Efficient Synthesis of a Family of Bifunctional Chelators Based on the PCTA[12] Macrocycle Suitable for Bioconjugation

Article abstract of DOI:10.1002/ejoc.201900280

PCTA[12] is a 12-membered tetraaza-macrocyclic ligand that incorporates a pyridine unit within the macrocyclic ring and three acetate pendant arms. Unlike DOTA and NOTA chelators, PCTA is a recent entry to the field of macrocyclic polyaminocarboxylate ligands available to complex a variety of M²⁺/M³⁺ ions for biomedical applications such as diagnostic and radiotherapeutic. Despite the promising properties of its chelates, only a few of bifunctional chelating agents (BFCAs) derived from PCTA have been described so far. Based on our very recent methodology for the preparation of PCTA[12] itself, we report here the efficient synthesis of several BFCAs derived from PCTA bearing a free reactive function group, mainly devoted to conjugation purposes: ester, carboxylic acid, alcohol, aliphatic amine, aromatic amine, maleimide, bromo or azide functions. These functions were introduced either on the 4-position of the pyridine ring or on the methylene carbon atom of the central acetate chelating arm, while keeping the three carboxylate groups available for metal chelation. Moreover, two of these BFCAs-PCTA were used for conjugation with a tetrapeptide (cholecystokinin analogue), a bioactive molecule (biotin), or a solid support (silica gel).

Full text of DOI:10.1002/ejoc.201900280

A Journal of
Accepted Article
Title: Efficient Synthesis of a Family of Bifunctional Chelators Based on
PCTA[12] Macrocycle Suitable for Bioconjugation
Authors: Nadine Leygue, Morgane Enel, abdel Diallo, Béatrice Mestre-
Voegtlé, Chantal Galaup, and Claude Picard
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10.1002/ejoc.201900280
European Journal of Organic Chemistry
Efficient Synthesis of a Family of Bifunctional Chelators Based on PCTA[12]
Macrocycle Suitable for Bioconjugation
Nadine Leygue*, Morgane Enel, Abdel Diallo, Béatrice Mestre-Voegtlé,‡ Chantal Galaup,
Claude Picard*
Abstract
PCTA[12] is a 12-membered tetraaza-macrocyclic ligand that incorporates a pyridine unit
within the macrocyclic ring and three acetate pendant arms. Unlike DOTA and NOTA
chelators, PCTA is a recent entry to the field of macrocyclic polyaminocarboxylate ligands
available to complex a variety of M2+/M3+ ions for biomedical applications such as diagnostic
and radiotherapeutic. Despite the promising properties of its chelates, only a few of
bifunctional chelating agents (BFCAs) derived from PCTA have been described so far. Based
on our very recent methodology for the preparation of PCTA[12] itself, we report here the
efficient synthesis of several BFCAs derived from PCTA bearing a free reactive function
group, mainly devoted to conjugation purposes: ester, carboxylic acid, alcohol, aliphatic
amine, aromatic amine, maleimide, bromo or azide functions. These functions were
introduced either on the 4-position of the pyridine ring or on the methylene carbon atom of
the central acetate chelating arm, while keeping the three carboxylate groups available for
metal chelation. Moreover, two of these BFCAs-PCTA were used for conjugation with a
tetrapeptide (cholecystokinin analogue), a bioactive molecule (biotin), or a solid support
(silica gel).
Dr N. Leygue, Dr M. Enel, A. Diallo, Dr B. Mestre-Voegtlé, Dr C. Galaup, Dr C. Picard.
SPCMIB, UMR 5068 CNRS-Université Paul Sabatier-Toulouse III, 118 route de Narbonne,
Toulouse F-31062, France. E-mail: leygue@chimie.ups-tlse.fr; picard@chimie.ups-tlse.fr
http://spcmib.univ-tlse3.fr
‡Present address: CNRS, Laboratoire de Chimie de coordination (LCC), UPR CNRS 8241,
205 route de Narbonne, 31077 Toulouse cedex 4, France.
Supporting information and ORCID(s) from the author(s) for this article are available on the
WWW under
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European Journal of Organic Chemistry
Introduction
Over a number of years, macrocyclic polyaminocarboxylate derivatives play a ubiquitous role
in biomedical applications such as diagnostic and therapeutic. This is a direct consequence of
their ability for complexing a variety of M2+/M3+ metal ions which drew a great attention
towards their applications in such fields. This is also due to the increased kinetic inertness of
related complexes of macrocyclic ligands compared to acyclic owing to the macrocyclic
effect minimizing transchelation or transmetallation reactions which is favourable for in vivo
applications. The resulting metal complexes have found a broad range of applications in
different imaging modalities such as magnetic resonance imaging (MRI, Gd3+ chelates),
single-photon emission computed tomography (SPECT, 111In3+ chelates), positron emission
tomography (PET, 64Cu2+ and 68Ga3+ chelates), or optical imaging (Ln3+ chelates) and in
90
internal radiotherapy for malignancies (177Lu3+, Y3+ chelates). In this family of ligands, the
vast majority of efforts have focused on use of DOTA (1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetate) and theirs
derivatives, which led to various and stable chelates available for medical imaging and
therapy (Figure 1).[1] They also play a pivotal role in the design of probes combining imaging
modalities (dual-imaging agent) and imaging/therapy (theranostic agents).[2]
PCTA macrocycle (Pyridine Containing TriAza; 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-
1(15),11,13-triene-3,6,9,-triacetic acid) is another noteworthy example of this family of
macrocyclic ligands and was assessed more recently as an efficient M2+/M3+ chelator in
various metal complexes. PCTA is a 12-membered tetraaza-macrocyclic ligand that
incorporates a pyridine unit within the macrocyclic ring and three acetate pendant arms
(Figure 1). This heptadentate ligand was originally synthesized and investigated for
complexation of alkaline earth metals (Ca2+, in particular) and transition metal ions (such as
Cu2+, Zn2+) by Stetter and Franck in 1981.[3] In the decade 1995-2005, several reports have
shown the promising properties of LnIII-PCTA complexes as relaxometric or luminescent
agents. Due to the presence of two water molecules in the first coordination sphere of the
metal, the [Gd-PCTA]0 complex displays a higher relaxivity (r1310K = 5.4 s-1 mM-1 at 20 MHz)
than the monoaqua [Gd-DOTA]- complex (r1310K = 3.5 s-1 mM-1 at 20 MHz), which makes it
an interesting alternative as MRI contrast agent.[4] This complex displays also a short
310K
residence lifetime of the inner-sphere water molecules (M
= 82 ns), a favourable factor
for providing high relaxivity once the chelate is immobilized by covalent binding to
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macromolecules or nano-sized systems. Besides, pyridine is a competent candidate to act as
an antenna/sensitizer for enhancing lanthanide ion luminescence in visible and near-infrared
regions with long emission lifetime, which mitigates the interference of background
fluorescence originating from biological samples. This chromophoric moiety was introduced
in several luminescent LnIII complexes and despite the presence of two coordinated water
molecules, Eu-PCTA complex is characterized by an emission lifetime of 370 s in aqueous
solution.[5,6] More recently, Tircso et al. reported detailed thermodynamic and
formation/dissociation kinetics of Ln3+-PCTA complexes.[7] These two seminal works
highlighted that Ln-PCTA complexes have suitable thermodynamic stabilities (20  log K 
21.5), favourably fast formation rates (faster than those observed for Ln-DOTA and Ln-
NOTA complexes) and favourable kinetic inertness toward acid-catalyzed dissociation. As a
consequence, PCTA was subsequently evaluated as a carrier for radioactive metal ions for
nuclear medicine applications, where a fast complexation of the radioisotope compared to its
half-life is an important concern. In vitro and in vivo experiments showed that PCTA and its
bioconjugated derivatives labelled with  (111In) and + (64Cu and 68Ga) emitters are promising
radiotracers for SPECT and PET imaging modalities in terms of radiolabeling kinetics, kinetic
inertness and positive in vivo properties such as biodistribution profiles.[8,9,10] For instance,
Ferreira et al. have undertaken a systematic comparison of three bifunctional chelates
68
containing PCTA, DOTA or NOTA chelating moieties and radiolabeled with Ga.[10a] PCTA
derivative has been shown to possess much faster radiolabeling kinetics under very mild
68
conditions (5 min, RT) than the corresponding DOTA derivative (10 min, 80°C). Ga-PCTA
stability was also found to be superior to 68Ga-DOTA, and comparable to 68Ga-NOTA,
especially with respect to transmetallation by transferrin. Additionally, the biodistribution
profile of 68Ga-PCTA revealed lower kidney retention than its 68Ga-NOTA counterpart, which
may be advantageous in some imaging applications. These results were confirmed by the
further study of RGD peptide conjugates derived from PCTA and NOTA.[10b] The utility of
PCTA as a chelating agent for 90Y and 177Lu was also explored in radiotherapy fields.[11]
Moreover, derivatives of PCTA where one, two or three carboxylate functions are substituted
by phosphonate, phosphonate ester, amide or picolinate coordinating groups were also
investigated with success as MRI contrast agents, luminescent probes or radiopharmaceuticals
for imaging and therapy.[5b,12] These data demonstrate that PCTA may be potentially
considered as a universal ligand for imaging or (and) radiotherapeutic applications involving
metallic complexes and make it a valuable addition to traditional ligands such as DOTA or
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NOTA. As emphasized by Maecke, the affinity towards biological receptors and
pharmacokinetics of bioconjugated metallic chelates may be strongly dependent on the
structure of chelators.[13] Thus, owing to the growing interest of the PCTA core, it is essential
to have in hand a family of PCTA derivatives with different reactive functions (bifunctional
chelating agents, BFCAs) in order to allow covalent coupling of the free ligand or its metal
complexes to bioactive molecules, biomolecules, macromolecules or nanosystems.[14]
Figure 1. Structures of DOTA, NOTA, PCTA and ligands discussed in this study.
Because of the importance of PCTA[12], we have recently reported a new synthetic
methodology for building the skeletal backbone of PCTA with high efficiency in the crucial
macrocyclization step.[15] Herein, we wish to demonstrate that this approach provides also an
easy access to new bifunctional PCTA ligands. The BFCAs-PCTA, described in this report,
display ester, carboxylic acid, alcohol, aliphatic amine, aromatic amine, maleimide, bromo or
azide functions to be eventually used for conjugation and leave the three carboxylate groups
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available for metal chelation (Figure 1). The ability of two of them for covalent attachment to
materials was also tested by their conjugation to a peptide (cholecystokinin analogue), a
bioactive molecule (biotin), or a solid support (silica gel).
Results and Discussion
Considering the parent ligand PCTA, there are several possible approaches to conjugate it to
a targeting moiety. The simplest conjugation strategy is to substitute one acetate donor with
an N-substituted amide donor. This strategy has been used for a long time in the field of
DOTA bioconjugates.[16] This approach does not affect the denticity of the ligand, but it does
affect the overall charge of the complex and may decrease in vitro and in vivo stabilities of the
resulting metallic complexes.[17] Three other approaches can be exploited which do not affect
the overall charge and the electron density of the metal ion. In these approaches, the
functional group can be attached to (i) one of the pyridine carbon (Cpy-functionalization), (ii)
one of the carbons of the ethylene group of the macrocyclic chelator backbone (C-
functionalization), (iii) one of the -positions to the carboxylic groups (N-functionalization).
Despite its promising properties, only few papers on bifunctional chelators based on the
PCTA structure have been published so far (Figure 2). Reactive bromophenyl, thiol, or ethyl
carboxyl ester ligands (L1-L3) were introduced into the 3-position of the pyridine ring.[18] This
3-position on the pyridine ring was selected because of synthetic facility to prepare the acyclic
pyridine precursor and was assumed to be sufficiently far away from the coordination cage to
limit any interference with the coordination cage.[18a] Aime et al. designed a centrally N-
functionalized ligand (L4) for which the natural amino acid tyrosine serves as a starting
material.[12c] Several other N-bifunctional PCTA derivatives (L5 as an example) were reported
in a patent, but only MS data were given for the characterization of these chelators.[19] Kovacs
and co-workers produced a C-benzylated derivative in which the aryl ring is functionalized
with a nitro group (L6).[7b] At the present time, the corresponding functionalized p-
aminobenzyl derivative is widely used for peptide, protein or antibody labelling due to its
commercial availability.[20]
In any case, the synthesis of these BFCAs-PCTA is based on the Richman-Atkins
methodology, i.e. the use of a diethylenetriamine derivative bearing two N-terminal
arysulfonamide groups (tosyl or nosyl). This route allows a good yield in the crucial
macrocyclization step, but requires two post-macrocyclisation steps: deprotection of the N-
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arylsulfonyl groups and the per N-carboxyalkylation reaction. These two steps may contribute
to a drop in the overall yield of the target products.[12c,18b,21]
In this work, our strategy involves the use of diethylenetriamine having pendant acetate
groups as precursor molecules for the macrocyclization process and an efficient sodium
template effect which controls the macrocyclization step.[15] For the synthesis of PCTA, this
approach led to a better overall yield (60%) compared with those previously reported
involving the Richman-Atkins methodology (26-36%).[3,22]
Figure 2. Structures of PCTA[12]-based BFCAs reported in the literature.
Synthesis of Cpy-bifunctionnal PCTA derivatives
The first bifunctional PCTA derivative was obtained introducing a methyl ester function on
the 4-position of the pyridine ring (Scheme 1, compound 4). This aromatic ester group was
selected because of its versatility with respect to a large range of transformations. For
example the ester functionality could be easily transformed into an alcohol group, a
carboxylic acid or amide groups in good yield under mild conditions. Moreover, methyl ester
can be selectively cleaved in the presence of tert-butyl esters by a judicious choice of
hydrolytic conditions. As reported in the introduction part, the preparation of the derivative 4
was based on our synthetic pathway for the access to PCTA itself. The trifunctionalized
pyridine synthon 1 was prepared from commercial methylisonicotinate in two steps according
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to our reported procedure: a Minisci reaction for inserting two hydroxymethyl groups on the
2,6-positions of the pyridine ring and a standard bromination of the corresponding diol with
PBr3 (overall yield 81%).[5d] The preparation of the second key compound, triamine 3, was
based on our synthetic procedure recently reported, involving the use of prealkylated amine
precursors, and relying on a coupling reaction of glycine tert-butyl ester and the bromine
derivative 2. This four step sequence afforded triamine 3 in a 70% yield from commercially
N-benzyl ethanolamine.[15] Condensation of 1 with 3 was carried out in acetonitrile at reflux
and in the presence of an excess of sodium carbonate as a base and by using a batchwise
procedure (reactants concentration 2  10-3 M). As observed previously for the tri tert-butyl
ester PCTA derivative,15 1H, 13C NMR and MS analyses of the crude macrocyclization
product evidenced the presence of a single major species, subsequently characterized as the
sodium monomeric complex Na-4. After purification by column chromatography on alumina
and treatment with a saturated EDTA solution to remove sodium species, the free macrocycle
4 was obtained in 90% yield. It can be noticed that free macrocycle 4 and its sodium complex
1
4.Na can be readily distinguished by their H NMR spectra.[23] Besides, a change in the
leaving group of the bifunctional electrophile (tosylate versus bromide) has a marked effect
upon the yield of the macrocyclization reaction, 55% vs. 90%.
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Scheme 1. Synthesis of macrocycle 4. Reagents and conditions: (a) (NH4)2S2O8, H2SO4 cat.,
MeOH, H2O, reflux, 2 h; (b) PBr3, CH2Cl2, reflux, 2 h; (c) i) BrCH2CO2tBu, K2CO3, CH3CN,
rt, 24 h, 100%; ii) N-bromosuccinimide, PPh3, CH2Cl2, rt 16 h, 85%; (d) i) NH2CH2CO2tBu
hydrochloride salt, K2CO3, CH3CN, reflux, 24 h, 82%; ii) Pd/C (10%), H2 gas (6 bar), MeOH,
rt, 12 h; 100%; (e) [reagents] = 210-3 M, Na2CO3, CH3CN, reflux, 64 h.
Several derivatives of PCTA can be obtained from the protected ligand 4 (Scheme 2).
Unmasking tert-butyl ester functions with trifluoroacetic acid afforded the corresponding
triacid 5 in a quantitative yield. Orthogonal removal of the methyl ester group was achieved in
mild basic conditions (Na2CO3, MeOH/H2O, room temperature) and provided the
corresponding acid 7 in 78% yield. Deprotection of the two kinds of ester functions (aqueous
6N HCl, 90°C) afforded tetraacid 6 as a tetrahydrochloride salt in a quantitative yield. The
introduction of an alcohol function from 4 can be cleanly achieved by reduction of the methyl
ester function with NaBH4 in methanol (compound 8). Besides, reaction of this ester function
with a large excess (30 equiv.) of neat ethylenediamine produces the N-(2-aminoethyl) amide
derivative 9 in a nearly quantitative yield. These hydroxyl and amino functions of 8 and 9
offer another potential source of further derivatization through biologically stable ether
linkage and by using amide, urea or carbamate linkages, respectively. We have also exploited
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the carboxylic acid function of 7 to access a BFCA containing a maleimide moiety (10), a
reactive group which is extensively used for bioconjugation because it can easily and
specifically react with thiols under mild conditions. The activation of the carboxylic acid
function of 7 was achieved in situ with a uronium-HOAt peptide coupling reagent (HATU)
and the subsequent coupling reaction with the heterobifunctional reagent, 6-(maleimido)
hexylamine (13, Scheme 3), afforded the desired compound 10 in 61% yield after
chromatographic purification. As shown in Scheme 3, the synthetic procedure for this
heterobifunctional cross-linking reagent 13 involves three steps starting from 6-
aminohexanol: the first is the protection of the amino function by a carbamate group; this is
followed by a Mitsunobu reaction to substitute the alcohol function with the maleimide
moiety and then the removal of the protecting group under standard conditions.
Scheme 2. Synthesis of PCTA derivatives from 4. Reagents and conditions: (a) CF3COOH,
CH2Cl2, rt, 16 h; (b) 6N HClaq, reflux, 24 h; (c) Na2CO3, MeOH-H2O, rt, 22 h; (d) NaBH4,
MeOH, rt, 7 days; (e) ethylenediamine (excess), rt, 16 h; (f) compound 13 (Scheme 3), HATU,
DIPEA, DMF, rt, 16 h.
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Scheme 3. Synthesis of heterobifunctional reagent 13. Reagents and conditions: (a)
(tBuOCO)2O, KOH, H2O-dioxane, rt, 3 h; (b) maleimide, PPh3, DIAD, THF, rt, 16 h; (c)
CF3COOH, CHCl3, rt, 2 h.
In order to have in hand a bifunctional derivative available for a click chemistry approach
and more specifically for the 1,3-dipolar Huisgen cycloaddition between alkyne and azide
functions, we planed the preparation of an azide PCTA derivative (Scheme 4). For this
purpose, we synthesized a macrocyclic precursor bearing a bromine atom at the C-4 position
of the pyridine ring, 15. Intermolecular cyclization of 4-bromo-2,6-bis(bromomethyl)pyridine
and triamine 3 was accomplished under batchwise procedure and in the presence of Na2CO3
in acetonitrile at reflux and led to the free macrocycle 15 in 62% isolated yield. As previously
observed for 4, this macrocyclization reaction was controlled by a sodium template effect.
Following a classical procedure reported for a similar acyclic pyridine ligand,[5e] bromine of
15 was firstly reacted with sodium azide (10 equiv.) in DMF at 90°C for 48 h to give the
targeted azide derivative 16 (40% yield). The experimental conditions of this reaction were
improved by the use of microwave (MW) irradiation technique and took place within 15 min
(MW conditions: 260 W, 100°C) in acetonitrile and in the presence of sodium azide (5 equiv.)
with an isolated yield of 58%. It is also interesting to note that the pyridyl bromide or pyridyl
azide functionalities of 15 or 16 offer the possibility to extend ligand -delocalization and
thus to improve the luminescence properties of Eu-PCTA complex.[24]
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Scheme 4. Synthesis of azido macrocycle 16. Reagents and conditions: (a) [reagents] = 210-3
M, Na2CO3, CH3CN, reflux, 64 h; (b) NaN3, CH3CN, MW (260 W, 100°C), 15 min.
Synthesis of N-bifunctionnal PCTA derivatives
Exploiting the synthetic procedure for the building of the triamine backbone 3, the access of
PCTA derivatives substituted at the central acetate -carbon was investigated. The use of -
amino acids such as L-glutamic acid, L-lysine, or L-p-nitrophenylalanine instead of glycine
allowed us to access to PCTA-like BFCAs displaying carboxylic acid, aliphatic amine, and
aromatic amine as reactive functions, respectively. The complete synthetic pathway for the
preparation of these three target molecules is shown in Schemes 5 and 6. In this approach, -
amino tert-butyl esters were used from commercial source (lysine derivative) or prepared by
using tert-butyl acetoacetate as the source of isobutene with catalysis by perchloric acid, as
reported by Taschner.[25] The polyamine substrates 19, 26, 28 were obtained upon dialkylation
of amino precursors with 2 molar equivalents of bromo derivative 2 in CH3CN at reflux and
in the presence of K2CO3 as the base; this was cleanly followed by debenzylation of the
intermediates by catalytic hydrogenation. It is noteworthy that dialkylation of L-glutamic acid
1-tert-butyl ester -5- methyl ester 21 instead of the corresponding di-tert-butyl ester 17 was
also carried out in these experimental conditions, but this reaction has not be successful.
During the alkylation of 21, intramolecular attack of primary amine of 21 and secondary
amine of reaction intermediate on the methyl ester bond leads to cyclization with the
formation of pyrrolidin-2-one derivatives yielding only impure product 22 with very low yield.
In the macrocyclization step, condensation of these intermediates 19, 26 and 28 with 2,6-
dibromopyridine let to fully protected macrocycles 29, 31 and 33 in good yields (60-85%),
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based on the efficient Na+ template effect. Finally, concomitant cleavage of BOC protecting
group and hydrolysis of tert-butyl ester functionalities were achieved under acid conditions
(6N aq HCl). Ligands were isolated as their hydrochloride salts in quantitative yields and in
good purity as evidenced by elemental analyses.
This route furnished BFCA 30 in five steps and 24% overall yield from glutamic acid. In
comparison, an overall yield of 3-4% in 30 has been previously reported by using the
traditional Richman-Atkins route and diethylenetriamine as starting material.[19] As the
protecting groups on the carboxylic acids in the precursor 29 are not orthogonal, 30 can only
be used as BFCA after metal complexation, because the reactivity of carboxylic acid groups is
very different after metal ion complexation, especially with Ln (vide infra). Besides, the
glutaric acid residue in 30 could be transformed into a six-membered cyclic anhydride which
can be selectively opened with different nucleophiles, offering interesting possibilities for
conjugation reactions. In this direction, DOTAGA-anhydride (GA = glutaric acid) is a
representative example of such BFCA containing an anhydride reactive moiety that has been
employed to access to other BFCA derivatives or various conjugated systems with proteins,
antibodies or silica nanoparticles.[26] Finally, we can also notice that the N-functionalized
derivative 32 was obtained with an overall yield twice as high as that reported for its C-
functionalized counter part (Figure 2, L6).[7b]
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Scheme 5. Synthesis of polyamines 19, 26, and 28. Reagents and conditions: (a) tert-butyl
acetate, HClO4, rt, 16 h; (b) bromide 2 (2 equiv), K2CO3, CH3CN, reflux, 24 h; (c) 10% Pd/C,
H2 gas (6 bar), MeOH, rt, 16 h; (d) Me3SiCl, MeOH, 0°C, 1 h; (e) tert-butyl acetate, HClO4, rt,
12 h; (f) bromide 2 (2 equiv), K2CO3, CH3CN, reflux, 24 h; (g) tert-butyl acetate, HClO4, rt,
16 h; (h) 10% Pd/C, H2 gas, MeOH, rt, 16 h; (i) Boc2O, 10% acetic acid in water, 1,4-dioxane,
rt, 16 h; (j) bromide 2 (2 equiv), Na2CO3, CH3CN, reflux, 24 h; (k) 10% Pd/C, H2 gas (6 bar),
MeOH, rt, 16 h; (l) bromide 2 (2 equiv), K2CO3, CH3CN, reflux, 24 h, ref [15]; (m) 10% Pd/C,
H2 gas (6 bar), MeOH, rt, 12 h, ref [15].
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Scheme 6. Synthesis of bifunctional PCTA derivatives 30, 32 and 34. Reagents and
conditions: (a) polyamine 19, [reagents] = 210-3 M, Na2CO3, CH3CN, reflux, 64 h; (b) 6N
HClaq, reflux, 24 h; (c) polyamine 26, [reagents] = 210-3 M, Na2CO3, CH3CN, reflux, 64 h;
(d) polyamine 28, [reagents] = 210-3 M, Na2CO3, CH3CN, reflux, 64 h.
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Synthesis of conjugates
As reported in Scheme 7, the triprotected derivative 7 was grafted at the N-terminus of an
Ahx-Ahx-CCK4 peptide (Ahx-Ahx-Trp-Nle-Asp-Phe-NH2, Ahx = 6-aminohexanoic acid, Nle
= Norleucine). This cholecystokinin tetrapeptide (CCK4) targets the cholecystokinin receptor
type 2 (CCK2R), which is expressed in various human cancers such as lung, ovarian,
pancreatic, medullary thyroid and numerous neuroendocrine/carcinoid tumors.[27] Thus, it
represents an ideal target for the diagnosis and radiotherapy of these kinds of tumours.[28] The
synthesis of bioconjugate 35 was achieved in two steps with an overall yield of 45%: (i) the
formation of an amide bond between the terminal amino function of the peptide derivative
and the activated carboxylic group of 7, using a classical PyBOP / HOBT protocol and (ii)
deprotection of tert-butyl ester functions of the resulting conjugate by trifluoroacetic acid in
the presence of scavengers of tert-butyl cations to avoid alkylation of the tryptophan unit of
the peptide. Assays were also performed to evaluate the ability of the peptide conjugate 35 to
rapidly form complex with naturally occurring 89Y. These experiments were performed at
room temperature with small amounts of peptide conjugate (0.4 mol) by the addition of 1
89
equiv. of Y at pH 5, since radiolabeling conditions are routinely performed in this range of
pH. These complexation assays were followed by MS monitored HPLC. In these conditions,
89
the conjugate 35 (tR = 19.9 min) has disappeared 15 min after addition of Y leading to a
single peak of the corresponding yttrium labeled conjugate 36 (tR = 17.6 min). By comparison
89
a complete Y labeling of the corresponding DOTA-(ahx)2-CCK4 conjugate takes place at
90°C after a 1 h incubation time. We have observed again the faster kinetics of complexation
of PCTA versus DOTA with this ion of interest.
As a second example, BFCA precursor 7 can be successfully coupled to a biotin derivative.
Biotinylated BFCAs are very valuable in the context of the pretargeting strategies developed
for cancer radioimmunotherapy (RIT). Such multistep strategies are based on the strong
avidin-biotin hydrophobic interaction between monoclonal antibody-avidin (or streptavidin)
conjugates and biotin derivatives carrying metallic chelates.[29] To synthesize the biotin-
conjugated derivative, the carboxylic acid of 7 was coupled with a commercial amino
derivative of biotin in the presence of the HATU / DIPEA system in DMF. After 48 h stirring
at room temperature, the reaction mixture was purified by column chromatography to afford
the pure product in 57% yield.
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European Journal of Organic Chemistry
Scheme 7. Synthesis of conjugates 36, 37 and SiO2-38. Reagents and conditions (a) i) PyBOP,
HOBt, DMF, rt, 24 h; ii) TFA/CH2Cl2 1/1, EDT, TES, rt, 3 h; (b) 89YCl3.6H2O, 0.1M sodium
acetate buffer pH 5; (c) HATU, DIPEA, DMF, rt, 72 h; (d) EuCl3.6H2O, H2O-NaOH, pH 5-6,
rt 16 h; (e) HBTU, Et3N, DMSO, rt, 16 h.
As a third illustration, the coupling of a metallic complex to a solid material was
exemplified by using a commercially available 3-aminopropyl silica gel (particle size 40-63
m).[30] In a first step, the EuIII complex of BFCA 30 was prepared by mixing slight excess
amount (10%) of EuCl3.6H2O salt and chelator in aqueous solution at pH 6.5. The purity of
the isolated complex was checked by UPLC analysis and its structure was determined by
HRMS. This EuIII complex was investigated by luminescence spectroscopy to evaluate the
16
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10.1002/ejoc.201900280
European Journal of Organic Chemistry
influence of the carboxylate function present in the side chain of the glutamic acid residue on
the nature of the inner sphere of metal. it is well established that the shape and splitting as
well as the relatives intensities of the 5D0  7FJ (J = 0 – 4) transitions are regarded as useful
parameters that may be used to assess changes in the Eu coordination environment.[31] At
room temperature and upon excitation at 270 nm, the luminescence spectrum of Eu(III)
complex 38 in Tris buffer (pH 7.4) is characterized by five bands in the 570 - 720 nm range
5
with the main band attributed to the D07F2 transition appearing in the 600 - 630 nm range
with a maximum at 616 nm. The fine structure and the relative intensities of the Eu emission
bands of 38 are similar to those of the parent complex Eu-PCTA. Particularly, the ratio
between the intensity of the 5D0  7F2 transition (electric dipole) and the intensity of the 5D0
7
 F1 transition (magnetic dipole) is approximately equal. On the other hand, the emission
lifetimes of europium complex 38 and Eu-PCTA are close (0.38 and 0.37 ms at room
temperature, respectively), showing that these two complexes share the same inner hydration
sphere. This was confirmed, as usually, by the evaluation of the number of metal-bound water
molecules (q) from the lifetime data in water and deuterated water.[32] The application of the
Sudnick and Horrocks equation reveals for 38 q = 2.05 in agreement with the bis hydrated
nature of Eu-PCTA.[33] These data strongly suggest that the carboxylic acid function of the
side chain of the glutamic acid residue is not coordinated to EuIII ion and therefore is available
for conjugation reaction. In a second step, luminescent silica particles were obtained by
reacting europium complex (2 equiv.) with commercially available 3-aminopropyl silica gel
(Sigma, 1 mmol NH2 per g) in DMSO at room temperature and using HBTU as the coupling
agent. By using UV and ICP-MS analyses, a 0.20 mmol amount of europium complex per
gram of support was found for the resulting conjugate. Luminescence experiments (Figure 3,
Table 1) showed that there is no significant difference on the luminescence spectrum pattern
and emission lifetime between the free lanthanide complex and the conjugated particles. The
temporal decay in the emission of the SiO2-38 hybrid was rigorously monoexponential
showing the presence of a single emission solution species. Moreover, the quenching by the
O-H high energy vibrational modes of the solvent and the luminescence quantum yield
observed for SiO2-38 (q = 1.83 and  = 3.5 %, respectively) were found be similar to those of
the precursor complex 38. These results indicate that the photophysical properties of the free
lanthanide complex are not affected by the silica networks after the complex is covalently
bound into the silica backbone.
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300
200
100
2
7
5D0  FJ
4
1
J=0
3
0
200
300
400
500
600
700
Wavelength (nm)
__
Figure 3. Corrected excitation (---) and emission ( ) spectra of SiO2-38 in Tris buffer (pH
7.4) at 298 K.The emission bands arise from 5D0  7FJ transitions; the J values are shown on
the spectrum.
Table 1. Photophysical data of europium complex 38 and SiO2-38 hybrid in aqueous Tris
buffer (pH 7.4).
[a]
Entry
qH2O
I 7F2 /
I 7F1[b]
I 7F4 /
I 7F1[b]
H2O
D2O

(%)[c]
(ms)
(ms)
38
0.38
0.40
2.13
1.87
2.05
1.83
2.7
3.0
2.1
2.1
3.2
3.5
SiO2-38
[a] Number of metal coordinated water molecules at 298K,
-1
-1
calculated using the equation[33]: qH2O(Eu) = 1.11[(H2O
) - (D2O)
7
5
– 0.31]with  in ms. [b] Integrated intensity ratio of D0 F2 or
5D0 F4 and D0 F1 transitions. [c] Quantum yield determined
7
5
7
by excitation of the pyridine moiety.
Conclusions
The metal-chelating agent PCTA and derivatives are increasingly exploited for various
applications in diagnostic imaging and therapeutic nuclear oncology. In the present paper, we
report the synthesis of a dozen of bifunctional derivatives bearing a free reactive functional
group on the 4-position of the pyridine ring or on the methylene carbon atom of the central
acetate chelating arm. Thanks to their free –COOMe, -COOH, -OH, -NH2, azido or
maleimido groups, all the novel PCTA derivatives may undergo coupling reactions with a
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10.1002/ejoc.201900280
European Journal of Organic Chemistry
wide range of biological vectors or supports. The synthetic methodology developed herein
provides a simple route to BFCAs-PCTA with high yield in the crucial macrocyclization step
and suitable overall yields. As demonstrated in the case of derivatives 7 and 30, conjugation
of these BFCAs to vectors can be performed with protected chelators, followed by
deprotection and complexation with a selected ion; or alternatively by using a preformed
metallic complex. In order to have in hand N-bifunctional PCTA chelators better suited to
luminescent materials for biological applications (shift towards lower excitation energies and
higher extinction coefficient of the chromophoric unit), judicious substitution of the pyridine
unit should be undertaken. This work is currently in progress.
Abbreviations
DIPEA: N-Ethyldiisopropylamine
EDT: 1,2-Ethanedithiol
DIAD: Diisopropyl azodicarboxylate
HATU: Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium; 1-
[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
hexafluorophosphate
HBTU: Hexafluorophosphate Benzotriazole Tetramethyl Uronium; N,N,N′,N′-Tetramethyl-
O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, O-(Benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate
HOBt: 1-Hydroxybenzotriazole hydrate
PyBOP: (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
TES: Triethylsilane
Experimental Section
Materials and methods
Reactions requiring an inert atmosphere were run under Argon. Reactions utilizing
microwave technology were conducted in a CEM Discover Benchmate microwave reactor
(model nr.908010). Acetonitrile was freshly distilled from P2O5, and CH2Cl2 was freshly
distilled from CaH2. Thin-layer chromatography was performed on Merck silica or alumina
plates with a fluorescence indicator. Column chromatography was carried out on silica gel
(Merck, 60-200 m, porosity 60 Å) and on alumina (Macherey-Nagel, activity IV, 50-200
m).
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13
1H and C spectra were recorded using a Bruker Avance 300 spectrometer operating at 300
1
MHz and 75 MHz for H and 13C respectively or on a Bruker Avance 500 spectrometer
operating at 500 MHz for 1H and 125 MHz for 13C. Chemical shifts are reported in ppm, with
residual protonated solvents as the internal references. Electrospray (ES) mass spectra were
obtained on a Q TRAP 2000 Applied Biosystems spectrometer and High-Resolution Mass
Spectra (HRMS) on a Xevo G2 QTof Waters spectrometer. DCI mass spectra were obtained
on a DSQ II Thermo Fisher and MALDI mass spectra on a MALDI micro MX Micromass
spectrometer. IR spectra were recorded on a Nexus ThermoNicolet spectrometer. Elemental
analyses were recorded on a Perkin Elmer 2400 series II Simultaneous CHN elemental
analyser at the “Service d’Analyse”, Laboratoire de Chimie de Coordination (Toulouse).
The HPLC analyses were performed on a Waters Alliance 2695 system with a PDA 2996
detector and using a reverse-phase column (Phenomenex Luna C18, 3 μm, 100 Ǻ, 100 x 3
mm) with a flow rate of 1 mL/min. Linear gradient system was HCOONH4 10 mM pH 4/
CAN 100/0 to 60/40 in 30 min (system 1) or 80/20 to 0/100 in 18 min then an isocratic
elution 0/100 for 4 min (system 2); H2O + 0.1% TFA/CH3CN 80/20 to 60/40 in 35 min
(sytem 3).
The UPLC analyses were performed on a Waters UPLC Acquity apparatus with PDA
(photodiode array) and SQ (simple quadripole) detectors, and using an Acquity BEH HILIC
column (1.7 m, 100  2.1 mm) with a flow rate of 0.4 mL.min-1. Linear gradient system was
H2O + 0.1% HCOOH (pH 2)/CH3CN + 0.1% HCOOH (pH 2) 05/95 to 50/50 in 7 min, then
an isocratic elution 50/50 for 10 min.
Absorption measurements were done with a Hewlett Packard 8453 temperature-controlled
spectrometer in 10 mm quartz cuvette. Emission, excitation spectra and luminescence decays
of europium complexes were measured using a Cary Eclipse spectrofluorimeter equipped with
a Xenon flash lamp source and a Hamamatsu R928 photomultiplier. Excitation spectra were
corrected for the excitation light intensity, while emission spectra were corrected for the
instrument response. Lifetimes  (uncertainty ≤ 5%) are made by monitoring the decay at 616
nm, a wavelength corresponding to the maximum intensity of the emission spectrum,
following pulsed excitation. They are the average values from at least five separate
measurements covering two or more lifetimes. The luminescence decay curves were fitted by
an equation of the form I(t) = I(0) exp(-t/) by using a curve-fitting program. Quantum yields
were determined by a comparative method with a standard reference ([Ru(bpy)3]Cl2 ( = 4%
in water) [34]; estimated experimental errors for quantum yield determination are  10%.
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Experimental procedure and characterization data of compounds
Compound 4: To a solution of triamine 3 (170 mg, 0.381 mmol) in anhydrous CH3CN (185
mL) was added Na2CO3 (404 mg, 3.81 mmol) and the suspension was heated at reflux for 1.5
h. Dibromide 1 (123 mg, 0.381 mmol) was added in one portion and the reaction mixture was
heated at reflux for 64 h and was then cooled to room temperature. The inorganic salts were
filtered off and the filtrate was concentrated under vacuum. The residue was subjected to
purification by column chromatography on alumina (CH2Cl2/MeOH, 100:0 to 98:02) to give a
mixture of sodium complex and free ligand. This mixture was dissolved in CH2Cl2 (30 mL)
and stirred overnight in the presence of a saturated Na2EDTA aqueous solution (10 mL). The
organic layer was dried (MgSO4) and evaporated under vacuum to give free compound 4 as a
yellow oil (208 mg, 90%). TLC (Al2O3, CH2Cl2/MeOH, 98:02) Rf = 0.3. Elemental analysis:
Found: C, 61.2; H, 8.2; N 9.1. C31H50N4O8 requires C, 61.4; H, 8.3; N, 9.2%. IR (neat):
1
max/cm-1 2977, 2934, 2856, 1729, 1676, 1571, 1368, 1224, 1153. H NMR (300 MHz,
CDCl3): δ = 1.36 (s, 9H), 1.41 (s, 18H), 2.83 (br s, 8H), 3.37 (s, 6H), 3.87 (s, 3H), 4.04 (s,
4H), 7.61 (s, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ = 28.0 (CH3), 28.1 (CH3), 50.2 (CH2),
52.5 (CH3), 58.2 (CH2), 59.6 (CH2), 81.2 (Cq), 81.4 (Cq), 121.1 (CH), 138.5 (Cq), 159.3 (Cq),
165.45 (CO), 170.4 (CO) ppm. HRMS (ESI+): m/z 629.3528 ([M+Na]+, 15%), 607.3712
([M+H]+, 100), 551.3083 ([M+2H-tBu]+, 62), 495.2456 ([M+3H-2tBu]+, 22), 439.1828
([M+4H-3tBu]+. HRMS (ESI+): m/z calcd for C31H51N4O8 [M+H]+ 607.3707, found 607.3712.
Sodium complex [Na-4]Cl: To a stirred solution of free ligand 4 (30 mg, 0.0494 mmol) in
CH3CN (25 mL) was added NaCl (29 mg, 0.496 mmol) and the suspension was heated at
reflux for 16 h. The inorganic salts were filtered off and the filtrate was concentrated under
vacuum to give [Na-4]Cl complex as a brown oil. IR (KBr pellet): max/cm-1 2974, 2933, 2856,
1
1725, 1671, 1572, 1369, 1228, 1158. H NMR (300 MHz, CDCl3): δ = 1.44 (s, 9H), 1.48 (s,
18H), 1.79-2.27 (m, 4H), 2.46-2.71 (m, 4H), 3.15 (s, 2H), 3.48 (d, JA-B = 17.7 Hz, A part of
AB system, 2H), 3.57 (d, JA-B = 17.7 Hz, B part of AB system, 2H), 3.82 (d, JA-B = 14.9 Hz, A
part of AB system, 2H), 3.94 (s, 3H), 4.22 (d, JA-B = 14.9 Hz, B part of AB system, 2H), 7.64
13
(s, 2H) ppm. C NMR (75 MHz, CDCl3): δ = 27.8 (CH3), 27.9 (CH3), 52.8 (CH3), 53.05
(CH2), 53.4 (CH2), 55.8 (CH2), 59.1 (CH2), 62.0 (CH2), 82.3 (Cq), 82.5 (Cq), 120.5 (CH),
139.2 (Cq), 159.5 (Cq), 165.0 (CO), 172.5 (CO), 172.9 (CO) ppm.
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Compound 5: A solution of free macrocycle 4 (30 mg, 0.05 mmol) in trifluoroacetic acid
(2mL) and anhydrous CH2Cl2 (2 mL) was stirred at room temperature for 16 h. The reaction
mixture was evaporated to dryness under vacuum, and the residue was taken up in 2 mL of
MeOH and 10 mL of ethyl ether. The resulting solid was collected and dried to yield triacid 5
(22 mg, 100%). Mp ~ 180°C (dec.). HPLC analysis: tR = 8.36 min (system 1). 1H NMR (300
MHz, D2O): δ = 2.95 (s, 4H), 3.63 (s, 6H), 3.96 (s, 3H), 4.38 (s, 4H), 5.00 (s, 4H), 7.97 (s,
13
2H) ppm. C NMR (75 MHz, D2O): δ = 50.6 (CH2), 53.5 (CH3), 54.4 (CH2), 55.1 (CH2),
56.75 (CH2), 59.7 (CH2), 121.6 (CH), 140.8 (Cq), 151.2 (Cq), 165.9 (CO), 168.5 (CO),
175.15 (CO) ppm. MS (ESI-): m/z 475.5 ([M+K-2H]-, 100%).
Compound 6: Free macrocycle 4 (100 mg, 0.165 mmol) in 6N HClaq (15 mL) was heated at
reflux for 24 h. The solvent was removed under vacuum to give the hydrochloride salt of 6 as
a pale yellow powder (102 mg, 100%). HPLC analysis: tR = 3.17 min (system 1). Elemental
analysis: Found: C, 34.3; H, 5.6; N 8.8. C18H24N4O8+4HCl+3H2O requires C, 34.6; H, 5.5; N,
9.0%. 1H NMR (300 MHz, D2O): δ = 2.90 (br s, 4H), 3.59 (s, 6H), 4.37 (s, 4H), 4.99 (s, 4H),
7.93 (s, 2H) ppm. 13C NMR (75 MHz, D2O): δ = 50.6 (CH2), 54.35 (CH2), 55.25 (CH2), 56.7
(CH2), 59.7 (CH2), 121.8 (CH), 141.4 (Cq), 151.0 (Cq), 166.9 (CO), 168.3 (CO), 175.2 (CO)
ppm. MS (ESI-): m/z 461.5 ([M+K-2H]-, 26%), 423.5 ([M-H]+, 100).
Compound 7: To a solution of free macrocycle 4 (70 mg, 0.115 mmol) in a mixture of
MeOH (3 mL) and H2O (1.5 mL) was added Na2CO3 (12 mg, 0.113 mmol). The mixture was
stirred at room temperature and the reaction was followed by TLC analysis. After 4 h, one
equivalent of Na2CO3 was added and the mixture was allowed to stir at room temperature for
18 h. The organic phase was evaporated under vacuum and the aqueous phase was adjusted to
pH 6 with 0.1 N HCl. The aqueous phase was then evaporated under vacuum and the residue
was subjected to purification by column chromatography on alumina (CH2Cl2/MeOH, 100:0
then 96:04) to give sodium complex [Na-7]Cl as a white solid (58 mg, 78%). TLC (Al2O3,
CH2Cl2/MeOH: 96/04) Rf = 0.10. HPLC analysis: tR = 10.36 min (system 2). Elemental
analysis: Found: C, 55.2; H, 7.4; N 8.45. C30H48N4O8+NaCl requires C, 55.3; H, 7.4; N, 8.6%.
1
IR (KBr pellet): max/cm-1 2974, 1727, 1616, 1564, 1454, 1369, 1233, 1159. H NMR (300
MHz, CDCl3): δ = 1.42 (s, 9H), 1.48 (s, 18H), 1.70-2.05 (m, 4H), 2.31-2.55 (m, 4H), 2.98 (s,
2H), 3.25 (d, JA-B = 17.5 Hz, A part of AB system, 2H), 3.42 (d, JA-B = 17.5 Hz, B part of AB
system, 2H), 3.53 (d, JA-B = 13.8 Hz, A part of AB system, 2H), 3.98 (d, JA-B = 13.8 Hz, B
13
part of AB system, 2H), 7.76 (s, 2H) ppm. C NMR (75 MHz, CDCl3): δ = 27.8 (CH3), 28.0
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(CH3), 53.0 (CH2), 53.7 (CH2), 55.9 (CH2), 59.6 (CH2), 62.5 (CH2), 82.6 (Cq), 82.7 (Cq),
122.4 (CH), 144.7 (Cq), 156.8 (Cq), 168.5 (CO), 172.6 (CO), 173.1 (CO) ppm. HRMS
(ESI+): m/z 615.3375 ([M+Na]+, 21%), 593.3554 ([M+H]+, 87), 537.2927 ([M+2H-tBu]+,
100), 481.2300 ([M+3H-2tBu]+, 32), 425.1670 ([M+4H-3tBu]+, 8). HRMS (ESI+): m/z calcd
for C30H49N4O8 [M+H]+ 593.3550, found 593.3554.
Compound 8: To a stirred solution of free macrocycle 4 (50 mg, 0.0824 mmol) in methanol
(4 mL) was added NaBH4 (223 mg, 5.9 mmol). The reaction was followed by TLC analysis.
The mixture was stirred at room temperature for 1 week and methanol was removed under
reduced pressure. A saturated aqueous solution of NH4Cl (2 mL) and water (4 mL) were
added to the residue and the mixture was extracted with CH2Cl2 (3 × 4 mL). The combined
organic layers were dried on MgSO4 and concentrated under vacuum. The residue was
subjected to purification by column chromatography on alumina (CH2Cl2/MeOH, 95:05) to
give sodium complex [Na-8]Cl as an oil (39 mg, 74%). TLC (Al2O3, CH2Cl2/MeOH: 95/05)
1
Rf = 0.37. H NMR (300 MHz, CDCl3): δ = 1.41 (s, 9 H), 1.47 (s, 18 H), 1.65-2.21 (m, 4H),
2.38-2.58 (m, 4H), 3.00 (s, 2H), 3.25 (d, JA-B = 17.5 Hz, A part of AB system, 2H), 3.41 (d,
JA-B = 17.5 Hz, B part of AB system, 2H), 3.54 (d, JA-B = 14.1 Hz, A part of AB system, 2H),
13
3.95 (d, JA-B = 14.1 Hz, B part of AB system, 2H), 4.73 (s, 2H), 7.24 (s, 2H) ppm. C NMR
(75 MHz, CDCl3): δ = 27.7 (CH3), 27.9 (CH3), 53.0 (CH2), 53.6 (CH2), 55.9 (CH2), 59.5
(CH2), 62.15 (CH2), 62.3 (CH2), 82.5 (Cq), 82.8 (Cq), 119.7 (CH), 155.7 (Cq), 157.0 (Cq),
172.7 (CO), 173.2 (CO) ppm. MS (ESI+): m/z 579.4 ([M+H]+, 100%). HRMS (ESI+): m/z
calcd for C30H51N4O7 [M+H]+ 579.3758, found 579.3760.
Compound 9: A mixture of [Na-4]Cl (55 mg, 0.0829 mmol) and ethylenediamine (148 mg,
2.47 mmol) was stirred for 16 h at room temperature. The reaction mixture was evaporated to
dryness under high vacuum and the crude product was purified on alumina (CH2Cl2/MeOH,
95:05) to give sodium complex [Na-9]Cl as a beige oil (52 mg, 90%).TLC (Al2O3,
CH2Cl2/MeOH, 90:10) Rf = 0.12. IR (ATR): max/cm-1 3362, 3285, 3070, 2976, 2928, 2854,
1
1728, 1658, 1609, 1562, 1457, 1368, 1229, 1158. H NMR (300 MHz, CDCl3): δ = 1.42 (s, 9
H), 1.49 (s, 18 H), 1.76-1.93 (m, 2H), 2.01-2.12 (m, 2H), 2.33-2.47 (m, 4H), 2.51 (s, 2H),
2.56-2.65 (m, 2H), 2.97-3.00 (m, 2H), 3.23 (d, JA-B = 17.4Hz, A part of AB system, 2H), 3.43
(d, JA-B = 17.4Hz, B part of AB system, 2H), 3.49-3.61 (m, 2H), 3.75 (d, JA-B = 14.4 Hz, A
part of AB system, 2H), 4.02 (d, JA-B = 14.4 Hz, B part of AB system 2H), 8.09 (s, 2H), 9.65
(t, J = 5.8 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3): δ = 27.8 (CH3), 27.9 (CH3), 41.3 (CH2),
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49.3 (CH2), 53.2 (CH2), 53.7 (CH2), 54.5 (CH2), 59.5 (CH2), 62.1 (CH2), 82.6 (Cq), 82.9 (Cq),
120.8 (CH), 144.3 (Cq), 158.1 (Cq), 165.65 (CO), 172.8 (CO), 173.2 (CO) ppm. MS (ESI+):
m/z 635.4 ([M+H]+, 96%), 579.3 ([M+2H-tBu]+, 39), 523.3 ([M+3H-2tBu]+, 100), 467.3
([M+4H-3tBu]+, 41). HRMS (ESI+): m/z calcd for C32H55N6O7 [M+H]+ 635.4132, found
635.4144.
Compound 10: A mixture of [Na-7]Cl (43 mg, 66.7 µmoles), compound 13 (62 mg, 266
µmol), HATU (101 mg, 266 µmol), and DIPEA (70 µL, 400 µmol) in DMF (3.3 mL) was
stirred for 16 h at room temperature. The reaction mixture was evaporated to dryness under
vacuum, CH2Cl2 (10 mL) was added to the residue and the resulting precipitated solid was
filtered. The organic phase was concentrated under vacuum. The oily residue was subjected to
purification by column chromatography on alumina (CH2Cl2/MeOH, 99:01) to give sodium
complex [Na-10]Cl as a beige oil (34 mg, 61%). IR (ATR): max/cm-1 3428, 2978, 2933, 2858,
1
1722 , 1705, 1665, 1566, 1368, 1230, 1157. H NMR (300 MHz, CDCl3): δ = 1.24-1.42 (m,
4H), 1.44 (s, 9 H), 1.51 (s, 18 H), 1.54-1.70 (m, 4H), 1.79-2.16 (m, 4 H), 2.36-2.66 (m, 4 H),
3.01 (s, 2H), 3.26 (d, JA-B = 17.6 Hz, A part of an AB system, 2H), 3.43 (d, JA-B = 17.6 Hz, B
part of an AB system, 2H), 3.37-3.44 (m, 2H), 3.50 (t, J = 7.2 Hz, 2H), 3.73 (d, JA-B = 14.5 Hz,
A part of an AB system, 2H), 4.00 (d, JA-B = 14.5 Hz, B part of an AB system, 2H), 6.67 (s,
2H), 7.22 (t, J = 5.9 Hz, 1H), 7.58 (s, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ = 26.27 (CH2),
26.30 (CH2), 27.8 (CH3), 28.0 (CH3), 28.4 (CH2), 28.8 (CH2), 37.75 (CH2), 40.2 (CH2), 53.3
(CH2), 53.8 (CH2), 56.1 (CH2), 59.6 (CH2), 62.0 (CH2), 82.6 (Cq), 83.0 (Cq), 119.8 (CH),
134.0 (CH), 144.9 (Cq), 158.6 (Cq), 165.4 (CO), 170.9 (CO), 172.9 (CO), 173.4 (CO) ppm.
MS (ESI+): m/z 771.5 ([M+H]+, 100%). HRMS (ESI+): m/z calcd for C40H63N6O9 [M+H]+
771.4656, found 771.4669.
Compound 15: Compound 15 was synthesized, as above for 4, from triamine 3 (264 mg,
0.592 mmol), Na2CO3 (630 mg, 5.94 mmol) and tribromide 14 (203 mg, 0.592 mmol) in
CH3CN (290 mL). After purification by column chromatography on alumina (CH2Cl2/MeOH,
99:01) and treatment with EDTA, free macrocycle 15 was obtained as a beige oil (230 mg,
62%). TLC (Al2O3, CH2Cl2/MeOH, 97:03) Rf = 0.3. IR (neat): max/cm-1 2978, 2934, 2871,
1
1730, 1673, 1635, 1568, 1368, 1227, 1153. H NMR (300 MHz, CDCl3): δ = 1.40 (s, 9 H),
1.45 (s, 18 H), 2.42-3.07 (m, 10H), 3.40 (s, 4H), 3.97 (s, 4H), 7.30 (s, 2H) ppm. 13C NMR (75
MHz, CDCl3): δ = 27.8 (CH3), 27.9 (CH3), 50.0 (CH2), 58.0 (CH2), 59.4 (CH2), 80.9 (Cq),
81.2 (Cq), 125.1 (CH), 133.4 (Cq), 159.2 (Cq), 170.2 (CO), 170.6 (CO) ppm. MS (ESI+): m/z
24
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10.1002/ejoc.201900280
European Journal of Organic Chemistry
629.2 ([M+H]+, 100%). 627.2 ([M+H]+, 96). 517.2 ([M+3H-2tBu]+,13), 515.2 ([M+3H-
79
2tBu]+,12). HRMS (ESI+): m/z calcd for C29H48N4O6 Br [M+H]+ 627.2757, found 627.2770.
1
Sodium complex [Na-15]Cl: H NMR (300 MHz, CDCl3): δ = 1.32 (s, 9 H), 1.37 (s, 18 H),
1.73-1.88 (m, 4H), 1.99-2.20 (m, 2H), 2.39-2.60 (m, 4H), 3.00 (s, 2H), 3.26 (d, JA-B = 17.7Hz,
A part of AB system, 2H), 3.41 (d, JA-B = 17.7Hz, B part of AB system, 2H), 3.63 (d, JA-B
=
14.9Hz, A part of AB system, 2H), 3.91 (d, JA-B = 14.9Hz, B part of AB system, 2H), 7.44 (s,
13
2H) ppm. C NMR (75 MHz, CDCl3): δ = 27.3 (CH3), 27.5 (CH3), 52.9 (CH2), 53.2 (CH2),
55.7 (CH2), 58.8 (CH2), 61.3 (CH2), 82.2 (Cq), 82.5 (Cq), 124.4 (CH), 134.3 (Cq), 159.1
(Cq), 172.5 (CO), 173.0 (CO) ppm.
Compound 16: To a solution of free macrocycle 15 (35 mg, 0.0557 mmol) in anhydrous
CH3CN (3 mL) was added NaN3 (18 mg, 0.277 mmol, 5 equiv.) and the mixture was
irradiated under MW (260 W, 100°C) for 15 min. The solvent was evaporated under vacuum
and the residue was subjected to purification by column chromatography on alumina
(CH2Cl2/MeOH, 97:03 then 95:05) to give the sodium complex. This sodium complex was
dissolved in CH2Cl2 (10 mL) and the solution was stirred overnight in the presence of a
saturated Na2EDTA aqueous solution (5 mL). The organic layer was dried (MgSO4) and
evaporated under vacuum to give free compound 16 as a beige oil (19 mg, 58%). IR (neat):
1
max/cm-1 2977, 2934, 2110, 1727, 1598, 1456, 1368, 1224, 1150. H NMR (300 MHz,
CDCl3): δ = 1.42 (s, 9 H), 1.48 (s, 18 H), 2.49-3.22 (m, 10H), 3.43 (s, 4H), 4.00 (s, 4H), 6.81
(s, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ = 28.1 (CH3), 28.2 (CH3), 50.2 (CH2), 58.3 (CH2),
59.9 (CH2), 81.2 (Cq), 112.65 (CH), 149.7 (Cq), 159.8 (Cq), 170.6 (CO) ppm. HRMS (ESI+):
m/z calcd for C29H48N7O6 [M+H]+ 590.3666, found 590.3679.
Sodium complex [Na-16]Cl: it was prepared, as described above for [Na-4]Cl. IR (neat):
1
max/cm-1 2977, 2932, 2112, 1723, 1598, 1456, 1369, 1227, 1161. H NMR (300 MHz,
CDCl3): δ = 1.44 (s, 9 H), 1.49 (s, 18 H), 1.83-2.06 (m, 2H), 2.15-2.29 (m, 2H), 2.50-2.67 (m,
4H), 3.14 (s, 2H), 3.43 (d, JA-B = 17.6 Hz, A part of AB system, 2H), 3.54 (d, JA-B = 17.6 Hz,
B part of AB system, 2H), 3.68 (d, JA-B = 14.7 Hz, A part of AB system, 2H), 4.10 (d, JA-B
=
13
14.7 Hz, B part of AB system, 2H), 6.79 (s, 2H) ppm. C NMR (75 MHz, CDCl3): δ = 27.9
(CH3), 28.0 (CH3), 53.2 (CH2), 53.7 (CH2), 56.0 (CH2), 59.3 (CH2), 62.1 (CH2), 82.4 (Cq),
82.6 (Cq), 111.7 (CH), 150.9 (Cq), 160.1 (Cq), 172.7 (CO), 173.25 (CO) ppm. MS (ESI+):
m/z 590.4 ([M+H]+, 100%), 534.3 ([M+2H-tBu]+, 34), 478.3 ([M+3H-2tBu]+, 9).
25
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10.1002/ejoc.201900280
European Journal of Organic Chemistry
CAUTION: Note that azido compounds are potentially explosive intermediates, and reactions
should be performed behind a plastic shield. Nevertheless, in our hands, we never observed
hazardous reactions with our experimental procedure.
Compound 17: To a solution of L-glutamic acid (896 mg, 6.1 mmol) in tert-butyl acetate (84
mL) was added HClO4 (0.6 mL). The mixture was stirred for 16 h at room temperature then
cooled down to 0 °C and extracted with a cold solution of HCl (0.5 N). The aqueous layer was
neutralized with NaHCO3 then extracted three times with EtOAc. The organic layers were
combined, dried over MgSO4, filtered and the solvent was removed under vacuum to give 17
as a semi-crystalline oil (805 mg, 51%). The crude product was used in the next step without
1
any purification. H NMR (300 MHz, CDCl3): δ = 1.41 (s, 9H), 1.43 (s, 9H), 1.69-1.82 (m,
1H), 1.91-2.02 (m, 1H), 2.32 (t, J = 7.6 Hz, 2H), 2.33 (s, 2H), 3.35 (dd, J = 5.4, 7.7 Hz, 1H)
13
ppm. C NMR (75 MHz, CDCl3): δ = 27.9 (CH3), 28.0 (CH3), 29.7 (CH2), 31.8 (CH2), 54.1
(CH), 80.4 (Cq), 81.3 (Cq), 172.5 (CO), 174.4 (CO) ppm. MS (DCI, NH3): m/z 260.1
([M+H]+, 100 %), 203.1 ([M-tBuOH+NH4]+, 60), 186.1 ([M-tBuOH+H]+, 21).
Compound 18: Di-tert-butyl L-glutamate 17 (118 mg, 0.46 mmol) and K2CO3 (630 mg, 4.56
mmol) in anhydrous CH3CN (14 mL) were heated at reflux and after 1h, bromide derivative 2
(300 mg, 0.91 mmol) was added. The reaction mixture was stirred for 24 h at reflux, filtered
and concentrated under vacuum. The residue was purified by column chromatography
(silicagel, CH2Cl2/EtOAc, 98:02) to give 18 as a yellow oil (198 mg, 57%). TLC (SiO2,
1
CH2Cl2/MeOH, 98:02) Rf = 0.31. H NMR (300 MHz, CDCl3): δ = 1.42 (s, 18H), 1.45 (s,
18H), 1.61-1.95 (m, 2H), 2.14-2.37 (m, 2H), 2.60-2.80 (m, 8H), 3.18 (s, 4H), 3.22 (dd, J = 3.9,
13
5.6 Hz, 1H), 3.74 (s, 4H), 7.10-7.36 (m, 10H) ppm. C NMR (75 MHz, CDCl3): δ = 25.0
(CH2), 28.08 (CH3), 28.16 (CH3), 28.23 (CH3), 32.0 (CH2), 50.0 (CH2), 53.4 (CH2), 55.2
(CH2), 58.3 (CH2), 63.4 (CH), 80.0 (Cq), 80.6 (Cq), 80.8 (Cq), 127.0 (CH), 128.2 (CH), 128.9
(CH), 139.1 (Cq), 170.7 (CO), 172.3 (CO), 172.8 (CO) ppm. MS (ESI+): m/z 776.5 ([M+Na]+,
32%), 754.5 ([M+H]+, 100), 698.4 ([M+2H-tBu]+, 24), 642.4 ([M+3H-2tBu]+, 35), 586.3
([M+4H-3tBu]+, 54), 530.3 ([M+5H-4tBu]+, 49). HRMS (ESI+): m/z calcd for C43H68N3O8
[M+H]+ 754.5007, found 754.5018.
Compound 19: Compound 18 (330 mg, 0.438 mmol) was solubilized in MeOH (15 mL) and
then palladium on carbon wt 10% (115 mg) was added. The black suspension was
hydrogenated under 6 bar of hydrogen pressure for 16 h at room temperature. The resulting
26
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10.1002/ejoc.201900280
European Journal of Organic Chemistry
mixture was filtered through a Celite® pad and the filtrate was concentrated under vacuum to
give the expected product 19 as a yellow oil (251 mg, 100%), which was engaged in the next
step without further purification. IR (neat): max/cm-1 2977, 2929, 1729, 1456, 1393, 1368,
1249, 1233, 1154. 1H NMR (300 MHz, CDCl3): δ = 1.35 (s, 9 H), 1.36 (s, 9 H), 1.37 (s, 18 H),
1.62-1.99 (m, 2H), 2.09-2.35 (m, 4H), 2.48-2.77 (m, 8H), 3.15-3.27 (m, 4H), 3.33 (s, 1 H)
13
ppm. C NMR (75 MHz, CDCl3): δ = 24.9 (CH2), 27.9 (CH3), 28.1 (CH3), 31.9 (CH2), 47.9
(CH2), 51.0 (CH2), 51.4 (CH2), 63.0 (CH), 79.9 (Cq), 80.7 (Cq), 80.8 (Cq), 171.3 (CO), 171.8
(CO), 172.5 (CO) ppm. MS (ESI+): m/z 574.4 ([M+H]+, 100%), 518.3 ([M+2H-tBu]+, 13),
462.3 ([M+3H-2tBu]+, 10).
Compound 20: To a solution of L-glutamic acid (1.84 g, 12.5 mmol) in MeOH (37 mL)
cooled at 0°C was added dropwise trimethylsilyl chloride (3.5 mL, 27.6 mmol). Once addition
was completed (1 h), MeOH was removed under vacuum to give the hydrochloride salt of 20
as a white solid (2.5 g, 100%). The crude product was used in the next step without any
purification. 1H NMR (300 MHz, D2O): δ = 2.14-2.34 (m, 2H), 2.64 (t, J = 7.6 Hz, 2H), 3.72
13
(s, 3H), 4.02-4.13 (m, 1H) ppm. C NMR (75 MHz, D2O): δ = 24.8 (CH2), 29.4 (CH2), 24.8
(CH2), 52.1 (CH), 52.4 (CH3), 171.5 (CO), 174.9 (CO) ppm. . MS (DCI, NH3): m/z 179.0
([M+NH4]+, 13 %), 162.0 ([M+H]+, 100).
Compound 21: To a solution of 20 (300 mg, 1.52 mmol) in tert-butyl acetate (21 mL) was
added HClO4 (0.15 mL). The mixture was stirred for 12h at room temperature then cooled
down to 0 °C and extracted with a cold solution of HCl 0.5 N (2 × 15 ml). The aqueous layer
was neutralized with NaHCO3 then extracted three times with EtOAc. The organic layers
were combined, dried over MgSO4, filtered and the solvent was removed under vacuum to
give 21 as a colorless oil (250 mg, 75%). 1H NMR (300 MHz, CDCl3): δ = 1.45 (s, 9H), 1.73-
1.86 (m, 3H), 1.94-2.08 (m, 1H), 2.44 (t, J = 7.6 Hz, 2H), 3.33 (dd, J = 5.3, 8.2 Hz, 1H), 3.67
(s, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ = 28.0 (CH3), 29.8 (CH2), 30.5 (CH2), 51.7 (CH),
54.3 (CH3), 81.3 (Cq), 173.7 (CO), 174.8 (CO) ppm. NMR data are similar to those reported
previously.[35]
Compound 23:
This compound was synthesized in two steps from 4-nitro-L-phenylalanine.
27
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10.1002/ejoc.201900280
European Journal of Organic Chemistry
L-phenylalanine, 4-nitro-, 1,1-dimethylethyl ester: This compound was synthesized as above
for 17 from 4-nitro-L-phenylalanine monohydrate (1 g, 4.38 mmol), tert-butyl acetate (60 ml)
and HClO4 (0.4 mL) and was obtained in 96% yield as a pale yellow solid (1.12 g). The crude
product was used in the next step without further purification. TLC (SiO2, EtOAc) Rf = 0.32.
1H NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 1.64 (s, 2H), 2.93 (dd, J = 7.7, 13.6 Hz, 1H),
3.12 (dd, J = 5.8, 13.6 Hz, 1H), 3.64 (dd, J = 5.8, 7.7 Hz, 1H), 7.38-7.43 (m, 2H), 8.14-8.19
(m, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ = 29.7 (CH3), 40.7 (CH2), 55.8 (CH), 81.7 (Cq),
+
123.5 (CH), 130.2 (CH), 145.6 (Cq), 146.8 (Cq), 173.6 (CO) ppm. MS (DCI/NH3 ): m/z 267.1
([M+H]+, 100%).
L-phenylalanine, 4-amino-, 1,1-dimethylethyl ester (23): A mixture of the previous nitro
derivative (0.55 g, 2.065 mmol) and palladium on carbon wt 10% (190 mg) in methanol (15
mL) was stirred under a dihydrogen atmosphere for 16 h at room temperature. The reaction
mixture was filtered through a Celite® pad and concentrated under vacuum to give 23 as a
1
beige powder (480 mg, 98%). TLC (SiO2, EtOAc) Rf = 0.13. H NMR (300 MHz, CDCl3): δ
= 1.44 (s, 9H), 2.80 (dd, J = 5.5, 13.9 Hz, 1H), 2.96 (dd, J = 5.5, 13.9 Hz, 1H), 3.63 (dd, J =
5.5, 7.3 Hz, 1H), 3.77 (s, 4H), 6.63 (d, J = 8.3 Hz, 2H), 6.98 (d, J = 8.3 Hz, 2H) ppm.13C
NMR (75 MHz, CDCl3): δ = 28.0 (CH3), 39.2 (CH2), 55.7 (CH), 81.7 (Cq), 115.4 (CH), 126.4
(Cq), 130.3 (CH), 145.0 (Cq), 173.2 (CO) ppm. . MS (DCI, NH3): m/z 237.1 ([M+H]+, 100 %),
181.0 ([M-tBu+2H]+, 10).
Compound 24: To a solution of 23 (250 mg, 1.058 mmol) in 10% acetic acid in water (10
mL) was added Boc2O (241 mg, 1.104 mmol) in 1,4-dioxane (10 mL). The reaction mixture
was stirred for 16 h at room temperature and then water (100 mL) was added. The mixture
was extracted with diethyl ether (30 mL). The aqueous layer was neutralized with a 2N NaOH
aqueous solution and extracted with diethyl ether (4 × 30 mL). The organic layers were
combined and concentrated under vacuum and the crude product was purified by column
chromatography on alumina (CH2Cl2/EtOAc, 95:05) to give 24 as a beige powder (230 mg,
1
65%). TLC (Al2O3, CH2Cl2/EtOAc, 95:5) Rf = 0.3. H NMR (300 MHz, CDCl3): δ = 1.40 (s,
9H), 1.48 (s, 9H), 2.36 (s, 2H), 2.78 (dd, J = 7.5, 13.6 Hz, 1H), 2.97 (dd, J = 7.5, 13.6 Hz, 1H),
3.57 (dd, J = 5.5, 7.5 Hz, 1H), 6.84 (s, 1H), 7.09 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H)
13
ppm. C NMR (75 MHz, CDCl3): δ = 27.9 (CH3), 28.2(CH3), 40.0 (CH2), 55.9 (CH), 80.3
(Cq), 81.3 (Cq), 118.6 (CH), 129.8 (CH), 131.5 (Cq), 137.1 (Cq), 152.8 (CO), 173.8 (CO)
ppm. MS (ESI+): m/z 359.2 ([M+Na]+, 6%), 337.2 ([M+H]+, 3), 281.2 ([M-tBu+2H]+, 8),
225.1 ([M-2tBu+3H]+ 100).
28
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10.1002/ejoc.201900280
European Journal of Organic Chemistry
Compound 25: Polyamine 25 was prepared, as described above for 18, from 24 (130 mg,
0.387 mmol), 2 (305 mg, 0.929 mmol) and Na2CO3 (410 mg, 3.87 mmol) in anhydrous
CH3CN (13 mL). The crude product was purified by column chromatography on alumina
(CH2Cl2/petroleum ether, 70/30) to give 25 as a yellow oil (199 mg, 62%). TLC (Al2O3,
CH2Cl2/petroleum ether, 70:30) Rf = 0.31. 1H NMR (300 MHz, CDCl3): δ = 1.35 (s, 9H), 1.45
(s, 18H), 1.51 (s, 9H), 2.57-3.95 (m, 10H), 3.19 (s, 4H), 3.44-3.51 (m, 1H), 3.74 (s, 4H), 6.59
(s, 1H), 7.02 (d, J = 8.4 Hz, 2H), 7.14-7.36 (m, 12H) ppm.13C NMR (75 MHz, CDCl3): δ =
28.12 (CH3), 28.15 (CH3), 28.3 (CH3), 35.6 (CH2), 50.1 (CH2), 53.1 (CH2), 55.3 (CH2), 58.3
(CH2), 66.2 (CH), 80.2 (Cq), 80.6 (Cq), 80.7 (Cq), 118.2 (CH), 126.9 (CH), 128.1 (CH),
128.9 (CH), 129.7 (CH), 133.3 (Cq), 136.3 (Cq), 139.1 (Cq), 152.7 (CO), 170.8 (CO), 171.7
(CO) ppm. MS (ESI+): m/z: 831.5 ([M+H]+, 100%).
Compound 26: This compound was prepared, as described above for 19, from 25 (190 mg,
0.228 mmol) and palladium on carbon wt 10% (65 mg) in MeOH (15 mL). 26 was obtained as
a yellow oil (147 mg, 100%) and the crude product was used for the next step without further
purification. 1H NMR (300 MHz, CDCl3): δ = 1.27 (s, 9H), 1.43 (s, 9H), 1.45 (s, 18H), 2.54-
13
3.83 (m, 15H), 6.59 (s, 1H), 7.10-7.24 (m, 4H), 7.98 (br s, 2H) ppm. C NMR (75 MHz,
CDCl3): δ = 27.9 (CH3), 28.0 (CH3), 28.2 (CH3), 35.5 (CH2 ), 45.9 (CH2), 47.7 (CH2), 48.0
(CH2), 64.8 (CH), 80.2 (Cq), 81.7 (Cq), 83.4 (Cq), 118.4 (CH), 130.0 (CH), 132.3 (Cq), 136.7
(Cq), 152.6 (CO), 166.2 (CO), 170.5 (CO) ppm. MS (ESI+): m/z 651.4 ([M+H]+, 100%).
Compound 29: This compound was synthesized, as described above for 4, from triamine 19
(220 mg, 0.383 mmol), Na2CO3 (406 mg, 3.83 mmol) and 2,6-dibromomethyl pyridine (101
mg, 0.383 mmol) in CH3CN (190 mL). After purification by column chromatography on
alumina (CH2Cl2/MeOH, 98:02) and treatment with EDTA, free macrocycle 29 was obtained
as a yellow oil (218 mg, 84%). TLC (Al2O3, CH2Cl2/MeOH, 98:02) Rf = 0.20. IR (neat):
1
max/cm-1 2977; 2929; 1729; 1456; 1393; 1368; 1249; 1233; 1154. H NMR (300 MHz,
CDCl3): δ = 1.36 (s, 9H), 1.37 (s, 9H), 1.43 (s, 18H), 1.57-1.85 (m, 2H), 1.98-2.26 (m, 2H),
2.49-2.81 (m, 8H), 2.87-3,00 (m, 1H), 3.34 (s, 4H), 3.96 (s, 4H), 7.10 (d, J = 7.7 Hz, 2H),
13
7.56 (t, J = 7.6 Hz, 1H) ppm. C NMR (75 MHz, CDCl3): δ = 25.4 (CH2), 28.0 (CH3), 28.1
(CH3), 31.7 (CH2), 49.0 (CH2), 51.4 (CH2), 58.1 (CH2), 59.75 (CH2), 65.0 (CH), 79.8 (Cq),
80.5 (Cq), 80.8 (Cq), 122.3 (CH), 136.9 (CH), 157.35 (Cq), 170.5 (CO), 172.71 (CO), 172.74
(CO) ppm. MS (ESI+): m/z 677.4 ([M+H]+, 100%), 621.4 ([M+2H-tBu]+, 6), 565.3 ([M+3H-
29
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