C-Functionalized chiral dioxocyclam and cyclam derivatives with 1,2,3-triazole units: Synthesis, complexation properties and crystal structures of copper(ii) complexes

Article abstract of DOI:10.1039/c5nj01927c

New C-functionalized syn- and anti-dioxocyclam and cyclam derivatives with 1,2,3-triazole units attached to carbon atoms within the skeleton were designed as valuable bifunctional chelating agents for applications in nuclear medicine. These macrocyclic chelators were prepared via a multi-step sequence involving α- and β-amino acids, and their copper(ii) complexation properties were evaluated. A solution structure in which the triazoles are in axially coordinating positions was proposed for the [Cu(anti-27)]²⁺ complex. Promising results have been obtained regarding the complexation kinetics (<10 s) and the pseudo-first order half-life for acid-assisted dissociation (t_1/2 = 3.21 d, 5 M HCl, 50°C).

Full text of DOI:10.1039/c5nj01927c

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C-Functionalized chiral dioxocyclam and cyclam
derivatives with 1,2,3-triazole units: synthesis,
complexation properties and crystal structures
of copper(II) complexes†
Cite this:DOI: 10.1039/c5nj01927c
A.-S. Felten,ab N. Petry,ab B. Henry,ab N. Pellegrini-Moıse*ab and K. Selmeczi*ab
¨
New C-functionalized syn- and anti-dioxocyclam and cyclam derivatives with 1,2,3-triazole units
attached to carbon atoms within the skeleton were designed as valuable bifunctional chelating agents
for applications in nuclear medicine. These macrocyclic chelators were prepared via a multi-step
sequence involving a- and b-amino acids, and their copper(II) complexation properties were evaluated.
A solution structure in which the triazoles are in axially coordinating positions was proposed for
the [Cu(anti-27)]2+ complex. Promising results have been obtained regarding the complexation kinetics
(o10 s) and the pseudo-first order half-life for acid-assisted dissociation (t1/2 = 3.21 d, 5 M HCl, 50 1C).
Received (in Montpellier, France)
22nd July 2015,
Accepted 24th November 2015
DOI: 10.1039/c5nj01927c
www.rsc.org/njc
toward certain metal ions.6 Due to their strong metal sequestration
capacities, N-polysubstituted cyclen and cyclam derivatives (e.g.
Introduction
Since the discovery of Cu-catalyzed azide–alkyne cycloaddition DOTA, TETA and CB-TE2A) are the most widely used molecular
(AAC), allowing the regioselective synthesis of 1,4-disubstituted systems in various domains, notably waste water treatment,7
1,2,3-triazoles, the latter have become a powerful tool in the catalysis,8 and many medical applications such as molecular
chemical ligation of two molecules and have found multiple imaging (PET and SPECT).9–14 The latter applications require
applications. Besides their use as stable linkers, the metal systems which contain two different kinds of functional groups,
complexation capacity of 1,2,3-triazoles has also been explored allowing fixation of the radiometal and connection to a biomolecule
by many research groups.1–4 Indeed, both N2 and N3 nitrogen via a coupling functional group, and thus these systems are
and C5 carbon atoms are capable of coordinating to metal ions. commonly called bifunctional chelators (BFCs).9–14 DOTA and
‘‘Regular’’ triazoles, in which the N3 nitrogen atom is involved NOTA are the most routinely used BFCs, despite the fact that
in coordination, are the most commonly investigated. Nevertheless, they have not been optimized for biomedical applications.
‘‘inverse’’ ligands, in which the less-electron rich N2 nitrogen Indeed, BFCs should have excellent coordination properties, i.e.
atom is involved in metal binding, have also been studied. selectivity towards endogenous cations to avoid transmetallation
1,4-disubstituted 1,2,3-triazole moieties in the ‘‘regular’’ or and high thermodynamic, kinetic, and electrochemical stability
‘‘inverse’’ form can be simply obtained by permutation of the to prevent transchelation in biological media.9–14 The develop-
azide and alkyne building blocks.2–4 We have previously ment of optimal BFCs is thus of considerable interest, and one
reported various diaza-crown ethers (cyclic ligands) and alkyl emerging approach is the anchoring of the coupling functional
diamines (acyclic ligands) armed with 1,2,3-triazole units which group onto a carbon atom within the carbon skeleton. These
coordinate to transition metal ions (Cu2+, Ni2+, Zn2+) via the N3 C-monofunctionalized systems can be obtained by two principal
nitrogen atoms.5
Moreover, tetraazamacrocycles, such as cyclam, cyclen and various electrophiles15 or by the bisaminal template approach.16
routes, namely by the cyclization of linear tetraamines with
their derivatives, display excellent coordination properties
In connection with our research on systems containing
1,2,3-triazole units as metal-complexing agents,5 we designed
a new macrocyclic ligand family based on chiral C-functionalized
cyclams engineered with two triazole heterocycles (Fig. 1). The
triazole units on the carbon atoms within the skeleton could act as
coordinating pendant arms and also as coupling functional
groups. The four free Nsecondary macrocyclic amines in these
systems could thus be more readily available for metal coordination.
Indeed, the properties and structures of macrocyclic ligand
a
´
`
Universite de Lorraine, UMR 7565 SRSMC, BP 70239, 54506 Vandœuvre-les-Nancy,
France. E-mail: Katalin.Selmeczi@univ-lorraine.fr
b
`
CNRS, UMR 7565 SRSMC, BP 70239, 54506 Vandœuvre-les-Nancy, France
† Electronic supplementary information (ESI) available: Crystallographic data for
[Cu(anti-23)] and [Cu(syn-25)], UV-vis and CD spectra, and ESI-MS for copper(II)
complexes. Characterization of enantiomers 1b–6b. NMR spectra for all
compounds. CCDC 1414285 and 1435292. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c5nj01927c
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Fig. 1 Planned chiral macrocyclic structures.
complexes critically depend on, among other factors, the type
and the intrinsic basicity of the donor atoms. The weakness of
metal–Ntertiary amine bonds relative to metal–Nsecondary amine
bonds in N- or C-methylated cyclams has been reported and
proven by the acid/base and metal complexation properties.17
A
Scheme 1 Preparation of precursors for macrocyclization: reagents and
conditions: (a) CbzCl, sat. aq. NaHCO3; (b) TBDMSCl, pyridine, DMF; (c) DibalH
(1.2 M in toluene), toluene, ꢀ78 1C, 1a: 47%, 1b: 51% (yields over 3 steps); (d) (1)
HClꢁH-bAla-OMe, DIEA, MeOH, 4 1C, (2) NaBH3CN, AcOH, MeOH, 2a: 67%, 2b:
61%; (e) Boc2O, TEA, CH2Cl2, 3a: 91%, 3b: 98%; (f) TBAF (1 M in THF), THF, 4a:
85%, 4b: 81%; (g) TsCl, TEA, DMAP (cat.), CH2Cl2, 5a: 84%, 5b: 91%; (h) NaN3,
DMF, 85 1C, 6a: 96%, 6b: 94%; (i) (1) trimethylsilylacetylene, CuSO4, sodium
ascorbate, H2O/tBuOH (1/1.5), (2) TBAF (1 M in THF), THF, 9: 72% (yield over
2 steps); (i) 3-phenylpropyne or N-tert-butyloxycarbonylpropargylamine,
CuSO4, sodium ascorbate, H2O/tBuOH (1/1.5), 7: 87%, 8: 78%.
few reports have already described tetraazamacrocycles, such
as cyclen, cyclam and derivatives, in which the macrocyclic
nitrogen atoms have been substituted with 1,2,3-triazole units.
These N-functionalized ligands were developed for ion-sensing,18–21
and molecular imaging, for example.22,23
The most appropriate pathway for our purposes appeared to
be the use of chiral amino acids as building blocks. A few
reports have described C-substituted cyclam derivatives which
were synthesized starting from L-phenylalanine, L-valine, L-leucine24
and L-proline,25 and acted as catalysts for alkene oxidation. To
the best of our knowledge, no reports have mentioned cyclams
bearing C-appended triazoles.
In this work, we developed the synthesis of 2,9-C-functionalized-
7,14-dioxocyclams based in part upon the macrocyclization of linear
precursors obtained from D- and L-serine and b-alanine building
blocks. The carbonyl group reduction led to the 2,9-C-functionalized
cyclams (Fig. 1). These macrocycles were engineered with two
1,2,3-triazole units linked in an ‘‘inverse’’ way. One of the
triazole heterocycles was substituted on position 4 with a benzyl
group, providing a model ligand useful for physico-chemical
studies (Fig. 1, R = Bn). Furthermore, an aminomethyl group
which could be used for linkage with a biomolecule was also
introduced (Fig. 1, R = CH2–NH2). Metal ion complexation
studies were performed in the presence of copper(II), which is
one of the most promising radiometals for PET imaging.9–14
with DibalH in toluene at ꢀ78 1C, were obtained in 49 and 51%
yields, respectively, over three steps. With the aldehydes 1a and
1b in hand, we turned our attention to the reductive amination
step. The formation of the imine intermediate was carried out
using a b-alanine methyl ester. Reduced dipeptides 2a and 2b
were obtained in 67 and 61% yields, respectively, and were then
treated with Boc2O in CH2Cl2 in order to protect the secondary
amine with a tert-butyloxycarbonyl group and facilitate further
chemical manipulation. According to the Cahn–Ingold–Prelog
priority rules, the C3 absolute configurations of the reduced
dipeptides 2a and 2b were R and S, respectively. The tert-
butyldimethylsilyl protecting group was removed by treatment
with TBAF in THF. The reaction of alcohols 4a and 4b with tosyl
chloride in dichloromethane gave the corresponding tosylates
5a and 5b in good yields. Substitution of the tosyl group with
sodium azide was carried out in hot DMF and led to azido
derivatives 6a and 6b in excellent yields. The synthesis of the
1,4-substituted-1,2,3-triazole moiety was next achieved by a
standard click chemistry protocol involving the generation of
Cu(I) from Cu(II) sulfate and sodium ascorbate in a 1/1.5 H2O/
Results and discussion
Synthesis
The linear precursors required for macrocyclization were prepared tBuOH mixture.2,26 Cycloaddition reactions were then performed
by a multi-step synthesis involving reductive amination and with three alkyne partners, i.e. trimethylsilylacetylene, 3-phenylprop-
peptidic coupling between D- and L-serine and b-alanine. Both 1-yne and N-tert-butyloxycarbonylpropargylamine. Derivatives 7 and
D- and L-serine were used to ensure that the two pendant arms 8 were obtained in good yields by cycloaddition reactions
would be oriented in an anti-arrangement, which would improve between azide 6a and 3-phenylprop-1-yne and N-tert-butyloxy-
the complexation properties.
carbonylpropargylamine, respectively. The 1,3-dipolar cycloaddition
Two parallel syntheses were performed using D- and L-serine, between trimethylsilylacetylene and 6b led to a mixture of
for which two protecting groups were required (Scheme 1). After TMS-protected and non-protected products. Without purification,
the preliminary experiments, we found that a benzyloxycarbonyl the crude mixture was treated with TBAF in THF to ensure
protecting group for the a-N atom and a tert-butyldimethylsilyl complete removal of the trimethylsilyl group, which afforded
protecting group for the hydroxymethyl side chain seemed compound 9 in 72% yield over two steps.
appropriate for our purposes. The aldehydes 1a (L-isomer) and
The next step consisted of a coupling reaction involving
1b (D-isomer), prepared by reduction of the corresponding ester compound 9 and building blocks 7 and 8 bearing 4-substituted
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triazoles (Scheme 2). Removal of the benzyloxycarbonyl group detected, but two other compounds, syn-21 and syn-22 were
in 7 and 8 was achieved by catalytic hydrogenation with formed in 27 and 23% yields, respectively. Careful analysis of
palladium on charcoal (Pd/C 10%) in EtOH and led to 10 the analytical data (NMR, MS and optical rotation) led us to the
and 11, respectively, in quantitative yields. The acid 12 was conclusion that syn-21 and syn-22 were stereoisomers resulting
obtained through saponification of the corresponding ester 9 from C9-epimerization during the cyclization (see Scheme 2
by treatment with potassium carbonate in a 10/1 MeOH/H2O for atom numbering). Epimerization was confirmed by the
mixture. Coupling of amines 10 and 11 with the carboxylic acid crystal structure of the [Cu(syn-25)] complex obtained by X-ray
12 in the presence of HBTU in THF afforded the compounds 13 diffraction (see Fig. S1 and Table S1, ESI†). It was clearly
and 14 in excellent yields (80 and 85%, respectively). Another established that this epimerization took place during the
set of C- and N-terminus deprotections led to the free amino cyclization process. Indeed, compound 19 did not undergo
acids 17 and 18, which could undergo macrocyclization C9-epimerization in the presence of cyclization reagents.
through treatment with HATU in a 5/1 CH2Cl2/DMF mixture
Amine and acid deprotections performed on 13 gave the
under high dilution conditions (5 mM). The corresponding corresponding amino acid 17 as a pure (2S,9R)-isomer, as
cyclic structures anti-19 and anti-20 were obtained in 20 and shown by the LC/MS spectrum. Furthermore, the protected
46% yields, respectively. No traces of dimerization product were linear compound 13 did not form its C9-isomer when it was
Scheme 2 Route to targeted macrocycles: reagents and conditions: (a) H2, Pd/C (10%), EtOH, quantitative yields; (b) K2CO3, MeOH/H2O (10/1),
quantitative yields; (c) HBTU, TEA, THF, rt, 18 h, 13: 80%, 14: 85%; (d) HATU, DIEA, CH2Cl2/DMF (5/1), rt, 60 h, anti-19: 20%, syn-21: 27%, anti-20: 46%,
syn-22: 23%; (e) TFA/DCM (1/9), quantitative yields; (f) (i) B2H6, THF, 0 1C to reflux, 18 h, (ii) 1 M HCl, reflux, 4 h, anti-27: 47%, syn-28: 67%, anti-29: 45%,
syn-30: 48%.
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placed under cyclization conditions (HATU, DIEA, DCM/DMF).
Because the epimerizable centre is not contiguous to the
activated carbonyl group, the observed epimerization cannot
be rationalized by the classical oxazolone formation which could
have occurred during acid activation. Thus the epimerization
process is believed to occur through H9 abstraction by the triazole
group itself, as observed for histidine derivatives with a methylene-
imidazole side chain.27 Two compounds, anti-19 and anti-20, were
thus obtained and had an anti-arrangement of the two triazole
moieties. Two others, syn-21 and syn-22, are C9-diastereoisomers of
compounds anti-19 and anti-20, respectively, and could be valuable
intermediates in the synthesis of cross-bridged derivatives.
In an attempt to improve the yield of the cyclization step,
other coupling conditions were tested for amino acid 18. HCTU
in CH2Cl2/DMF, PyBop in CH2Cl2 and DPPA in CH3CN were
used for this purpose. Compounds anti-20 and syn-22 were
isolated with ratios ranging from 1/1 to 1/1.2 in every case, but
without improvement of the macrocyclization yields (38, 39
and 30% global yields, respectively). The two diastereoisomers
anti-19/syn-21 and anti-20/syn-22 were separated by column
chromatography on silica gel. Removal of the tert-butyloxycarbonyl
group was achieved by treatment with 1/9 TFA/DCM mixture to
afford the water soluble salts anti-23, anti-24, syn-25 and syn-26
in quantitative yields and with high purity. Finally, diborane
reduction of dioxocylams anti-23, anti-24, syn-25 and syn-26
was performed in refluxing THF and led to a borane–amine
intermediate which was cleanly cleaved under acidic conditions
by treatment with refluxing aqueous 1 M HCl.28 The successful
isolation of the pure amine ligands anti-27 and syn-28 was
achieved by basification to pH 11–12 with KOH pellets and
extraction with CH2Cl2. Isolation of compounds anti-29 and syn-30
required treatment of the acid aqueous phase with an anionic
exchange resin, and pure amines were obtained between pH = 13
and pH = 9.
Fig. 2 ORTEP diagram of the [Cu(anti-23)] complex with thermal
ellipsoids at 50% probability. H atoms have been omitted for clarity except
on the C2 and C9 atoms. Averaged bond lengths and bond angles around
the metal ion: N1–Cu 1.964 Å, N4–Cu 2.015 Å, N8–Cu 1.973 Å, N11–Cu
2.007 Å, N3b0–Cu 2.543 Å, N1–Cu–N11 94.551, N11–Cu–N8 84.861,
N8–Cu–N4 94.801, N4–Cu–N1 85.381, N1–Cu–N3b0 98.921.
Table 1 Crystal data and X-ray experimental parameters for [Cu(anti-23)]
Compound reference
Chemical formula
Formula mass
Crystal system
a/Å
b/Å
c/Å
a/1
[Cu(anti-23)]
C23H30CuN10O2
542.11
Trigonal
24.1919(6)
24.1919(6)
12.7495(2)
90
a
b/1
g/1
90
120
6461.9(3)
100(2)
Unit cell volume/Å3
Temperature/K
Space group
R3
9
MoKa
0.797
52 499
6598
0.0414
0.0295
0.0742
0.0335
0.0768
1.024
No. of formula units per unit cell, Z
Radiation type
Absorption coefficient, m/mmꢀ1
No. of reflections measured
No. of independent reflections
Rint
Coordination properties of C-functionalized dioxocyclam and
cyclam derivatives
Final R1 value (I > 2s(I))
Final wR(F2) value (I > 2s(I))
Final R1 value (all data)
Final wR(F2) value (all data)
Goodness of fit on F2
Flack parameter
CCDC number
Physico-chemical studies were performed on anti-23 and anti-27, in
which one of the pendant arms is directed toward the upper face
and the other pendant arm is directed toward the lower face of the
macrocyclic structure. This arrangement of triazole units can
assure a more favourable coordination arrangement, which is
required for the development of BFCs for PET applications,
for example. The coordination properties, complexation kinetics
ꢀ0.006(5)
1414285
a
Not including disordered solvent, presumably a mixture of MeOH and
MeCN.
and kinetic inertness in acidic media of anti-23 and anti-27 were coordination, with two deprotonated amide nitrogen atoms
studied by UV-vis and CD spectroscopies, ESI-MS spectrometry and two amine nitrogen atoms in the equatorial plane. The
and X-ray crystallography.
axial position is occupied by the N3b0 nitrogen atom of
the triazole unit of the neighbouring molecule, which links
adjacent complexes (Fig. 3). The four donor atoms N1, N4, N8
Structural characterization of complex [Cu(anti-23)]
Solid state structure. X-ray quality crystals were obtained and N11 are nearly coplanar with the copper(II) ion, which is
after recrystallization from a MeOH/MeCN mixture at room 0.085 Å outside of the plane and is shifted towards the N3b0 atom.
temperature. The ORTEP diagram of [Cu(anti-23)] is shown
The N–Cu–N angles for which the N atoms are separated by an
in Fig. 2 with selected bond lengths and angles, and the ethylene chain are smaller by about 91 than those for which the N
corresponding crystallographic data are presented in Table 1.
atoms are separated by a propylene chain (B851 versus B94.71).
[Cu(anti-23)] crystallized in the trigonal R3 space group.
The Cu–Namide bonds (with N1 and N8) are slightly shorter
The copper(II) ion has a slightly distorted square pyramidal than the Cu–Namine bonds (with N4 and N11), B1.96 Å versus
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kinetics and proton-assisted dissociation of the [Cu(anti-23)]
complex were not investigated in the following studies.
Structural characterization of complex [Cu(anti-27)]2+
Spectroscopic studies in aqueous solution. After addition of
1 equivalent of Cu(II) to a 5 mM aqueous solution of anti-27
ligand without adjustment of the pH (pH = 5.83), the colourless
solution immediately turned into a pink mixture. The visible
spectrum of the formed complex shows one band at l = 530 nm
(e = 75 Mꢀ1 cmꢀ1), corresponding to the d–d transition of
the copper(II) ion (Fig. 4). The identity of this species was
determined by high resolution ESI mass spectrometry in the
positive ion mode. The mass spectra show four major peaks, all
corresponding to the complex [Cu(anti-27)]2+. Three peaks with
z = 1 were observed, corresponding to the [M + ClO4ꢀ]+,
[M + Clꢀ]+ and [M ꢀ H]+ molecular ions, as well as a peak with
z = 2, corresponding to the [M]2+ molecular ion (Fig. S5 and S6,
ESI† with isotopic patterns and errors in mDa). The CD
spectrum of the [Cu(anti-27)]2+ complex shows a weak negative
Cotton effect in the 400–650 nm region, while the ligand syn-28
exhibits a stronger effect with the opposite sign in the same
range in the presence of Cu2+ ion (Fig. S7, ESI†). After varying
the pH of the aqueous solution of the [Cu(anti-27)]2+ complex
between pH = 2 and 11, neither the visible absorption spectrum
nor the CD spectrum in the range of 300–800 nm showed
any variation, suggesting that the Cu(II) complex remained
unaltered in a wide pH range (Fig. S8 and S9, ESI†). To further
characterize the [Cu(anti-27)]2+ complex, the visible spectra of
the copper(II) complexes of the non-substituted commercial
cyclam, dioxocyclam and compound anti-23 were obtained at
298 K, and are presented in Fig. 4 with the corresponding lmax
values. The spectrum of the [Cu(dioxocyclam)] complex was
measured at a basic pH in order to fully deprotonate the
two peptide functions required for metal coordination. A
hypsochromic shift of lmax for [Cu(anti-27)]2+ - [Cu(anti-23)],
and also for [Cu(cyclam)]2+ - [Cu(dioxocyclam)], was observed.
Billo proposed an empirical model, which was later improved
by Prenesti, for the estimation of the visible spectrophotometric
Fig. 3 Partial ORTEP representation of the crystal packing diagram of the
[Cu(anti-23)] complex.
B2.01 Å, as might be expected due to the increased charge
attraction between the deprotonated Namide atoms and the
metal cation. The second triazole arm with the benzyl substituent
group remains non-coordinated to the metal ion. It is suggested
that the trans amide groups (at the 7 and 14 positions, Scheme 2)
significantly reduce the ability of the macrocyclic ring to adjust the
coordination sphere around the copper(II) ion to give an octahedral
geometry with two Ntriazole atoms in the axial positions.
Spectroscopic studies in aqueous solution. Circular dichroism
and visible spectrophotometric titrations were performed in 5 mM
solutions between pH 3 and 4.5 at room temperature (rt), and the
spectra are presented in Fig. S2 and S3 (ESI†). Above pH 4.5, slow
precipitation of [Cu(anti-23)] was observed because of the weak
solubility of this neutral complex obtained by deprotonation of two
amide groups. One band at 490 nm appeared in the visible
absorption spectra upon increasing the pH, and an isosbestic
point was observed, suggesting the formation of one species. This
change observed in the d–d transition of the copper(II) ion can be
attributed to the coordination of two deprotonated Namide and two
secondary Namine atoms. The same change was observed in the CD
spectra in the range of 300–800 nm, with a weak negative Cotton
effect at 530 nm. Generally, the sign of a Cotton effect associated
with a chromophore is dependent on the conformation of the
asymmetric environment as well as the absolute configuration.
The measured CD spectrum is the result of contributions from all
conformations present in solution.29 If the hexadecant rule holds
in our case, the predominant Cotton effect in the d–d transition
region must have the opposite sign for anti-23 and syn-25, and for
anti-27 and syn-28. Accordingly, a stronger Cotton effect with
the opposite sign was observed for the [Cu(syn-25)] complex. The
[Cu(anti-23)] complex was also identified by high resolution ESI
mass spectrometry in the positive ion mode. The mass spectrum
showed one major peak with z = 1, corresponding to the [M + H]+
molecular ion (Fig. S4, ESI† with isotopic pattern).
Dioxocyclam derivative anti-23 was able to bind copper(II)
with simultaneous dissociation of the two amide protons; thus, the
metal binding was highly pH-sensitive, which is not compatible with
Fig. 4 Visible absorption spectra of [Cu(anti-27)]2+ (pH = 6.5), [Cu(anti-23)]
(pH = 4.5), [Cu(cyclam)]2+ (pH = 6.9) and [Cu(dioxocyclam)] (pH = 10.5) in
radiopharmaceutical applications. Consequently, the complexation aqueous solution at 298 K.
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Table 2 EPR parameters of [Cu(anti-27)]2+ and [Cu(cyclam)]2+ in frozen
solution (water/ethylene glycol, 70/30) at 100 K
(Diamsar) can be radiolabelled under mild conditions at ambient
temperature in less than 60 min and are therefore more
compatible with biological vectors.35,36 The complexation
kinetics of the anti-27 macrocycle were studied in 0.4 M ammonium
acetate buffer at pH 6.5 and at room temperature. After the addition
of 1 equivalent of CuCl2 in 0.05 M HCl, the visible spectrum of the
mixture was measured over time (every 0.5 s). The variation in
intensity of the band at 530 nm corresponds to the rapid formation
a
a
Compound
g>
gJ
A>
AJ
[Cu(anti-27)]2+
2.052
2.047
2.190
2.179
37.5
30
195
210
[Cu(cyclam)]2+
a
In 10ꢀ4 cmꢀ1
.
lmax values of copper(II) complexes in aqueous solution.30 of the [Cu(anti-27)]2+ complex, which is complete (>99%) within
According to this model, the individual ligand field contribution 10 s (Fig. S10 and S11, ESI†). This instantaneous complex
of metal–Namide bonds is stronger than that of metal–Namine formation is very promising, since fast radiolabelling at room
bonds, leading to higher ligand field splitting Do and lower lmax temperature is crucial for sensitive biomolecules, which are
values. A bathochromic shift of lmax for [Cu(cyclam)]2+
-
degraded at high temperature.
[Cu(anti-27)]2+ was detected, which can be explained by the
coordination of the triazoles in axial positions in the [Cu(anti-27)]2+
complex, since axial ligands decrease the d–d transition energy
according to Billo’s model. No bathochromic shift of lmax for
[Cu(dioxocyclam)] - [Cu(anti-23)] was observed, showing that
in the [Cu(anti-23)] complex, the triazoles didn’t coordinate
to the metal ion in solution. This is consistent with the X-ray
structures of the [Cu(syn-25)] and [Cu(anti-23)] complexes
presented in Fig. S1 (ESI†) and Fig. 2, respectively. In both
cases, the triazoles are unavailable for metal coordination
because of the rigidity induced by the two deprotonated amide
bonds in the macrocycle. Despite numerous crystallization
efforts, we have been unable to obtain crystals of the [Cu(anti-
27)]2+ complex suitable for determination of the solid state structure
by X-ray diffraction. Thus, on the basis of the above considerations,
an axially coordinated structure can be proposed for the
[Cu(anti-27)]2+ complex in solution.
Pseudo-first order half-life for acid-assisted dissociation of the
[Cu(anti-27)]2+ complex
The kinetic stability of copper complexes for radiopharmaceutical
applications is commonly evaluated by their proton-assisted
dissociation under pseudo-first order conditions.9,12 The half-
life value obtained from this experiment is used, among other
parameters, to predict the in vivo viability of a radiometallated
chelator. The dissociation of [Cu(anti-27)]2+ was monitored by
following the changes in the visible absorption spectrum at
50 1C in 5 M HCl solution over 1 month (Fig. S12, ESI†). The
experimental data were fitted with an exponential curve of pseudo-
first order kinetics using the program Berkeley Madonna 8.3.18.
This gave a half-life value of t1/2 = 3.21 days (Fig. S13, ESI†). This
result indicated the degree of kinetic stability of the complex
relative to the most common cyclam-based systems in the
literature (t1/2 = 3.20 h for Cu–TETA at 50 1C in 5 M HCl,
t1/2 = 6.40 days for Cu–CB-TE2A at 90 1C in 5 M HCl).37–39
Electron paramagnetic resonance corroborates the axial
coordination of the triazole group. The X-band EPR spectra
of [Cu(anti-27)]2+ and [Cu(cyclam)]2+ in water/ethylene glycol
(70/30 v/v%) were recorded at 100 K, and the simulated anisotropic
parameters are reported in Table 2. The g values, gJ > g> > 2, are
Conclusion
typical of axially elongated d9 copper(II) complexes in the dx2ꢀy2 The stepwise synthesis of new syn- and anti-dioxocyclam and
ground state.31a The g and A values of [Cu(anti-27)]2+ are close to cyclam derivatives functionalized with two 1,2,3-triazole units
those of [Cu(cyclam)]2+, suggesting a principally square-planar on carbon atoms within the carbon skeleton was carried out
coordination geometry.31b Furthermore, the slight increase in the starting from D- and L-serine and b-alanine. These new ligands
gJ value and the decrease in AJ on moving from [Cu(cyclam)]2+ to provide four free Nsecondary macrocyclic amines, which are
[Cu(anti-27)]2+ are associated with the red shift of lmax (vide supra). available for metal coordination. The triazole units could thus
Both observations are in agreement with the fact that the planar act as an integral part of the metal chelator, via the N2 nitrogen
ligand field becomes weaker when the axial ligand field becomes atoms. One of the 1,2,3-triazole moieties was functionalized
stronger, according to ligand field theory.31c–e
with a benzyl or an aminomethyl group; the latter could
be useful for conjugation with model peptides. During the
cyclization step, a C9-epimerization phenomenon occurred,
Complexation kinetics of complex [Cu(anti-27)]2+
During the evaluation of a new bifunctional chelator, some and syn-dioxocyclam and syn-cyclam derivatives were unexpectedly
characteristics, such as the fast room temperature radiolabelling obtained via this stepwise route.
kinetics (on-rate) and the slow acid dissociation kinetics (off-
Regarding the physico-chemical properties, dioxocyclam
rate), should be a priority. However, within the radiometallation derivative anti-23 was able to bind to copper(II) with simultaneous
protocol for 64Cu, there are a large variety of labelling conditions dissociation of the two amide protons. Consequently, the metal
for many different chelators.9 Here are a few representative binding was highly pH-sensitive, which is not compatible with
examples: DOTA conjugates are typically radiolabelled at pH 5–8 radiopharmaceutical applications. Despite the fact that both
with temperatures in the range of 40–80 1C,32,33 CB-TE2A conjugates triazole arms are not involved in the formation of the [Cu(anti-
typically require high temperatures and long reaction times 23)] complex, they play a key role in the stabilization of the
(60 min at 90 1C),33,34 and TETA and sarcophagine-like ligands [Cu(anti-27)]2+ complex, as revealed by the different chemical
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coordination behaviour. Fast complexation occurred under with a low temperature device using liquid N2 to perform XRD
physiological/radiolabelling conditions (o10 s, rt), and the intensity measurements at near 100 K. The X-ray source was a
formed complex remained unaltered in a wide pH range (pH sealed tube emitting MoKa radiation (l = 0.71073 Å, [Cu(anti-
2–11). Moreover, the complex was found to be inert towards acid 23)]) and also CuKa radiation (l = 1.5418 Å, [Cu(syn-25)]).
assisted dissociation (t1/2 = 3.21 d, 5 M HCl, 50 1C). From UV-vis Diffraction pattern frames were processed with CrysAlis PRO,
and EPR spectroscopic studies, an axially coordinated structure Agilent Technologies, Version 1.171.37.31 software. The structures
was proposed for the [Cu(anti-27)]2+ complex in solution.
We are continuing this work to investigate the bioconjugation and refined on F2 by full matrix least-squares using SHELXL
were solved by direct methods using the SHELXT software package
reactions and radiolabelling of these chelators.
software.41 Crystal data, data collection parameters, and results
of the analyses are listed in Table 1 and Table S1 (ESI†).
Supplementary crystallographic data for the [Cu(anti-23)] structure
(atomic coordinates, thermal parameters, and intramolecular
bond distances and angles) are provided in the CIF.
Experimental section
General
Kinetic studies
TLC analyses were performed on Kieselgel 60 F254 plates
(Merck) using standard procedures. Compounds were visualized
using UV light (254 nm) and/or 30% methanolic H2SO4/heat as
the developing agent. Column chromatography was performed
using silica gel SI 60 (63–200 mm) (Merck). High pressure column
chromatography was performed using an axially compressed
20 mm id steel column (Separex-Novasep). FT-IR spectra were
recorded on a Perkin-Elmer spectrum 1000 (NaCl window or
KBr pellet, cmꢀ1). ATR-FT-IR spectra of Cu(II) complexes were
obtained on an IRAffinity-1 Shimadzu spectrophotometer.
Melting points were determined with a Tottoli apparatus and
are uncorrected. Optical rotations were measured on an Anton
The formation of the copper(II) complex of the anti-27 ligand
was studied under conditions used for radiolabelling: 0.4 M aq.
ammonium acetate buffer at pH 6.5 and at 25 1C. The solution
of anti-27 (5 mM) was prepared in the buffer, and the kinetic
measurements were started on the addition of 20 mL of a 0.25 M
CuCl2 solution prepared in 0.05 M HCl. Visible spectra between
300 and 900 nm were measured on a MOS-450 spectrophotometer
from Bio-Logic equipped with a diode-array detector, allowing the
spectrum to be recorded every 0.5 s.
The acid-assisted dissociation of the [Cu(anti-27)]2+ complex
was studied under pseudo-first order conditions in 5 M aq. HCl
containing the complex at a concentration of 2.35 mM and at
50 1C. After addition of the concentrated acid to the sample
solution, the mixture was maintained in a sealed tube at 50 1C
and absorption spectra were measured over time at 0, 3.5, 20.5,
44, 96 and 720 hours. The experimental data were fitted with an
exponential curve of pseudo-first order kinetics using the
program Berkeley Madonna 8.3.18.
1
Paar MC300 polarimeter (deg mL gꢀ1 dmꢀ1). H and 13C NMR
spectra were recorded on a Bruker DPX250 spectrometer
(250 MHz and 62.9 MHz, respectively) and a DRX400 spectro-
meter (400 MHz and 100.6 MHz, respectively). For complete
assignment of 1H and 13C signals, two-dimensional 1H,1H COSY
and 1H,13C correlation spectra were recorded. The chemical
shifts are reported in ppm (d) relative to residual solvent peaks.
Elucidations of the chemical structures were based on 1H,
COSY, HSQC, 13C and HMBC experiments (see Schemes 1 and
2 for atom numbering). UV-visible spectra were recorded on a
Perkin-Elmer Lambda1050 UV-vis-NIR spectrophotometer using
a 1 cm optical path length cell at T = 298 K. The circular
dichroism (CD) spectra and also some visible absorption spectra
were recorded on a MOS-450 spectrometer from Bio-Logic,
coupled with a Metrohm 716 titrator combined with a Fisher
Bioblock electrode for pH adjustments and controlled by a
program written in Labview. ESI-HRMS spectra were obtained
using a Bruker Daltonics MicrOTOFQ mass spectrometer. EPR
spectra were recorded using a continuous-wave Bruker EMXplus
10/12 spectrometer (X-band frequency 9.412 GHz). Measurements
were carried out in frozen (T = 100 K) water/ethylene glycol
(70/30 v/v%) solution, and the concentration of the anti-27
and cyclam ligands was 5 mM. CHN elemental analyses were
performed using a Thermo Finnigan EA 1112 Series Flash
Elemental Analyser. Reduced peptides 2–5 are named according
to the literature.40
[Cu(anti-23)]
To a solution of 10.6 mg of anti-23 ligand (0.015 mmol) in
1500 mL of water, a solution of 0.01 M Cu(ClO4)2 (1500 mL, 1 eq.
of Cu2+) was added. The solution was colourless and transparent.
After addition of 1 M NaOH solution, the mixture turned pink in
colour, and above pH 4.5 the slow formation of a pink precipitate
was observed. The precipitate was dissolved on addition of 1 M
HCl solution. Total precipitation of the complex was achieved by
addition of 50 mL of 1 M NaOH. After filtration, the crude
product was recrystallized from a MeOH/MeCN mixture by slow
evaporation at rt to yield violet crystals. ESI-HRMS+ (H2O) m/z
calculated for [C23H31N10O2Cu]+, z = 1, [M + H]+ 542.1922, found
542.1946 (ꢀ2.4 mDa). Anal. calcd for CuC23H30N10O2ꢁ2H2Oꢁ
3MeOH C, 46.31; H, 6.88; N, 20.77; found C, 46.51; H, 6.35; N,
20.35. IR (cmꢀ1) n 3142, 2916, 2870, 1547, 1427, 1327, 1224,
1055, 976, 870, 791, 719, 700.
[Cu(anti-27)]2+
To a solution of 6.8 mg of anti-27 ligand (0.015 mmol) in
1500 mL of water, a solution of 0.01 M Cu(ClO4)2 (1500 mL,
Crystal structure determination
CCDC 1414285 and 1435292. X-ray structures were determined 1 eq. of Cu2+) was added. The solution immediately turned
using a SuperNova, Dual, Cu at zero, Atlas diffractometer equipped pink in colour. The pH of the solution was approximately 6.
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At this pH, very little precipitate could be formed due to the (dd, 1H, Jgem = 12.2 Hz, J3,4 = 5.7 Hz, H4), 2.80–2.91 (m, 3H, H4,
possible excess of copper(II), which precipitated as copper H5), 3.72–3.80 (m, 4H, H20, H8), 3.69–3.72 (m, 2H, H3, H20),
hydroxide. If present, this precipitate was filtered on cotton 5.06 (s, 2H, OCH2Ph), 5.27 (d, 1H, J2,3 = 5.7 Hz, H2), 7.23–7.34
13
and the solvent was evaporated. The complex was obtained as a (m, 5H, H-Ar); C NMR (62.9 MHz, CDCl3): d = ꢀ5.6 (2 ꢂ C30),
pink powder. UV-vis lmax (H2O)/nm 530 (e/dm3 molꢀ1 cmꢀ1 75). 18.1 (OSiC(CH3)3), 25.8 (C40), 34.6 (C6), 45.0 (C5), 50.4 (C4),
ESI-HRMS+ (H2O) m/z calculated for [C23H36N10CuCl]+, z = 1, 51.5 (C3, C8), 63.2 (C20), 66.5 (OCH2Ph), 128.0, 128.4 (CH-Ar),
[M
+
Clꢀ]+ 550.2103, found 550.2145 (ꢀ4.2 mDa); for 136.5 (C-Ar), 156.1 (C1), 173.0 (C7); IR: 3338, 2953, 2929, 2896,
[C23H35N10Cu]+, z = 1, [M ꢀ H]+ 514.2337, found 514.2375 1726, 1500; MS (HR-ESI) m/z calcd for C21H37N2O5 [M + H]+
(ꢀ3.9 mDa); z = 2, [M]2+ 257.6205, found 257.6238 (ꢀ3.3 mDa). 425.2466, found: 425.2476.
IR (cmꢀ1) n 3138, 2918, 1556, 1394, 1211, 1147, 1053, 1010.
Cbz-(L)-Ser(OTBDMS)W[CH2NBoc]bAla-OMe 3a
Cbz-(L)-Ser(OTBDMS)-H 1a
A stirred solution of 2a (12.4 g, 29 mmol) in anhydrous
A stirred solution of Cbz-(L)-Ser(OTBDMS)-OMe (12.8 g, 34.8 mmol) DCM (250 mL) was cooled to 0 1C under an argon atmosphere.
in anhydrous toluene (250 mL) was cooled to ꢀ78 1C under argon. Di-tert-butyl dicarbonate (14.0 g, 64 mmol) and Et3N (8.90 mL,
DibalH (1.2 M solution in toluene, 40 mL, 48 mmol) was added 64 mmol) were successively added. The resulting solution
dropwise via a syringe and the reaction mixture was stirred at was allowed to warm to room temperature and stirred until
ꢀ78 1C for 2 h. The reaction was quenched by the addition of completion (2 h, monitored by TLC), after which it was
EtOAc and the temperature was allowed to warm up. A saturated quenched by the addition of 1 N aqueous HCl. The two phases
aqueous solution of Rochelle’s salt was poured portion-wise into were separated and the organic layer was washed successively
the mixture under vigorous stirring until two clear layers were with sat. aq. NaHCO3 and sat. aq. NaCl, and dried over MgSO4.
obtained and then separated. The aqueous layer was extracted with The solvent was evaporated under reduced pressure and the
EtOAc (4 ꢂ 60 mL), and the organic phases were combined and residue was purified by column chromatography (silica, from
washed with brine (40 mL), dried over MgSO4, filtered and cyclohexane to cyclohexane/EtOAc 80/20) to afford 13.90 g
evaporated under reduced pressure. The crude product was (91%) of compound 3a as a viscous liquid. [a]2D0 4.7 (c 1.16,
1
purified by column chromatography (silica, cyclohexane/EtOAc CHCl3); H NMR (250 MHz, CDCl3): d = 0.03 (s, 6H, H30), 0.88
90/10) to furnish the pure aldehyde (7.94 g, 23.5 mmol, 67%) as (s, 9H, H40), 1.42 (s, 9H, C(CH3)3), 2.55 (br s, 2H, H6), 3.15–3.20
a colourless viscous liquid. [a]2D0 7.6 (c 1.20, CHCl3); 1H NMR (m, 1H, H4), 3.34–3.64 (m, 8H, H4, H5, H8, H20), 3.87 (br s, 1H,
(250 MHz, CDCl3): d = 0.04 (s, 6H, H30), 0.86 (s, 9H, H40), H3), 5.07 (br s, 2.5H, H2, OCH2Ph), 5.58 (br s, 0.5H, H2),
3.88 (dd, 1H, Jgem = 10.4 Hz, J4,5 = 4.0 Hz, H20), 4.21 (dd, 1H, 7.24–7.35 (m, 5H, H-Ar); 13C NMR (62.9 MHz, CDCl3): d = ꢀ5.6
Jgem = 10.4 Hz, J4,5 = 2.5 Hz, H20), 4.30–4.35 (m, 1H, H3), 5.14 (s, (2 ꢂ C30), 18.1 (OSiC(CH3)3), 25.8 (C40), 28.2 (C(CH3)3), 32.9, 33.5
2H, OCH2Ph), 5.67 (d, 1H, J3,4 = 6.1 Hz, H2), 7.36 (br s, 5H, H-Ar), (C6), 44.0 (C5), 48.4 (C4), 51.5 (C8), 52.4 (C3), 63.1 (C20), 66.3
9.65 (s, 1H, H aldehyde); 13C NMR (62.9 MHz, CDCl3): d = ꢀ5.7 (OCH2Ph), 80.2 (C(CH3)3), 127.8, 128.3 (CH-Ar), 136.5 (C-Ar),
(2 ꢂ C30), 18.1 (OSiC(CH3)3), 25.6 (C40), 61.2 (C3), 61.8 (C20), 67.1 155.1, 155.8, 156.2, 156.5 (C1, CO), 171.9, 172.3 (C7); IR: 3443,
(OCH2Ph), 128.1, 128.2, 128.5 (CH-Ar), 136.1 (C-Ar), 156.0 (C1), 3352, 2954, 2930, 2898, 2857, 1728, 1698, 1513; MS (HR-ESI) m/z
198.7 (C4); IR: 3442, 3342, 2954, 2930, 2884, 1717, 1508.
calcd for C26H44N2NaO7 [M + Na]+ 547.2816, found: 547.2810.
Cbz-(L)-Ser(OTBDMS)W[CH2NH]bAla-OMe 2a
Cbz-(L)-Ser(OH)W[CH2NBoc]bAla-OMe 4a
A stirred solution of 1a (17.00 g, 50.40 mmol) in dry MeOH A stirred solution of 3a (13.87 g, 26.4 mmol) in dry THF
(600 mL) was cooled to 0 1C under an argon atmosphere. In a (160 mL) was treated with N-tetrabutylammonium fluoride
separate round bottom flask, b-alanine methyl ester hydro- (1.0 M solution in THF, 55 mL, 55 mmol). The resulting
chloride (13.95 g, 100.80 mmol) was dissolved in MeOH solution was stirred overnight at room temperature and then
(60 mL) and DIEA (17.14 mL, 100.80 mmol). The solutions diluted with H2O (150 mL). THF was removed under vacuum
were then mixed and stirred at 4 1C for 4 h, at which point and the product was extracted with EtOAc (3 ꢂ 80 mL). The
acetic acid (8.68 mL, 156.24 mmol) was added, followed by extracts were combined and washed with sat. aq. NaCl (50 mL),
NaBH3CN (4.48 g, 77.50 mmol). The resulting solution was dried over MgSO4, filtered, and concentrated under reduced
stirred for another hour. Completion of the reaction was pressure. The crude product was purified by column chromato-
monitored by TLC (silica, hexane/EtOAc 80/20). MeOH was then graphy (silica, cyclohexane/EtOAc 50/50) to afford 9.20 g
removed under reduced pressure and the residue was dissolved (22.4 mmol, 85%) of compound 4a as a viscous liquid. [a]2D0 5.5
in EtOAc (600 mL). The organic layer was washed successively (c 1.24, CHCl3); 1H NMR (400 MHz, CDCl3): d = 1.43 (s, 9H,
with 10% aq. NaHCO3 (200 mL) and brine (200 mL), and C(CH3)3), 2.56 (t, 2H, J5,6 = 6.9 Hz, H6), 3.15 (dd, 1H, Jgem = 13.9
dried over MgSO4. The solvent was evaporated under reduced Hz, J3,4 = 5.1 Hz, H4), 3.36–3.84 (m, 10H, H3, H4, H5, H8, H20,
pressure and the residue was purified by column chromato- H30), 5.07 (s, 2H, OCH2Ph), 5.57 (d, 1H, J2,3 = 7.3 Hz, H2),
graphy (silica, cyclohexane/EtOAc from 70/30 to 20/80) to afford 7.24–7.34 (m, 5H, H-Ar); 13C NMR (100 MHz, CDCl3): d = 28.1
16.4 g (67%) of 2a as a viscous liquid. [a]2D0 4.6 (c 0.56, CHCl3); (C(CH3)3), 33.4 (C6), 44.0 (C5), 46.7 (C4), 51.1 (C3), 51.6 (C8),
1H NMR (250 MHz, CDCl3): d = 0.00 (s, 6H, H30), 0.84 (s, 9H, 61.2 (C20), 66.6 (OCH2Ph), 81.1 (C(CH3)3), 127.8, 127.9, 128.4
H40), 1.48 (s, 1H, NH), 2.46 (t, 2H, J6,5 = 6.4 Hz, H6), 2.66 (CH-Ar), 136.3 (C-Ar), 156.1(C1), 156.9 (CO), 171.6 (C7);
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IR: 3431, 3360, 2975, 2953, 1722, 1697, 1674, 1522; MS (HR-ESI) NaCl, dried over MgSO4, filtered and evaporated. The crude
m/z calcd for C20H30N2NaO7 [M + Na]+ 433.1945, found: 433.1949. product was purified by column chromatography (silica, cyclo-
hexane/EtOAc 70/30 to 40/60) to afford 3.06 g (5.54 mmol, 87%)
of 7 as a colourless oil. [a]2D0 ꢀ2.3 (c 0.56, CHCl3); 1H NMR
Cbz-(L)-ser(OTs)W[CH2NBoc]bAla-OMe 5a
A stirred solution of 4a (9.20 g, 22.4 mmol) in dry CH2Cl2 (400 MHz, CDCl3): d = 1.40 (s, 9H, C(CH3)3), 2.52 (br s, 2H, H6),
(150 mL) was cooled to 0 1C under an argon atmosphere. Et3N 3.18–3.56 (m, 4H, H4, H5), 3.64 (s, 3H, H8), 4.06 (s, 2H, H50),
(9.40 mL, 67 mmol), tosyl chloride (6.48 g, 34 mmol) and DMAP 4.10 (br s, 1H, H3), 4.43 (m, 2H, H20), 5.04 (m, 2H, OCH2Ph),
(275 mg, 2.24 mmol) were successively added and the reaction 5.42, 5.99 (2 br s, 1H, H2), 7.19–7.35 (m, 11H, H-Ar, H30); 13C
mixture was stirred overnight at room temperature. The NMR (100 MHz, CDCl3): d = 28.4 (C(CH3)3), 32.3 (C50), 33.7 (C6),
solution was diluted with CH2Cl2 (50 mL), and washed with 44.4 (C5), 48.8 (C4), 51.2 (C20), 51.9 (C8, C3), 66.9 (OCH2Ph), 81.2
sat. aq. NaHCO3 (2 ꢂ 70 mL) and sat. aq. NaCl (50 mL). After (C(CH3)3), 122.8 (C30), 126.6 (CH-Ar), 128.0 (CH-Ar), 128.3, 128.6,
drying over MgSO4, filtration and evaporation of the solvent 128.7 (CH-Ar), 128.8 (CH-Ar), 136.4 (C-Ar), 139.1 (C-Ar), 147.8
yielded the crude product, which was purified by column (C40), 156.4 (C1), 156.9 (CO), 172.0 (C7); IR: 3319, 2922, 1719,
chromatography (silica, cyclohexane/EtOAc 75/25) to afford 1690, 1522; MS (HR-ESI) m/z calcd for C29H38N5O6 [M + H]+
10.65 g (18.8 mmol, 84%) of compound 5a as a viscous colourless 552.2817, found: 552.2818.
liquid. [a]2D0 3.5 (c 0.65, CHCl3); 1H NMR (250 MHz, CDCl3): d = 1.41
(s, 9H, C(CH3)3), 2.42 (s, 3H, H40), 2.51–2.56 (m, 2H, H6), 3.19–3.65
Compound 8
(m, 7H, H4, H5, H8), 4.04 (br s, 3H, H20, H3), 4.99–5.17 (m, 2.5H, Prepared following the procedure described for compound 7
OCH2Ph, H2), 5.67 (s, 0.5H, H2), 7.26–7.33 (m, 7H, H-Ar, H30), 7.77 using tert-butyloxycarbonylpropargylamine.
(d, 2H, J = 8.2 Hz, H30); 13C NMR (100 MHz, CDCl3): d = 21.6 (C40),
Yield: 78%, colourless foam. [a]2D0 1.0 (c 1.03, CHCl3);
28.2 (C(CH3)3), 32.9, 33.5 (C6), 44.2 (C5), 48.0 (C4), 49.5, 50.1 (C3), 1H NMR (400 MHz, CDCl3): d = 1.41 (s, 9H, C(CH3)3), 1.43 (s,
51.7 (C8), 66.6, 66.8 (OCH2Ph), 69.2 (C20), 80.8 (C(CH3)3), 127.8 9H, C(CH3)3), 2.52 (br s, 2H, H6), 3.18–3.56 (m, 4H, H4, H5),
(C30), 127.9, 128.4 (CH-Ar), 129.9 (C30), 132.3 (C-Ar), 136.3 (C-Ar), 3.65 (s, 3H, H8), 4.14 (br s, 1H, H3), 4.37 (d, 2H, J = 3.7 Hz, H50),
145.0 (C-Ar), 154.8, 156.0 (C1), 156.0, 156.5 (CO), 171.9, 172.4 (C7); 4.48 (m, 2H, H20), 5.07 (m, 2H, OCH2Ph), 5.13 (br s, 1H,
IR: 3338, 2975, 2954, 1726, 1698, 1527; MS (HR-ESI) m/z calcd for NHBoc), 5.98 (br s, 1H, H2), 7.28–7.37 (m, 5H, H-Ar), 7.59 (br
C27H36N2NaO9S [M + Na]+ 587.2034, found: 587.2044.
s, 1H, H30); 13C NMR (100 MHz, CDCl3): d = 28.4 (C(CH3)3, C80),
33.6 (C6), 36.1 (C50), 44.3 (C5), 48.7 (C4), 51.2 (C20), 51.9 (C3,
C8), 66.8 (OCH2Ph), 79.7, 81.1 (2 ꢂ C(CH3)3), 123.2 (C30), 128.0
Compound 6a
To a solution of 5a (10.65 g, 18.8 mmol) in DMF was added (CH-Ar), 128.2 (CH-Ar), 128.5 (CH-Ar), 136.3 (C-Ar), 145.4 (C40),
sodium azide (4.88 g, 75 mmol). The reaction mixture was 155.8 (CO), 156.3 (C1), 156.7 (CO), 172.0 (C7); IR: 3335, 2974,
heated to 85 1C overnight. The solvent was then removed by 2934, 1687, 1510, 1366, 1244; MS (HR-ESI) m/z calcd for
evaporation under high vacuum and the residue was partitioned
between EtOAc (90 mL) and water (450 mL). The aqueous phase
was extracted with EtOAc (4 ꢂ 90 mL). The combined organic
C
28H42N6NaO8 [M + Na]+ 613.2956, found: 613.2964.
Compound 9
extracts were washed with sat. aq. NaCl (2 ꢂ 60 mL), dried over To a stirred solution of 6b (4.15 g, 9.53 mmol) in tBuOH (45 mL)
MgSO4, filtered and evaporated under reduced pressure. The and H2O (30 mL), trimethylsilylacetylene (5.40 mL, 38 mmol,
crude product was obtained as a yellow oil and used without 4 eq.), anhydrous copper sulfate (305 mg, 1.90 mmol, 0.2 eq.)
further purification (7.92 g, 18.2 mmol, 96%). [a]2D0 3.5 (c 0.54, and sodium ascorbate (755 mg, 3.80 mmol, 0.4 eq.) were
1
CHCl3); H NMR (250 MHz, CDCl3): d = 1.45 (s, 9H, C(CH3)3), successively added. The reaction mixture was heated to 80 1C
2.58 (br s, 2H, H6), 3.16–3.51 (m, 6H, H4, H5, H20), 3.68 (s, 3H, for 60 h, after which EtOAc was added and the two phases were
H8), 3.98 (br s, 1H, H3), 5.10–5.26 (m, 2.4H, OCH2Ph, H2), 5.76 separated. The organic layer was washed twice with sat. aq.
(br s, 0.6H, H2), 7.29–7.35 (m, 5H, H-Ar); 13C NMR (62.9 MHz, NaCl, dried over MgSO4, filtered and evaporated. The crude
CDCl3): d = 28.2 (C(CH3)3), 33.5 (C6), 44.1 (C5), 48.5 (C4), 50.8 product was purified by column chromatography (silica, cyclo-
(C3), 51.7 (C8), 52.4 (C20), 66.6 (OCH2Ph), 80.7 (C(CH3)3), 127.9, hexane/EtOAc 100/0 to 25/75) to afford on the one hand 1.10 g
128.0, 128.4 (CH-Ar), 136.3 (C-Ar), 156.1 (C1), 156.6 (CO), 172.0 (2.0 mmol) of silylated cycloadduct and on the other hand
(C7); IR: 3336, 2975, 2102, 1728, 1697, 1525; MS (HR-ESI) m/z 2.97 g (6.4 mmol, 88%) of 9. In a second step, the silylated
calcd for C20H29N5O6 [M + H]+ 458.2010, found 458.2023.
triazole (1.10 g, 2.0 mmol) was treated with an excess of TBAF
(1 M solution in THF, 10 mL, 10 mmol) in THF (20 mL) for 40 h.
The reaction mixture was quenched with water and the solvent
Compound 7
To a stirred solution of azide 6a (2.55 g, 5.85 mmol) in tBuOH was removed under reduced pressure. The crude residue
(20 mL) and H2O (15 mL), 3-phenylpropyne (1.50 mL, was taken up in EtOAc and was washed with brine, dried
11.7 mmol, 2 eq.), anhydrous copper sulfate (187 mg, 1.17 mmol, over MgSO4 and evaporated. Purification by column chromato-
0.2 eq.) and sodium ascorbate (463 mg, 2.34 mmol, 0.4 eq.) were graphy (silica, cyclohexane/EtOAc 100/0 to 25/75) yielded
successively added. The reaction mixture was heated to 80 1C 353 mg of 9 (0.76 mmol, 72% for 2 steps) as a colourless oil.
1
overnight. EtOAc and H2O were added and the layers were [a]2D0 ꢀ2.4 (c 0.31, CHCl3); H NMR (400 MHz, CDCl3): d = 1.42
separated. The organic layer was washed twice with sat. aq. (s, 9H, C(CH3)3), 2.54 (br s, 2H, H6), 3.29–3.50 (m, 4H, H4, H5),
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3.66 (s, 3H, H8), 4.18 (br s, 1H, H3), 4.55 (m, 2H, H20), 5.08 Compound 14
(s, 2H, OCH2Ph), 5.54 (br s, 0.5H, H2), 6.03 (br s, 0.5H, H2),
Prepared following the procedure described for compound 13
starting from 8 and 9.
7.28–7.38 (m, 5H, H-Ar), 7.60–7.73 (m, 2H, H30, H40); 13C NMR
(100 MHz, CDCl3): d = 28.2 (C(CH3)3), 33.5 (C6), 44.3 (C5), 48.6
(C4), 50.9 (C20), 51.7 (C3), 51.8 (C8), 66.8 (OCH2Ph), 81.0
(C(CH3)3), 124.7 (C40), 127.9, 128.1, 128.4, 128.5 (CH-Ar),
133.9 (C30), 136.3 (C-Ar), 156.2 (C1), 156.7 (CO), 172.0 (C7);
IR: 3290, 2950, 1717, 1688, 1522; MS (HR-ESI) m/z calcd for
1
Yield: 85%, white foam. [a]2D0 ꢀ2.5 (c 1.0, CHCl3); H NMR
(400 MHz, CDCl3): d = 1.38 (s, 9H, (CH3)3), 1.40 (s, 9H, (CH3)3),
1.41 (s, 9H, C(CH3)3), 2.33 (m, 2H, H6), 2.52 (t, 2H, J = 6.1 Hz,
H13), 3.16–3.59 (m, 4H, H3, H10), 3.32–3.55 (m, 4H, H5, H12),
3.64 (s, 3H, OCH3), 4.14–4.60 (m, 6H, H2, H9, H6b, H6a),
4.35 (d, 2H, J = 4.4 Hz, CH2NH), 5.03 (s, 2H, OCH2Ph), 5.39
(m, 1H, NHBoc), 6.10 (br s, 1H, NH), 7.04 (br s, 1H, NH),
7.25–7.33 (m, 5H, H-Ar), 7.64 (br s, 3H, H5a, H4a, H5b);
13C NMR (62.5 MHz, CDCl3): d = 28.3 (2 ꢂ C(CH3)3), 28.4
(C(CH3)3), 33.5 (C13), 35.7 (C6), 36.1 (CH2NH), 44.3, 44.6 (C5,
C12), 48.6, 48.8 (C3, C10), 50.2 (C2 or C9), 50.7, 51.0 (C6b, C6a),
51.4 (C2 or C9), 51.8 (OCH3), 66.8 (OCH2Ph), 79.6, 80.9, 81.0
(3 ꢂ C(CH3)3), 123.3 (C5a), 124.8 (C5b), 127.9, 128.1, 128.5
(CH-Ar), 133.8 (C4b), 136.3 (C-Ar), 145.6 (C4a), 155.9 (CO), 156.3
(CO), 156.8 (CO), 171.5, 172.0 (C7, C14); IR: 3327, 2974, 1675,
1505, 1477, 1416, 1365, 1244, 1157; MS (HR-ESI) m/z calcd for
C
22H31N5O6 [M + H]+ 462.2347, found: 462.2348.
Compound 13
Compound 7 (3.01 g, 5.45 mmol) and 10% Pd/C (500 mg,
16% w/w) were mixed in anhydrous EtOH (90 mL). The flask
was flushed with H2 and the mixture was stirred overnight at
room temperature under a H2 atmosphere. The catalyst was
removed by filtration through a pad of celite, and washed with
EtOH. The filtrate was concentrated under vacuum to afford the
free amine 10 as a colourless wax which was used without
further purification. To a solution of 9 (2.40 g, 5.19 mmol) in
MeOH (25 mL) and H2O (2.5 mL) was added K2CO3 (1.79 g,
13 mmol, 2.5 eq.). The mixture was stirred for 20 h at room
temperature and then acidified to pH = 4 by addition of
Amberlite resin IR120. After filtration, the resin was washed
with EtOAc. The organic layer was washed twice with sat. aq.
NaCl and dried over MgSO4. The solvent was removed under
C
41H64N11O1 [M + H]+ 886.4781, found: 886.4776.
(2S,9R)-4,11-N-tert-Butyloxycarbonyl-9-[[4-(phenylmethyl)-1H-
1,2,3-triazol-1-yl]methyl]-2-[(1H-1,2,3-triazol-1-yl)methyl]-7,14-
dioxo-1,4,8,11-tetraazacyclotetradecane anti-19
reduced pressure and the product was dried under high To a stirred solution of compound 13 (3.41 g, 4.0 mmol) in MeOH
vacuum to yield pure 12 as a white foam. A solution of the free (50 mL) and H2O (5 mL) was added K2CO3 (1.69 g, 12.2 mmol,
amine 10 (2.15 g, 5.14 mmol, 1.03 eq.) in anhydrous THF 3 eq.). The mixture was stirred for 20 h at room temperature,
(30 mL) was added under argon via a cannula to an ice-cold acidified to pH = 1–2 by addition of 2 M aq. HCl and extracted with
stirred solution of acid 12 (2.22 g, 5.0 mmol, 1 eq.) and HBTU EtOAc (4 ꢂ 40 mL). The combined organic layers were washed with
(2.19 g, 5.50 mmol, 1.1 eq.) in anhydrous THF (80 mL). Et3N sat. aq. NaCl, dried over MgSO4 and evaporated under vacuum.
(765 mL, 5.50 mmol, 1.1 eq.) was then added dropwise and the The expected acid was obtained as a white foam and used without
temperature was allowed to warm to room temperature. further purification. The obtained acid (3.33 g, 3.99 mmol) and
The reaction mixture was stirred overnight. The THF was 10% Pd/C (660 mg, 20% w/w) were mixed in anhydrous methanol
evaporated under reduced pressure, and the residue was taken (75 mL). The flask was flushed with H2 and the mixture was stirred
up in EtOAc and successively washed with 1 M aq. HCl, sat. aq. at room temperature under a H2 atmosphere for 20 h. The catalyst
NaHCO3 and sat. aq. NaCl. After drying over MgSO4 and was removed by filtration through a pad of celite and washed with
evaporation of the solvent under reduced pressure, the crude MeOH. Evaporation of the solvent under vacuum afforded the
product was obtained and purified by column chromatography linear precursor amino acid as a colourless wax in quantitative
(silica, CH2Cl2/MeOH, 100/0 to 96/4). The desired compound 13 yield. To a solution of the linear deprotected precursor 17 (2.68 g,
was obtained as a white foam (3.41 g, 4 mmol, 80%). [a]D20 ꢀ4.3 3.83 mmol) in anhydrous CH2Cl2 (700 mL) at 0 1C was added DIEA
(c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3): d = 1.41 (s, 9H, dropwise (4.20 mL, 24 mmol, 10 eq.), followed by a solution
(CH3)3), 1.44 (s, 9H, (CH3)3), 2.33 (m, 2H, H6), 2.54 (t, 2H, of HATU (3.02 g, 7.94 mmol, 2 eq.) in DMF (80 mL). At the end
J = 6.0 Hz, H13), 3.16–3.59 (m, 8H, H3, H5, H10, H12), 3.65 of the addition, the temperature was allowed to warm to room
(s, 3H, OCH3), 4.07 (s, 2H, OCH2Ph), 4.12–4.34, 4.35–4.45, temperature and the reaction mixture was stirred for 60 h. The
4.47–4.61 (3m, 6H, H2, H9, H6a, H6b), 5.06 (s, 2H, OCH2Ph), solvents were evaporated under reduced pressure, and the residue
7.18–7.36 (m, 10H, H-Ar), 7.41 (s, 1H, H5b), 7.64 (br s, 2H, H5a, was taken up in CH2Cl2 (150 mL) and successively washed
H4a); 13C NMR (100 MHz, CDCl3): d = 28.4 (2 ꢂ C(CH3)3), 32.2 with 1 M aq. HCl solution (2 ꢂ 30 mL), sat. aq. NaHCO3 solution
(CH2Ph), 33.5 (C13), 35.8 (C6), 44.4, 44.6 (C5, C12), 48.6 (2 ꢂ 30 mL) and sat. aq. NaCl (40 mL). After drying over MgSO4
(C3, C10), 50.6, 50.7 (C6a, C6b), 50.9, 51.1 (C2, C9), 51.9 and evaporation of the solvent under reduced pressure, the crude
(OCH3), 66.8 (OCH2Ph), 81.0, 81.1 (2 ꢂ C(CH3)3), 123.0 (C5a), product was obtained and purified by column chromatography
124.9 (C5b), 126.6, 128.0, 128.2, 128.6, 128.7, 128.8 (CH-Ar), (silica, CH2Cl2/MeOH, 100/0 to 90/10). Two diastereoisomers
133.9 (C4b), 136.4 (C-Ar), 139.1 (C-Ar), 147.7 (C4a), 156.3 (CO), were isolated (19, 528 mg, 0.78 mmol, 20% and 21, 713 mg,
156.9 (CO), 171.5, 172.0 (C7, C14); IR: 3283, 2972, 2930, 1675, 1.05 mmol, 27%).
1530, 1414, 1366, 1244, 1157; MS (HR-ESI) m/z calcd for
Compound 19: White foam; [a]3D2 = ꢀ2.2 (c 1.00, CHCl3);
C42H59N10O9 [M + H]+ 847.4461, found: 847.4453.
1H NMR (400 MHz, CD3OD): d = 1.41 (s, 18H, 2 ꢂ C(CH3)3),
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2.17–2.53 (m, 4H, H6, H13), 2.84–3.90 (m, 8H, H3, H10, H5, H12), 3.13 (br s, 2H, H3, H10), 3.35 (m, 2H, H5, H12), 3.66 (m, 4H, H5,
4.02 (s, 2H, CH2Ph), 4.19–4.78 (m, 6H, H6a, H6b, H2, H9), H12, H3, H10), 4.30 (s, 2H, CH2NH), 4.40–4.54 (m, 4H, H6a,
7.18–7.30 (m, 5H, H-Ar), 7.65 (s, 1H, H5a), 7.71 (s, 1H, H4b), 7.97 H6b, H2, H9), 4.61–4.74 (m, 2H, H6a, H6b), 7.74 (s, 1H, H4b),
(s, 1H, H5b); 13C NMR (100 MHz, CD3OD): d = 28.7 (2 ꢂ C(CH3)3), 7.77 (s, 1H, H5a), 7.96 (s, 1H, H5b); 13C NMR (100 MHz,
32.5 (CH2Ph), 36.9, 37.8 (C6, C13), 47.5 (C5, C12), 51.8, 52.4 (C2, C9, CD3OD): d = 28.8 (3 ꢂ C(CH3)3), 36.7 (CH2NH2), 37.0 (C6,
C3, C10, C6a, C6b), 82.0 (2 ꢂ C(CH3)3), 124.7 (C5a), 126.7 (C5b), C13), 46.7 (C5, C12), 50.8, 51.2 (C2, C9, C6a, C6b, C3, C10),
127.5 (CH-Ar), 129.6 (CH-Ar), 134.4 (C4b), 140.5 (C-Ar), 148.5 (C4a), 80.4, 82.5 (3 ꢂ C(CH3)3), 125.2 (C5a), 127.1 (C5b), 134.4 (C4b),
157.9 (2 ꢂ CO), 174.5 (C7, C14); IR: 3337, 2980, 2930, 1697, 1655, 147.1 (C4a), 158.2 (3 ꢂ CO), 174.1 (C7, C14); IR: 2972, 2930,
1524, 1466, 1412, 1367, 1286, 1250, 1219, 1157; MS (HR-ESI) 1661, 1516, 1408; MS (HR-ESI) m/z calcd for C32H54N11O8
m/z calcd for C33H49N10O6 [M + H]+ 681.3831, found: 681.3845.
[M + H]+ 720.4151, found: 720.4137.
(2S,9S)-4,11-N-tert-Butyloxycarbonyl-9-[[4-(phenylmethyl)-1H-
1,2,3-triazol-1-yl]methyl]-2-[(1H-1,2,3-triazol-1-yl)methyl]-7,14-
dioxo-1,4,8,11-tetraazacyclotetradecane syn-21
(2S,9R)-9[[4-(Phenylmethyl)-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-
1,2,3-triazol-1-yl)methyl]-7,14-dioxo-1,4,8,11-tetraazacyclotetra-
decane, trifluoroacetate salt anti-23
White foam; [a]3D2 ꢀ49.4 (c 1.00, CHCl3). Two rotamers exist for TFA (2 mL, 26.5 mmol) was added dropwise to a stirred solution
21; only the major one is described. 1H NMR (400 MHz, CDCl3): of 19 (340 mg, 0.50 mmol) in CH2Cl2 (25 mL) at 0 1C under an
d = 1.30, 1.34 (2s, 18H, 2 ꢂ C(CH3)3), 1.91–2.63 (m, 4H, H6, argon atmosphere. At the end of the addition, the temperature
H13), 2.76–3.04 (m, 2H, H3, H10), 3.20–3.39 (m, 2H, H5, H12), was allowed to warm to room temperature and the mixture was
3.63–3.90 (m, 4H, H5, H12, H3, H10), 4.05, 4.11 (s, 2H, CH2Ph), stirred for 3 h, at which point the reaction was complete
4.26–4.48 (m, 2H, H6a, H6b), 4.45–4.58 (m, 2H, H2, H9), (monitored by TLC). Water was added (20 mL) to quench the
4.60–4.78 (m, 2H, H6a, H6b), 6.22, 6.30 (2d, 2H, NH), 7.06 reaction and the two layers were separated. The organic phase
(s, 1H, H5a), 7.19–7.33 (m, 5H, H-Ar), 7.52 (s, 1H, H5b), 7.70 (s, was extracted once with distilled water (10 mL). The combined
1H, H4b); 13C NMR (100 MHz, CDCl3): d = 28.4 (2 ꢂ C(CH3)3), aqueous layers were then co-evaporated three times with Et2O
32.2 (CH2Ph), 35.4, 35.5 (C6, C13), 47.5, 47.6 (C5, C12), to remove residual TFA and freeze-dried to furnish the desired
49.1, 49.3, 49.4, 49.6 (C2, C9, C6a, C6b), 51.0, 51.1 (C3, product as a TFA salt (350 mg, quantitative yield) as a white
C10), 82.0, 82.1 (2 ꢂ C(CH3)3), 122.9 (C5a), 124.7 (C5b), powder. [a]2D7 3.9 (c 0.80, H2O); 1H NMR (400 MHz, D2O):
126.6 (CH-Ar), 128.6, 128.7 (CH-Ar), 133.7 (C4b), 138.9 (C-Ar), d = 2.19–2.29 (m, 1H, H6 or H13), 2.42–2.52 (m, 2H, H6,
147.7 (C4a), 154.9 (2 ꢂ CO), 172.1, 172.2 (C7, C14); IR: 3402, H13), 2.54–2.62 (m, 1H, H6 or H13), 3.07–3.15 (m, 1H, H5 or
2970, 2932, 1663, 1518, 1462, 1408, 1380, 1350, 1250, 1159; H12), 3.15–3.23 (m, 1H, H5 or H12), 3.32–3.38 (m, 4H,
MS (HR-ESI) m/z calcd for C33H49N10O6 [M + H]+ 681.3831, H3, H10), 3.39–3.53 (m, 2H, H5, H12), 3.96 (s, 2H, CH2Ph),
found: 681.3866.
4.39–4.48 (m, 1H, H6a), 4.53–4.68 (m, 5H, H2, H9, H6a, H6b),
7.16–7.31 (m, 5H, H-Ar), 7.69 (s, 1H, H5a), 7.74 (s, 1H, H4b),
7.93 (s, 1H, H5b); 13C NMR (100 MHz, D2O): d = 29.7, 29.8 (C6,
C13), 30.7 (CH2Ph), 42.4, 42.5 (C5, C12), 46.5, 46.7 (C2, C9),
46.9, 47.0 (C3, C10), 49.8 (C6b), 50.0 (C6a), 124.2 (C5a), 126.4
(2S,9R)-4,11-N-tert-Butyloxycarbonyl-9-[4-[[(N-tert-butyloxycarbonyl)-
aminomethyl]-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-1,2,3-triazol-1-
yl)methyl]-7,14-dioxo-1,4,8,11-tetraazacyclotetradecane anti-20
Prepared following the procedure described for compound 19 (C5b), 126.7 (CH-Ar), 128.5 (CH-Ar), 128.9 (CH-Ar), 134.0 (C4b),
starting from 14. Two diastereoisomers were isolated (20, 139.1 (C-Ar), 147.9 (C4a), 174.0, 174.6 (C7, C14); IR: 2995, 1666,
800 mg, 1.11 mmol, 46% and 22, 400 mg, 0.55 mmol, 23%).
1547, 1454; MS (HR-ESI) m/z calcd for C23H33N10O2 [M + H]+
Compound 20: Yield: 46%, white foam. [a]2D0 ꢀ0.3 (c 0.92, 481.2782, found: 481.2780.
1
MeOH); H NMR (250 MHz, CD3OD): d = 1.43 (s, 9H, C(CH3)3),
(2S,9S)-9[[4-(Phenylmethyl)-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-
1,2,3-triazol-1-yl)methyl]-7,14-dioxo-1,4,8,11-tetraazacyclotetra-
decane, trifluoroacetate salt syn-25
1.44 (s, 18H, 3 ꢂ C(CH3)3), 2.38–2.48 (m, 4H, H6, H13),
2.97–3.87 (m, 8H, H3, H10, H5, H12), 4.28 (s, 2H, CH2NH),
4.25–4.72 (m, 6H, H6a, H6b, H2, H9), 7.71 (s, 1H, H4b), 7.80
(s, 1H, H5a), 7.97 (s, 1H, H5b); 13C NMR (62.5 MHz, CD3OD): Prepared following the procedure described for compound 23
d = 28.7 (3 ꢂ C(CH3)3), 36.7 (C6, C13), 47.3 (C5, C12), 51.7, 52.3, starting from 21. Quantitative yield, white foam. [a]2D7 ꢀ40.9
52.5 (C2, C9, C6a, C6b, C3, C10), 80.4, 82.0 (3 ꢂ C(CH3)3), 124.8 (c 0.83, H2O); 1H NMR (400 MHz, D2O): d = 2.19–2.28 (m, 1H, H6
(C5a), 126.7 (C5b), 134.4 (C4b), 147.2 (C4a), 158.0 (3 ꢂ CO), or H13), 2.44–2.67 (m, 3H, H6, H13), 3.01–3.08 (dt, 1H, J = 3.8,
174.6 (C7, C14); IR: 2978, 2937, 1668, 1540, 1417; MS (HR-ESI) 13.4 Hz, H5 or H12), 3.09–3.17 (dt, 1H, J = 3.8, 13.4 Hz, 1H, H5
m/z calcd for C32H54N11O8 [M + H]+ 720.4151, found: 720.4124. or H12), 3.24–3.35 (m, 3H, H3, H10, H5 or H12), 3.37–3.61 (m,
3H, H3, H10, H5 or H12), 3.97 (s, 2H, CH2Ph), 4.34–4.43 (m, 1H,
(2S,9S)-4,11-N-tert-Butyloxycarbonyl-9-[(1H-1,2,3-triazol-1-yl)methyl]-
2-[4-[[(N-tert-butyloxycarbonyl)aminomethyl]-1H-1,2,3-triazol-1-
H2 or H9), 4.44–4.52 (m, 2H, H2 or H9, H6a), 4.55–4.66 (m, 3H,
H6a, H6b), 7.18–7.30 (m, 5H, H-Ar), 7.74 (s, 1H, H5a), 7.77 (s,
1H, H4b), 7.96 (s, 1H, H5b); 13C NMR (100 MHz, D2O): d = 28.7,
yl]methyl]-7,14-dioxo-1,4,8,11-tetraazacyclotetradecane syn-22
Yield: 23%, white foam. [a]2D0 ꢀ19.8 (c 0.80, CHCl3); 1H NMR 29.0 (C6, C13), 30.6 (CH2Ph), 42.3, 42.5 (C5, C12), 45.6 (C3,
(400 MHz, CD3OD): d = 1.41 (s, 18H, 2 ꢂ C(CH3)3), 1.44 (s, 9H, C10), 47.5, 48.2 (C2, C9), 49.7 (C6b), 50.1 (C6a), 124.4 (C5a),
C(CH3)3), 2.32 (br d, 2H, H6, H13), 2.54 (br t, 2H, H6, H13), 126.5 (C5b), 126.8 (CH-Ar), 128.5 (CH-Ar), 128.9 (CH-Ar),
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133.9 (C4b), 138.9 (C-Ar), 147.9 (C4a), 174.0, 174.8 (C7, C14); IR: H7, H14), 2.58 (dd, 1H, J = 11.5 Hz, 5.5 Hz, H3), 2.59 (dd, 1H,
3028, 1665, 1547, 1452; MS (HR-ESI) m/z calcd for C23H33N10O2 J = 11.5 Hz, 5.5 Hz, H10), 2.88 (m, 2H, H7, H14), 3.06 (m,
[M + H]+ 481.2782, found: 481.2798.
2H, H2, H9), 3.07–3.19 (m, 2H, H5, H12), 4.04 (s, 2H, CH2Ph),
4.35 (dd, 1H, J = 14.0 Hz, 6.5 Hz, H6a), 4.44 (dd, 1H, J =
14.0 Hz, 6.5 Hz, H6b), 4.48 (dd, 1H, J = 14.0 Hz, 4.5 Hz, H6a),
4.55 (dd, 1H, J = 14.0 Hz, 4.5 Hz, H6b), 7.17–7.30 (m, 5H, H-Ar),
7.71 (s, 1H, H5a), 7.74 (d, 1H, J = 1.0 Hz, H4b), 8.00 (d, 1H,
(2S,9R)-9[4-[(Aminomethyl)-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-
1,2,3-triazol-1-yl)methyl]-7,14-dioxo-1,4,8,11-tetraazacyclotetra-
decane, trifluoroacetate salt anti-24
Prepared following the procedure described for compound 23 J = 1.0 Hz, H5b); 13C NMR (100 MHz, CD3OD): d = 29.6 (C6,
starting from 20. Quantitative yield, white foam. [a]2D0 ꢀ0.4 C13), 32.5 (CH2Ph), 48.7, 48.8 (C5, C12), 51.4, 51.6 (C6a, C6b),
1
(c 0.66, H2O); H NMR (250 MHz, D2O): d = 2.47–2.56 (m, 2H, 51.9 (C7, C14), 53.4, 53.5 (C3, C10), 59.1 (C2, C9), 124.8 (C5a),
H6, H13), 2.57–2.68 (m, 2H, H6, H13), 3.14–3.28 (m, 2H, H5, 126.8 (C5b), 127.5 (CH-Ar), 129.6 (CH-Ar), 134.4 (C4b), 140.5
H12), 3.31–3.46 (m, 4H, H3, H10), 3.49–3.59 (m, 2H, H5, H12), (C-Ar), 148.6 (C4a); IR: 3246, 3182, 2916, 2806, 1639, 1547; MS
4.25 (s, 2H, CH2NH2), 4.55–4.75 (m, 6H, H2, H9, H6a, H6b), (HR-ESI) m/z calcd for C23H37N10 [M + H]+ 453.3197, found:
7.75 (s, 1H, H4b), 7.94 (s, 1H, H5b), 8.06 (s, 1H, H5a); 13C NMR 453.3209.
(62.5 MHz, D2O): d = 29.8, 29.9 (C6, C13), 33.8 (CH2NH2), 42.5
(C5, C12), 46.7 (C2, C9), 46.8 (C3, C10), 49.9, 50.2 (C6a, C6b),
125.8 (C5a), 126.3 (C5b), 134.1 (C4b), 140.0 (C4a), 174.5 (C7,
(2S,9S)-9[[4-(Phenylmethyl)-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-
1,2,3-triazol-1-yl)methyl]-1,4,8,11-tetraazacyclotetradecane syn-28
C14); IR: 2978, 1668, 1543; MS (HR-ESI) m/z calcd for
C
17H30N11O2 [M + H]+ 420.2578, found: 420.2568.
Prepared following the procedure described for compound 27
starting from 25. Yield: 67%, white foam. [a]2D0 ꢀ15.5 (c 0.60,
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.54–1.65, 1.66–1.79 (2m,
4H, H6, H13), 2.32–2.38 (m, 2H, H3, H10), 2.52–2.66 (m, 6H,
H5, H12, H3, H10, H7, H14), 2.71–2.84 (m, 4H, H7, H14, H5,
(2S,9S)-9-[4-[(Aminomethyl)-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-
1,2,3-triazol-1-yl)methyl]-7,14-dioxo-1,4,8,11-tetraazacyclotetra-
decane, trifluoroacetate salt syn-26
Prepared following the procedure described for compound 23 H12), 2.99–3.09 (m, 2H, H2, H9), 4.07 (s, 2H, CH2Ph), 4.21 (dd,
starting from 22. Quantitative yield, white foam. [a]2D0 ꢀ0.6 1H, J = 14.0 Hz, 6.5 Hz, H6a), 4.30 (dd, 1H, J = 14.0 Hz, 5.0 Hz,
1
(c 0.90, H2O); H NMR (400 MHz, D2O): d = 2.49–2.57 (m, 2H, H6a), 4.32 (dd, 1H, J = 14.0 Hz, 6.5 Hz, H6b), 4.44 (dd, 1H,
H6, H13), 2.62–2.73 (m, 2H, H6, H13), 3.14 (m, 1H, H5 or H12), J = 14.0 Hz, 5.0 Hz, H6b), 7.19–7.31 (m, 5H, H-Ar), 7.23 (s, 1H,
3.17 (m, 1H, H5 or H12), 3.32 (m, 1H, H3 or H10), 3.35 (m, 1H, H5a), 7.62 (s, 1H, H5b), 7.67 (s, 1H, H4b); 13C NMR (100 MHz,
H3 or H10), 3.44–3.52 (m, 2H, H5, H12), 3.52–3.62 (m, 2H, H3, CDCl3): d 29.2 (C6, C13), 32.4 (CH2Ph), 48.5 (C5, C12), 50.4, 50.5
H10), 4.24 (s, 2H, CH2NH2), 4.42–4.56 (m, 2H, H2, H9), (C7, C14), 51.1 (C3, C10), 51.8 (C6b), 52.0 (C6a), 57.4 (C2, C9),
4.58–4.68 (m, 4H, H6a, H6b), 7.76 (s, 1H, H4b), 7.95 (s, 1H, 122.7 (C5a), 124.6 (C5b), 126.6 (CH-Ar), 128.7, 128.8 (CH-Ar),
H5b), 8.06 (s, 1H, H5a); 13C NMR (100 MHz, D2O): d = 29.0 (C6, 133.8 (C4b), 139.3 (C-Ar), 147.6 (C4a); IR: 3278, 3238, 2918,
C13), 33.8 (CH2NH2), 42.5 (C5, C12), 45.8 (C3, C10), 47.6, 48.0 2816, 1655, 1547, 1452, 1431, 1219; MS (HR-ESI) m/z calcd for
(C2, C9), 49.7, 50.0 (C6a, C6b), 125.8 (C5a), 126.4 (C5b), 134.0
(C4b), 140.0 (C4a), 174.5, 174.6 (C7, C14); IR: 3013, 1661, 1547;
MS (HR-ESI) m/z calcd for C17H30N11O2 [M + H]+ 420.2578,
found: 420.2574.
C
23H37N10 [M + H]+ 453.3197, found: 453.3212.
(2S,9R)-9-[4-[(Aminomethyl)-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-
1,2,3-triazol-1-yl)methyl]-1,4,8,11-tetraazacyclotetradecane anti-29
(2S,9R)-9[[4-(Phenylmethyl)-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-
1,2,3-triazol-1-yl)methyl]-1,4,8,11-tetraazacyclotetradecane anti-27
Prepared following the procedure described for compound 27
starting from 24. After cooling at room temperature, the THF
was removed under vacuum and the resulting aqueous phase
was washed with CH2Cl2 (2 ꢂ 10 mL) and evaporated to dryness.
The acidic aqueous phase was subjected to anionic exchange
column chromatography and the resulting pure amine was
collected from pH = 13 to pH = 9. Yield: 45%, white foam.
[a]2D0 ꢀ0.1 (c 0.48, H2O); 1H NMR (400 MHz, D2O): d = 1.45–1.55,
1.61–1.68 (2m, 4H, H6, H13), 2.24–2.30 (m, 2H, H3, H10),
2.37–2.54 (m, 6H, H5, H12, H3, H10, H7, H14), 2.71–2.78,
2.82–2.88 (m, 4H, H7, H14, H5, H12), 3.05–3.13 (m, 2H, H2,
H9), 3.79 (s, 2H, CH2NH2), 4.32–4.51 (m, 4H, H6a, H6b), 7.73
(s, 1H, H5a), 7.79 (s, 1H, H5b), 7.90 (s, 1H, H4b); 13C NMR
(100 MHz, D2O): d = 27.5 (C6, C13), 35.6 (CH2NH2), 45.2, 45.3
(C5, C12), 48.0 (C7, C14), 50.6, 50.7 (C6a, C6b), 50.9 (C3, C10),
56.2 (C2, C9), 123.8 (C5a), 126.4 (C5b), 133.9 (C4b), 148.8 (C4a);
IR: 3417, 3251, 3189, 2952, 2905, 1578; MS (HR-ESI) m/z calcd for
C17H34N11 [M + H]+ 392.2993, found: 392.2950.
To a suspension of the TFA salt of anti-23 (490 mg, 0.69 mmol)
in dry THF (3 mL) under an argon atmosphere was slowly
added a borane tetrahydrofuran complex solution (1 M in THF,
10 mL, 10 mmol, 15 eq.) at 0 1C. The mixture was stirred at
room temperature for 3 h and then heated under reflux for 18 h.
After cooling at 0 1C, the excess borane was hydrolyzed with a
1 : 1 mixture of water and THF (15 mL), after which the THF was
evaporated under reduced pressure. A 6 N HCl solution (18 mL)
was added and the mixture was heated to reflux for 4 h. After
cooling at room temperature, KOH pellets were added until
pH > 12. The solution was extracted with CH2Cl2 (6 ꢂ 15 mL),
and the combined organic layers were washed with sat. aq. NaCl
and dried over MgSO4. After evaporation, compound 27 (138 mg,
47%) was obtained as a white foam. [a]2D0 ꢀ1.1 (c 0.8, MeOH);
1H NMR (400 MHz, CD3OD): d = 1.55–1.68, 1.70–1.82 (2m, 4H, H6,
H13), 2.21 (t, 2H, J = 11.5 Hz, H3, H10), 2.43–2.52 (m, 4H, H5, H12,
New J. Chem.
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Paper
12 T. J. Wadas, E. H. Wong, G. R. Weisman and C. J. Anderson,
Chem. Rev., 2010, 110, 2858.
(2S,9S)-9-[4-[(Aminomethyl)-1H-1,2,3-triazol-1-yl]methyl]-2-[(1H-
1,2,3-triazol-1-yl)methyl]-1,4,8,11-tetraazacyclotetradecane syn-30
13 P. S. Donnelly, Dalton Trans., 2011, 40, 999.
14 S. V. Smith, J. Inorg. Biochem., 2004, 98, 1874.
Prepared following the procedure described for compound 27
starting from 26. After cooling at room temperature, the THF
was removed under vacuum and the resulting aqueous phase
was washed with CH2Cl2 (2 ꢂ 10 mL) and evaporated to
dryness. The acidic aqueous phase was subjected to anionic
exchange column chromatography and the resulting pure
amine was collected from pH = 13 to pH = 9. Yield: 48%, white
foam. [a]2D0 ꢀ34.7 (c 0.30, H2O); 1H NMR (400 MHz, D2O):
d = 1.59–1.68 (m, 4H, H6, H13), 2.48–2.75 (m, 10H, H5, H12,
H14, H3, H10), 3.17–3.25 (m, 2H, H2, H9), 3.60–3.62 (m, 2H,
H7), 3.87 (s, 2H, CH2NH2), 4.38–4.59 (m, 4H, H6a, H6b), 7.73
(s, 1H, H5a), 7.80 (s, 1H, H5b), 7.92 (s, 1H, H4b). 13C NMR
(100 MHz, D2O): d = 27.1, 27.8 (C6, C13), 35.6 (CH2NH2), 44.9,
45.0, 47.7, 47.8, 49.4 (C14, C5, C12, C3, C10), 51.1, 51.3 (C6a,
C6b), 55.8 (C9, C2), 61.5 (C7), 123.7 (C5a), 126.3 (C5b),
134.0 (C4b), 148.8 (C4a); IR: 3421, 3259, 3178, 2947, 2912,
1605; MS (HR-ESI) m/z calcd for C17H34N11 [M + H]+ 392.2993,
found: 392.2991.
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Acknowledgements
´
The authors thank the Region Lorraine for the post-doctoral
funding of A.-S. Felten. The authors acknowledge S. Parant,
`
B. Fernette, F. Lachaud and P. Lemiere for technical assistance,
NMR and ESI-MS, and E. Wenger for X-ray measurements at
´
Universite de Lorraine.
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