Bifunctional ligands based on the DOTA-monoamide cage

Article abstract of DOI:10.1039/b715844k

Efficient routes to DOTA-monoamide ligands bearing amino, hydroxyl, aldehyde and maleimido groups are described. These functional groups, which can be spaced at will from the coordination cage, will readily react with suitable groups of targeting moieties

Full text of DOI:10.1039/b715844k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Bifunctional ligands based on the DOTA-monoamide cage
Alessandro Barge,a Lorenzo Tei,b Dharita Upadhyaya,a Franco Fedeli,c Lorena Beltrami,c Rachele Stefan`ıa,c
Silvio Aime*c and Giancarlo Cravotto*a
Received 15th October 2007, Accepted 28th January 2008
First published as an Advance Article on the web 22nd February 2008
DOI: 10.1039/b715844k
Efficient routes to DOTA-monoamide ligands bearing amino, hydroxyl, aldehyde and maleimido
groups are described. These functional groups, which can be spaced at will from the coordination cage,
will readily react with suitable groups of targeting moieties. Bioconjugates obtained in this way can be
used for diagnostic imaging and therapeutic applications.
It is therefore important to develop BFCAs whose chelating
moiety will coordinate metal ions more tightly than DTPA-amide.
Introduction
In this regard, DTPA,6 DOTA7–10 and DOTA-monoamide11–15
systems have been considered as good alternatives. The synthesis
of functionalised DOTA derivatives as BFCAs was a hot topic at
the end of the ’80s and beginning of the ’90s. Several patents and
papers were published by the groups of Meares, Parker, Gansow
and Sherry.16,17 More recent applications to magnetic resonance
molecular imaging and nuclear medicine required the use of
new BFCAs, which entailed the development of more efficient
synthetic strategies and the optimization of previous protocols.
Thus, the commercially available triprotected DOTA-tris(tert-
butyl) ester,11 which forms DOTA-monoamide derivatives when it
reacts with amines, is one of the most frequently used bifunctional
agents.12 Conjugation to biological vectors has also been exploited
with the benzyl-protected analogue DOTA-tris(benzyl) ester,13
the isothiocyanate-functionalized p-NCS-Bz-DOTA both on the
macrocyclic backbone and on the pendant arm (C-DOTA and
PA-DOTA, respectively),7 DOTAGA(t-BuO)4,8 which contains
an additional unprotected carboxylic group, 4-acetylphenyl-,
ethynylphenyl-9 and vinyl sulfone-cysteineamido,14 maleimido-
cysteinamido15 DOTA derivatives containing a methyl ketone, an
alkyne, a vinyl sulfone and a maleimido group, respectively.
Herein we report the syntheses of BFCAs based on DOTA-
monoamide that display amino, alcohol, aldehyde or maleimido
functions to be eventually used for conjugation to biological
vectors of interest. Each of these functions may be spaced at will
from the chelating moiety by choosing the number of interposed
methylene carbons (Scheme 1).
In the last few decades many acyclic and macrocyclic compounds
have found important applications in diagnostic and therapeutic
medicine in the form of metal complexes. These chelates are used
both as contrast agents for magnetic resonance imaging (MRI)1
and as radiopharmaceuticals for the diagnosis and therapy of
tumours.2 A very important goal of current research is to achieve
a precise delivery of these compounds to specific cellular targets.
The general strategy is to insert them into bioconjugates that
will bind to specific receptors that are overexpressed in certain
tissues or act as markers to visualize a cell product that is the
“signature” of a specific disease. In such conjugates the moiety
encapsulating the metal ion is a bifunctional chelating agent
(BFCA) that contains a suitable function through which it can be
covalently bound to the specific biological carrier. In this regard,
the design and development of synthetic BFCAs for connecting
these targeting vehicles to metal complexes, and improvements
in related conjugation and chelation procedures are topics of
outstanding importance in molecular medicine.
In the field of polyaminopolycarboxylic ligands a straightfor-
ward route to bifunctional chelating agents is the reaction of
DTPA anhydride with amines to form DTPA mono- and bis-amide
derivatives.3,4 The latter chelating moiety can readily coordinate
a number of metal ions (transition, group III or lanthanide) of
biomedical interest. Relevant examples are found in the use of In or
Ga for SPECT studies and Y and Lu for therapeutic applications.3
However, most recent work on DTPA-amide-containing agents
has dealt with the use of Gd complexes for MRI investigations.4
Evidence has emerged from in vitro studies that Gd complexes of
DTPA-amide may not have a sufficient thermodynamic/kinetic
stability for the intended purposes.5 This concern may weigh
even more when DTPA-amide BFCAs are used in molecular
imaging applications for which long contact times with biological
structures are required.
Results
It is well known that DOTA-monoamide derivatives are not the
best ligands for Gd(III) for use in MRI because of their long water
coordinated exchange time. However, this turns out not to be a
real limitation, especially in cell labeling procedures, because it
has been reported18 that when a Gd(III) complex is internalized
into the cell by endocytosis its relaxivity is dramatically decreased
(and its contrast-enhancing power consequently decreased),
both for fast- and slow-water-exchanging complexes. Thus
DOTA-monoamide based contrast agents can be successfully used
in molecular imaging applications.
aDipartimento di Scienza e Tecnologia del Farmaco, Universita` di Torino,
Via Giuria, 9-10125, Torino, Italy. E-mail: giancarlo.cravotto@unito.it;
Fax: +39 0116707687; Tel: +39 0116707684
bDipartimento di Scienze dellꢀAmbiente e della Vita, Universita` del Piemonte
Orientale, via Bellini 25, G-Alessandria, Italy
cDipartimento di Chimica IFM, Universita` di Torino, Via Giuria, 7-10125,
Torino, Italy. E-mail: silvio.aime@unito.it; Fax: +39 0116707855; Tel: +39
0116706451
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R
in the presence of K2CO3. Alternatively, Amberliteꢀ IRA 410 was
used as base instead of K2CO3 in the N-alkylation, thus dispensing
with the preliminary treatment of DO3A(t-BuO)3 hydrobromide
with the ion exchange resin. In this procedure the resin, being
added directly to the reaction mixture, played a double role, both
deprotonating the ammonium salt and acting as a proton scav-
enger. As the two protocol variants approximately resulted in the
same reaction time and yield, the one-pot reaction was preferred.
After the alkylation, the Cbz protecting group was removed by
hydrogenation at room pressure (Pd/C in MeOH). When carried
out under magnetic stirring, this cleavage was unsatisfactory (still
incomplete after 24 h); it went however to completion in 5 hours
when a Venturi-effect stirrer was used. Reaction rates and yields
were further improved working under power ultrasound20 (up
to 95% in about 2 h). To the best of our knowledge this is the
first report of Cbz cleavage under sonochemical conditions. The
free NH2 group of bifunctional chelates 5 and 6 can be exploited
while the tert-butyl ester protecting groups are maintained on the
carboxyl groups; alternatively the latter can be deprotected with
TFA to yield ligands 7 and 8. Another synthetic strategy for the
preparation of BCFA 5 has been described by Andre´ and co-
workers.21 They started from the monoalkylation of ‘cyclen’ with
ethyl bromoacetate, then N-alkylated the remaining amino groups
with tert-butyl bromoacetate and ended with the aminolysis of
the ethyl ester with ethylendiamine; the overall yield was 45%.
Although our procedure is one step longer, it was optimized
starting from DO3A(t-BuO)3 and had a higher overall yield
(∼ 54%) (Scheme 2).
Scheme 1 DOTA-monoamide derivatives bearing different functional
groups.
a) Synthesis of NH2-functionalized DOTA-monoamide bifunc-
tional agents.
In the present work mono-Cbz alkyldiamino derivatives were
converted to bromoacetamido derivatives 1 and 2 by reaction with
bromoacetyl bromide. Compounds 1 and 2 were then used for
the N-alkylation of DO3A(t-BuO)3 (1,4,7-tris-tert-butoxycarbo-
nylmethyl-1,4,7,10-tetraazacyclododecane) which was prepared
as the hydrobromide according to a reported procedure.19 The
secondary ammonium salt of DO3A(t-BuO)3 is not nucleophilic
enough to react with a primary halide; indeed the reaction gave
negligible or poor yields even in the presence of K2CO3. This
hurdle was overcome by treating DO3A(t-BuO)3 hydrobromide
b) Synthesis of maleimido-functionalized DOTA-monoamide
bifunctional agents.
Following the procedure reported by Ondrus et al.,22 which
entails the use of a 7-exo-Diels–Alder adduct of furan and
maleic anhydride (7-exo-oxohimic anhydride 9) and working
under microwave irradiation, we converted in good yields ligands
7 and 8 to the maleimido derivatives 10 and 11 (Scheme 3).
R
with the strongly basic ion-exchange resin Amberliteꢀ IRA 410 in
its OH form, that easily deprotonated the ammonium salt to give
DO3A(t-BuO)3 as free amine. The latter, being more nucleophilic
than the hydrobromide salt, reacted better with primary halides
Scheme 2 Synthesis of DOTAMA(t-BuO)3en (5), DOTAMA(t-BuO)3C6NH2 (6), DOTAMAen (7), DOTAMAC6NH2 (8)
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Scheme 3 Microwave-assisted synthesis of DOTA-monoamide maleimido derivatives.
Scheme 4 Synthesis of bifunctional agent 14.
c) Synthesis of OH-functionalized DOTA-monoamide bifunc-
tional agents.
In a more straightforward way, the amino alcohols were
reacted with chloroacetyl bromide in water at pH 10 to prevent
ester formation. Further alkylation of DO3A(t-BuO)3 gave the
derivatives containing a free OH with an overall yield of 47% and
55% for 14 and 17, respectively (Scheme 5).
d) Synthesis of CHO-functionalized DOTA-monoamide bi-
functional agents.
Swern oxidation23 of 14 and 17 afforded the corresponding
aldehydes in good yields (Scheme 6).
Derivative14, bearing a free alcohol function, was prepared with
a 42% overall yield from 6-aminohexanol; this was protected with
tetrahydropyran and converted to the bromoacetamido derivative
12. DO3A(t-BuO)3 alkylation with 12 and subsequent removal of
the THP group with sodium cyanoborohydride and BF3·etherate
in dry THF under power ultrasound gave the final product
(Scheme 4).
Scheme 5 Synthesis of bifunctional agents 14 and 17 without OH protection.
Scheme 6 Swern oxidation of 14 and 17 to yield aldehyde-containing DOTA-monoamide bifunctional agents 18 and 19.
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Oxidation of 14 and 17 with pyridinium dichromate (PDC),
pyridinium chlorochromate (PCC) or Dess–Martin periodinane
gave very poor results. Moderate yields were only achieved using
PDC under power ultrasound.
The OH group present on bifunctional agents 14 and 17 could be
transformed into a sulfonyl derivative (e.g. mesyl, tosyl, nosyl), to
be subsequently reacted with a variety of nucleophilic species. Still
more interestingly, it can form a carboxylic ester. This type of ester
could be easily cleaved even in vivo by hydrolysis with esterase; this
could be an advantage in terms of detoxification and catabolism.
After target recognition has taken place, it could be advantageous
for the imaging probe to be cleaved off from its vector. In this
way each of the two moieties would follow its metabolic pathway
independently from the other, thus reducing overall toxicity.
Aldehyde derivatives 18 and 19 can easily react by reductive
amination with free amino groups present on the vector surface.
This reaction would be especially useful when the vector is a small
molecule, whose surface modification could dramatically affect
interaction with the target site. Reductive amination preserves the
overall charge of the vector, which is not the case when conjugation
is achieved through an amide bond.
Finally, it should be pointed out that for each functional group
two different BFCAs were synthesized, with spacers consisting of
either two or six methylene groups. The possibility of choosing
between different spacers affords a control of the complex–vector
distance. The distance between the DOTA coordination cage and
the vector may strongly affect both the conjugation reaction and
bioconjugate interaction with the target. In fact, the DOTA-
monoamide cage, being often bulkier than the vector itself, may
hinder the interaction very considerably. As shown in our previous
work on the glutamine vector,28 such hindrance can be minimized
by increasing the complex–vector distance.
Discussion
Thanks to their free -OH, -NH2, -CHO or maleimido groups, all
our novel DOTA-monoamide derivatives can undergo coupling
reactions with a wide range of biological vectors.
As already reported for many bifunctional agents, especially
DOTA-tris(tert-butyl) ester,12 they can be used in solid-phase
Fmoc peptide synthesis (SPPS) by exploiting the orthogonal
protecting group strategy and thus be attached to peptides. For
example,
4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-protected
lysine (lysine-ivDde) can be joined to the DOTA-tris(tert-butyl)
ester BFCA after removal of the ‘ivDde’ group with 2% hydrazine
in DMF (solid phase reaction). Likewise, amino derivatives 5 and
6 can be coupled by a similar procedure with carboxylic groups on
the side chains of glutamic and aspartic acids. In fact, Glu and Asp,
orthogonally protected e.g. with Dmab (2-{1-[4-(hydroxymethyl)-
phenylamino]-3-methylbutylidene}-5,5-dimethyl-1,3-cyclohexan-
edione) ester, could be selectively deprotected with 2% hydrazine
in DMF and subsequently coupled with 5 and 6. Aldehydes can
also be introduced in solid-phase peptide synthesis, as they will
form secondary amines by reductive amination with primary
amino groups present in the peptide. A reductive amination
protocol for solid-phase peptide synthesis has already been
published24 and works well with our BFCAs 18 and 19 (data
not shown). It should be noted that reductive amination must
be the last step before cleavage, otherwise Boc protection of the
secondary amine would be required. tert-Butyl ester protecting
groups are easily removed, together with side-chain protecting
groups, during peptide cleavage with the usual reagents.
The maleimido group of 10 and 11 is one of the most useful
linkers in bioconjugate chemistry because the Michael addition
of a thiol to the maleimide ring takes place under very mild
conditions and is highly specific.25 BFCAs 10 and 11 can work
either in Fmoc SPPS or in aqueous solution, the maleimido group
becoming attached to a cysteine thiol. As in 10 and 11 the carboxyl
groups of the chelating moieties are unprotected, their coupling
should be the last step of the SPPS before cleavage is carried
out. Maleimide is particularly useful in aqueous solution since at
neutral or slightly acidic pH it reacts about 1000-fold faster with
thiols than with amines, which are mostly protonated.26 These
conditions are most appropriate when the vector is unstable in an
organic solvent or under the acidic conditions that are required
for tert-butyl ester cleavage, a common occurrence with many
proteins, monoclonal antibodies and some peptides. It is also
possible to start the assembly right away with the metal complex
rather than the free ligand; this will avoid problems arising from
complex formation by peptides or proteins.
Conclusions
In the present paper we have reported the synthesis of a series of bi-
functional building blocks to be exploited for various applications
in diagnostic and therapeutic medicine. Their chelating moieties
are based on the DOTA-monoamide cage which forms highly
stable coordination complexes with both the Gd(III) ion (used in
MRI) and several radioactive metal ions used in nuclear medicine
(PET, SPECT). They offer a wide range of reactive groups and the
possibility of choosing between two spacer lengths; these features
are useful toward optimizing BFCA conjugation to the vector and
eventually synthesizing a large library of specific molecular probes.
While most of the published BFCAs are normally prepared on a
small scale, the present protocols are well suited for multigram
preparations. Finally, conjugation of these BFCAs to targeting
vectors can be performed with different procedures: in organic
solvents with protecting groups still present on the chelating
moieties, followed by deprotection and complexation with the
desired metal ion; or alternatively in aqueous media, using the
preformed complex if this proves more convenient.
Experimental
We reported the use of the Gd(III) complex of 7, converted to
the emisquarate derivative (4-alkylamino-3-ethoxy-3-cyclobuten-
1,2-dione), for conjugation with a polyornithine.27 Similarly, 7
and 8 can easily be converted to the emisquarate derivatives for
conjugation with any NH2-containing vector. As in the example
reported, 7 and 8 can be first complexed with the appropriate metal
ion, then conjugated to a protein or specific antibody.
All chemicals were purchased either from Sigma-Aldrich Co. or
Lancaster Synthesis GmbH and were used without purification
unless otherwise stated. NMR spectra were recorded on a Bruker
Avance 300 (operating at 7 Tesla) and on a JEOL Eclipse Plus
400 (operating at 9.4 Tesla). ESI mass spectra were recorded
on a Waters Micromass ZQ and elemental analyses on a Flash
EA-1112 CHNS-O Elemental Analyzer (Thermoquest Inc.).
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MW-promoted reactions were carried out in a MicroSYNTH
oven (Milestone, Bergamo). Sonochemical apparatus used in
the present work was developed in the authors’ laboratory in
collaboration with Danacamerini sas (Torino).29 DO3A(t-BuO)3
was prepared following a reported procedure.16
General procedure for the attachment of a chloroacetamide
pendant arm to DO3A(t-BuO)3
DO3A(t-BuO)3 free base (0.1 mol) was dissolved in acetonitrile
(970 mL) and the chloroacetamido derivative (0.1 mol) was added.
After 1 hour stirring at room temperature, DIPEA (0.1 mol) was
added and the mixture was stirred for another 24 hours. The
solvent was removed under vacuum, the residue solubilized in
MeOH and purified over a XAD 1600 chromatographic column
by gradient elution (water–MeOH). After solvent evaporation a
white solid was obtained.
General procedure for bromo-acetamidation of monoprotected
alkanediamines
In a round-bottom flask mono-Cbz-alkanediamine (10 mmol)
(Cbz = benzyloxycarbonyl) and K2CO3 (20 mmol) were mixed
in 20 mL of CH3CN. The mixture was cooled down to 0 ◦C,
then a solution of bromoacetyl bromide (1.1 mol eq. in 10 mL
CH3CN) was slowly added over 1 hour. After 4 hours stirring
at room temperature, the solid was removed by filtration and the
organic phase evaporated under reduced pressure. The residue was
dissolved in CH2Cl2 (50 mL) and washed with 5% Na2CO3 (2 ×
20 mL), H2O (20 mL), 0.01 N HCl (2 × 20 mL), and finally again
with H2O (20 mL). The organic phase was dried with Na2SO4,
filtered and evaporated under reduced pressure. The residue was
then dried under vacuum to yield the product as a white solid.
Hydrogenation of the Cbz group (benzyloxycarbonyl) on a mmol
scale (general)
The Cbz-protected DOTA-monoamide derivative (2 mmol) was
dissolved in MeOH (40 mL) and 10% its weight of Pd/C-10%
was added. The reaction was carried out under H2 at atmospheric
pressure and room temperature for 2 hours, using a high-power
ultrasound bath operating at 21 kHz and 80 W. The mixture was
then filtered and the solvent evaporated. The residue was washed
with diethyl ether (2 × 20 mL) and dried under vacuum to yield a
white solid.
General procedure for chloroacetamidation of unprotected amino
alcohols
Hydrogenation of the Cbz group (benzyloxycarbonyl) on a mol
scale (general)
To a solution◦of amino alcohol (0.20 mol) in water (50 mL),
cooled to 5–7 C, chloroacetyl chloride (16.3 mL; 0.20 mol) was
slowly added while pH 10 was being maintained by adding 10
N NaOH. The solution was stirred for 30 min, then neutralized
with HCl and concentrated under vacuum; inorganic salt was
eliminated by adding methanol. After evaporation of the solvent,
the product was recovered as a colourless oil.
The Cbz-protected DOTA-monoamide derivative (0.1 mol) was
dissolved in MeOH (50 mL) and 10% its weight of Pd/C-10% was
added. Hydrogenation was carried out for 5 h at room pressure and
temperature, under stirring with a Venturi-effect stirrer (MRK1
Buddenberg) spinning at 2000 rpm. Then the mixture was filtered,
the solvent evaporated and the residue dissolved in 30 mL of 0.5 N
HCl (this concentration did not affect the tert-butyl esters, while
eliminating carbamic acid residues). After stirring under vacuum
for 10 min until the evolution of CO2 had ceased, the pH was raised
General procedure for the attachment of a bromoacetamide
pendant arm to DO3A(t-BuO)3 using K2CO3 as base (a)
◦
to about 10 by adding NaOH 1 N at 0 C and the product was
DO3A(t-BuO)3 free base was obtained by dripping DO3A(t-
R
extracted with CH2Cl2 (4 × 50 mL). The organic phase was dried
with Na2SO4, filtered and evaporated under reduced pressure. The
residue was dried under vacuum to yield the product as a white
solid.
BuO)3 HBr over Amberliteꢀ IRA 410 in its basic form and eluting
it with water/ethanol 1:1. The bromoacetamide derivative (10
mmol) was dissolved in CH3CN (30 mL) and slowly dripped,
over 1 h, into a mixture of DO3A(t-BuO)3 free base (10 mmol)
and solid K2CO3 (15 mmol) suspended in CH3CN (50 mL). The
mixture was stirred at room temperature for 4 h. After filtration,
the solvent was removed by evaporation under reduced pressure
to yield the product as a white solid.
General procedure for alcohol oxidation (Swern)
A mixture of dichloromethane (4 mL) and oxalyl chloride
(2.08 mmol, 178 ll) was placed in a 10 mL two-necked round-
bottom flask and cooled down to −60 ◦C. DMSO (4.16 mmol, 295
ll) was then added and the mixture was stirred for 10 min. Then
the alcohol, dissolved in DMSO–CH2Cl2 (0.74 mmol, 500 ll–
500 ll), was added over 5 min and the mixture stirred for
45 min. Triethylamine (1.20 mL, 8.6 mmol) was added and the
mixture was stirred for 2 h at room temperature. It was then
washed with water (5 mL) and the aqueous layer extracted with
dichloromethane (3 × 3 mL). The organic layers were combined,
washed first with saturated NH4Cl, then with brine and dried
over anhydrous sodium sulfate. The aldehyde was collected after
solvent evaporation as a pale yellow oil.
General procedure for the attachment of a bromoacetamide
R
pendant arm to DO3A(t-BuO)3 using Amberliteꢀ IRA 410 as
base (b)
Bromoacetamide (8 mmol) and DO3A(t-BuO)3 HBr (8 mmol)
R
were dissolved in CH3CN (150 mL); then 16 mL of Amberliteꢀ
IRA 410, previously activated with 10% NaOH, washed with H2O
to neutral pH and finally conditioned with CH3CN, were added
to the solution. The mixture was stirred at room temperature for
4 h. After filtration, the solvent was removed by evaporation under
reduced pressure to yield the product as a colourless oil.
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General procedure for tert-butyl ester deprotection
ESI-HRMS (m/z): 671.5065 (M + H+), (calcd. 671.5071).
C34H66N6O7 (670.92) calcd. C, 60.87; H, 9.92; N, 12.53; found
C, 61.18; H, 10.50; N, 12.90%.
The compound protected with tert-butyl ester groups (8 mmol)
was dissolved in dichloromethane (50 mL) and the mixture
cooled down to 0–5 ◦C. Trifluoroacetic acid (TFA, 48 mmol) was
then slowly added under magnetic stirring and the temperature
slowly increased to RT. The mixture was concentrated, 3%
triisopropylsilane in TFA (18 mL) was added and the solution
stirred for 48 h. A white solid was collected after addition of diethyl
ether (50 mL), washed three times with diethyl ether (10 mL) and
dried under vacuum.
Synthesis of 1-(9-amino-3-aza-2-oxononyl)-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane—
DOTAMA-C6-NH2 (8)
Compound 6 was fully deprotected with trifluoroacetic acid (TFA)
according to the above-reported procedure. DOTAMA-C6-NH2
(8) was obtained as trifluoroacetate salt in quantitative yield. By
acidimetric titration, and CHN analysis, 2.5 TFA molecules were
found for each DOTAMA-C6-NH2 molecule. 1H NMR (DMSO-
d6, 300 MHz): d = 8.33 (b, 1H), 7.92 (b, 2H), 4.40 (s, 2H), 4.22
(s, 2H), 3.69 (s, 4H), 3.60–2.70 (b, 20H), 1.85 (t, 2H, J = 7.3 Hz),
1.60 (m, 2H), 1.50 (m, 2H), 1.40 (m, 2H). 13C NMR (DMSO-d6,
75 MHz): d = 171.6, 171.1, 168.8, 57.9, 56.3, 55.8, 53–48 (b), 39.4,
38.7, 28.0, 26.4, 25.3, 25.1. IR (mmax/(cm−1), liquid film): 3443,
3250, 1729, 1654, 1463, 1425, 1202, 1138
Synthesis of N-(6-benzyloxycarbonylaminohexyl)bromoacetamide
(2)
6-Benzyloxycarbonylaminohexanamine (3.0 g, 10.45 mmol),
K2CO3 (2.89 g, 20.9 mmol) and bromoacetyl bromide (2.34 g,
11.5 mmol) were reacted according to the above-reported general
procedure for bromoacetamidation of monoprotected alkanedi-
ESI-HRMS (m/z): 503.3208 (M + H+) (calcd. 503.3193).
C22H42N6O7·2.5 C2HO2F3 (791.46) calcd. C, 40.97; H, 5.67; N,
10.62; found C, 40.72; H, 5.38; N, 10.30%.
1
amines, giving 3.52 g (9.48 mmol, 90.7% yield) of product 2. H
NMR (CDCl3, 300 MHz): d = 7.35 (s, 5H), 6.51 (b, 1H), 5.08 (s,
2H), 4.75 (b, 1H), 3.87 (s, 2H), 3.26 (m, 2H), 3.18 (m, 2H), 1.50
(m, 4H), 1.34 (m, 4H). 13C NMR (CDCl3, 75 MHz): d = 165.6,
156.5, 136.8, 128.7, 128.3, 128.2, 66.8, 40.1, 29.9, 29.3, 29.1, 26.3,
26.1. ESI-MS (m/z): 371.30 (M + H+) (calc. 371.09).
Synthesis of 1-(9-maleimido-3-aza-2-oxononyl)-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane—
DOTAMA-C6-Nmal (11)
Compound 8 (87 mg, 0.11 mmol) was dissolved in water (20 mL)
and 7-exo-oxohimic anhydride (9) (19 mg, 0.11 mmol) was added.
The mixture was placed in a two-necked round-bottom flask which
was inserted in the cavity of the professional microwave oven.
The flask was equipped with a fiber-optic thermometer and a
condenser. The mixture was irradiated with microwave for 5 min
(30 s at 600 W, 4.5 min at 350 W) the reaction temperature
Synthesis of 1-(3,10-diaza-2,11-dioxo-11-phenyloxyundecyl)-
4,7,10-tris(tert-butyloxycarbonylmethyl)-1,4,7,10-
tetraazacyclododecane (4)
DO3A(t-BuO)3 free base (4.71 g, 9.15 mmol), K2CO3 (1.57 g,
11.4 mmol) and 2 (3.52 g, 9.48 mmol) were reacted according to
the general procedure for the attachment of a bromoacetamide
pendant arm to DO3A(t-BuO)3 using K2CO3 as base, and gave
7.08 g (8.8 mmol, 95% yield) of 4. The alternative method: using
◦
remaining constant at 110 C. The solvent was then evaporated
under vacuum and the residue triturated with diethyl ether to give
a white solid. The crude material was dissolved in water, loaded
over an Amberlite XAD 1600 column and eluted with a water–
methanol gradient (100 : 0 to 0 : 100). After solvent evaporation
product 11 was collected as a white solid (yield 60%).1H NMR
(DMSO-d6, 300 MHz): d = 8.33 (b, 1H), 6.90 (s, 2H), 4.40 (s,
2H), 4.22 (s, 2H), 3.69 (s, 4H), 3.60–2.70 (b, 20H), 1.85 (t, 2H,
J = 7.3 Hz), 1.60 (m, 2H), 1.50 (m, 2H), 1.40 (m, 2H). 13C NMR
(DMSO-d6, 75 MHz): d = 171.6, 171.1, 170.7, 168.8, 134.2, 57.9,
56.3, 55.8, 53–48 (b), 38.7, 32.7, 28.0, 26.4, 25.3, 25.1. ESI-HRMS
(m/z): 583.3077 (M + H+) (calc. 583.3092). C26H42N6O9 (582.64)
calcd. C, 53.60; H, 7.27; N, 14.42; found C, 53.27; H, 6.90; N,
14.13.
R
Amberliteꢀ IRA 410 as base instead of K2CO3 gave 4 in 90% yield.
1H NMR (CDCl3, 300 MHz): d = 7.35 (s, 5H), 7.29 (b, 1H), 5.07
(s, 2H), 5.02 (b, 1H), 4.37–2.75 (b, 28H), 1.55 (s, 9H), 1.49 (s, 9H),
1.44 (s, 9H), 1.73 (m, 2H), 1.41 (m, 2H), 1.25 (m, 4H). 13C NMR
(CDCl3, 75 MHz): d = 171.9, 171.7, 171.5, 156.5, 136.9, 128.4,
127.9, 66.3, 58.2, 56.8, 56.5, 53–48 (b), 40.9, 39.1, 29.2, 28.2, 26.3,
26.1, 28.1, 28.0. ESI-MS (m/z): 805.7 (M + H+), (calc. 805.5).
Synthesis of 1-(9-amino-3-aza-2-oxononyl)-4,7,10-tris(tert-
butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane—
DOTAMA(t-BuO)3-C6-NH2 (6)
Product 4 (0.88 g, 1.09 mmol) was deprotected by hydrogenation
at room pressure, accordingly to the above-reported procedure
(mmol scale), yielding 0.68 g (1.01 mmol, yield 93%) of product 6.
Gram-scale deprotection of 4, carried out using a Venturi-effect
Synthesis of N-(2-benzyloxycarbonylaminoethyl)bromoacetamide
(1)
2-(Benzyloxycarbonylamino)ethanamine (24.7 g, 0.127 mol),
K2CO3 (0.150 mol, 20.7 g) and bromoacetyl bromide (0.127 mol,
25.6 g, 11.0 mL) were reacted according to the above-reported
general procedure for bromoacetamidation of monoprotected
alkanediamines, giving product 4 (29.4 g, 0.093 mol) in 73.4%
yield. 1H NMR (CDCl3, 300 MHz): d 7.34 = (s, 5H), 6.55 (b, 1H),
5.10 (s, 2H), 4.80 (b, 1H), 3.85 (s, 2H), 3.31 (m, 2H), 3.22 (m,
2H). 13C NMR (CDCl3, 75 MHz): d = 166.2, 158.0, 136.9, 128.8,
1
stirrer, gave 6 in 90% yield. H NMR (CDCl3, 300 MHz): d =
8.54 (b, 1H), 8.37 (b, 2H), 4.44 (s, 2H), 4.25 (s, 2H), 3.71 (s, 4H),
3.60–2.70 (b, 20H), 1.89 (t, 2H, J = 7.3 Hz), 1.60 (m, 2H), 1.50
(m, 2H), 1.44 (s, 27H), 1.40 (m, 2H). 13C NMR (CDCl3, 75 MHz):
d = 171.8, 171.6, 170.2, 81.9, 58.2, 56.7, 56.2, 53–48 (b), 39.8, 39.1,
28.4, 26.9, 25.8, 25.7, 28.2, 28.1, 28.0. IR (mmax/(cm−1), liquid film):
3451, 3266, 1732, 1654, 1537, 1456, 1368, 1155.
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128.4, 128.3, 67.0, 40.1, 38.0, 29.9. ESI-MS (m/z): 315.27 (M +
obtained as a white solid in 65% yield.1H NMR (DMSO-d6,
300 MHz): d = 8.30 (b, 1H), 6.90 (s, 2H), 3.63 (b, 4H), 3.38
(s, 4H), 3.24 (s, 4H), 3.10–2.60 (b, 16H). 13C NMR (DMSO-d6,
75 MHz): d = 171.6, 171.0, 10.7, 168.5, 134.2, 57.2, 55.5, 55.2, 53–
48 (b), 38.0, 32.7. ESI-HRMS (m/z): 527.2484 (M + H+) (calcd.
527.2466). C22H34N6O9 (526.54) calcd. C, 50.18; H, 6.51; N, 15.96;
found C, 50.45; H, 6.87; N, 16.32%.
H+) (calcd. 315.03).
Synthesis of 1-(3,6-diaza-2,7-dioxo-7-phenyloxyheptyl)-4,7,10-
tris(tert-butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane
(3)
14.5 g (0.028 mol) of DO3A(t-BuO), 4.64 g (0.034 mol) of
K2CO3 and 8.82 g (0.028 mol) of 1 were reacted according to
the above-reported general procedure for the attachment of a
bromoacetamide pendant arm to DO3A(t-BuO)3 using K2CO3
as base, giving product 3 in 85% yield. The alternative method
Synthesis of 2-(6-aminohexyloxy)tetrahydropyran (12)
Dihydropyran (DHP, 25 mmol) was added to a solution of 6-
amino-1-hexanol hydrochloride (8.5 mmol) in CH2Cl2 (50 mL).
R
using Amberliteꢀ IRA 410 as base instead of K2CO3 gave 3 in
◦
The liquid was cooled down to 0 C, p-toluenesulfonic acid (9.3
1
80% yield. H NMR (CDCl3, 300 MHz): d = 7.30 (s, 5H), 6.80
◦
mmol) was added, and the mixture stirred at 0 C for 1 h. After
(b, 2H), 5.05 (s, 2H), 3.50–2.55 (b, 28H), 1.45 (s, 9H), 1.41 (s,
9H), 1.38 (s, 9H). 13C NMR (CDCl3, 75 MHz): d = 172.1, 171.8,
171.5, 157.0, 137.9, 128.4, 127.9, 82.9, 67.3, 59.2, 57.1, 56.5, 53–48
(b), 40.9, 39.1, 29.2, 29.0. ESI-MS (m/z): 749.3 (M + H+), (calcd.
749.7).
another 8 h at room temperature the reaction was complete. Ice
was then added and the phases were separated. The organic phase
was washed with saturated NaHCO3 solution (2 × 10 mL) and
dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure to give the protected alcohol 12 in quantitative
yield as a colourless oil. 1H NMR (CDCl3, 300 MHz): d = 4.48 (t,
J = 4.4 Hz, 1H), 3.83 (m, 1H), 3.69 (m, 1H), 3.45 (m, 1H), 3.33
(m, 1H), 2.65 (t, J = 8 Hz, 2H), 1.32–1.69 (m, 14H). 13C NMR
(CDCl3, 75 MHz) d 165.0, 99.2, 67.1, 62.5, 40.2, 19.8, 30.8, 29.4,
29.3, 26.7, 25.9, 25.5. ESI-MS (m/z): 202.10 (M + Na+), (calcd.
202.17).
Synthesis of 1-(5-amino-3-aza-2-oxopentyl)-4,7,10-tris(tert-
butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane—
DOTAMA(t-BuO)3-en (5)
22.5 g (0.03 mol) of 3 and 2.0 g of catalyst were subjected to the
general procedure for hydrogenating the Cbz group on the molar
scale. 17.5 g (0.028 mol) of 5 were obtained, a 95% yield. 1H NMR
(CDCl3, 300 MHz): d = 8.80 (b, 1H), 8.71 (b, 2H), 3.60 (b, 4H),
3.40 (s, 4H), 3.21 (s, 4H), 3.20–2.70 (b, 16H), 1.44 (s, 27H). 13C
NMR (CDCl3, 75 MHz): d = 171.8, 171.6, 170.2, 81.9, 59.2, 57.5,
56.2, 53–48 (b), 40.8, 39.7, 28.8, 28.5, 28.3. IR (mmax/(cm−1), liquid
film): 3452, 3259, 1730, 1655, 1540, 1455, 1370. ESI-HRMS (m/z):
615.4425 (M + H+) (calcd. 615.4445), C30H58N6O7 (614.81) calcd.
C, 58.61; H, 9.51; N, 13.67; found C, 58.26; H, 9.18; N, 13.50%.
Synthesis of bromoacetamidohexyl tetrahydropyran-2-yl ether
(12a)
2-(6-Aminohexyloxy)tetrahydropyran (12) (14.9 mmol), K2CO3
(20.9 mmol) and bromoacetyl bromide (1.36 mL, 15.6 mmol)
were reacted under the above-reported general conditions for
bromoacetamidation of monoprotected alkanediamines. By silica-
gel column chromatography (CH2Cl2 : CH3OH 98 : 2, Rf 0.32)
10.44 mmol of product were obtained (70% yield).1H NMR
(CDCl3, 300 MHz): d = 6.49 (s, 1H), 4.53 (t, 1H, J = 4.0 Hz),
3.85 (s, 2H), 3.83 (m, 1H), 3.71 (m, 1H), 3.28 (m, 1H), 3.24 (m,
1H), 1.34–1.57 (m-overlapped, 14H). 13C NMR (CDCl3, 75 MHz)
d 165.0, 99.2, 67.1, 62.5, 40.2, 19.8, 30.8, 29.7, 29.4, 29.3, 26.7,
25.9, 25.5. ESI-MS (m/z): 322.4 (M + Na+), (calcd. 322.1).
Synthesis of 1-(5-amino-3-aza-2-oxopentyl)-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane—
DOTAMA-en (7)
Compound 3 was fully deprotected with trifluoroacetic acid
according to the above-reported procedure. DOTAMA-en (7)
was obtained as trifluoroacetate salt in quantitative yield. By
acidimetric titration, and CHN analysis, 2.5 TFA molecules were
found for each DOTAMA-en molecule.1H NMR (DMSO-d6,
300 MHz): d = 8.30 (b, 1H), 7.90 (b, 2H), 3.63 (b, 4H), 3.38
(s, 4H), 3.24 (s, 4H), 3.10–2.60 (b, 16H). 13C NMR (DMSO-d6,
75 MHz): d = 171.6, 171.0, 168.5, 57.2, 55.5, 55.2, 53–48 (b), 38.8,
37.7. IR (mmax/(cm−1), liquid film): 3443, 3248, 1728, 1650, 1459,
1427, 1208, 1140. ESI-HRMS (m/z): 447.2585 (M + H+) (calcd.
447.2567). C18H34N6O7 2.5 C2HO2F3 (731.23) calcd. C, 37.76; H,
5.03; N, 11.49; found C, 38.53; H, 5.39; N, 12.07%.
Synthesis of 1-(tetrahydropyran-2-yloxyhexylcarbamoylmethyl)-
4,7,10-tris(tert-butyloxycarbonylmethyl)-1,4,7,10-
tetraazacyclododecane (13)
DO3A-tris-tert-butyl ester free base (3.7 mmol), K2CO3 (14.8
mmol) and bromoacetamidohexyl tetrahydropyran-2-yl ether (12)
(1.19 g, 3.7 mmol) were reacted under the above-reported general
conditions (a) for substitution of DO3A(t-BuO)3, giving 13 in 76%
1
yield. H NMR (CDCl3, 300 MHz): d = 8.5 (s, 1H), 4.56 (t, 1H,
J = 4.0 Hz), 3.86 (m, 1H), 3.73 (m, 1H), 3.50 (m, 1H), 3.38 (m,
1H), 3.28 (s, 2H), 3.23 (s, 2H), 3.22 (s, 4H), 3.20 (m, 2H), 3.00 (s,
2H), 2.89–2.51 (b, 16H), 1.71–1.38 (m-overlapped, 14H), 1.45 (s,
27H). 13C NMR (CDCl3, 400 MHz): d = 171, 98.91, 81.03, 81.00,
67.6, 62.4, 58.0, 56.7, 56.2, 54.9, 53.4, 52.7, 52,0, 39.4, 30.8, 30.0,
29.8, 28.3, 27.2, 26.2, 25.5, 19.7. ESI-MS (m/z): 778.50 (M + Na+)
(calcd. 778.53).
Synthesis of 1-(5-maleimido-3-aza-2-oxopentyl)-4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraaza cyclododecane—
DOTAMA-en-mal (10)
Compound 7 (80 mg, 0.11 mmol) was dissolved in water (20 mL)
and 7-exo-oxohimic anhydride (9) (19 mg, 0.11 mmol) was added.
By the procedure described for product 11, product 10 was
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Synthesis of N-(6-hydroxyhexyl)chloroacetamide (15)
chloroacetamide pendant arm. 1H NMR (CDCl3, 400 MHz): d =
4.07 (s, 1H), 3.74 (m, 2H), 3.46 (m, 2H), 3.35–3.27 (b, 8H), 3.0–2.8
(b, 16H), 1.45 (s, 27H).13C NMR (CDCl3, 400 MHz): d = 173.1,
170.6, 169.7, 167.1, 81.8, 81.6, 60.8, 58.0, 55.6, 51.3, 49.3, 47.4,
28.2. IR (mmax/(cm−1), KBr): 5480, 1741, 1722, 1469, 1452, 1361,
1255, 1151, 1168. ESI-HRMS (m/z): 616.4300 (M + H+) (calcd.
616.4285). C30H57N5O8 (615.80) calcd. C, 58.51; H, 9.33; N, 11.37;
found C, 58.83; H, 9.84; N, 11.62%.
Product 15 was obtained in 85% yield from chloroacetyl bromide
and 6-aminohexanol following the general procedure for chloroac-
etamidation. 1H NMR (CDCl3, 300 MHz): d = 9.95 (b, 1H), 4.50
(b, 1H), 4.05 (s, 2H), 3.50 (t, 2H, J = 7.0 Hz), 3.19 (t, 2H, J =
7.2 Hz), 1.50 (m, 4H), 1.30 (m, 4H). 13C NMR (CDCl3, 75 MHz)
d 165.0, 62,0, 52.3, 40.3, 30.3, 32.1, 26.5, 25.6. ESI-MS (m/z):
194.15 (M + Na+), (calcd. 194.09).
Synthesis of 1-(2-oxoethylcarbamoylmethyl)-4,7,10-tris(tert-
butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (18)
Synthesis of 1-(6-hydroxyhexylcarbamoylmethyl)-4,7,10-tris(tert-
butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (14)
By the general procedure for alcohol oxidation, compound 14 was
Sodium borocyanohydride (5.7 mmol) was added to a solution
of the THP ether (compound 13, 2.9 mmol) and BF3·OEt2 (14.5
mmol) in dry THF (10 mL). The mixture was irradiated with
ultrasound (21 kHz, 50 W) for 2 hours, then filtered to give 14
as a white solid in 79% yield. Alternatively, 14 was obtained
in 55% yield by direct N-alkylation of DO3A(t-BuO)3 with
N-(6-hydroxyhexyl)chloroacetamide (15) following the general
procedure for the attachment of a chloroacetamide pendant arm.
1H NMR (CDCl3, 300 MHz): d = 8.5 (s, 1H), 4.27 (b, 4H), 4.13 (b,
2H), 4.0 (b, 2H), 3.61 (m, 2H), 3.67–3.40 (b, 16H), 3.21 (m, 2H),
1.55–1.27 (m-overlapped, 35H). 13C NMR (CDCl3, 75 MHz): d =
169.0, 166.0, 164.0, 84.8, 82.4, 68.2, 62.3, 55.4, 55.0, 54.2, 51.6,
48.9, 48.6, 39.5, 32.3, 28.9, 28.0, 27.9, 26.5, 25.2. IR (mmax/(cm−1),
liquid film): 3453, 3252, 1728, 1652, 1543, 1451, 1154, 1223.
ESI-HRMS (m/z): found 672.4895 (M + H+) (calcd. 672.4911).
C34H65N5O8 (671.90) calcd. C, 60.78; H, 9.75; N, 10.42; found C,
60.37; H, 9.32; N, 10.21%.
1
converted to 19 in 90% yield. H NMR (CDCl3, 400 MHz): d =
4.07 (1H), 3.81 (m, 2H), 3.32–3.20 (b, 8H), 2.7–2.5 (b, 16H), 1.36
(s, 27H). 13C NMR (CDCl3, 400 MHz): d = 201.0, 173.1, 171.1,
170.2, 81.0, 82.1, 55.7, 52.8, 49.3, 46.1, 28.2. IR (mmax/(cm−1), liquid
film): 3277, 3249, 1738, 1675,1553, 1452, 1390, 1371, 1223, 1150.
ESI-HRMS (m/z): found 614.4145 (M + H+) (calcd. 614.4129).
C30H55N5O8 (613.79) calcd. C, 58.70; H, 9.03; N, 11.41; found C,
59.11; H, 9.47; N, 11.81%.
Acknowledgements
We gratefully acknowledge the support of MIUR (FIRB and
PRIN), Regione Piemonte (CIPE project) and EU (EMIL and
DiMI NoEs, Meditrans). DJU acknowledges ASP (Associazione
per lo Sviluppo Scientifico e Technologico del Piemonte), Torino
for a research fellowship. We are indebted to Dr M. Boccasile
(Waters Italia S.p.A., Vimodrone, Italy) for HRMS analyses and
to Professor F. Trotta (University of Torino, Italy) for CHN
elemental analyses.
Synthesis of 1-(6-oxohexylcarbamoylmethyl)-4,7,10-tris(tert-
butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (19)
By the general procedure for alcohol oxidation, compound 14
was converted to 19 in 90% yield. H NMR (CDCl3, 300 MHz):
Notes and references
1
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overlapped, 35H). 13C NMR (CDCl3, 75 MHz): d = 203.0, 173.1,
172.2, 171.0, 81.6, 81.3, 72.9, 62.0, 56.1, 55.7, 55.6, 52.0, 46.1,
43.8, 38.8, 29.1, 28.1, 27.9, 26.4, 21.7. IR (mmax/(cm−1), liquid film):
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Synthesis of N-(2-hydroxyethyl)chloroacetamide (16)
16 was obtained in 85% yield from chloroacetyl bromide and
ethanolamine following the general procedure for chloroacetami-
dation of amino alcohols.
1H NMR (CDCl3, 400 MHz): d = 9.95 (b, 1H), 4.26 (b, 1H),
3.90 (s, 2H), 3.66 (t, 2H, J = 7.0 Hz), 3.26 (t, 2H, J = 7.3 Hz). 13
C
NMR (CDCl3, 75 MHz) d 165.0, 60.8, 52.0, 47.5. ESI-MS (m/z):
138.35 (M + H+) (calcd. 138.02).
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Synthesis of 1-(2-hydroxyethylcarbamoylmethyl)-4,7,10-tris(tert-
butyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (17)
Product 17 was obtained in 65% yield by direct N-alkylation
of DO3A(t-BuO)3 free base with N-(2-hydroxyethyl)chloro-
acetamide following the general procedure for the attachment of a
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