TAME-Hex A - A novel bifunctional chelating agent for radioimmunoimaging

Article abstract of DOI:10.1002/ejoc.200400224

Two novel (bifunctional) chelating agents, TAME-Hex A and B, which are polyaminopolycarboxylic acids based on the tripodal TAME [tris(aminomethyl) ethane] structure, have been designed and synthesized. The chelators show very good stability with gallium(I

Full text of DOI:10.1002/ejoc.200400224

FULL PAPER
TAME-Hex A ؊ A Novel Bifunctional Chelating Agent for
Radioimmunoimaging
Enver Arslantas,[a] Peter M. Smith-Jones,[b] Gerd Ritter,[c] and Richard R. Schmidt*[a]
Keywords: Chelating agents / Radioimmunoimaging / Positron emission tomography / Gallium chelate / TAME derivative
Two novel (bifunctional) chelating agents, TAME-Hex A and
B, which are polyaminopolycarboxylic acids based on the tri-
podal TAME [tris(aminomethyl)ethane] structure, have been
candidates for use in radioimmunoimaging (RII), especially
in positron emission tomography (PET).
designed and synthesized. The chelators show very good ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
stability with gallium(III) ions and thus are highly effective
Germany, 2004)
Introduction
Bifunctional chelating agents continue to be an interest-
ing topic in medicinal and chemical research because of
their usefulness in the diagnosis and therapy of cancer dis-
eases.[1] Radioimmunoimaging (RII) and radioimmunother-
apy (RIT) rely on the ability of a molecule with a chelating
functionality, which will sequester a radioactive metal ion,
to combine with a monoclonal antibody or any other recep-
tor-specific substrate, like an oligopeptide[2] or a Lewisx
structure analog.[3] This approach allows radiopharmaceut-
icals to be delivered specifically to malignant tissue while
minimizing the risk of unspecific irradiation of sane tissue.
Chelating agents useful for RII and RIT should be able
to form metal-chelates with high thermodynamic stability
as well as high kinetic inertness in vivo to reduce intoxi-
cation arising from the loss of the radioactive (heavy-)metal
ion. Derivatives of the chelating structures of DTPA (dieth-
ylenetriaminetetraacetic acid)[4] and DOTA (1,4,7,10-tetra-
azacyclododecane-N,NЈ,NЈЈЈ,NЈЈЈ-tetraacetic acid), like
BAD[5] (Figure 1), are most frequently used for this purpose
at present, but new chelators with improved or altered
physical properties for different biological applications are
desirable.
Figure 1. The structures of BAD, TAME, H3[(5-MeOsal)3TAME]
and TAME-Hex
1,1,1-Tris(aminomethyl)ethane (TAME) (Figure 1) is a
tridentate ligand which is used as a starting material in the
synthesis of more complex ligands.[6,7] Salicylaldimines of
TAME are used as chelating agents in the complete encap-
sulation of Ga3ϩ ions.[8] Together with the positron emitting
isotope 68Ga, which is available from a parent/daughter
generator system (68Ge/68Ga) independently of an in-house
cyclotron, these compounds are useful in positron emission
tomography (PET), a very powerful technique used in medi-
cal diagnosis. An example of such a chelating agent, H3[(5-
MeOsal)3TAME], which can be used to assess myocardial
blood flow, is shown in Figure 1.[9]
Astonishingly, no bifunctional chelating agents based on
the tripodal TAME structure, which could be useful for
radioimmuno-imaging or -therapy, have been reported in
the literature. In this paper, we report the synthesis of two
novel, tripodal chelators, one bifunctional and the other
monofunctional, which are derivatives of tris(aminomethyl)-
[a]
Fachbereich Chemie, Universität Konstanz,
Fach M 725, 78457 Konstanz, Germany
Fax: (internat.) ϩ 49-(0)7531-88-3135
E-mail: Richard.Schmidt@uni-konstanz.de
[b]
Radiology Department, Memorial Sloan Kettering Cancer
Center,
1275 York Avenue, New York, NY 10021, USA
E-mail: smithjop@mskcc.org
[c]
Ludwig Institute for Cancer Research,
Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10021, USA
Fax: (internat.) ϩ 1-212-717-3100
E-mail: gritter@licr.org
Eur. J. Org. Chem. 2004, 3979Ϫ3984
DOI: 10.1002/ejoc.200400224
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E. Arslantas, P. M. Smith-Jones, G. Ritter, R. R. Schmidt
FULL PAPER
ethane-N,N,NЈ,NЈ,NЈЈ,NЈЈ-hexaacetic acid (TAME-Hex) the subsequent reduction of the triazide 5 to the triamine 6
(Figure 1). The Ga3ϩ complexes of these chelators should by use of the Staudinger reaction.[11]
be very stable and are expected to be very useful in positron
emission tomography.
The carboxymethyl functionalities were introduced next.
Although direct alkylation of 6 with bromoacetic acid
should, in principle, be possible, the isolation of the highly
polar product from the reaction mixture as well as the
monitoring of the progress of the reaction would be diffi-
cult. Thus, the use of a bromoacetic ester is preferable. tert-
Butyl bromoacetate was the reagent of choice because it
will not undergo intramolecular lactam formation like
many other esters. The alkylation of triamine 6 with tert-
butyl bromoacetate and sodium carbonate in DMF deliv-
ered the corresponding sixfold tert-butyl ester. However, to
achieve greater purity, deprotection of the ester groups by
TFA and subsequent reaction with diazomethane to yield
the hexamethyl ester 7 was necessary. Pure 7 was then sa-
ponified with 1  NaOH in methanol/water, acidified with
HCl to pH 1.5 and submitted to RP-18 column chromatog-
raphy to obtain the chelator 8 in the free-acid form. Finally,
the transformation of 8 to the isothiocyanate form 9
(TAME-Hex A) was performed by catalytic reduction and
subsequent reaction with thiophosgene.
Results and Discussion
TAME-Hex A
To obtain a bifunctional chelating agent, it was decided
to introduce the p-nitrobenzyloxy moiety into the 2-posi-
tion of TAME-Hex, which could then be easily transformed
into the corresponding isothiocyanate for the purpose of
conjugation to a monoclonal antibody or peptide. Starting
from commercially available pentaerythritol, it was neces-
sary to differentiate between one of the four chemically
equivalent hydroxy groups of this compound. This was ac-
complished by acylation of pentaerythritol with three
equivalents of benzoyl chloride in pyridine (Scheme 1). The
threefold benzoylated product 1 could easily be isolated
from the other over- and underacylated products by silica
gel chromatography. As O-alkylation with p-nitrobenzyl
bromide is usually not possible with sodium hydride as
base, silver() oxide[10] was used for the transformation of 1
into the alkylated compound 2. Deacylation of 2 with Na-
OMe delivered the desired O-(4-nitrobenzyl)pentaerythritol
(3) in 28% yield over three steps.
Tame-Hex B
Furthermore, a monofunctional derivative of TAME-
Hex, TAME-Hex B, was synthesized (Scheme 2), which is
also of interest for use in imaging methods like PET.
Scheme 2. Synthesis of TAME-Hex B (15). Reagents and con-
ditions: i) NaN3, DEG, 120 °C; ii) H2/Pd-C, MeOH, HCl; iii) ben-
zaldehyde, MeOH; iv) NaBH4, CHCl3/MeOH; v) BnBr, DMF, DI-
PEA; vi) 1.5  HCl; vii) BrCH2CO2tBu, Na2CO3, DMF, 65 °C;
viii) TFA; ix) CH2N2, MeOH; x) 1  NaOH, MeOH/H2O; xi) HCl;
xii) RP-18 column
Scheme 1. Synthesis of TAME-Hex A (9). Reagents and con-
ditions: i) BzCl (3 equiv.), Py; ii) p-nitrobenzyl bromide, Ag2O,
CH2Cl2; iii) NaOMe, MeOH/CH2Cl2; iv) TosCl, Py, DMAP; v)
NaN3, DMSO, 90 °C; vi) Ph3P, THF/H2O; vii) BrCH2CO2tBu,
Na2CO3, DMF, 65 °C; viii) TFA; ix) CH2N2, MeOH; x) 1  NaOH,
MeOH/H2O; xi) HCl; xii) RP-18 column; xiii) H2/Pd-C, H2O,
Na2CO3; xiv) Cl2CS, CHCl3/H2O
The advantage of this synthetic procedure is that one of
The next crucial step was the transformation of the three the four amino groups in tetrakis(aminomethyl)methane
hydroxy groups of compound 3 into amines. This goal was (11),[12] which is obtained from the tetratosylate of pentaer-
best achieved by the threefold tosylation of 3 (Ǟ 4), nucleo- ythritol (10),[13] can be differentiated by the reaction with
philic substitution with sodium azide in DMSO (Ǟ 5) and four equivalents of benzaldehyde and subsequent reduction
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TAME-Hex A Ϫ A Novel Bifunctional Chelating Agent for Radioimmunoimaging
FULL PAPER
of the resulting aldimine to the triazaadamantane 12.[14]
Compound 12 was then benzylated and the benzylidene
protecting groups removed by 1.5  hydrochloric acid to
afford the TAME derivative 13 in 77% yield. Alkylation
with tert-butyl bromoacetate as mentioned above, depro-
tection with TFA and esterification with diazomethane
delivered the hexamethyl ester 14 in 70% yield. Alkaline
saponification and work up as described above for com-
pound 9 finally led to the desired chelating agent 15
(TAME-Hex B).
Note that this synthesis can be is easily varied to give a
bifunctional derivative of TAME-Hex B. To achieve this, it
is necessary to use an alkylating or acylating reagent that
contains a potential anchoring group for the derivatization
of compound 12.
Radiolabeling and Stability Testing of New Chelators
Figure 3. Stability of 67Ga-15 to trans-chelation by a 1000-fold
The stabilities of the 111In3ϩ- and 67Ga3ϩ-chelates of 8
and 15 were tested by performing trans-chelation experi-
ments using DTPA (diethylenetriaminepentaacetic acid) as
the competing ligand. Compound 9 could not be employed
in these investigations because the isothiocyanate group
transforms under the experimental conditions.
excess of DTPA at an ambient temperature
cation within nuclear medicine to stably chelate 67Ga and
68Ga.
Both chelators could be radiolabeled with either 111In or
67Ga with less than 0.1% unchelated radiometal remaining
at the end of the radiolabeling process. 111In-8 and 111In-15
had similar stabilities with about 10% of the metal-chelates
remaining intact after 24 hours.
Conclusions
The stabilities of the 67Ga chelates were significantly bet-
ter: 67Ga-8 was very stable with 94% remaining intact after
10 days (Figure 2). 67Ga-15 was even more stable with
around 99% still intact after 10 days (Figure 3).
TAME-Hex B 15 has nine potential donors to fully satu-
rate the coordination spheres of either GaIII or InIII; the
higher stability of GaIII chelates is probably due to its
smaller ionic radius and higher charge density. This new
TAME-Hex derivative is now being evaluated for appli-
In conclusion, we have designed and synthesized two no-
vel chelating agents belonging to the class of polyaminopo-
lycarboxylic acids based on the tripodal TAME structure.
The first stability tests showed that the new gallium chelates
are stable against trans-chelation by a 1000-fold excess of
DTPA and thus are potentially highly effective candidates
for use in radioimaging. Further investigations into the ex-
act nature of the thermodynamics, kinetics and biological
stabilities of the chelates are currently under investigation.
Experimental Section
General Remarks: Twice-distilled water was used for the reactions
leading to and with chelating agents. Merck ‘‘Plastikfolien Kieselgel
60 F254’’ plates were used for thin layer chromatography. Detection
was achieved by UV absorption at λ ϭ 254 nm or by dipping the
plates in a solution of ammonium molybdate (20 g) and cerium(IV)
sulfate in 10% sulfuric acid (Mostain, 400 mL) or a 1% solution of
ninhydrin in ethanol and subsequent heating to 150 °C. Preparative
flash column chromatography was performed with a pressure of
1.3Ϫ1.4 bar using Silica 60 (40Ϫ63 µm, 230Ϫ400 mesh ASTM)
from Baker Co. or Knauer Europrep C18 (600pm, 35Ϫ70 µ, irregu-
lar). Melting points were determined in a Gallenkamp apparatus
and are uncorrected. NMR spectra were measured with a Bruker
AC 250 (250 MHz) or Bruker DRX 600 (600 MHz) spectrometer.
MALDI mass spectra were measured with a KRATOS Analytical
Kompact MALDI 2 spectrometer. 2,5-Dihydroxybenzoic acid
(DHB) was used as the matrix in the positive mode unless other-
wise stated, and azathiothymine (ATT) in the negative mode.
Figure 2. Stability of 67Ga-8 to trans-chelation by a 1000-fold ex-
cess of DTPA at an ambient temperature
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E. Arslantas, P. M. Smith-Jones, G. Ritter, R. R. Schmidt
FULL PAPER
Radiolabeling: The chelators were radiolabeled with both 111In and
67Ga by heating them at elevated temperatures at a neutral pH and
by using a transfer ligand of ammonium acetate. 111In (2 µL, 1
(8.14 g, 11.1 mmol, 99%) as a colorless solid, m.p. 134 °C. 1H NMR
(250 MHz, CDCl3): δ ϭ 2.44 (s, 9 H, 3 CH3 {Tos}), 3.40 (s, 2 H,
CH2OCH2Ar), 4.42 (s, 2 H, OCH2Ar), 3.94 [s, 6 H, C(CH2O)3],
mCi, 0.05  HCl) or 67Ga (2 µL, 1mCi, 0.05  HCl), chelator 8 or 7.30Ϫ8.18 (m, 16 H, Ar, 3 Tos) ppm. C33H35NO12S3 (733.8): calcd.
15 (5 µL, 2 mg·mLϪ1) and 3  NH4OAc (50 µL, pH 7.0) were
heated at 100 °C for 10 minutes. The radiochemical yield was as-
sayed by mixing an aliquot of the reaction mixture with 5 m
DTPA (pH 7.0) immediately prior to C18 RP HPLC analysis with
an eluent gradient of 50 m NH4OAc (pH 7.0) to 50 m NH4OAc
C 54.01, H 4.81, N 1.91; found C 54.01, H 4.94, N 1.61.
3-Azido-2,2-bis(azidomethyl)-1-(4-nitrobenzyloxy)propane (5): So-
dium azide (5.3 g, 81 mmol) was added to a solution of the tritosyl-
ate 4 (4.96 g, 6.76 mmol) in dry DMSO (100 mL) and the mixture
was stirred for 20 h at 90 °C. After cooling to room temperature,
the mixture was poured into cold water (300 mL) and extracted
with diethyl ether twice (200 and 100 mL). The organic layer was
washed twice with brine and dried with Na2SO4. Concentration
under reduced pressure afforded triazide 5 (2.03 g, 5.10 mmol,
75%) as a yellowish oil which could be used without further purifi-
cation in the next step. 1H NMR (250 MHz, CDCl3): δ ϭ 3.39 [s, 6
H, C(CH2O)3], 3.50 (s, 2 H, CH2OCH2Ar), 4.62 (s, 2 H, OCH2Ar),
7.45Ϫ8.24 (m, 4 H, Ar) ppm.
(pH 7.0)/80% acetonitrile over 10 minutes at 0.7 mL·minϪ1
.
Stability Testing: The 111In or 67Ga chelates were diluted to 5 µM
with 5 m DTPA (pH 7.0) and stored at ambient temperature.
Periodically 10 µL samples were removed and analyzed by C18 RP
HPLC as outlined above.
Tri-O-benzoylpentaerythritol (1): Benzoyl chloride (3.6 mL,
31 mmol) was slowly added to a well-stirred suspension of pentaer-
ythritol (1.3 g, 9.5 mmol) in dry pyridine (15 mL) at room tempera-
ture (gentle cooling was necessary after a while). After 3 h of stir-
ring, water (120 mL) was added and the mixture was extracted with
ethyl acetate (120 mL). The organic layer was washed with 5% HCl
and water (100 mL each), dried with MgSO4 and the solvents eva-
porated. The residue was purified by flash chromatography on a
silica column (toluene/acetone, 10:1) to afford 1.9 g (4.3 mmol,
45%) of 1 as a viscous, colorless oil. 1H NMR (250 MHz, CDCl3):
δ ϭ 2.76 (t, 3J ϭ 6.8 Hz, 1 H, CH2OH), 3.68 (d, 3J ϭ 6.8 Hz, 2 H,
CH2OH), 4.60 [s, 6 H, C(CH2O)3], 7.38Ϫ8.04 (m, 15 H, 3 Ph) ppm.
C26H24O7·H2O (466.5): calcd. C 66.94, H 5.62; found C 66.47, H
5.30.
2-(Aminomethyl)-2-[(4-nitrobenzyl)oxymethyl]propane-1,3-diamine
(6): Triazide 5 (347 mg, 0.87 mmol) was dissolved in THF (5 mL)
and triphenylphosphane (913 mg, 3.5 mmol) in THF (5 mL) was
added to the stirred, ice-cooled solution under an Ar atmosphere.
Water (2 mL) was added and the mixture was stirred at room tem-
perature for 12 h and for a further 6 h at 50 °C. The solution was
cooled and then neutralized by addition of conc. HCl (0.2 mL)
and then concentrated under reduced pressure. The residue was
partitioned between CH2Cl2 (40 mL) and 0.5  HCl (40 mL), and
the aqueous phase was extracted twice with CH2Cl2 (30 mL each).
The aqueous phase was made alkaline (pH ϭ 9Ϫ10) with NaOH
and the product re-extracted with CH2Cl2 (4 ϫ 30 mL). The or-
ganic layer was dried (Na2SO4) and concentrated to afford triamine
6 (211 mg, 0.66 mmol, 76%) as a colorless oil, and was used directly
in the next step.
Tri-O-benzoyl-O-(4-nitrobenzyl)pentaerythritol (2): A mixture of 1
˚
(4.25 g, 9.47 mmol), 3-A molecular sieves (4.0 g), silver oxide
(3.30 g, 14.2 mmol) and 4-nitrobenzyl bromide (67 mg, 0.31 mmol)
in dry CH2Cl2 (35 mL) was stirred for 40 h, after which it was fil-
tered and the filtrate evaporated. The residue was applied on a silica
column and chromatography was performed using CH2Cl2 as elu-
ent, to give 2 (4.60 g, 7.88 mmol, 83%) as a nearly colorless, glassy
mass. 1H NMR (250 MHz, CDCl3): δ ϭ 3.77 (s, 2 H, CH2O-
CH2Ar), 4.60 (s, 2 H, OCH2Ar), 4.64 [s, 6 H, C(CH2O)3],
7.37Ϫ8.02 (m, 19 H, Ar, 3 Ph) ppm. C33H29NO9 (583.6): calcd. C
67.92, H 5.01, N 2.40; found C 67.18, H 5.04, N 2.53.
Tetramethyl 2-[Bis(methoxycarbonylmethyl)aminomethyl]-2-[(4-
nitrobenzyl)oxymethyl]propylene-1,3-dinitrilotetraacetate (7): Tria-
mine 6 (211 mg, 0.66 mmol) was dissolved in dry DMF (5 mL)
and, after saturation of the solution with Ar, alkylated with tert-
butyl bromoacetate (0.97 mL, 6.5 mmol) and Na2CO3 (687 mg,
6.5 mmol) at 65 °C for 18 h. The reaction mixture was stirred for
12 h at room temperature and then diluted with diethyl ether
(50 mL) and water (50 mL). The organic layer was washed with
brine (50 mL), dried with Na2SO4, concentrated and submitted to
flash chromatography (petroleum ether/ethyl acetate, 10:1) to af-
O-(4-Nitrobenzyl)pentaerythritol (3): The tribenzoate 2 (4.55 g,
7.80 mmol) was dissolved in a mixture of CH2Cl2 (20 mL) and
MeOH (40 mL) and NaOMe (300 mg, 5.5 mmol) was added to the
well-stirred solution. After stirring for 24 h at room temperature,
the mixture was neutralized with acidic resin (Amberlite IR-120),
filtered and concentrated under reduced pressure. The residue was
crystallized from toluene (40 mL) to afford 3 (1.56 g, 5.77 mmol,
74%) as a colorless solid, m.p. 107 °C. 1H NMR (250 MHz,
ford the hexaalkylated product as
a colorless oil (253 mg,
0.27 mmol, 41%); Rf ϭ 0.68 (petroleum ether/ethyl acetate, 4:1).
To achieve greater purity, this product was converted into the corre-
sponding hexa(methyl ester) by cleavage of the tert-butyl ester
groups with TFA (3 mL, overnight) and by subsequent methylation
of the vacuum-concentrated residue with diazomethane. Therefore,
an excess amount of CH2N2 in ether was added to the solution of
the reactant in 90% MeOH. After stirring for several minutes, the
excess of CH2N2 was destroyed by addition of acetic acid. The
mixture was concentrated in vacuo and the residue purified by flash
3
CDCl3): δ ϭ 2.23 (t, J ϭ 6.7 Hz, 3 H, 3 CH2OH), 3.56 (s, 2 H,
3
CH2OCH2Ar), 3.75 [d, J ϭ 6.7 Hz, 6 H, C(CH2OH)3], 4.60 (s, 2
3
H, OCH2Ar), 7.46 (d, J ϭ 8.8 Hz, 2 H, Ar), 8.80 (d, 3J ϭ 8.8 Hz,
2 H, Ar) ppm. C12H17NO6 (271.3): calcd. C 53.13, H 6.32, N 5.16;
found C 53.23, H 5.92, N 4.74.
O-(4-Nitrobenzyl)-tris[O-(4-methylphenylsulfonyl)]pentaerythritol chromatography (toluene/acetone, 10:1, ϩ0.5% Et3N) to yield 7
(4): Compound 3 (3.03 g, 11.2 mmol) was dissolved in dry pyridine (150 mg, 0.21 mmol, 81%) as a colorless oil. Total yield from 7 (3
(90 mL) and tosyl chloride (8.60 g, 45.1 mmol) was added, followed steps): 32%. 1H NMR (250 MHz, CDCl3): δ ϭ 2.78 [s, 6 H,
by DMAP (140 mg, 1.15 mmol). The reaction mixture was stirred C(CH2N)3], 3.52 (s, 2 H, 2-H) 3.59 (s, 12 H, 6 NCH2CO2), 3.67 (s,
3
for 8 h at room temperature and for a further 8 h at 40 °C, after 18 H, 6 OMe), 4.49 (s, 2 H, OCH2Ar), 7.47 (d, J ϭ 8.7 Hz, 2 H,
which it was poured into ice-water (250 mL) and extracted with
CH2Cl2 (250 mL). The organic layer was washed with water
Ar), 8.21 (d, 3J ϭ 8.7 Hz, 2 H, Ar) ppm. MALDI-MS (positive
mode): m/z ϭ 701.6 [MH]ϩ (calcd. 701.7), 722.6 [MNa]ϩ (calcd.
(250 mL), dried with MgSO4 and concentrated. The solid residue 723.7). C30H44N4O15·2H2O (736.0): calcd. C 50.13, H 6.73, N 7.80;
was purified by treatment with warm MeOH (100 mL) to afford 4 found C 50.17, H 6.39, N 7.68.
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TAME-Hex A Ϫ A Novel Bifunctional Chelating Agent for Radioimmunoimaging
FULL PAPER
2-[Bis(carboxymethyl)aminomethyl]-2-[(4-nitrobenzyl)oxymethyl]-
propylene-1,3-dinitrilotetraacetic Acid (8): Hexakis(methyl ester) 7
(70 mg, 0.10 mmol) was dissolved in MeOH (2 mL), and a solution
methane (80 mL) and water (60 mL). The organic layer was washed
with brine (50 mL), dried with Na2SO4, concentrated and submit-
ted to flash chromatography (petroleum ether/ethyl acetate, 10:1)
of NaOH (160 mg, 4.0 mmol) in twice-distilled water (1 mL) was to afford the hexaalkylated product as a colorless oil (1.12 g,
added. After stirring for 24 h, the mixture was acidified with HCl
to approximately pH 1.5, and concentrated under reduced pressure.
The solid residue was dissolved in a minimum amount of MeOH/
H2O (1:3), loaded onto a RP-18 column and eluted with MeOH/
H2O (1:3 Ǟ 1:1, ϩ1% HOAc). Lyophilization of the collected and
partially concentrated fractions afforded a fluffy, pale-yellow solid
1.12 mmol, 44%); Rf ϭ 0.67 (petroleum ether/ethyl acetate, 4:1).
To achieve greater purity, this product was converted into the corre-
sponding hexa(methyl ester) by cleavage of the tert-butyl ester
groups with TFA (10 mL, overnight) and by subsequent meth-
ylation of the vacuum-concentrated residue with diazomethane.
Therefore, an excess amount of CH2N2 in diethyl ether was added
to the solution of the reactant in 90% MeOH. After stirring for
several minutes, the excess of CH2N2 was destroyed by addition of
acetic acid. The mixture was concentrated in vacuo and the residue
purified by flash chromatography (toluene/acetone, 10:1, ϩ1%
1
(51 mg, 0.08 mmol, 80%). H NMR (250 MHz, D2O): δ ϭ 3.36 [s,
6 H, C(CH2N)3], 3.74 (s, 12 H, 6 NCH2CO2), 3.95 (s, 2 H, 2-H),
4.73 (s, 2 H, OCH2Ar), 7.62 (d, 3J ϭ 8.2 Hz, 2 H, Ar), 8.26 (d,
3J ϭ 8.2 Hz, 2 H, Ar) ppm. MALDI-MS (positive mode): m/z ϭ
639.5 [MNa]ϩ (calcd. 639.5). C24H32N4O15·H2O (634.6): calcd. C
45.43, H 5.40, N 8.83; found C 45.43, H 5.71, N 8.81.
1
Et3N) to yield 14 (570 mg, 0.77 mmol, 70%) as a colorless oil. H
NMR (250 MHz, CDCl3): δ ϭ 2.72 (s, 2 H, 2-H), 2.90 [s, 6 H,
C(CH2N)3], 3.59 [s, 16 H, 6 NCH2CO2, N(CH2Ph)2], 3.65 (s, 18 H,
6 OMe), 7.27Ϫ7.34 (m, 10 H, 2 Ph) ppm. C37H52N4O12·H2O
(762.9): calcd. C 58.26, H 7.14, N 7.34; found C 58.32, H 7.03,
N 7.25.
2-[Bis(carboxymethyl)aminomethyl]-2-[(4-isothiocyanatobenzyl)oxy-
methyl]propylene-1,3-dinitrilotetraacetic Acid (9): Compound
8
(6.2 mg, 9.8 µmol) and sodium carbonate (4.3 mg, 40 µmol) were
dissolved in water (1.5 mL) and the mixture added to a suspension
of Pd on charcoal (10%, 6 mg) in water (1.5 mL), which had been
previously stirred under hydrogen. After 4.5 h of hydrogenation un-
der a slight pressure (balloon), the mixture was filtered through
a pad of Celite (which had previously been washed with water).
Thiophosgene (5 µL, 25 µmol) in CHCl3 (3 mL) was added to the
filtrate with vigorous stirring. After stirring for 1 h, the CHCl3 layer
was separated and the aqueous layer was partially evaporated in
vacuo (30 °C) to remove any volatiles and lyophilized to afford 9
(9.8 mg, quant.) as an amorphous, nearly colorless solid. 1H NMR
(250 MHz, D2O): aromatic protons: δ ϭ 7.31Ϫ7.51 (m, 4 H) ppm.
MALDI-MS (positive mode, ATT-matrix) of an acidified (HCl)
sample: m/z ϭ 630.2 [MH]ϩ (calcd. 629.5), 652.3 [MNa]ϩ (calcd.
651.5).
2-[Bis(carboxymethyl)aminomethyl]-2-[(dibenzylamino)methyl]-
propylene-1,3-dinitrilotetraacetic Acid (15): Hexa(methyl ester) 14
(350 mg, 0.46 mmol) was dissolved in MeOH (5 mL), and a solu-
tion of NaOH (480 mg, 12.0 mmol) in twice-distilled water (6 mL)
was added. After stirring for 3 d in a Teflon vessel, the mixture was
acidified with HCl to approximately pH 1 and concentrated under
reduced pressure. The solid residue was dissolved in a minimum
amount of MeOH/H2O (1:4) containing 1% of Et3N (without Et3N
the residue would not dissolve), loaded onto a RP-18 column and
eluted with MeOH/H2O (1:4 Ǟ 10:1, ϩ1% HOAc). Lyophilization
of the collected fractions afforded a colorless solid (171 mg,
0.24 mmol, 52%), which contained 1 equiv. of HOAc (and 0.1
equiv. of Et3N). Crystallization of a part of this product from a
1
2-(Aminomethyl)-2-[(dibenzylamino)methyl]propane-1,3-diamine
Tetrahydrochloride (13): DIPEA (1.22 mL, 7.0 mmol) and benzyl
bromide (0.71 mL, 6.0 mmol) were added to a stirred solution of
compound 12 (1.64 g, 3.37 mmol) at room temperature. After stir-
ring overnight, the reaction mixture turned to a white suspension,
which was diluted with diethyl ether and toluene (80 mL each) and
washed with water (80 mL) and brine (80 mL). The organic layer
was dried with CaCl2 and concentrated under reduced pressure.
The residue was suspended in a small amount of diethyl ether, fil-
tered and washed with methanol to yield 7-[(dibenzylamino)me-
thyl]-2,4,6-triphenyl-1,3,5-triazaadamantane (1.66 g, 2.88 mmol,
85%) as a white powder. This was dissolved in freshly distilled THF
(40 mL) and 1.5  HCl (50 mL) was added under vigorous stirring,
which was continued for 30 min. The THF was removed by rotary
evaporation, and the resulting aqueous solution was extracted with
CH2Cl2 (2 ϫ 50 mL). Evaporation of the aqueous layer under re-
duced pressure delivered 13 (1.27 g, 2.57 mmol, 77%) as an
amorphous, colorless solid. 1H NMR (250 MHz, [D6]DMSO): δ ϭ
little water delivered 15 free of HOAc. H NMR (250 MHz, D2O;
15·HOAc): δ ϭ 2.07 (s, 3 H, OAc), 3.17 [br. s, 6 H, C(CH2N)3],
3.41 (br. s, 2 H, 2-H), 3.54 (br. s, 12 H, 6 NCH2CO2), 4.40 [br. s,
4 H, N(CH2Ph)2], 7.53 (br. s, 10 H, 2 Ph) ppm. 1H NMR
(250 MHz, D2O; pH ϭ 12): δ ϭ 1.92 (s, 3 H, OAc), 2.88 (br. s,
2 H, 2-H), 2.98 [br. s, 6 H, C(CH2N)3], 3.37 (br. s, 12 H, 6
NCH2CO2), 3.70 [br. s, 4 H, N(CH2Ph)2], 7.27Ϫ7.48 (m, 10 H, 2
Ph) ppm. MALDI-MS (positive mode): m/z ϭ 661.4 [MH]ϩ (calcd.
660.7), 683.5 [MNa]ϩ (calcd. 683.7). C31H40N4O12 (660.7): calcd.
C 56.36, H 6.10, N 8.48; found C 56.01, H 6.53, N 8.31.
Acknowledgments
This work was supported by the Ludwig Institute for Cancer
Research, New York, and by the Fonds der Chemischen Industrie.
3.15 [br. s, 6 H, C(CH2N)3], 3.21 (br. s, 2 H, 2-H), 3.67 [br. s, 4 H,
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ϩ
N(CH2Ph)2], 7.29Ϫ7.43 (m, 10 H, 2 Ph), 8.55 (br. s, 9 H, 3 NH3
)
ppm. C19H22N4·4HCl·2H2O (494.3): calcd. C 46.10, H 7.34, N
11.33; found C 46.00, H 7.20, N 11.43.
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benzylamino)methyl]propylene-1,3-dinitrilotetraacetate (14): Com-
pound 13 (1.25 g, 2.53 mmol) was dissolved in dry DMF (15 mL)
and, after saturation of the solution with Ar, alkylated with tert-
butyl bromoacetate (3.2 mL, 21 mmol) and Na2CO3 (2.8 g,
26 mmol) at 65 °C for 8 h. The reaction mixture was stirred for a
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3966Ϫ3973.
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Received April 2, 2004
3984
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 3979Ϫ3984