Synthesis of two novel mixed bifunctional chelating agents: DO2AP(t Bu) 4and DO3AP(t Bu)4

Article abstract of DOI:10.1055/s-0040-1707893

A convenient synthesis of two novel macrocyclic bifunctional chelating agents (BFCAs), formally derived from the well-known ligands DO3A and DOTA by selective replacement of one carboxymethyl side arm with a phosphonomethyl residue, is reported.

Full text of DOI:10.1055/s-0040-1707893

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© Georg Thieme Verlag Stuttgart · New York
2020, 31, A–D
letter
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F. Travagin et al.
Letter
Synlett
Synthesis of Two Novel Mixed Bifunctional Chelating Agents:
DO2AP(tBu)4 and DO3AP(tBu)4
Fabio Travagina,b
Luca Biondia
Luciano Lattuada*c
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Previous works
This work
tBuOOC
COOtBu
(tBuO)2OP
tBuOOC
COOtBu
N
N
N
N
N
N
N
0
0
0
0-0
0
0
1-6
5
7
7-
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9
5
Giovanni B. Giovenzana*b
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N
tBuOOC
R
R
a Bracco Imaging Spa, Bracco Research Centre, Via Ribes 5,
10010 Colleretto Giacosa (TO), Italy
R = H, CH2COOH
b Dipartimento di Scienze del Farmaco, Università degli Studi
del Piemonte Orientale, Largo Donegani 2/3, 28100 Novara
(NO), Italy
c Bracco SpA, Innovation Hub, Via Caduti di Marcinelle 13,
20134 Milano (MI), Italy
giovannibattista.giovenzana@uniupo.it
luciano.lattuada@bracco.com
Received: 22.03.2020
Accepted after revision: 08.05.2020
Published online: 16.04.2020
Many popular and widely applied BFCAs are based on
polyaminopolycarboxylic acids due to their versatility and
ability to coordinate a broad variety of metal ions, generat-
ing stable complexes.5 High thermodynamic and kinetic
stabilities of metal complexes are usually achieved with
macrocyclic chelating agents owing to the preorganized
structure of these ligands. The typical example of this class
is DOTA (Scheme 1), which has been exploited extensively
for the preparation of metal complexes to be used in vivo.6,7
The macrocyclic derivatives DO3A tri-tert-butyl ester 18
and DOTA(tBu)3 29 (Scheme 1), both based on the 1,4,7,10-
tetrazacyclododecane (‘cyclen’), are among the most used
BFCAs. Both are commercially available and can be easily
prepared through relatively simple procedures.8–10 Com-
pound 1 has been used in solid-phase synthesis by derivat-
ization of the peptide on the resin with bromoacetyl bro-
mide (Scheme 1, route a), which is then reacted with the
BFCA.11 Compound 2 can be directly conjugated to mole-
cules of interest bearing an amino group (e.g. a protected
peptide) by means of a suitable coupling agent.12 Moreover,
DOI: 10.1055/s-0040-1707893; Art ID: st-2020-b0163-l
Abstract A convenient synthesis of two novel macrocyclic bifunctional
chelating agents (BFCAs), formally derived from the well-known ligands
DO3A and DOTA by selective replacement of one carboxymethyl side
arm with a phosphonomethyl residue, is reported.
Key words bifunctional chelating agents, DO3A, DOTA, phosphonic
group, protecting group
Bifunctional chelating agents (BFCAs) are small mole-
cules containing a metal chelating unit and a reactive func-
tional group.1 They find widespread applications in chemis-
try, biochemistry and medicine because they can be easily
conjugated to biomolecules, such as peptides2 or antibod-
ies,3 and then loaded with metals or radiometals to obtain
diagnostic, therapeutic or theranostic agents, which are ex-
tensively employed nowadays in research, preclinical and
clinical studies.4
Commercially
This work
available BFCAs
tBuOOC
PO(OtBu)2 tBuOOC
COOtBu
COOtBu
COOtBu
N
N
N
N
N
N
Base
O
Conjugated
metal complex
Chelating agents
a
NH
NH
Br
N
H
-OOC
O
COO-
HOOC
FG
3
1
N
N
N
N
N
N
N
N
1) Deprotection
2) Complexation
DO2AP(tBu)4
DO3A(tBu)3
Mn+
O
Br
COO- HOOC
COOH
Br
N
H
FG = COOH
FG = PO3H2
DOTA
DO3AP
tBuOOC
PO(OtBu)2
COOtBu
tBuOOC
COOtBu
COOtBu
N
N
N
N
N
N
N
N
NH2
b
O
O
Condensing
agent
Targeting
vector
Functional group
for conjugation
HO
HO
2
4
DOTA(tBu)3
DO3AP(tBu)4
Scheme 1 Macrocyclic BFCAs: conjugation strategies, commercially available examples, and synthesized compounds
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–D
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

B
F. Travagin et al.
Letter
Synlett
1) P(OtBu)3,
(CH2O)n, 70
°C, 36 h
Cbz
Cbz
1) BrCH2COOtBu,
DIPEA, MeCN, rt, 22.5 h
COOtBu
NH HN
N
N
N
N
HN
·2 HCl
NH
CbzCl
2) NH2OH·HCl, EtOH,
reflux, 48 h
(88%, 2 steps)
dioxane, H2O
pH 3
2) H2, Pd/C,
MeOH, rt, 8 h
(65%, 2 steps)
NH
N
HN
N
tBuOOC
CHO
CHO
5
6
7
HO
COOtBu
COOtBu
BnOOC
COOtBu
NH
N
N
N
N
N
N
N
N
N
H2, Pd/C
BrCH2COOBn
O
MeOH, rt, 8 h
(quant.)
K2CO3, MeCN
rt, 37 h
N
N
tBuOOC
PO(OtBu)2
tBuOOC
PO(OtBu)2
tBuOOC
PO(OtBu)2
(58%)
8
4 DO3AP(tBu)4
3 DO2AP(tBu)4
Scheme 2 Synthesis of monophosphonic BFCAs DO2AP(tBu)4 3 and DO3AP(tBu)4 4
compound 2 has been used extensively in the solid-phase
synthesis of DOTA-functionalized peptides (Scheme 1,
route b).13
We have been interested and involved for several years
in the design and synthesis of novel BFCAs, ranging from
linear DTPA to mesocyclic AAZTA14 and to macrocyclic
DOTA or HP-DO3A chelating agents.14,15
erodiprotected derivative 6 was the key intermediate for
the synthesis of both BFCAs. Alkylation of the secondary
amines with tert-butyl bromoacetate in the presence of
N,N-diisopropylethylamine (DIPEA) as the base, followed by
selective removal of the formyl protective group by reflux-
ing in ethanol in the presence of hydroxylamine hydrochlo-
ride, led to the advanced synthon 7 in 88% yield. The key
phosphonic moiety was introduced by applying a modified
Kabachnik–Fields reaction;22 namely, treating 7 with tri-
tert-butyl phosphite and paraformaldehyde at 70 °C for 36
hours.23 The resulting protected mixed phosphonic-carbox-
ylic ester was not isolated due to observed instability. The
Cbz protective group was directly and smoothly removed
by hydrogenolysis (Pd/C in methanol), to obtain the mono-
phosphonic DO2AP tetra-tert-butyl ester 3 in 65% yield over
two steps.24
The second BFCA bears an additional side arm contain-
ing the free carboxylic acid acting as the remote reactive
functional group. The side arm is introduced by alkylation
of the secondary amine 3 with benzyl bromoacetate in ace-
tonitrile at room temperature in the presence of K2CO3 as
the base. Selective removal of the benzyl ester by catalytic
hydrogenolysis completes the preparation of the desired bi-
functional chelating agent 4 (DO3AP(tBu)4, Scheme 2).25
The preparation of both BFCAs was performed on a
gram scale, testifying to the robustness of this concise syn-
thetic approach and allowing suitable amounts to be ob-
tained for further applications.26,27
Here, we report the synthesis of the novel bifunctional
chelating agents 3 and 4 (Scheme 2), differing from 1 and 2
by the replacement of a carboxylic moiety with a phos-
phonic group. The phosphonic group is commonly used as a
(bio)isostere of carboxylic acid in medicinal chemistry.16 At
physiological pH values the phosphonate tetrahedral
deprotonated form (PO32–) offers multiple coordination
modes, leading to stabilities of the metal complexes compa-
rable to those of the carboxylic counterpart or even higher.6
DO3AP (Scheme 1) forms complexes with lanthanide (Ln3+)
with thermodynamic stabilities higher than those of the
corresponding Ln3+-DOTA chelates.17 Moreover, the residual
anionic charges left after complexation of the dianionic
phosphonate group provide a beneficial effect on the solva-
tion of the corresponding chelate, due to the establishment
of an extended hydrogen-bond network. This is extremely
important in Gd3+ complexes, which are currently used as
MRI contrast agents, and is clearly demonstrated in the
comparison of Gd3+-DO3AP, with the carboxylic congener
Gd3+-DOTA.18 The former, by virtue of the higher hydration
of the paramagnetic complex, shows an improved relaxivity
(i.e. contrast efficiency), pointing out the importance of this
specific isosteric substitution of metal-coordinating groups.
N-Formyl-cyclen 5 was selected as the starting material
for the synthesis of the new monophosphonic derivatives 3
and 4, since it can be easily prepared in two steps from cy-
clen in almost quantitative yield.19 Compound 5 was regi-
oselectively protected at the 7-position by reaction with
benzylchloroformate in 1,4-dioxane/water at pH 3.20 The
regioselective protection relies on a modification of the
protocol originally proposed by Kovacs and Sherry,21 and
exploits a pH-controlled procedure for the selective func-
tionalization of polyazamacrocycles. The orthogonally het-
In summary, a concise entry to two macrocyclic bifunc-
tional chelating agents, formally derived from the widely
used DOTA/DO3A ligands, has been described in this work.
The synthesis of the two new BFCAs starts from a readily
available mono-protected cyclen and involves simple steps,
requiring common and inexpensive reagents and exploiting
a recently reported and very effective protection method.20
Future development of this work will deal with the conju-
gation of the two new BFCAs to molecular vectors and eval-
uation of the corresponding conjugated metal complexes
for diagnostic or therapeutic purposes.26
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–D

C
F. Travagin et al.
Letter
Synlett
Supporting Information
(13) (a) Azad, B. B.; Rota, V. A.; Breadner, D.; Dhanvantari, S.; Luyt, L.
G. Bioorg. Med. Chem. 2010, 18, 1265. (b) Mishra, R.; Su, W.;
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Bioconjugate Chem. 2009, 20, 1860. (c) Sosabowski, J. K.; Mather,
S. J. Nat. Protoc. 2006, 1, 972. (d) De León-Rodríguez, L. M.; Ortiz,
A.; Weiner, A. L.; Zhang, S.; Kovacs, Z.; Kodadek, T.; Sherry, A. D.
J. Am. Chem. Soc. 2002, 124, 3514. (e) Graham, K. A. N.; Wang,
Q.; Eisenhut, M.; Haberkorn, U.; Mier, W. Tetrahedron Lett. 2002,
43, 5021.
Supporting information for this article is available online at
https://doi.org/10.1055/s-0040-1707893.
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L. E.; Bornhop, D. J. Tetrahedron Lett. 2005, 46, 4707.
(24) Di-tert-butyl
4-[[Di(tert-butoxy)phosphoryl]methyl]-
1,4,7,10-tetrazacyclododecane-1,7-diacetate (3): A mixture of
7 (13.9 g, 26 mmol, 1 equiv), tri-tert-butyl phosphite (7.6 g, 28.6
mmol, 1.1 equiv) and paraformaldehyde (0.9 g, 30 mmol, 1.15
equiv) was stirred and heated at 70 °C. After 16 h, additional tri-
tert-butyl phosphite (1 g, 3.8 mmol, 0.15 equiv) and parafor-
maldehyde (0.1 g, 3.3 mmol, 0.13 equiv) were added and the
mixture was heated for a further 20 h. The mixture was evapo-
rated under vacuum at 80 °C to eliminate volatile by-products.
The oily residue was dissolved in methanol (145 mL), 5% Pd/C
(2.6 g) was added and the mixture was stirred under hydrogen
atmosphere at room temperature for 8 h. The mixture was fil-
tered through a Millipore FT 0.45 m filter, evaporated, and the
residue was purified by flash chromatography (CH2Cl2 then
CH2Cl2/MeOH = 9:1) to give compound 3 (10.3 g, 65% over two
steps) as a brown oil. IR (neat): 2977, 2927, 1730, 1368, 1223,
1152, 973, 917, 849, 692 cm–1 1H NMR (300 MHz, CDCl3, 333
.
K):  = 3.02–2.91 (m, 5 H), 2.71–2.38 (m, 18 H), 1.09 (s, 18 H),
1.04 (s, 18 H) ppm. 13C NMR (75 MHz, CDCl3, 333 K):  = 169.8
(C), 82.2 (d, J = 9.8 Hz, C), 80.6 (C), 57.5 (CH2), 51.2 (CH2), 50.9
(CH2), 48.9 (CH2), 46.8 (CH2), 45.3 (d, J = 145 Hz, CH2), 29.9 (d,
J = 3.7 Hz, CH3), 27.5 (CH3) ppm. 31P NMR (121 MHz, CDCl3, 333
K):  = 18.1 (s) ppm. MS (ESI+): m/z (%) = 607.24 (100) [M + H]+,
551.20 (21), 495.16 (35), 439.15 (34), 383.18 (20). HRMS (ESI+):
m/z calcd for C29H59N4O7P: 606.41214; found: 607.41886 (100)
[M + H]+, 629.40094 (46) [M + Na]+.
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–D

D
F. Travagin et al.
Letter
Synlett
(25) 7-[[Di(t-butoxy)phosphoryl]methyl]-1,4,7,10-tetrazacyclo-
dodecane-1,4,10-triacetic acid 4,10-di-t-butyl ester (4): A
mixture of compound 8 (1.38 g, 1.83 mmol), 10% Pd/C (0.28 g)
and MeOH (10 mL) was stirred under hydrogen atmosphere at
room temperature for 10 h. The mixture was filtered through
Celite and evaporated to give compound 4 (1.20 g, 99%) as a
pale-yellow oil. IR (neat): 3381, 2975, 1725, 1650, 1367, 1151,
1070, 976, 567, 433 cm–1. 1H NMR (300 MHz, CD3OD, 313 K):  =
3.79–2.82 (m, 24 H), 1.52–1.48 (m, 36 H) ppm. 13C NMR (75
MHz, CD3OD, 298 K):  = 170.9 (C), 169.1 (C), 82.7 (C), 79.9 (d,
J = 8.1 Hz, C), 57.4 (CH2), 54.2 (CH2), 52.4 (d, J = 140.6 Hz, CH2),
52.3 (CH2), 51.8 (6 CH2), 30.1 (d, J = 3.8 Hz, CH3), 27.6 (CH3)
ppm. 31P NMR (121 MHz, CD3OD, 313 K):  = 5.85 (s) ppm. MS
(ESI+): m/z (%) = 703.2 (36) [M + K]+, 687.4 (100) [M + Na]+, 630.9
(12). HRMS (ESI+): m/z calcd for C31H61N4O9P: 664.41762;
found: 665.42279 (100) [M + H]+, 687.40452 (55) [M + Na]+.
(26) Lattuada, L.; Cappelletti, E.; Linder, K. E.; Nunn, A. D.
US20110250133A1, 2011.
(27) Lattuada, L.; Napolitano, R.; Boi, V.; Visigalli, M.; Aime, S.;
Giovenzana, G. B.; Mingo, A. F. WO2017178301A1, 2017.
© 2020. Thieme. All rights reserved. Synlett 2020, 31, A–D