture was stirred for 2 h at 0 ꢁC and for another
2 h at rt. The solvent was evaporated; the residue was
dissolved in EtOAc (30 mL) and was washed with water
(30 mL). The aqueous phase was extracted with EtOAc
(20 mL), the combined organic extract was dried, con-
centrated, and the residue was subjected to FCC on
50 g SiO2 (CH2Cl2/MeOH, 9:1). From chromatography
375 mg of N-chloroacetyl-Gly-Trp-OMe (13) was iso-
lated (74%, based on O-hydroxysuccinimidyl N-chloro-
4.1.3. Acylation of H-Phe-OEtÆHCl (19) and H-Lys(-
Boc)-OMeÆHCl (22) with chloroacetyl chloride (18). To
separate suspensions of H-Phe-OHÆHCl (19, 574 mg,
2.5 mmol) and H-Lys(Boc)-OMeÆHCl (22, 742 mg,
2.5 mmol) in dry DMF (2 mL each) was added Na2CO3
(530 mg, 5 mmol). The individual mixtures were stirred
for 30 min at rt followed by dropwise addition (over a
1 min period) of chloroacetyl chloride (18, 200 lL,
2.5 mmol). The mixtures were then stirred for 1 h at rt
after which time they were diluted with brine (50 mL)
and extracted with EtOAc (2· 30 mL). The combined
organic extracts were washed with brine (2· 50 mL),
dried, and concentrated to give N-chloroacetyl-Phe-
OEt (20) and N-chloroacetyl-Lys(Boc)-OMe (23),
respectively. Compound 20 was purified by crystalliza-
tion from CH2Cl2/hexane solution (524 mg, 78%), com-
pound 23 (809 mg, 96%) was used for the next step
without further purification.
acetylglycinate; 52%, based on N-chloroacetylglycine).
25
D
Colorless solid; ½aꢂ +26 (c 0.38, MeOH). HPLC: tR
1
7.3 min; H NMR (DMSO-d6) d 10.88 (s, D2O exch.,
1H); 8.39 (m, D2O exch., 1H); 7.47 (d, J = 8 Hz, 1H);
7.33 (d, J = 8 Hz, 1H); 7.15 (d, J = 2 Hz, 1H); 7.06
(dd, J = 8, 8 Hz, 1H); 6.98 (dd, J = 8, 8 Hz, 1H); 4.53
(m, 1H); 4.11 (s, 2H); 3.80 (dd, J = 16.5, 5.5 Hz, 1H);
3.73 (dd, J = 16.5, 5.5 Hz, 1H); 3.57 (s, 3H); 3.16 (dd,
J = 14.5, 5 Hz, 1H); 3.05 (dd, J = 14.5, 8 Hz, 1H); 13C
NMR (DMSO-d6) d 172.1, 168.3, 166.1, 136.1, 127.0,
123.8, 121.0, 118.4, 117.9, 111.4, 109.2, 53.1, 51.9,
42.5, 41.9, 27.1. HRMS (EI) m/z: found 351.0982
N-Chloroacetyl-Phe-OEt (20), white crystals, mp 55–
25
D
1
57 ꢁC (61–64 ꢁC, lit.11); ½aꢂ +55 (c 0.64, CH2Cl2).
HPLC: tR 9.1 min; For H NMR (CDCl3) and HRMS
(EI) data see reference11; 13C NMR (CDCl3) d 170.6,
165.4, 135.3, 129.0, 128.3, 127.0, 61.4, 53.2, 42.1, 37.5,
13.8.
N-Chloroacetyl-Lys(Boc)-OMe (23), colorless oil.
20
1
HPLC: tR 10.3 min; For ½aꢂ , H NMR (CDCl3), 13C
D
NMR (CDCl3), and HRMS (FAB) data see Ref. 12.

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M. Suchy et al. / Bioorg. Med. Chem. 16 (2008) 6156–6166
6162
4.1.4. Reaction of N-chloroacetyl-Gly-Trp-OMe (13), N-
chloroacetyl-Gly-Tyr(t-Bu)-OMe (16), N-chloroacetyl-
Phe-OEt (20) and N-chloroacetyl-Lys(Boc)-OMe (23)
with NaI. To separate solutions of N-chloroacetyl-Gly-
Trp-OMe (13, 718 mg, 2 mmol), N-chloroacetyl-Gly-
Tyr(t-Bu)-OMe (16, 770 mg, 2 mmol), N-chloroacetyl-
Phe-OEt (20, 539 mg, 2 mmol) and N-chloroacetyl-Lys(-
Boc)-OMe (23, 674 mg, 2 mmol) in acetone (12 mL) was
added NaI (899 mg, 6 mmol). The individual mixtures
were stirred for 18 h at rt and then were concentrated
to approximately 1/3 of their original volumes. The mix-
tures were each diluted with EtOAc (30 mL) and then
washed with 10% Na2SO3 solution (20 mL). The aque-
ous phases were extracted with EtOAc (20 mL) and
the combined organic extracts were dried and concen-
trated to give N-iodoacetyl compounds 14, 17, 21, and
24. These were purified as follows: N-iodoacetyl-Gly-
Trp-OMe (14), crystallization from acetone/hexane solu-
tion; N-iodoacetyl-Gly-Tyr(t-Bu)-OMe (17), FCC on
40 g SiO2 (CH2Cl2/MeOH, 19:1); N-iodoacetyl-Phe-
OEt (21), crystallization from dichloromethane/hexane
solution; N-iodoacetyl-Lys(Boc)-OMe (24), FCC on
40 g SiO2 (CH2Cl2/MeOH, 19:1) followed by crystalliza-
tion from dichloromethane/hexane solution.
J = 7 Hz, 3H); 13C NMR (CDCl3) d 171.0, 166.4,
135.4, 129.4, 128.6, 127.2, 61.7, 53.8, 37.7, 14.1. HRMS
(EI) m/z: found 361.0173 (361.0175 calcd for
C13H16INO3). LRMS (EI) m/z (rel abundance): 361
[M+H]+ (10), 288 (10), 176 (100), 131 (19), 120 (24).
N-Iodoacetyl-Lys(Boc)-OMe (24, 655 mg, 77%), color-
25
less crystals; mp 68–70 ꢁC; ½aꢂ +23 (c 0.64, CH2Cl2).
D
1H NMR (CDCl3) d 6.64 (m, D2O exch., 1H); 4.55 (m,
1H); 3.75 (s, 3H); 3.74 (d, J = 11.5 Hz, 1H); 3.70 (d,
J = 11.5 Hz, 1H); 3.11 (d, J = 6 Hz, 1H); 3.08 (d,
J = 6 Hz, 1H); 1.89 (m, 1H); 1.72 (m, 1H); 1.42 (s,
9H); 1.40 (m, 4H); 13C NMR (CDCl3) d 172.4, 167.4,
156.0, 79.0, 52.4, 39.9, 31.5, 29.4, 28.3, 22.2. HRMS
(EI) m/z: found 429.0873 (429.0842 calcd for
C14H25IN2O5). LRMS (EI) m/z (rel abundance): 429
[M+H]+ (10), 355 (23), 245 (45), 184 (33), 142 (100),
84 (67).
4.1.5. Synthesis of N-iodoacetyl-Gly-Phe-Lys(Boc)-OMe
(26). To a stirred suspension of N-chloroacetylglycine
(11, 303 mg, 2 mmol) and NHS (230 mg, 2 mmol) in
dry CH2Cl2 (10 mL) at 0 ꢁC was added DCC (454 mg,
2.2 mmol). The cooling bath was removed and the mix-
ture was stirred for 18 h at rt. The solvent was evapo-
rated, the residue was suspended in dry THF (8 mL),
and then H-Phe-OH (25, 330 mg, 2 mmol) and Et3N
(560 lL, 4 mmol) were added. The mixture was stirred
for 2 h at rt after which time it was diluted with 1 M
HCl (50 mL) and was extracted with EtOAc (40 + 3·
20 mL). The combined organic extract was dried, con-
centrated, and the residue was dissolved in dry THF
(20 mL). The solution thus obtained was cooled to
0 ꢁC, NHS (230 mg, 2 mmol) and DCC (495 mg,
2.4 mmol) were added, and the mixture was stirred for
30 min at 0 ꢁC, followed by the addition of H-Lys(-
Boc)-OMeÆHCl (22, 594 mg, 2 mmol) and Et3N
(560 lL, 4 mmol). The cooling bath was removed and
the mixture was stirred for 18 h at rt. The mixture was
concentrated to about 1/3 of its original volume, diluted
with brine (50 mL), and extracted with CHCl3 (40 + 2·
20 mL). The combined organic extracts were dried, con-
centrated, and the residue obtained [N-chloroacetyl-
Gly-Phe-Lys(Boc)-OMe containing ca. 20% of DCU
(by 1H NMR)] was dissolved in acetone (15 mL). To this
mixture was added NaI (899 mg, 6 mmol) and the resul-
tant suspension was stirred for 18 h at rt. The mixture
was then concentrated to ca. 1/3 of its original volume,
diluted with EtOAc (30 mL), and washed with 10%
Na2SO3 solution (20 mL). The aqueous phase was ex-
tracted with EtOAc (20 mL), and the combined organic
extract was dried and concentrated to give crude N-iod-
oacetyl compound 26. Compound 26 was purified by
FCC on 50 g SiO2 (CH2Cl2/MeOH, 19:1) followed by
crystallization from dichloromethane/hexane solution.
N-Iodoacetyl-Gly-Trp-OMe (14, 664 mg, 75%), color-
25
less crystals; mp 175–177 ꢁC; ½aꢂ +21 (c 0.48, MeOH).
D
HPLC: tR 7.9 min; 1H NMR (DMSO-d6) d 10.87 (s,
D2O exch., 1H); 8.45 (t, D2O exch., J = 5.5 Hz, 1H);
8.35 (d, D2O exch., J = 7.5 Hz, 1H); 7.46 (d, J = 8 Hz,
1H); 7.32 (d, J = 8 Hz, 1H); 7.15 (d, J = 2 Hz, 1H);
7.05 (dd, J = 8, 8 Hz, 1H); 6.97 (dd, J = 8, 8 Hz, 1H);
4.52 (m, 1H); 3.75 (dd, J = 16.5, 5.5 Hz, 1H); 3.69 (m,
3H); 3.15 (dd, J = 14.5, 6 Hz, 1H); 3.04 (dd, J = 14.5,
8 Hz, 1H); 13C NMR (DMSO-d6) d 172.1, 168.4,
167.8, 136.1, 127.0, 123.8, 121.0, 118.4, 117.9, 111.4,
109.1, 53.1, 51.8, 42.1, 30.7, 27.1. HRMS (EI) m/z:
found 443.0347 (443.0342 calcd for C16H18IN3O4).
LRMS (EI) m/z (rel abundance): 443 [M+H]+ (10),
201 (42), 130 (100).
N-Iodoacetyl-Gly-Tyr(t-Bu)-OMe (17, 884 mg, 93%),
25
colorless solid; ½aꢂ +50 (c 0.60, CH2Cl2). HPLC: tR
D
7.8 min; 1H NMR (CDCl3) d 7.11 (t, D2O exch.,
J = 5 Hz, 1H); 7.00 (d, J = 8 Hz, 2H); 6.90 (d,
J = 8 Hz, 2H); 6.75 (d, D2O exch., J = 8 Hz, 1H); 4.81
(dd, J = 6, 6 Hz, 1H); 3.95 (dd, J = 16.5, 5 Hz, 1H);
3.88 (dd, J = 16.5, 5 Hz, 1H); 3.71 (s, 2H); 3.68 (s,
3H); 3.08 (dd, J = 14, 6 Hz, 1H); 3.03 (dd, J = 14,
6.5 Hz, 1H); 1.31 (s, 9H); 13C NMR (CDCl3) d 171.7,
168.4, 168.3, 154.2, 130.5, 129.6, 124.1, 78.4, 53.6,
52.3, 43.4, 37.1, 28.7. HRMS (EI) m/z: found 476.0801
(476.0808 calcd for C18H25IN2O5). LRMS (EI) m/z (rel
abundance): 476 [M+H]+ (10), 420 (10), 243 (37), 178
(100), 107 (42).
N-Iodoacetyl-Phe-OEt (21, 568 mg, 82%), colorless crys-
N-Iodoacetyl-Gly-Phe-Lys(Boc)-OMe [26, 670 mg, 52%
based on N-chloroacetylglycine (11)], colorless crystals;
25
D
tals; mp 60–61 ꢁC; ½aꢂ +68 (c 0.44, CH2Cl2). HPLC: tR
25
D
1
1
10.3 min; H NMR (CDCl3) d 7.27 (m, 3H); 7.14 (m,
2H), 6.48 (d, D2O exch., 6.5 Hz, 1H); 4.83 (dd, J = 6,
5.5 Hz, 1H); 4.19 (q, J = 7 Hz, 2H); 3.69 (d,
J = 11.5 Hz, 1H); 3.65 (d, J = 11.5 Hz, 1H); 3.17 (dd,
J = 14, 6 Hz, 1H); 3.12 (dd, J = 14, 6 Hz, 1H); 1.26 (t,
mp 110–112 ꢁC; ½aꢂ +36 (c 0.42, CH2Cl2). H NMR
(DMSO-d6) d 8.42 (m, D2O exch., 2H); 8.28 (m, D2O
exch., 1H); 8.13 (m, D2O exch., 1H); 7.21 (m, 4H);
6.79 (m, 1H); 4.56 (m, 1H); 4.18 (m, 1H); 3.67 (s, 2H);
3.65 (m, 2H); 3.60 (s, 3H); 2.99 (m, 1H); 2.86 (m, 2H);

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M. Suchy et al. / Bioorg. Med. Chem. 16 (2008) 6156–6166
6163
2.71 (m, 1H); 1.61 (m, 3H); 1.34 (s, 9H); 1.29 (m, 3H);
13C NMR (DMSO-d6) d 172.3, 171.2, 168.5, 168.1,
167.8, 155.5, 137.6, 129.2, 128.0, 126.2, 77.3, 53.5,
52.1, 51.8, 42.2, 42.1, 37.6, 30.7, 30.5, 29.1, 28.3, 22.7.
HRMS (ESI) m/z: found 655.1596 [M+Na]+ (655.1605
calcd for C25H37IN4O7Na).
14, 17, 21, 24, 26 or 17 mg, 0.1 mmol for electrophile
29) and DIPEA (14, 17, 21, 24, and 26, 175 lL, 1 mmol;
29, 70 lL, 0.4 mmol) in MeCN (14, 17, 21, 24, and 26,
5 mL; 29, 3 mL) were added four equivalents of the N-
iodoacetyl compounds 14, 17, 21, 24, 26 (1 mmol), and
29 (0.4 mmol). The reaction mixtures were stirred at ele-
vated temperature as follows: N-iodoacetyl-Gly-Trp-
OMe (14), 72 h at 50 ꢁC; N-iodoacetyl-Gly-Tyr(t-Bu)-
OMe (17) and N-iodoacetyl-Gly-Phe-Lys(Boc)-OMe
(26), 24 h at 70 ꢁC; N-iodoacetyl-Phe-OEt (21) and N-
iodoacetyl-Lys(Boc)-OMe (24) 18 h at 50 ꢁC; N-iodo-
acetyl-Lys(Boc)-Phe-OEt (29), 48 h at 70 ꢁC. The reac-
tion mixture obtained after alkylation of cyclen with
N-iodoacetyl-Gly-Trp-OMe (14) was cooled to rt,
poured on crushed ice (100 mL), and the resulting pre-
cipitate was isolated by vacuum filtration. After washing
the precipitate with water, tetraacetyl-Gly-Trp-OMe cy-
clen (31) of satisfactory purity was obtained. The reac-
tion mixtures obtained from alkylation of cyclen with
N-iodoacetyl compounds 17, 21, 24, and 26 were cooled
to rt, diluted with EtOAc (30 mL), and washed with
water (30 mL). The aqueous phases were extracted with
EtOAc (20 mL) and combined organic extracts were
dried and concentrated. The peralkylated cyclens 32–
35 were purified by repeated trituration (twice) in hex-
anes. The reaction mixture obtained after alkylation of
cyclen with N-iodoacetyl-Lys(Boc)-Phe-OEt (29) was
cooled to rt and the precipitate was isolated by filtration.
The precipitate was washed with cold acetonitrile to give
tetraacetyl-Lys(Boc)-Phe-OEt cyclen (36) of satisfactory
purity.
4.1.6. Synthesis of N-iodoacetyl-Lys(Boc)-Phe-OEt (29).
To a stirred suspension of chloroacetic acid (27, 190 mg,
2 mmol) and NHS (230 mg, 2 mmol) in dry CH2Cl2
(10 mL) at 0 ꢁC was added DCC (495 mg, 2.4 mmol).
The cooling bath was removed and the mixture was stir-
red for 18 h at rt. The solvent was evaporated, the resi-
due was suspended in dry THF (10 mL), and H-
Lys(Boc)-OH (28, 493 mg, 2 mmol) and Et3N (560 lL,
4 mmol) were added. The resultant mixture was stirred
for 4 h at rt after which it was diluted with 1 M HCl
(30 mL) and was extracted with EtOAc (2· 20 + 3·
10 mL). The combined organic extract was dried, con-
centrated, and the residue was dissolved in dry THF
(10 mL). The solution thus obtained was cooled to
0 ꢁC, NHS (230 mg, 2 mmol) and DCC (495 mg,
2.4 mmol) were added, and the mixture was stirred for
30 min at 0 ꢁC and for 18 h at rt. The mixture was di-
luted with brine (30 mL) and then extracted with EtOAc
(30 + 2· 20 mL). The organic extract was dried and con-
centrated and the residue was dissolved in dry THF
(10 mL) followed by the addition of H-Phe-OEtÆHCl
(19, 459 mg, 2 mmol) and Et3N (1.12 mL, 8 mmol).
The mixture was stirred for 4 h at rt, was diluted with
saturated NaHCO3 solution (30 mL) and was extracted
with EtOAc (30 + 2· 20 mL). The combined organic ex-
tract was dried, concentrated and the residue was sub-
jected to FCC on 30 g SiO2 (hexane/acetone, 2:1). The
product obtained [N-chloroacetyl-Lys(Boc)-Phe-OEt
Tetraacetyl-Gly-Trp-OMe cyclen (31, 335 mg, 93%);
25
D
colorless solid; ½aꢂ +26 (c 0.39, MeOH). HPLC: tR
1
24.7 min; H NMR (DMSO-d6) d 10.86 (s, D2O exch.,
4H); 8.38 (m, D2O exch., 2H); 8.24 (m, D2O exch.,
4H); 8.05 (m, D2O exch., 2H); 7.44 (d, J = 7.5 Hz,
4H); 7.30 (m, 4H); 7.12 (s, 4H); 7.04 (dd, J = 7.5,
7.5 Hz, 4H); 6.95 (dd, J = 7.5, 7.5 Hz, 4H); 4.51 (m,
4H); 3.73 (m, 8H); 3.54 (m, 20H); 3.15–2.84 (br m,
24H); 13C NMR (DMSO-d6) d 172.2, 171.0, 169.3,
169.0, 168.8, 136.1, 127.1, 123.8, 121.1, 118.6, 118.0,
111.5, 109.2, 53.2, 53.1, 51.9, 50.9, 41.6, 27.2. HRMS
(ESI) m/z: found 1433.6708 [M+H]+ (1433.6642 calcd
for C72H89N16O16).
1
containing ca. 10% of DCU (by H NMR)] was dis-
solved in acetone (7 mL) and NaI (513 mg, 3.42 mmol)
was added. The mixture was stirred for 18 h at rt and
was diluted with EtOAc (30 mL). The organic phase
was washed with 10% Na2SO3 solution (20 mL) and
aqueous phase was extracted with EtOAc (20 mL).
The combined organic extract was dried and was con-
centrated to give crude N-iodoacetyl compound 29,
which was subsequently purified by FCC on 30 g SiO2
(hexane/acetone, 2:1).
N-Iodoacetyl-Lys(Boc)-Phe-OEt [29, 467 mg, 40% based
Tetraacetyl-Gly-Tyr(t-Bu)-OMe cyclen (32, 381 mg,
25
D
25
D
on chloroacetic acid (27)], colorless solid; ½aꢂ +36 (c
99%); colorless solid; ½aꢂ +38 (c 0.66, CH2Cl2). HPLC:
1
1
0.56, CH2Cl2). H NMR (CDCl3) d 7.26 (m, 3H); 7.11
(m, 2H); 6.78 (d, D2O exch., J = 7.5 Hz, 1H); 6.47 (d,
D2O exch., J = 7.5 Hz, 1H); 4.81 (m, 1H); 4.68 (br s,
D2O exch., 1H); 4.37 (m, 1H); 4.17 (q, J = 7 Hz, 2H);
3.66 (s, 2H); 3.16–3.01 (m, 4H); 1.82 (m, 1H); 1.61 (m,
1H); 1.46 (m, 2H); 1.42 (s, 9H); 1.33 (m, 2H); 1.23 (t,
J = 7 Hz); 13C NMR (CDCl3) d 171.3, 171.0, 167.9,
156.0, 135.7, 129.2, 128.4, 127.0, 78.8, 61.4, 53.5, 53.2,
39.9, 37.7, 32.1, 29.4, 28.4, 22.5, 22.2, 14.0. HRMS
(ESI) m/z: found 612.1537 [M+Na]+ (612.1547 calcd
for C24H36IN3O6Na).
tR 25.8 min; H NMR (CDCl3) d 8.03 (br s, D2O exch.,
4H); 7.77 (br s, D2O exch., 4H); 7.04 (m, 8H); 6.87 (d,
J = 8 Hz, 8H); 4.73 (m, 4H); 3.84 (m, 8H); 3.63 (s,
12H); 3.62–3.64 (m, 8H); 3.05–2.60 (br m, 24H); 1.29
(s, 36H); 13C NMR (CDCl3) d 172.2, 169.1, 154.1,
129.6, 124.1, 114.9, 78.3, 53.6, 52.2, 46.3, 42.4, 36.9,
28.7. HRMS (ESI) m/z: found 1565.8562 [M+H]+
(1565.8507 calcd for C80H117N12O20).
Tetraacetyl-Phe-OEt cyclen (33, 246 mg, 89%); colorless
25
D
solid; ½aꢂ +46 (c 0.44, CH2Cl2). HPLC: tR 27.0 min; 1H
NMR (CDCl3) d 7.57 (br s, D2O exch., 4H); 7.23 (m,
16H); 7.10 (m, 4H); 4.83 (m, 4H); 4.13 (q, J = 7 Hz,
8H); 3.18–2.95 (m, 12H); 2.57–2.33 (m, 12H); 1.21 (t,
J = 7 Hz, 12H); 13C NMR (CDCl3) d 171.4, 171.1,
4.1.7. Peralkylation of cyclen (30) with N-iodoacetyl
compounds 14, 17, 21, 24, 26, and 29. To separate solu-
tions of cyclen 30 (43 mg, 0.25 mmol for electrophiles

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M. Suchy et al. / Bioorg. Med. Chem. 16 (2008) 6156–6166
6164
170.3, 136.4, 136.0, 129.2, 128.8, 128.0, 126.5, 61.0, 56.3,
52.7, 51.8, 37.0, 13.7. HRMS (ESI) m/z: found 1127.5836
[M+Na]+ (1127.5793 calcd for C60H80N8O12Na).
0.1 mmol) in CH2Cl2 (2 mL) was added TFA (1 mL).
The mixture was stirred for 3 h at rt, then the solvent
was evaporated, and the residue was dissolved in THF
(2 mL). To this solution was added NaOH (1 M,
2 mL) and the mixture was vigorously stirred for 1 h
at rt. The THF was evaporated and the aqueous residue
was cooled to 0 ꢁC. The pH was adjusted to 6 (1 M HCl)
and the mixture was set aside for 1 h at 0 ꢁC. After the
aqueous phase was decanted, an oily precipitate that
had deposited on the flask walls was triturated with hex-
Tetraacetyl-Lys(Boc)-OMe cyclen (34, 340 mg, 99%);
25
D
colorless solid; ½aꢂ +12 (c 0.41, CH2Cl2). 1H NMR
(CDCl3) d 7.72 (br s, D2O exch., 4H); 4.91 (m, D2O
exch., 4H); 4.44 (m, 4H); 3.85–3.65 (br m, 8H); 3.72 (s,
12H); 3.22–2.67 (br m, 24H); 1.83 (m, 12H); 1.56–1.26
(m, 12H); 1.43 (s, 36H); 13C NMR (CDCl3) d 172.5,
156.0, 78.8, 56.5, 53.3, 52.3, 51.6, 39.9, 30.7, 29.3, 28.4,
28.3, 22.8. HRMS (ESI) m/z: found 1373.8469 [M+H]+
(1373.8507 calcd for C64H117N12O20).
anes to afford tetraacetyl-Gly-Trp-OH cyclen (6, 70 mg,
25
51%). Colorless solid; ½aꢂ +65 (c 0.31, DMSO). HPLC:
D
Method A: tR 5.7 min; Method B: tR 3.7 min; 1H NMR
(DMSO-d6) d 8.25 (br s, D2O exch., 4H); 8.27 (br s, D2O
exch., 4H); 8.19 (m, D2O exch., 4H); 6.98 (d, J = 8.5 Hz,
8H); 6.63 (d, J = 8.5 Hz, 8H); 4.35 (m, 4H); 3.74 (m,
8H); 3.49–3.23 (m, 8H); 3.15–2.71 (br m, 24H); 13C
NMR (DMSO-d6) d 172.9, 171.8, 168.8, 156.1, 130.1,
127.3, 115.2, 57.0, 54.0, 46.3, 41.7, 36.2. HRMS (ESI)
m/z: found 1285.5415 [M+H]+ (1285.5377 calcd for
C60H77N12O20).
Tetraacetyl-Gly-Phe-Lys(Boc)-OMe cyclen (35, 466 mg,
25
D
94%); colorless solid; ½aꢂ +10 (c 0.49, CH2Cl2). 1H
NMR (DMSO-d6) d 8.41–8.20 (br m, D2O exch.,
12H); 7.19 (m, 16H); 6.74 (m, 4H); 4.58 (br s, D2O exch.,
4H); 4.19 (m, 4H); 3.81–3.54 (br m, 16H); 3.59 (s, 12H);
3.21–2.49 (br m, 32H); 1.77–1.57 (br m, 12H); 1.49–1.12
(br m, 12H); 1.34 (s, 36H); 13C NMR (DMSO-d6) d
172.5, 172.3, 170.1, 169.8, 169.5, 155.6, 129.2, 128.0,
115.8, 77.4, 52.0, 51.8, 41.4, 37.6, 34.3, 33.0, 31.1, 30.7,
29.2, 28.3, 22.3. HRMS (ESI) m/z: found 2212.1941
[M+Na]+ (2212.1922 calcd for C108H164N20O28Na).
4.1.10. Tetraacetyl-Phe-OH cyclen (7). A solution of
NaOH (2.5 M, 1.6 mL) was added to a solution of tetra-
acetyl-Phe-OEt cyclen (33, 180 mg, 0.16 mmol) in THF
(1.6 mL). The mixture was vigorously stirred for 2 h at
rt, the THF was evaporated and the aqueous residue
was cooled to 0 ꢁC. The pH was adjusted to 5 (1 M
HCl) and the mixture was transferred to a centrifuge
tube and was centrifuged for 5 min at 1000 rpm. The
supernatant was decanted and the pellet was triturated
Tetraacetyl-Lys(Boc)-Phe-OEt cyclen (36, 129 mg,
25
D
64%); colorless solid; ½aꢂ ꢀ8 (c 0.65, CH2Cl2). 1H
NMR (CDCl3) d 7.70 (br s, D2O exch., 8H); 7.28–7.12
(m, 20H); 4.93 (m, 4H); 4.75 (br s, D2O exch., 4H);
4.43 (m, 4H); 4.12 (m, 8H); 3.11–3.00 (br m, 28H);
2.62 (br s, 12H); 1.76–1.65 (br m, 8H); 1.41 (s, 36H);
1.35–1.13 (m, 28H); 13C NMR (CDCl3) d 171.5, 171.3,
155.9, 135.9, 129.1, 128.4, 126.9, 78.8, 61.3, 58.7, 53.4,
53.2, 53.1, 40.0, 37.7, 31.5, 29.3, 28.4, 22.8, 13.9. HRMS
(ESI) m/z: found 2018.1862 [M+H]+ (2018.1870 calcd
for C104H161N16O24).
with hexanes to afford tetraacetyl-Phe-OH cyclen (7,
25
D
110 mg, 69%). Colorless solid; ½aꢂ ꢀ37 (c 0.81,
CH2Cl2). HPLC: Method A: tR 17.1 min; Method B:
tR 4.1 min; 1H NMR (DMSO-d6) d 8.36 (br s, D2O
exch., 4H); 7.16 (m, 20H); 4.43 (m, 4H); 3.74–2.56 (br
m, 32H); 13C NMR (DMSO-d6) d 173.0, 169.8, 137.7,
129.2, 128.2, 126.1, 56.2, 52.4, 50.6, 48.2, 37.4. HRMS
(ESI) m/z: found 993.4673 [M+H]+ (993.4722 calcd for
C52H65N8O12).
4.1.8. Tetraacetyl-Gly-Trp-OH cyclen (5). A solution of
NaOH (1 M, 1.58 mL) was added to a solution of tetra-
acetyl-Gly-Trp-OMe cyclen (31, 113 mg, 0.079 mmol) in
THF (2.5 mL). The mixture was vigorously stirred for
1 h at rt and then the THF was evaporated leaving an
aqueous residue that was cooled to 0 ꢁC. The pH was
adjusted to 2 and the mixture was set aside for 1 h at
0 ꢁC after which a precipitate was isolated by filtration,
washed with water, and dried to afford tetraacetyl-Gly-
4.1.11. Boc-deprotection and hydrolysis of peralkylated
cyclens 34–36. Tetraacetyl-Lys(Boc)-OMe (34, 137 mg,
0.1 mmol),
tetraacetyl-Gly-Lys(Boc)-Phe-OEt
(35,
150 mg, 0.068 mmol) and tetraacetyl-Lys(Boc)-Phe-
OEt (36, 151 mg, 0.075 mmol) were dissolved in TFA
(1.5 mL, compounds 34 and 35) or in CH2Cl2 (2 mL)
and TFA (1 mL, compound 36) and the reaction mix-
ture was stirred for 15 min at rt. The solvents were evap-
orated, the residues were dissolved in MeOH (34, 1 mL;
35, 700 lL) or THF (36, 1.5 mL), and NaOH solutions
(2.5 M; 34, 1 mL; 35, 700 lL or 1 M; 36, 1.5 mL) were
added. The mixtures were stirred for 2 h at 60 ꢁC (com-
pounds 34 and 35) or for 1 h at rt (compound 36). The
hydrolysis mixtures were then cooled to 0 ꢁC and the pH
was adjusted to 6 (1 M HCl). The resultant aqueous
solutions were subjected for size exclusion chromatogra-
phy; fractions containing the products (identified by nin-
hydrin test) were combined and were lyophilized to
afford ligands 8–10.
Trp-OH cyclen (5, 81 mg, 74%) of sufficient purity. Col-
25
orless solid; ½aꢂ +31 (c 0.32, DMSO). HPLC: Method
D
A: tR 19.7 min; Method B: tR 2.6 min; 1H NMR
(DMSO-d6) d 10.84 (s, D2O exch., 4H); 8.28 (m, D2O
exch., 4H); 8.21 (m, D2O exch., 4H); 7.49 (d, J = 8 Hz,
4H); 7.30 (d, J = 8 Hz, 4H); 7.12 (s, 4H); 7.03 (dd,
J = 8, 8 Hz, 4H); 6.94 (dd, J = 8, 8 Hz, 4H); 4.48 (m,
4H); 3.74 (m, 8H); 3.46 (m, 8H); 3.17–2.98 (br m,
12H); 2.82 (m, 12H); 13C NMR (DMSO-d6) d 173.2,
170.8, 168.6, 136.1, 127.3, 123.8, 121.0, 118.5, 118.2,
111.5, 109.6, 53.1, 51.1, 51.0, 41.7, 27.2. HRMS (ESI)
m/z: found 1377.6010 [M+H]+ (1377.6016 calcd for
C68H81N16O16).
4.1.9. Tetraacetyl-Gly-Tyr-OH cyclen (6). To a solution
of tetraacetyl-Gly-Tyr(t-Bu)-OMe cyclen (32, 157 mg,
Tetraacetyl-Lys-OH cyclen (8, 78 mg, 85%); colorless
oil; ½aꢂ ꢀ9 (c 0.55, H2O). H NMR (D2O) d 4.00 (m,
25
D
1

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M. Suchy et al. / Bioorg. Med. Chem. 16 (2008) 6156–6166
6165
4H); 3.83–2.72 (br m, 24H); 1.72–1.59 (m, 16H); 1.37–
1.24 (m, 8H); 13C NMR (D2O) d 163.1, 54.0, 38.1,
37.3, 30.0, 25.2, 22.6, 21.2, 13.4; HRMS (ESI) m/z:
found 917.5826 [M+H]+ (917.5784 calcd for
C40H77N12O12).
24.61 (s); 22.45 (s); 17.77 (s); 17.55 (s); 10.04 (s); 7.88
(s); 7.21–6.90 (m); 6.42–6.29 (m); 5.97 (s); 4.04–0.68
(m); ꢀ1.73 (s); ꢀ2.60 (s); ꢀ2.82 (s); ꢀ3.98 (s); ꢀ5.81
(s); ꢀ8.59 (s); ꢀ9.22 (s); ꢀ9.64 (s); ꢀ10.32 (s); ꢀ11.45
(s); ꢀ12.01 (s); ꢀ13.03 (s); ꢀ13.94 (s); ꢀ19.91 (s).
1
Tetraacetyl-Gly-Phe-Lys-OH cyclen (9, 78 mg, 66%);
Tm3+ DOTAM-Lys-OH (40). Colorless oil. H NMR
25
D
1
colorless oil; ½aꢂ +5 (c 0.92, H2O). H NMR (D2O) d
(D2O) d 45.27 (s); 34.30 (s); 3.78–1.92 (m); 1.43–1.01
(m); ꢀ0.08 (s); ꢀ1.19 (s); ꢀ4.67 (s); ꢀ8.50 (s); ꢀ10.13
(s); ꢀ14.23 (s); ꢀ81.56 (s); ꢀ94.42 (s).
7.13 (m, 20H); 3.98–3.69 (m, 8H); 3.40–2.63 (br m,
48H); 1.61–1.44 (m, 16H); 1.26–1.12 (m, 8H); 13C
NMR (D2O) d 173.2, 173.1, 163.6, 163.2, 162.9, 162.5,
129.5, 128.9, 121.0, 118.1, 115.2, 112.3, 55.2, 55.1,
53.2, 42.5, 39.5, 34.5, 31.4, 31.2, 26.6, 22.6, 22.4, 22.3,
22.1; HRMS (ESI) m/z: found 1734.9384 [M+H]+
(1734.9457 calcd for C84H126N20O20).
Eu3+ DOTAM-Gly-Phe-Lys-OH (41). Colorless oil. H
1
NMR (D2O) d 8.17 (s); 7.21–6.81 (m); 3.94–2.30 (m);
1.86 (s); 1.70–0.72 (m); ꢀ3.05 to ꢀ3.57 (m); ꢀ8.82 to
ꢀ9.08 (m); ꢀ12.13 to ꢀ12.69 (m).
Tetraacetyl-Lys-Phe-OH cyclen (10, 100 mg, 88%); color-
Eu3+ DOTAM-Lys-Phe-OH (42). Colorless solid. 1H
NMR (D2O) d 19.04 (s); 8.39 (s); 7.67 (s); 7.27–6.98
(m); 3.18 (s); 2.85–2.44 (m); 2.03 (s); 1.58 (s); 1.20–0.95
(m); 0.22–0.09 (m); ꢀ1.63 (s); ꢀ3.09 (s); ꢀ4.45 (s);
ꢀ6.36 (s); ꢀ9.80 (s); ꢀ10.28 (s); ꢀ11.33 (s); ꢀ12.20 (s).
25
D
less solid; ½aꢂ ꢀ17 (c 0.60, H2O). 1H NMR (D2O) d 7.45–
7.23 (m, 20H); 4.48–4.45 (m, 4H); 3.92–3.88 (m, 4H);
3.60–2.64 (br m, 40H); 1.81–1.50 (m, 12H); 1.28–1.07
(m, 12H); 13C NMR (D2O) d 163.7, 163.4, 163.0, 162.7,
130.0, 128.9, 118.1, 115.2, 55.2, 39.5, 39.3, 38.3, 30.6,
26.8, 26.5, 22.4, 21.6; HRMS (ESI) m/z: found
1506.8600 [M+2H]+ (1506.8599 calcd for C76H114N16O16).
Acknowledgments
4.1.12. Metalation of ligands 5–10. Separate solutions of
tetraacetyl-Gly-Trp-OH cyclen (5, 58 mg, 0.042 mmol),
tetraacetyl-Gly-Tyr-OH cyclen (6, 70 mg, 0.054 mmol),
tetraacetyl-Phe-OH cyclen (7, 60 mg, 0.06 mmol), tetra-
acetyl-Lys-OH cyclen (8, 92 mg, 0.1 mmol), tetraacetyl-
Gly-Phe-Lys-OH cyclen (9, 119 mg, 0.068 mmol) and
tetraacetyl-Lys-Phe-OH cyclen (10, 90 mg, 0.06 mmol)
in water (5 and 6, 5 mL; 7, 4 mL; 8and 10, 3 mL; 9,
2 mL) were treated with lanthanide(III) chloride hexa-
hydrates as follows: 5, EuCl3Æ6H2O, 77 mg, 0.21 mmol;
6, EuCl3Æ6H2O, 24 mg, 0.065 mmol; 7, EuCl3Æ6H2O,
44 mg, 0.12 mmol; 8, TmCl3Æ6H2O, 58 mg, 0.15 mmol;
9, EuCl3Æ6H2O, 25 mg, 0.068 mmol; 10, EuCl3Æ6H2O,
22 mg, 0.06 mmol. The reaction mixtures were treated
with NaOH (2.5 M solution) to adjust the pH to 9 and
were stirred for 18 h at 50 ꢁC (ligand 5) or at rt (ligands
6–10) followed by size exclusion chromatography. The
fractions containing the lanthanide(III) complexes
(identified by UV/I2 vapors, complexes 37–39; ninhydrin
test, complexes 40–42) were combined and were freeze-
dried to afford complexes 37–42. Selected data are re-
ported in Table 1.
The authors thank the Ontario Research and Develop-
ment Challenge Fund, the Canadian Institutes of Health
Research Strategic Training Program in Cancer Re-
search and Technology Transfer, and The Ontario Insti-
tute for Cancer Research for supporting this work. Mr.
Doug Hairsine of the Department of Chemistry Mass
Spectrometry facility at the University of Western On-
tario is thanked for expert assistance with collection
and analysis of mass spectral data. We also thank Mr.
Filip Wojciechowski for generously providing com-
pound 19.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2008.
04.038.
References and notes
1. Zhang, S.; Merritt, M.; Woessner, D. E.; Lenkinski, R. E.;
Sherry, A. D. Acc. Chem. Res. 2003, 36, 783–790.
2. Ward, K. M.; Aletras, A. H.; Balaban, R. S. J. Magn.
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Eu3+ DOTAM-Gly-Trp-OH (37). Colorless solid.
HPLC (Method B): tR 4.0 min. HPLC analysis indicated
the presence of ca. 15% of free ligand 5. The H NMR
1
3. Zhang, S.; Malloy, C. R.; Sherry, A. D. J. Am. Chem. Soc.
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Eu3+ DOTAM-Gly-Tyr-OH (38). Colorless solid.
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44.88 (s); 34.15 (s); 24.42 (s); 22.41 (s); 8.24 (s); 7.24
(s); 6.70–6.61 (m); 3.25–2.82 (m); 2.39–2.03 (m); 1.73–
1.02 (m); ꢀ3.01 (s); ꢀ3.52 (s); ꢀ3.94 (s); ꢀ4.95 (s);
ꢀ8.14 (s); ꢀ8.84 (s); ꢀ9.03 (s); ꢀ9.72 (s); ꢀ12.03 (s);
ꢀ12.92 (s); ꢀ81.04 (s).
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Angew. Chem., Int. Ed. 2002, 41, 4334–4336; (b) Aime, S.;
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1
(Method B): tR 6.2 min. H NMR (D2O) d 27.11 (s);

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