Introduction of Peripheral Carboxylates to Decrease the Charge on Tm3+ DOTAM-Alkyl Complexes: Implications for Detection Sensitivity and in Vivo Toxicity of PARACEST MRI Contrast Agents

Article abstract of DOI:10.1021/acs.jmedchem.5b00621

A series of structurally modified Tm³⁺ DOTAM-alkyl complexes as potential PARACEST MRI contrast agents has been synthesized with the aim to decrease the overall positive charge associated with these molecules and increase their biocompatibility. Two types of structural modification have been performed, an introduction of terminal carboxylate arms to the alkyl side chains and a conjugation of one of the alkyl side chains with aspartic acid. Detailed evaluation of the magnetic resonance imaging chemical exchange contrast associated with the structurally modified contrast agents has been performed. In contrast to the acutely toxic Tm³⁺ DOTAM-alkyl complexes, the structurally modified compounds were found to be tolerated well during in vivo MRI studies in mice; however, only the aspartic acid modified chelates produced an amide proton-based PARACEST signal. (Figure Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00621

Article
pubs.acs.org/jmc
Introduction of Peripheral Carboxylates to Decrease the Charge on
3+
Tm DOTAM-Alkyl Complexes: Implications for Detection Sensitivity
and in Vivo Toxicity of PARACEST MRI Contrast Agents
†
,‡
†
†
‡
‡
‡
Mojmír Suchy,
́
Mark Milne, Adam A. H. Elmehriki, Nevin McVicar, Alex X. Li, Robert Bartha,
,†,§
and Robert H. E. Hudson*
†
Department of Chemistry, The University of Western Ontario, Chemistry Building, London, Ontario N6A 5B7, Canada
Department of Medical Biophysics, The University of Western Ontario, London, Ontario N6A 5K8, Canada
‡
§
Centre for Advanced Materials and Biomaterials Research, The University of Western Ontario, London, Ontario N6A 5B7, Canada
S Supporting Information
*
ABSTRACT: A series of structurally modified Tm3+
DOTAM-alkyl complexes as potential PARACEST MRI
contrast agents has been synthesized with the aim to decrease
the overall positive charge associated with these molecules and
increase their biocompatibility. Two types of structural
modification have been performed, an introduction of terminal
carboxylate arms to the alkyl side chains and a conjugation of
one of the alkyl side chains with aspartic acid. Detailed evaluation of the magnetic resonance imaging chemical exchange contrast
associated with the structurally modified contrast agents has been performed. In contrast to the acutely toxic Tm3 DOTAM-
alkyl complexes, the structurally modified compounds were found to be tolerated well during in vivo MRI studies in mice;
however, only the aspartic acid modified chelates produced an amide proton-based PARACEST signal.
+
INTRODUCTION
part to concomitant magnetization transfer (MT) from
■
14
endogenous macromolecules. The MT effect is a competing
mechanism that saturates the bulk water spins and
consequently lowers the contrast efficiency of PARACEST
MRI CAs within the MT frequency range (ca. −100 to 100
ppm). Several research groups including ours have investigated
the use of PARACEST MRI CAs for in vivo studies with
limited success. To enhance in vivo PARACEST contrast
detection, various pulse sequence modifications have been
proposed including a method called on resonance paramagnetic
Currently, there are several groups of magnetic resonance
1
imaging (MRI) contrast agents (CAs) available, including
3
+
2
2+
3
Gd chelates, Mn chelates, superparamagnetic iron oxide-
4
5
particles (SPIO), and hyperpolarized nuclei such as Xe.
Paramagnetic chemical exchange saturation transfer (PARAC-
6
EST) MRI CAs are complexes of multidentate ligands with
3+
3+
3+
3+
3+
3+
lanthanides (Dy , Eu , Nd , Tb , Tm , Yb ) or transition
2+
2+
2+
7
metals (Co , Fe , Ni )] that offer several advantages
compared to the aforementioned groups of MRI CAs. The
contrast associated with PARACEST MRI CAs is generated by
selective radiofrequency saturation pulses applied to water
bound to the metal center or other exchangeable protons
within the close proximity of the paramagnetic cation. These
exchangeable protons are in dynamic chemical exchange with
15
chemical exchange effects (OPARACHEE), along with the
use of different pulse sequences such as fast low angle shot
1
6
17
(
FLASH), fast imaging with steady state precession (FISP),
18
and frequency labeled exchange transfer (FLEX). Another
feasible alternative leading to in vivo contrast enhancement is to
increase the payload of PARACEST MRI CAs by utilizing
nanoparticle- or dendrimer-based CAs or discrete “small
molecule-based” targeted CAs (e.g., glucose targeted CA 1,
6
bulk water. PARACEST MRI CAs possess an excellent
19
20
environmental sensitivity and are capable of sensing important
21
8
9,10
physiological parameters (e.g., temperature, pH ), the
concentration of physiologically important cations and anions
Figure 1, detected in mouse liver). Considering the effective
range of endogenous MT effects (ca. −100 to 100 ppm), the
design of PARACEST MRI CAs with exchangeable proton
frequencies shifted near or outside of this range might offer a
solution to the in vivo sensitivity problems caused by
endogenous MT. Toward this end, Aime’s group has recently
2
+
11
(
e.g., Zn , phosphate), the concentration of primary
12
metabolites (e.g., glucose), and various enzymatic activities
1
3
(
e.g., caspase 3). Moreover the MRI contrast generated when6
using PARACEST MRI CAs can be turned on and off at will,
offering a significant advantage in comparison to the widely
3+
3+
investigated Yb -HPDO3A (2, Figure 1), which has two
used Gd -based CAs, where the contrast is always on.
Despite the many advantages listed above, the in vivo use of
PARACEST MRI CAs is hampered by severe limitations. Upon
administration to biological system, the PARACEST MRI CAs
suffer a significant sensitivity decrease attributable in a large
22
exchangeable protons at ca. 66 and 92 ppm. This CA, a
Received: April 23, 2015
Published: July 27, 2015
©
2015 American Chemical Society
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Figure 1. Structures of MRI CAs 1−6.
chemical analogue (Yb versus Gd) of the clinically approved
CA (Gadoteridol or ProHance) was recently used to map the
extracellular pH in murine melanoma. The favorable outcome
of these studies support the potential clinical translation of
Yb -HPDO3A (2) for human use. However, promising this
approach is, it would be instructive to examine lanthanides that
can produce larger hyperfine shifts to avoid overlap with the
MT frequency range.
temperature and pH mapping as shown upon injection of 3
directly into a mouse leg muscle.
10
Unfortunately, the in vivo sensitivity associated with CA 3
(amide-based CEST effect at −47 ppm) is severely attenuated
by the endogenous MT effect. We have been investigating
alternative molecular designs with the aim to increase the
chemical shift of the exchangeable amide-proton. Our efforts
3+
22
3+
led to the preparation of two CAs Tm DOTAM-n-hexyl (4a,
Figure 1) and Tm3+ DOTAM-t-butyl (4b, Figure 1) with
exchangeable proton chemical shifts (4a, −87 ppm, 6% and
−94 ppm, 17%; 4b, −68 ppm, 10% and −102 ppm, 21%)
located near the border of the endogenous MT frequency range
26
As an alternative approach to the aforementioned, we have
recently developed a dipeptide decorated PARACEST MRI CA
3+
23
Tm DOTAM-Gly-Lys-OH (3, Figure 1) and investigated its
potential in vivo application. We have detected the CA (upon
intravenous administration of 150 μL of 50 mM solution) in
26
in bovine serum albumin (BSA) phantom studies. The
presence of two exchangeable proton frequencies was attributed
to the SAP/TSAP isomerism (SAP, square antiprism; TSAP,
2
4
25
mouse kidneys and xenograft mouse brain tumors.
Moreover, 3 can also be utilized for simultaneous in vivo
6
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Scheme 1. Preparation of Electrophiles 8a−8c, 9a−9c, 12a−12c, and 13a−13c
twisted square antiprism) as previously described for other
water exchange kinetics. Conjugation of Asp-OH with CA 4c
was also performed. The overall charge of these Asp-modified
CAs was positive (+1) presuming complete ionization;
however, we have previously shown that Tm DOTAM-Gly-
Lys-OH (3, Figure 1), bearing an overall positive charge at pH
Tm3 DOTAM-derived complexes.
+
27
Although the detailed BSA phantom studies performed with
CA 4b indicated that this agent may be a good candidate for
3+
26a
measuring in vivo temperature; the results obtained after in
vivo administration were quite disappointing. Contrast agent
6
−7, is compatible with in vivo studies and can be administered
4b, which possesses a +3 overall charge, was found to be acutely
10,24,25
safely in relatively high (50 mM) concentrations.
The
toxic to mice, causing death within seconds of intravenous
administration or direct injection into leg muscle. This
observation is consistent with the results reported by other
researchers, wherein a small series of overall positively charged
structures of Asp-modified CAs 6a−6c are depicted in Figure 1.
In addition to 6a−6c, we have also prepared the conjugate 6d
because the unmodified “parent” Tm3+ DOTAM-propargyl
28
CAs was found to be acutely toxic. We decided to modify the
original chemical structure of agents 4a and 4b by introduction
of carboxylate functionality on the termini of each side chain.
This modification would produce CAs with overall negative
(
4d) exhibits a relatively strong CEST effect (16% at −54
29
ppm), comparable in magnitude to the CEST effect
associated with 3. Complex 4d also exhibits nonexchangeable
proton signals associated with the cyclen moiety whose
(
−1) charge presuming a completely ionized state. Similarly, we
3+
chemical shift respond sensitively to temperature (1.05 ppm/
have also modified the Tm DOTAM-benzyl (4c, Figure 1)
26b
1
°
C), offering the potential for H magnetic resonance
that exhibits a strong amide-based CEST effect (27% at −51
ppm). The structures of modified CAs 5a−5c are depicted in
Figure 1. As shown later, this structural modification had a
detrimental effect on chemical exchange magnetic properties
30
spectroscopy (MRS) temperature mapping.
Chemical synthesis of CAs 5a−5c and 6a−6d is described
along with detailed investigation of the chemical exchange
mediated contrast associated with these compounds. As
described below, Asp-modified CAs 6a−6d are compatible
with in vivo studies, moreover, the CEST sensitivities of 6a−6d
are similar to those of the unmodified “parent” CAs 4a−4d,
suggesting, that conjugation with Asp-OH might be used in the
future as an efficient tool to decrease the overall charge of the
CAs while maintaining desirable CEST contrast.
(no CEST effect observed for 5a−5c), presumably due to
significant changes in water exchange kinetics. Other structural
modifications have been considered and the conjugation of one
of the four side chains with L-aspartic acid (Asp-OH) presented
itself as a feasible alternative. We speculated that this remote
modification may, to a large extent, preserve the original
coordination geometry (both SAP and TSAP isomers present)
of 4a and 4b as well as cause less of a change to the favorable
6
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RESULTS AND DISCUSSION
Scheme 2. Preparation of CAs 5a−5c
■
Chemistry. The approach to CAs 5a−5c is based on the
methodology previously developed in our laboratory based on
the alkylation of cyclen or its derivatives by amino acid N-
23,26b
haloacetyl derivatives, Scheme 1.
-Aminohexanoic acid (7a, Scheme 1), 3-amino-3-methyl-
butyric acid (7b, Scheme 1), and 4-aminomethyl benzoic acid
7c, Scheme 1) were converted in quantitative yields to the
6
(
corresponding esters by treatment with SOCl in MeOH.
2
Acylation of the esters derived from acids 7a and 7b with
chloroacetyl chloride under previously established conditions
was found to be troublesome (i.e. resulting in complex mixtures
of products), therefore peptide coupling between chloroacetic
31
acid and corresponding esters was used and provided the N-
chloroacetyl derivatives 8a in 82% yield (based on 7a, Scheme
1) and 8b (57%, based on 7b).
Acylation of 4-aminomethyl benzoic acid (7c) with
chloroacetyl chloride produced the desired N-chloroacetyl
derivative 8c (Scheme 1), albeit in poor yield (28%).
Conversion 8a−8c to the N-iodoacetyl-derived electrophiles
2
3
9
a−9c via the Finkelstein reaction (NaI/acetone) proceeded
1 13
smoothly and in good yield (58−86%, Scheme 1). H and
NMR spectra associated with N-haloacetyl derivatives 8a−8c
and 9a−9c can be found in the Supporting Information. With
electrophiles 9a−9c in hand, the peralkylation of cyclen (10,
Scheme 2) was carried out, followed by treatment of the
corresponding ligands with TmCl ·H O first under slightly
C
23
3
2
basic conditions (NaOH, pH ∼ 9) to achieve the hydrolysis of
the esters and then under slightly acidic conditions (HCl, pH ∼
9
6
) to achieve the quantitative metalation. CAs 5a−5c were
obtained in reasonable yields (37−53%, based on 10, Scheme
), they were purified as described in the Experimental Section,
and were characterized by high resolution mass spectrometry
HR-MS) as shown in Experimental and in the Supporting
2
(
Information.
A slightly different methodology has been utilized to prepare
the Asp-modified CAs 6a−6c. Amino acids 7a−7c were
acylated with chloroacetyl chloride (Scheme 1) as described
3
2
in the literature. The N-chloroacetyl amino acids 11a and 11c
were obtained as colorless solids, while 11b, known to be an
oil, was obtained by solvent extraction (see Experimental
Section for details) along with a small amount of chloroacetic
acid (formed upon hydrolysis of chloroacetyl chloride), which
was ultimately removed by flash column chromatography
3
3
results of these studies were rather disappointing. The problem
36
was solved by use of tri-Boc-cyclen (14, Scheme 3).
Alkylation of 14 with electrophiles 12b, 13a, and 13c
proceeded smoothly affording the intermediates 15a−15c in
high yields (70−94%, Scheme 3). The Boc groups were
removed by trifluoroacetic acid (TFA)/triethylsilane (TES)
(
FCC). Amino acids 11a−11c were subjected to peptide
coupling with L-Asp-(OMe) ·HCl using N-hydroxysuccini-
mide/dicyclohexylcarbodiimide (NHS/DCC, 12a and 12b,
2
promoted acidolysis (Scheme 3), followed by alkylation with N-
23
26b
iodoacetyl-n-hexylamine
(to prepare the ligand 16a), N-
34
Scheme 1) or propanephosphonic acid anhydride (T3P, 12c,
Scheme 1). N-Chloroacetyl derivatives 12a−12c were prepared
in moderate yields (43−66%), and characterization data for
26b
iodoacetyl-t-butylamine (to prepare the ligand 16b), or N-
26b
iodoacetyl-benzylamine (to prepare the ligand 16c, Scheme
3
3
). Treatment of ligands 16a−16c with TmCl ·H O (Scheme
3
2
1
2a−12c can be found in the Experimental Section and
) was performed as described above, leading to CAs 6a−6c in
23
Supporting Information. Finkelstein reaction of 12a and 12c
gave the desired N-iodoacetyl derivatives 13a and 13c (Scheme
moderate yields (16−48%) and quantities sufficient for the
detailed testing of magnetic properties associated with 6a−6c.
The intermediates 15a−15c and 16a−16c were characterized
1). The same reaction was carried out with 12b, and the desired
1
N-iodoacetyl derivative 13b was detected by HR-MS analysis of
the crude reaction mixture but decomposed during FCC
purification (Scheme 1). Fortunately, the N-chloroacetyl
derivative 12b was found to be sufficiently reactive for the
subsequent step. After having prepared the electrophiles, we
first investigated the possibility of selective monoalkylation of
by H NMR spectroscopy and HR-MS, and CAs 6a−6c were
characterized by HR-MS (see Experimental Section and
Supporting Information for further details).
A different synthetic methodology has been utilized to
37
prepare the propargyl-containing CA 6d. Literature protocols
have been applied to convert but-2-yne-1,4-diol (17) to 1,4-
dichlorobut-2-yne (18, Scheme 4), followed by nucleophilic
3
5
cyclen 10 with electrophiles 12b, 13a, and 13c; however, the
6
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Scheme 3. Preparation of CAs 6a−6c
39
Scheme 4. Preparation of Intermediates 20 and 22
but-2-yne-1,4-diamine (19, Scheme 4) in 18% overall yield
based on 17). Acylation of 19 with chloroacetyl chloride
(
provided an electrophile 20 in 64% yield (Scheme 4, see
Experimental Section and Supporting Information for charac-
terization). Our initial intention was to assemble the CA 6d in
fashion similar to CAs 6a−6c. To proceed, we first removed the
Boc group from 20 (proceeded without difficulties, data not
shown), followed by treatment with racemic 2-bromosuccinic
acid methyl ester. Unfortunately, this later step proved difficult,
and we were unable to obtain the desired alkylation product in
reasonable yield. Our results indicated that an alternative
́
synthetic strategy leading to CA 6d was required. Delepine
reaction of 1,4-dichlorobut-2-yne (18, Scheme 4) with
hexamethylenetetraamine, followed by hydrolysis of the
40
quarternary ammonium salt and protection of the amine
4
1
(
4
Boc), afforded N-Boc-4-chlorobut-2-ynylamine (21, Scheme
) in 45% overall yield (based on 18). LiOH-mediated
42
alkylation of Asp(OMe) ·HCl with chloroderivative 21
2
afforded the conjugate 22 in 46% yield (Scheme 4). t-Butyl
43
cyclen-N-monoacetate (23) was alkylated with N-chloroace-
9
44
tyl-propargylamine, affording the ligand 24 in moderate yield
48%, Scheme 5). The acid labile protecting groups (Boc in 22
(
and t-butyl ester in 24) were removed by TFA-mediated
acidolysis to give intermediates 25 and 26, which were coupled
together by means of 1-benzotriazolyl-N,N,N′,N′-tetramethy-
luronium hexafluorophosphate/1-hydroxybenzotriazole
replacement of the chlorine atoms with NaN , PPh -mediated
reduction and selective Boc protection to afford mono-N-Boc
3
3
3
8
6
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Scheme 5. Preparation of CA 6d
possessing the same ligand framework as 4b and 5b have been
investigated, albeit for direct water exchange at the metal
46
center. The CEST effect due to the metal-bound water of the
3+
Eu DOTAM-t-butyl complex was found to be rather weak
due to the faster-than-optimal exchange rate between bound
and bulk pools. When carboxylate functionalities were
introduced (Eu3+ analogue of 5b), the exchange rate decreased
to a more favorable regime and the CEST effect became more
47
prominent. In our case, although we are considering the
exchange at amide protons, the change of exchange rate
precipitated by the peripheral modification and attendant
changes in solvation appeared to be detrimental, consequently,
the CAs 5a−5c did not exhibit any observable CEST effect.
To our delight, more favorable results have been obtained for
aspartic acid-modified CAs 6a−6d. CEST spectra associated
with 6a−6d have been acquired in water (37 °C, pH 7.0) as
described in the General Experimental Procedures. The results
(
Figure 2, Table 1) have been compared to those obtained for
26,29
unmodified congeners 4a−4d.
observed when comparing the CEST effects associated with
Some general trends were
4
a−4d to those associated with 6a−6d.
In general, conjugation of the Asp-OH moiety causes a slight
decrease in the chemical shift of the exchangeable proton
relative to bulk water that is set to 0 ppm. Although this
decrease is quite small for almost all CEST effects (1−4 ppm,
Table 1), it is more prominent in the case of n-hexyl decorated
CAs 4a and 6a, wherein the chemical shift of the exchangeable
proton closer to bulk water is shifted by 30 ppm upon
conjugation with Asp-OH. The chemical shift of the
exchangeable proton further from bulk water, on the other
hand, follows the general trend outlined above (3 ppm shift).
The magnitude of the CEST effect associated with Asp-OH
modified agents is also significantly reduced to ca. one-third
(
Table 1), when compared to the unmodified congener CAs
with two notable exceptions. The CEST effects (with the
chemical shift closer to bulk water) of 4a and 6a have the same
intensity (6%, Table 1). Only a small decrease of the CEST
effect associated with CA 6d is observed upon comparison with
29
the “parent” CA 4d (22% for 6d versus 25% for 4d, Table 1).
Overall, the results indicate that Asp-OH conjugates 6a and 6b
produced appreciable CEST contrast using exchangeable
protons with chemical shifts near the limit of the endogenous
MT effect. The conjugation with Asp-OH maintained the
sensitivity of propargyl-decorated CA 6d, while it was decreased
for benzyl-decorated CA 6c. As described below, CAs 6a−6d
were found to be compatible with in vivo studies, suggesting
that the conjugation with Asp-OH might be used as general
strategy for the preparation of CAs with diminished overall
charged while maintaining a degree of their desirable magnetic
properties.
4
5
(
HBTU/HOBt)-mediated peptide coupling (Scheme 5).
Ligand 27 was purified by high pressure liquid chromatography
HPLC) and was obtained in 14% yield. Treatment of 27 with
TmCl ·H O was carried out as described above, providing the
(
3
2
CA 6d in 47% yield (Scheme 5, see Experimental Section and
Supporting Information for the spectral characterization).
CEST Spectra. After having the CAs 5a−5c and 6a−6d in
hand, we acquired the corresponding CEST spectra with the
intention to compare them to the CEST spectra from the
We first attempted to
acquire the CEST spectra associated with tetracarboxylate-
Rates of Chemical Exchange. The exchange rate constant
of the amide protons (kex) with bulk water were measured by
26,29
48
unmodified “parent” CAs 4a−4d.
the Ω-plot method and are reported as residence lifetimes (τ
= 1/k ) in Table 2. The longer the residence lifetime of the
ex
modified CAs 5a−5c. Despite of varying the temperature (20−
amide proton represents slower exchange with the bulk water.
The rate of exchange is an important factor for being able to
observe a CEST signal and must satisfy the slow-to-
intermediate exchange regime under the conditions of
measurement as expressed by τ*Δω > 1, where Δω represents
the frequency difference between exchanging pools of
protons. Chelates 4a−4d showed clear CEST signals but
were acutely toxic to mice. Modification of the ligand structure
to include peripheral carboxylate groups, presumably reduced
3
7 °C), concentration (10−100 mM) and pH (phosphate
buffered saline pH 5.0−7.5), we were unable to observe any
CEST effect associated with CAs 5a−5c. A possible explanation
of this rather discouraging result is based on alteration of amide
proton exchange rate, which is presumably strongly influenced
by the change in second sphere solvation due to the presence of
terminal COOH functionalities. This notion is supported by
49
3
+
recent work from Sherry’s group, wherein Eu complexes
6
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Figure 2. CEST spectra of CAs 4a−4d (left) and 6a−6d (right). The spectra were acquired at 37 °C, saturation power 15 μT, concentration 10 mM,
pH 7.0, at 600 MHz.
the overall charge of the complexes from +3 to −1 (considering
a fully ionized species) and eliminated the acute toxicity but
also undesirably eliminated the CEST signals. Clearly, the
peripheral modification had changed the amide proton
exchange rate to outside the range that is useful for generating
a CEST signal. Because no CEST signal was observed, we could
not apply the Ω-plot method to determine the exchange rates.
However, when only one arm of the ligand was modified with
an asparate residue to produce chelates 6a−6d and reduce the
overall charge to +1 (for the fully ionized species), CEST
signals were observable and exchange rates were measured. The
exchanges rates are reported in Table 2 for each signal observed
with the exception of the weaker signals in 4a and 6b, which
could not be accurately measured. These measurements were
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Table 1. Magnitudes and Chemical Shifts (ppm) of CEST
Effects Associated with CAs 4a−4d and 6a−6d at 600 MHz
effect on the exchange of amide protons ascribed the high-
shifting isomer. Compound 4c (benzyl-type amide substituent)
shows a larger effect when compared to 6c, that is, a significant
increase in the exchange reflected by a decreased lifetime.
However, 4d (propargyl amide substituent), that like 4c shows
only a CEST effect associated with a SAP conformer, shows a
slight decrease of the exchange rate. Sherry and co-workers
have evaluated the effect of polar amide substituents on the
a
CA no.
CEST (%)
6
δ (ppm)
4a
−87
−94
−68
−102
−51
−54
−57
−91
−66
−100
−50
−49
1
7
4b
10
2
1
4
4
6
c
d
a
27
25
6
3+
water exchange of some similar Eu chelates and determined
that more polar substituents slow the exchange rate,
46
presumably by structuring second-sphere water of solvation.
5
The present case involves amide−proton exchange rather than
direct water exchange at the metal center; however, factors that
affect the rate of exchange of the bulk solvent with metal-bound
water might also reasonably be expected to affect water access
and exchange rates at the amide group in a parallel fashion, yet
this is not observed. Besides a change in the exchange rate that
could diminish the observed CEST signal intensity, compounds
6b
3
7
6
6
c
10
22
d
a
[
CA] = 10 mM, 37 °C, pH 7.0, saturation power 15 μT.
Table 2. Chemical Shift (δ), Lifetime (τ), and CEST Signal
Strength (at 18μT) for Amide Protons Assigned to Low
6
a−6d are unsymmetrical and it is a possibility that the overall
CEST signal is divided over several peaks, some that may be
too weak to observe.
Shifting (LS) or High Shifting (HS) Complexes at pH 7.0,
a
[CA] = 20 mM
Relaxation Evaluation. Another recent study by Sherry
and co-workers has shown that PARACEST CAs containing
inner sphere water can produce sizable relaxation effects
(
mainly T ), resulting in image darkening during in vivo
2
50
PARACEST imaging. To overcome this problem, we have
recently studied a small series of Tm based anilide
PARACEST MRI CAs, which do not contain inner sphere
water or the water exchange rates lie outside the optimum
exchange regime. An in vitro phantom study clearly
demonstrated, that T2 relaxation effects are negligible for
Tm3+ based anilide PARACEST MRI CAs, while significant
3
+
51
3+
image darkening is observed for Tm DOTAM-Gly-Lys-OH
(
51
3, Figure 1), known to possess inner sphere water.
Bearing this knowledge in mind, we decided to evaluate the
3+
relaxivities associated with Tm DOTAM-alkyl-based CAs 4a−
4
Table 3. Tm DOTAM complexes are known to be poor T
d, 5a−5c, and 6a−6d. The results of our studies are listed in
3+
1
Table 3. Longitudinal (r ) and Transverse (r ) Relaxivities in
1
2
−1
−1
(
s
mM ) Associated with CAs 4a−4d, 5a−5c, and 6a−6d
compd
r1
r2
4
4
4
4
a
b
c
0.04
0.01
0.05
0.05
0.06
0.02
0.01
0.05
0.03
0.02
0.06
0.74
0.15
2.28
2.43
1.26
0.16
0.42
0.26
0.28
0.53
3.46
d
a
5a
*At 18 μT.
5
5
6
6
6
6
b
c
made as detailed in the Experimental Section; however, it is key
to note that the agent concentration and saturation powers for
these measurements are different than used for Table 1, and
hence the magnitude of the CEST signal varies.
a
b
c
d
The CEST effect signals observed fall into two arbitrary
chemical shift ranges labeled as “low shift” (δ < 90 ppm), which
is characteristic of known Tm DOTAM derivatives in the
3
+
relaxation agents (low longitudinal relaxivity r ) for water; our
1
−1
square antiprismatic (SAP) conformation, and “high shift” (δ >
findings are fully consistent with this observation (r ≤ 0.06 s
1
26
3+
−1
9
0 ppm), which, by analogy, may be produced from Tm
DOTAM derivatives in the twisted square antiprismatic
TSAP) conformation. Unfortunately, a simple trend cannot
mM , Table 3). Transverse relaxivities (r ) of water for
2
chelates 4a−4d, 5a−5c, and 6a−6d range between 0.15 and
3.46 s− mM (Table 3), indicating that inner sphere water
1
−1
(
be elucidated from the data in Table 2. For instance,
introduction of a charged carboxylate group into a hydrophobic
alkyl amide (e.g., 4a, hexyl; or 4b, t-butyl) had only a small
might be present in some of these CAs (high r , short T ) while
2
2
absent or exchanging at rates unfavorable to produce T effects
2
in other members of the series (low r , long T ). The
2
2
6
523
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Journal of Medicinal Chemistry
Article
propargyl-decorated CAs 4d and 6d possess high r values,
imaging, wherein darkening of kidneys (Figure 3) was observed
20 min post injection (tail vein) despite the fact that 6a is a
2
suggesting the presence of inner sphere water, it appears to be
absent (or the water exchange rates lie outside the optimum
exchange regime) in t-butyl decorated CAs 4b, 5b, and 6b. The
difference in water exchange rates at the metal center can be
attributed to different coordination geometries; the Tm3+
center is likely significantly more accessible in the presence of
rather small propargyl groups as opposed to bulky lipophilic t-
butyl groups. In the case of hexyl- (4a, 5a and 6a) and benzyl-
decorated (4c, 5c, and 6c) complexes, the r values appear to be
2
more dependent on the overall structure of the complex as
indicated by results in Table 3. This effect is more prominent
−
1
for benzyl-decorated CAs, possessing high r value (2.28 s
2
−1
3+
mM ) for unmodified Tm DOTAM-benzyl (4c, Table 3),
while introduction of COOH functionalities or conjugation
with Asp-OH results in marked decrease of r values (5c, 0.42
2
−1
−1
−1
−1
s
mM ; 6c, 0.53 s mM ; Table 3). Overall, our results
indicate that T relaxation effects during in vivo studies should
2
be negligible for CAs 6a−6c, while significant T related signal
2
loss can be expected for propargyl-decorated conjugate 6d.
3
+
In Vivo Toxicity. As noted previously, Tm DOTAM-
alkyl-based CAs 4a−4d were found to be acutely toxic, causing
the death of laboratory mice within seconds of administration
by either tail vein or intramuscular (leg) injection of a 10 mM
solution. The toxicity was attributed to the overall 3+ charge
associated with these complexes; similar problems with
28
positively charged MRI CAs have been observed previously.
Tetracarboxylate modified CAs 5a−5c (presumed overall
charge −1) have been found to be compatible with potential in
vivo studies, as the mice injected with 50 mM (phosphate
buffered saline) solutions of 5a−5c remained alive and
appeared healthy for an extended period of time (up to 1
month). Although no imaging studies were performed with
these CAs because they did not produced measurable CEST
contrast, the ligand framework present in 5a−5c might
Figure 3. T weighted image of mouse kidneys before the injection of
2
6
a (top left) and 20 min post injection of 6a (top right). Wash-in
curves of 6a (bottom).
3
+
however serve for the preparation of Dy based T exchange
2
1
52
CAs or H MRS thermometry probes.
rather poor T exchange CA (Table 3). This result indicates
2
The conjugation of Asp-OH to 4a−4d to produce 6a−6d
eliminated the acute toxic effects associated with the former
compounds. CAs 6a−6d bear an overall charge of +1
that the CEST effects associated with 6a (6% at −57 ppm and
5% at −91 ppm, Table 1) are too weak to be directly detected
in vivo. Considering the similar intensity of CEST effects (3%
at −66 ppm and 7% at −100 ppm, Table 1), it is not surprising
that similar negative results have been observed upon
administration (tail vein) of 50 mM solution of t-butyl-
modified CA 6b in phosphate buffered saline. Higher
concentrations of 6b have not been administered due to the
limited amount of material available at the time.
(
completely ionized species) and appear to be fully compatible
with in vivo studies, suggesting that charge distribution in 6a−
6d plays a significant role. Tail vein or leg muscle injection of
50 mM solution (phosphate buffered saline) of 6a−6d was
tolerated well, and mice remained alive and appeared healthy
for a period of several weeks after administration. Herein we
suggest that conjugation with Asp-OH or related dianionic
moieties (e.g., Glu-OH) might be used as a general strategy for
the preparation of in vivo compatible CAs bearing decreased
overall positive charge.
The in vivo CEST effect associated with the benzyl-
decorated CA 6c, which displays only a modest 10% CEST
at −50 ppm in vitro, was observed when a 50 mM solution
(phosphate buffered saline) of 6c was injected into leg muscle,
Figure 4. Despite the modest intensity of the signal, this result
is a clear demonstration of the observation of a PARACEST
effect in vivo. This result also highlights the challenge of
PARACEST imaging in the presence of endogenous MT.
A similar study with CA 6d bearing propargyl side chains did
not result in the in vivo detection of CEST contrast, likely due
In Vivo Imaging. Our in vivo PARACEST imaging studies
started with CA 6a bearing hexyl side chains. Among the two
CAs (6a and 6b) possessing CEST effects around −100 ppm,
the synthesis of 6a proceeded with higher overall yield and
produced sufficient amounts of 6a to carry out more detailed
investigation. However, no in vivo CEST effect (1 h duration of
the imaging session) was observed upon tail vein injection of 50
mM solution of 6a in phosphate buffered saline. Neither
changes in administration route (leg muscle injection) nor
increased concentration (100 mM of 6a in phosphate buffered
saline, higher concentrations were not attempted due to
solubility issues) improved the outcome of this study. The
50
to the image darkening caused by T
exchange (Table 3).
2
CONCLUSIONS
■
In the present work, we have synthesized a series of
carboxylate- or aspartic acid (Asp-OH)-modified PARACEST
MRI CAs based on DOTAM-alkyl-based structures present in
4a−4d. The synthesis was performed either by applying the
presence of 6a in kidneys was confirmed by T weighted
2
6
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Journal of Medicinal Chemistry
Article
analogous manner failed, likely due to the T related image
2
50
darkening.
Herein we propose that the conjugation with Asp-OH (or
related moiety) might be used as a general strategy for
producing in vivo compatible CAs bearing decreased overall
positive charge while retaining the ability to produce CEST
contrast associated with their simpler, highly positively charged
counterparts.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Reagents were commercially
available and all solvents were HPLC grade except for water (18.2 MΩ
−
1
cm , Millipore), CH Cl , dioxane, DMF, Et O, and THF (dried by
2
2
2
passing through columns of activated Al O ). Solvents were removed
2
3
under reduced pressure in a rotary evaporator, aqueous solutions were
lyophilized, and organic extracts were dried over Na SO . Flash
2
4
column chromatography (FCC) was carried out using silica gel (SiO2),
mesh size 230−400 Å. Thin-layer chromatography (TLC) was carried
out on Al backed silica gel plate with compounds visualized by I
2
vapors, anisaldehyde stain, 5% ninhydrin stain, phosphomolybdic acid
stain, and UV light. Melting points were obtained on Fisher−Johns
apparatus and are uncorrected. Ultra performance liquid chromatog-
raphy (UPLC, Waters Acquity) was performed using a BEH C18
column (particle size 1.7 μm; 1.0 id ×100 mm) and HR-ESI-MS
detector (Waters/Micromass LCT Premier XE). Mobile phase:
method A, 100% H O−100% MeCN (both solvents containing
2
0
.1% HCOOH) over 5 min, linear gradient, flow rate 0.1 mL/min.
HPLC purification (method B) was performed using a Delta-Pak C18
3
00 Å column (particle size 15 μm; 8 mm × 100 mm radial-
compression cartridge). Mobile phase for method B was 0 min, 90%
H O−10% MeCN (both solvents containing 0.1% TFA) to 8 min,
2
4
1% H O−59% MeCN, 3 mL/min. Size exclusion chromatography
2
(
0
SEC) was carried out on Bio-Gel P2, 45−90 μm mesh resin (8 g, per
.1 mmol of compound). Ten fractions (10 mL each) were collected
3+
and identified with I vapors. Absence of free Tm was verified by
xylenol orange test. NMR spectra were recorded on a 400 MHz
spectrometer (Varian MERCURY) for H NMR spectra δ values were
2
5
3
1
recorded as follows: CDCl (7.27 ppm), DMSO-d (2.49 ppm), D O
3
6
2
(
(
(
4.75 ppm); for 13C (125 MHz) δ CDCl (77.0 ppm), DMSO-d
3
6
39.5 ppm). Mass spectra (MS) were obtained using electron impact
Figure 4. In vivo CEST signal observed for 6c intramuscular leg
injection. Top and bottom regions of interest in the mouse leg
EI, Finnigan MAT 8200) or electrospray ionization (ESI, Micromass
1
LCT). The purity of compounds (≥95%) was verified by H NMR
spectroscopy (small molecules) or UPLC (method A, ligands and
complexes).
(
circled) show a weak signal at approximately −50 ppm, indicated by
the arrowheads.
Nuclear Magnetic Resonance Experiments. Chemical ex-
change saturation transfer (CEST) spectra were acquired on a 600
MHz vertical bore NMR spectrometer (Varian INOVA). The CEST
effect was measured by recording the bulk water signal intensity as a
function of presaturation frequency. The basic pulse sequence
2
3
“
cyclen tetraalkylation” strategy (5a−5c) or by use of suitably
35
protected cyclen derivatives such as tri-Boc-cyclen (for CAs
a-6c) or t-butyl cyclen-N-monoacetate (for CA 6d).
The primary goal of this work was to modify the chemical
6
consisted of a 2 s presaturation pulse (B = 650 Hz) followed by
1
hard 20° pulse and a 1 s data acquisition. All CEST spectra were
recorded in steps of 1 ppm from −110 to +110 ppm at concentration
of 10 mM in Millipore, pH = 7.0 and at 37 °C. Each sample was pH
adjusted using either 10 mM HCl or 10 mM NaOH; the volumes of
acid or base were negligible relative to the total sample volume. T1
relaxation time constant measurements were made for four different
concentrations (1, 2, 4, 8 mM, averaged n = 3) of CA (in pH adjusted
water) using an inversion recovery sequence comprised of 12 inversion
times in the range of 10 ms to 10 s with a 20 s repetition time to
structure of acutely toxic CAs 4a−4d while retaining their
interesting and potentially useful magnetic properties. Both
chemical modifications lead to CAs, which appear to be
nontoxic and are well tolerated during in vivo studies. The
presence of terminal carboxylate groups on each arm leads to a
complete obliteration of the CEST effect associated with CAs
5
a−5c, presumably due to the unfavorable water exchange
kinetics. On the other hand, the CEST effect (CAs 6a−6d) is
partially retained upon conjugation with the Asp-OH moiety.
In vivo imaging studies were performed with CAs 6a−6d.
Although both 6a and 6b produced highly chemical shifted
exchangeable protons near the boundary of the endogneous
ensure full recovery, pH 7.0 and 37 °C. r reported are taken from the
1
slope of the linear fit. T relaxation time constant measurements were
2
made for four different concentrations (1, 2, 4, 8 mM, averaged n = 3)
of CA (in pH adjusted water) using a CPMG pulse sequence
comprised of 12 train echo times in the range of 10 ms to 10 s with a
2
6
MT effect, their in vivo detection by CEST imaging was not
possible, presumably due to low local accumulation of agent.
Benzyl-decorated Asp-OH conjugate 6c was detected (CEST
imaging) upon injection of the CA into the leg muscle. The
detection of propargyl-substituted CA 6d administered in an
2
0 s repetition time to ensure full recovery, pH 7.0 and 37 °C. r2
reported are taken from the slope of the linear fit.
All proton exchange rates were measured by using a small animal
9.4 T Agilent MRI scanner (Agilent, Santa Clara, CA) at 37 °C. Plots
of CEST effect as a function of B1 amplitude were produced by
6
525
DOI: 10.1021/acs.jmedchem.5b00621
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Journal of Medicinal Chemistry
Article
varying the amplitude (6, 9, 12, 15, and 18 μT) of a 5 s saturation
pulse that preceded a standard fast spin−echo pulse sequence (TR =
ration of eluates afforded N-chloroacetyl ω-aminocaproic acid methyl
ester (8a) and N-chloroacetyl 3-amino-3-methylbutyric acid methyl
ester (8b). A small amount of dicyclohexyl urea (DCU) eluted along
with desired products 8a and 8b; it was removed by dissolving the
separate residues in ice-cold acetone (ca. 5−10 mL) and filtering the
insoluble DCU off by using a Pasteur pipet with a cotton plug.
A suspension of the carboxylic acid ester hydrochloride derived
from 7c in dry DMF (2.5 mL) was cooled to 0 °C, followed by the
4
000 ms, echo-train length 4, effective echo time 10 ms, averages 5,
dummy scans 2, matrix 64 × 64, FOV 30 mm × 30 mm). The results
were then fitted by using Matlab, and the exchange rates were
calculated based on the linear fit parameters. These were acquired at
2
0 mM chelate concentration in 20 mM NaH PO buffer (pH 7.0, 37
2 4
°
C).
In Vivo Imaging. T *-weighted MRI images of the mouse kidneys
2
addition of Na CO (636 mg, 6 mmol). Chloroacetyl chloride (310
2 3
were acquired on a 9.4 T horizontal bore Agilent MRI scanner
equipped with a 30 mm millipede radio frequency coil (Agilent, Santa
Clara, CA). Images were acquired using a two-dimensional fast low-
μL, 3.9 mmol) was added dropwise (over ca. 1 min), the cooling bath
was removed, and the mixture was stirred for 18 h at RT. The mixture
was taken up into brine (50 mL) and was extracted with EtOAc (30
mL + 20 mL), and the combined organic extract was consecutively
2
angle shot (FLASH) pulse sequence (field of view 32.0 × 32.0 mm ,
data matrix 128 × 128, pulse repetition time (TR) 40 ms, echo time
washed with saturated NaHCO solution (2 × 50 mL) and brine (2 ×
3
(
TE) = 8 ms, slice thickness = 1 mm). The contrast agent, 200 μL, 2
5
3
0 mL) and was concentrated. The residue was subjected to FCC on
mg, was injected over 5 min (40 uL/min) into the tail vein. Imaging
began 3 min after injection.
0 g of SiO eluting 8c with hexanes/acetone (2:1), concentration of
2
eluate resulted in crystallization, the mixture was diluted with hexanes,
and was set aside for 1 h at −10 °C. Separated crystals were filtered off,
washed with hexanes, and dried to leave N-chloroacetyl p-amino-
methylbenzoic acid methyl ester (8c).
In vivo CEST imaging was performed on an Agilent (Santa Clara,
CA, USA) 9.4 T MRI scanner equipped with a 3 cm diameter custom-
built radio frequency surface coil. A C57BL/6 mouse (3 months of
age, weighing 25 g) was anesthetized (induced using 4% isoflurane in
oxygen and maintained using 1.5%−2.5% isoflurane in oxygen). The
mouse was secured on a magnetic resonance imaging (MRI)-
compatible stage with a rectal temperature probe and a respiratory
sensor pad for monitoring during MRI. Body temperature was
maintained at 37 °C during the imaging procedure by blowing warm
air over the animal using a model 1025 small-animal monitoring and
gating system (SA Instruments Inc., Stony Brook, NY). Approximately
N-Chloroacetyl ω-Aminocaproic Acid Methyl Ester (8a, 545 mg,
1
8
4
1
4
2%). Colorless solid. H NMR (CDCl ) δ 6.65 (s, D O exch, 1H),
3
2
.05 (s, 2H), 3.66 (s, 3H), 3.31 (m, 2H), 2.32 (m, 2H), 1.65 (m, 2H),
13
.55 (m, 2H), 1.36 (m, 2H). C NMR (CDCl ) δ 173.9, 166.0, 51.5,
3
2.6, 39.6, 33.8, 28.9, 26.2, 24.4. HRMS (EI) m/z; found 221.0815
+
[
M] (calcd 221.0819 for C H ClNO ). LRMS (EI) m/z (rel
9
16
3
+
abundance): 221 [M ] (5), 186 (100), 172 (18), 144 (45).
N-Chloroacetyl 3-Amino-3-methylbutyric Acid Methyl Ester (8b,
15 min before acquisition of CEST images, 50 μL of 100 mM 6c
1
3
1
55 mg, 57%). Colorless oil. H NMR (CDCl ) δ 7.01 (s, D O exch,
3
2
dissolved in phosphate buffered saline was injected intramuscularly (at
a depth of 2 mm) in the left leg. Injection was performed at a rate of
13
H), 3.95 (s, 2H), 3.68 (s, 3H), 2.73 (s, 2H), 1.46 (s, 6H). C NMR
(
CDCl ) δ 171.4, 165.4, 52.5, 51.6, 43.9, 42.8, 26.7. HRMS (EI) m/z;
3
25.0 μL/min using a 27-gauge needle and a syringe pump (Harvard
+
found 207.0655 [M] (calcd 207.0662 for C H ClNO ). LRMS (EI)
m/z (rel abundance): 207 [M ] (14), 192 (13), 176 (24), 160 (60),
8
14
3
Apparatus, Holliston, MA). The mouse was recovered following
imaging. The animal procedure was performed according to a protocol
approved by the Western University Animal Use Subcommittee.
CEST images were acquired using a 2 mm thick single slice positioned
+
1
34 (82), 114 (100), 83 (68).
N-Chloroacetyl p-Aminomethylbenzoic Acid Methyl Ester (8c,
1
2
8
1
06 mg, 28%). Colorless crystals; mp 90−92 °C. H NMR (CDCl ) δ
3
through the injection site that was identified using T -weighted scout
2
.01 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.03 (s, D O exch,
2
images. For in vivo CEST imaging, a standard fast spin echo pulse
13
H), 4.55 (d, J = 6.0 Hz, 2H), 4.12 (s, 2H), 3.91 (s, 3H). C NMR
2
sequence was used (field of view = 19.2 × 19.2 mm , matrix = 64 × 64,
(
CDCl ) δ 166.7, 166.0, 142.4, 130.0, 129.5, 127.5, 52.1, 43.4, 42.5.
3
repetition time (TR) = 5000 ms, echo train length (ETL) = 32,
effective echo time = 10 ms, 2 pre scans and 1 average) preceded by a
continuous wave presaturation pulse (amplitude = 14 μT, duration =
+
HRMS (ESI) m/z: found 242.0572 [M + H] (calcd 242.0584 for
C H ClNO ).
11
13
3
Acylation of Carboxylic Acids 7a−7c with Chloroacetyl
5.0 s). CEST spectra were acquired using saturation frequency offsets
Chloride. Carboxylic acids 7a (1.31 g, 10 mmol), 7b (117 mg, 1
mmol), and 7c (1.51 g, 10 mmol) were dissolved in separate solutions
of NaOH (800 mg, 20 mmol, acids 7a and 7c; 80 mg, 2 mmol, acid
−
1000 ppm, −65 to −30 ppm with 1 ppm increments along with −1,
−
0.5 to +0.5 ppm with 0.1 ppm increments, + 35 to +65 ppm with 1
ppm increments and +1000 ppm) in ascending order. In vivo CEST
7
b) in water (7a, 5 mL; 7b, 500 μL; 7c, 10 mL). Resulting solutions
spectra were B -corrected on a pixel-by-pixel basis by first interpolating
0
were cooled to 0 °C, followed by a dropwise addition (over ca. 5 min)
of the solution of chloroacetyl chloride (950 μL, 12 mmol, acids 7a
the water peak to 1 Hz resolution, then fitting it to a twelfth-order
polynomial. Water resonance (0 ppm) was defined at the frequency
where the interpolated fitted polynomial was lowest.
and 7c; 95 μL, 1.2 mmol, acid 7b) in dry Et O (2.5 mL, acids 7a and
2
7
c; 250 μL, acid 7b). The mixtures were stirred for 10 min at 0 °C and
Esterification of Carboxylic Acids 7a−7c and Subsequent
Reactions with Chloroacetic Acid or Chloroacetyl Chloride.
Separate solutions of carboxylic acids 7a−7c (7a, 394 mg; 7b, 352 mg;
4
0 min at RT. The mixtures were cooled to 0 °C, the pH was adjusted
to ca. 2−3 (1 M HCl), and Et O was evaporated. N-Chloroacetyl ω-
2
aminocaproic acid (11a) and N-chloroacetyl p-aminomethylbenzoic
acid (11c) precipitated upon acidification; mixtures were set aside for
7
c, 453 mg; 3 mmol each) in MeOH (3 mL) were cooled to 0 °C,
followed by a dropwise addition (over ca. 1 min) of SOCl (265 μL,
2
1
h at 0 °C, separated precipitates were filtered off with suction,
washed with water, and dried. The mixture containing N-chloroacetyl
-amino-3-methylbutyric acid (11b) was extracted with Et O (4 × 5
3
.6 mmol). The cooling baths were removed, and the mixtures were
stirred for 24 h at room temperature (RT). Volatiles were evaporated
3
to leave corresponding carboxylic acid esters as hydrochloride salts
2
mL), and the combined organic extract was dried and concentrated.
(
quantitative yields). Chloroacetic acid (285 mg, 3 mmol) was
The residue was subjected to FCC on 15 g SiO , eluting with hexanes/
dissolved in dry CH Cl (15 mL), NHS (345 mg, 3 mmol) was added,
2
2
2
acetone (5:1). Evaporation of the eluate afforded N-chloroacetyl 3-
and the mixture was cooled to 0 °C. DCC (743 mg, 3.6 mmol) was
added, the cooling bath was removed, and the stirring continued for 24
h at RT. The solvent was evaporated; the solid residue was treated
separately with solutions (in dry THF, 15 mL) of carboxylic acid ester
amino-3-methylbutyric acid (11b).
3
2
N-Chloroacetyl ω-Aminocaproic Acid (11a, 995 mg, 48%).
1
Colorless solid. H NMR (DMSO-d
(s, D O exch, 1H), 4.02 (s, 2H), 3.06 (m, 2H), 2.19 (m, 2H), 1.48 (m,
2H), 1.40 (m, 2H), 1.25 (m, 2H). C NMR (DMSO-d
165.9, 42.8, 38.9, 33.7, 28.7, 26.0, 24.3. HRMS (EI) m/z; found
) δ 12.02 (s, D O exch, 1H), 8.20
6
2
hydrochlorides derived from 7a and 7b. Et N (840 μL, 6 mmol) was
2
3
13
added, and the mixtures were stirred for 18 h at RT. Resulting reaction
mixtures were diluted with 1 M HCl (30 mL) and were extracted with
EtOAc (3 × 20 mL). Combined organic extracts were concentrated;
) δ 174.5,
6
+
207.0659 [M] (calcd 207.0662 for C H14ClNO ). LRMS (EI) m/z
8 3
+
the residues were subjected to FCC on 40 g of SiO eluting 8a with
(rel abundance): 207 [M ] (5), 172 (100), 140 (34), 112 (35), 69
(29).
2
hexanes/acetone (2:1) and 8b with hexanes/acetone (3:1). Evapo-
6
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DOI: 10.1021/acs.jmedchem.5b00621
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Journal of Medicinal Chemistry
Article
N-Chloroacetyl 3-Amino-3-methylbutyric Acid (11b, 76 mg,
N-Chloroacetyl p-Aminomethylbenzoic Acid-Asp-(OMe) (12 c,
2
33
1
1
3
9%). Colorless oil. H NMR (DMSO-d ) δ 12.35 (s, D O exch,
438 mg, 59%). Colorless solid. H NMR (CDCl ) δ 7.76 (d, J = 8.5
6
2
3
1
H), 7.93 (s, D O exch, 1H), 3.97 (s, 2H), 2.66 (s, 2H), 1.33 (s, 6H).
Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.03 (d, D O exch, J = 8.0 Hz, 1H),
2
2
1
3
C NMR (DMSO-d ) δ 172.1, 165.2, 51.5, 43.3, 42.9, 26.5. HRMS
7.10 (s, D
O exch, 1H), 5.03 (m, 1H), 4.53 (d, J = 6.0 Hz, 2H), 4.10
2
6
+
(
EI) m/z; found 193.0507 [M] (calcd 193.0506 for C H ClNO ).
(s, 2H), 3.78 (s, 3H), 3.69 (s, 3H), 3.13 (dd, J = 17.0, 4.5 Hz, 1H),
7
12
3
+
13
LRMS (EI) m/z (rel abundance): 193 [M ] (30), 178 (21), 160 (65),
2.97 (dd, J = 17.0, 4.5 Hz, 1H). C NMR (CDCl ) δ 171.6, 171.1,
3
134 (91), 94 (100).
166.4, 166.0, 141.5, 132.9, 127.7, 127.6, 52.9, 52.1, 48.8, 43.3, 42.5,
+
N-Chloroacetyl p-Aminomethylbenzoic Acid (11c, 1.46 g, 64%).
35.9. HRMS (ESI) m/z: found 371.1011 [M + H] (calcd 371.1010
1
for C H ClN O ).
Colorless solid. H NMR (DMSO-d ) δ 11.78 (s, D O exch, 1H), 8.93
16 20
2
6
6
2
Finkelstein Reaction of N-Chloroacetyl Derivatives 8a−8c,
12a and 12c. NaI (450 mg, 3 mmol) was added to separate solutions
of N-chloroacetyl derivatives (8a, 222 mg; 8b, 207 mg; 8c, 242 mg;
12a, 351 mg; 12c, 371 mg; 1 mmol each) in acetone (8 mL, 8a−8c,
12a; 15 mL, 12c). The mixtures were stirred for 18 h at RT, the
acetone was evaporated, and separate residues were partitioned
between 10% solution of Na SO (40 mL) and EtOAc (2 × 30 mL).
(
t, D O exch, J = 6.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0
2
13
Hz, 2H), 4.38 (d, J = 6.0 Hz, 2H), 4.16 (s, 2H). C NMR (DMSO-d6)
δ 167.3, 166.3, 144.0, 129.7, 129.5, 127.3, 42.7, 42.3. HRMS (EI) m/z;
found 227.0345 [M] (calcd 227.0349 for C H ClNO ). LRMS (EI)
m/z (rel abundance): 227 [M ] (24), 192 (100), 151 (22), 135 (26),
1
+
10
10
3
+
07 (27).
Conjugation of Carboxylic Acids 11a−11c with L-Asp-
2
3
Combined organic extracts were dried and concentrated. The residues
(
OMe) ·HCl. NHS (136 mg, 1.18 mmol, acid 11a; 62 mg, 0.54
2
were either subjected to FCC on 30 g of SiO , eluting 9a with
mmol, acid 11b) was added to separate stirred solutions of N-
chloroacetyl ω-aminocaproic acid (11a, 246 mg, 1.18 mmol) and N-
chloroacetyl 3-amino-3-methylbutyric acid (11b, 105 mg, 0.54 mmol)
in dry THF (10 mL, acid 11a; 4 mL, acid 11b). The solutions were
cooled to 0 °C, followed by the addition of DCC (317 mg, 1.54 mmol,
acid 11a; 145 mg, 0.7 mmol, acid 11b) and subsequent stirring for 20
2
hexanes/acetone (5:1), eluting 9b with hexanes/acetone (2:1), eluting
1
2a with CH Cl /MeOH (98:2), or crystallized from acetone/hexanes
2 2
(
9c) or CH Cl /hexanes (12c). Evaporation of eluates afforded N-
2 2
iodoacetyl ω-aminocaproic acid methyl ester (9a), N-iodoacetyl 3-
amino-3-methylbutyric acid methyl ester (9b), and N-iodoacetyl ω-
aminocaproic acid-Asp-(OMe) (13a). The crystallizations were set
min at 0 °C. L-Asp-(OMe) ·HCl (246 mg, 1.24 mmol, acid 11a; 107
mg, 0.54 mmol, acid 11b) and Et N (330 μL, 2.36 mmol, acid 11a;
1
removed, and the mixtures were stirred for 18 h at RT. The mixtures
were diluted with CH Cl (50 mL, acid 11a; 20 mL, acid 11b) and
were washed with 1 M HCl (30 mL, acid 11a; 20 mL, acid 11b). The
aqueous phases were extracted with an additional amount of CH Cl2
2
2
aside for 3 h at −10 °C, and the crystals were filtered off, washed with
hexanes, and dried to leave N-iodoacetyl p-aminomethylbenzoic acid
methyl ester (9c) and N-iodoacetyl p-aminomethylbenzoic acid-Asp-
3
50 μL, 1.08 mmol, acid 11b) were added, the cooling baths were
(
OMe) (13c).
2
2
2
N-Iodoacetyl ω-Aminocaproic Acid Methyl Ester (9a, 241 mg,
1
7
3
1
4
7%). Colorless solid. H NMR (CDCl ) δ 6.17 (s, D O exch, 1H),
3
2
2
.70 (s, 2H), 3.68 (s, 3H), 3.28 (m, 2H), 2.33 (m, 2H), 1.66 (m, 2H),
(30 mL, acid 11a; 20 mL, acid 11b), and the combined organic
.55 (m, 2H), 1.38 (m, 2H). 13C NMR (CDCl ) δ 174.0, 167.0, 51.5,
3
extracts were dried and concentrated. The residues were subjected to
FCC on 30 g of SiO eluting 12a and 12b with CH Cl /MeOH
(
aminocaproic acid-Asp-(OMe) (12a) and N-chloroacetyl 3-amino-3-
methylbutyric acid-Asp-(OMe) (12b) containing small amount of
DCU, which was removed by dissolving the separate residues in ice-
cold acetone (ca. 5−10 mL) and filtering the insoluble DCU off by
using a Pasteur pipet with a cotton plug.
0.0, 33.7, 28.8, 26.1, 24.3, −0.4. HRMS (EI) m/z; found 313.0174
2
2
2
+
[
M] (calcd 313.0175 for C H INO ). LRMS (EI) m/z (rel
9
16
3
95:5). Evaporation of the eluates afforded N-chloroacetyl ω-
+
abundance): 313 [M ] (5), 240 (20), 112 (72), 72 (27).
2
N-Iodoacetyl 3-Amino-3-methylbutyric Acid Methyl Ester (9b,
2
1
2
1
56 mg, 86%). Colorless oil. H NMR (CDCl ) δ 6.43 (s, D O exch,
3
2
13
H), 3.71 (s, 3H), 3.64 (s, 2H), 2.73 (s, 2H), 1.45 (s, 6H). C NMR
(
CDCl ) δ 171.6, 166.4, 52.7, 51.7, 43.7, 26.6, −0.6. HRMS (EI) m/z;
3
+
found 299.0022 [M] (calcd 299.0018 for C H INO ). LRMS (EI)
m/z (rel abundance): 299 [M ] (47), 114 (100), 73 (38).
8
14
3
A stirred solution of N-chloroacetyl p-aminomethylbenzoic acid
+
(11c, 455 mg, 2 mmol) and L-Asp-(OMe) ·HCl (395 mg, 2 mmol) in
2
N-Iodoacetyl p-Aminomethylbenzoic Acid Methyl Ester (9c, 193
dry DMF (2 mL) was cooled to 0 °C, followed by the addition of N-
methylmorpholine (NMM, 990 μL, 9 mmol). A commercial solution
of T P in DMF (1.75 mL, 3 mmol) was the added dropwise over ca. 5
min. The mixture was stirred for 1 h at 0 °C and for 18 h at RT. The
mixture was then diluted with saturated NaHCO solution (50 mL)
and was extracted with EtOAc (3 × 30 mL). Combined organic extract
was washed with brine (4 × 50 mL), dried, and concentrated. The
residue was subjected to FCC on 40 g of SiO , eluting 12c with
CH Cl /MeOH (98:2) later replaced with CH Cl /MeOH (95:5).
1
mg, 58%). Colorless crystals; mp 142−144 °C. H NMR (CDCl ) δ
3
7
.97 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 6.78 (s, D O exch,
1H), 4.48 (d, J = 6.0 Hz, 2H), 3.90 (s, 3H), 3.74 (s, 2H). C NMR
2
13
3
(
CDCl ) δ 167.3, 166.7, 142.8, 130.0, 129.4, 127.4, 52.1, 43.9, −1.0.
3
+
3
HRMS (ESI) m/z: found 333.9952 [M + H] (calcd 333.9940 for
C H INO ).
11
13
3
N-Iodoacetyl ω-Aminocaproic Acid-Asp-(OMe) (13a, 252 mg,
2
1
2
57%). Colorless solid. H NMR (CDCl ) δ 6.66 (s, D O exch, 1H),
3 2
2
2
2
2
6.62 (d, D O exch, J = 11.5 Hz, 1H), 4.86 (m, 1H), 3.74 (s, 3H), 3.69
2
Evaporation of the eluate afforded N-chloroacetyl p-aminomethylben-
(s, 2H), 3.68 (s, 3H), 3.25 (m, 2H), 3.01 (dd, J = 17.0, 4.5 Hz, 1H),
2.84 (dd, J = 17.0, 4.5 Hz, 1H), 2.24 (m, 2H), 1.65 (m, 2H), 1.53 (m,
zoic acid-Asp-(OMe) (12c).
2
2H), 1.36 (m, 2H). 13C NMR (CDCl ) δ 172.7, 171.5, 171.2, 167.2,
N-Chloroacetyl ω-Aminocaproic Acid-Asp-(OMe) (12a, 276 mg,
2
3
1
6
6%). Colorless solid. H NMR (CDCl ) δ 6.68 (s, D O exch, 1H),
52.8, 52.0, 48.3, 39.9, 36.0, 35.9, 28.5, 25.9, 24.7, −0.4. HRMS (ESI)
3
2
+
6
.53 (d, D O exch, J = 8.0 Hz, 1H), 4.85 (m, 1H), 4.03 (s, 2H), 3.74
m/z: found 443.0664 [M + H] (calcd 443.0679 for C H IN O ).
2
14 24
2
6
(
s, 3H), 3.68 (s, 3H), 3.29 (m, 2H), 3.02 (dd, J = 17.0, 4.5 Hz, 1H),
N-Iodoacetyl p-Aminomethylbenzoic Acid-Asp-(OMe) (13 c, 384
2
1
2
2
5
.83 (dd, J = 17.0, 4.5 Hz, 1H), 2.23 (m, 2H), 1.65 (m, 2H), 1.55 (m,
mg, 83%). Colorless crystals; mp 127−129 °C. H NMR (CDCl ) δ
3
H), 1.36 (m, 2H). 13C NMR (CDCl ) δ 172.5, 171.5, 171.2, 165.8,
7.70 (d, J = 8.0 Hz, 2H), 7.32 (s, D O exch, 1H), 7.29 (d, J = 8.5 Hz,
3
2
2.7, 52.0, 48.3, 42.6, 39.5, 36.0 (2 × C), 28.9, 26.1, 24.8. HRMS (ESI)
2H), 7.06 (t, D O exch, J = 6.0 Hz, 1H), 5.04 (m, 1H), 4.46 (d, J = 6.0
2
+
m/z: found 351.1321 [M + H] (calcd 351.1323 for C H ClN O ).
Hz, 2H), 3.79 (s, 3H), 3.75 (s, 2H), 3.70 (s, 3H), 3.13 (dd, J = 17.5,
14
24
2
6
4.5 Hz, 1H), 2.97 (dd, J = 17.5, 4.5 Hz, 1H). 13C NMR (CDCl ) δ
N-Chloroacetyl 3-Amino-3-methylbutyric Acid-Asp-(OMe) (12b,
2
3
1
7
1
1
9 mg, 43%). Colorless oil. H NMR (CDCl ) δ 7.20 (s, D O exch,
171.6, 171.1, 167.5, 166.7, 141.9, 132.6, 127.6, 127.5, 52.9, 52.1, 48.9,
3
2
+
H), 6.70 (d, D O exch, J = 8.0 Hz, 1H), 4.87 (m, 1H), 4.07 (d, J =
43.6, 36.0, −0.9. HRMS (ESI) m/z: found 463.0360 [M + H] (calcd
2
5.0, 1H), 3.97 (d, J = 15.0, 1H), 3.76 (s, 3H), 3.70 (s, 3H), 3.04 (dd, J
463.0366 for C H IN O ).
16
20
2
6
=
17.0, 4.5 Hz, 1H), 2.82 (dd, J = 17.0, 4.5 Hz, 1H), 2.77 (d, J = 13.5,
N-Chloroacetyl-N′-Boc 1,4-Diaminobut-2-yne (20). A solution
13
1H), 2.55 (d, J = 13.5, 1H), 1.49 (s, 3H), 1.47 (s, 3H). C NMR
of but-2-yne-1,4-diol (17, 6.5 g, 75.5 mmol) in pyridine (11 mL) was
(
3
CDCl ) δ 171.4, 170.9, 170.2, 166.2, 53.2, 52.8, 52.1, 48.2, 46.0, 43.0,
5.9, 27.1 (2 × C). HRMS (ESI) m/z: found 337.1155 [M + H]
cooled to 0 °C, followed by a dropwise addition (over 1 h) of SOCl
(13.2 mL, 181.2 mmol). The cooling bath was removed, and the
3
2
+
37
(
calcd 337.1166 for C H ClN O ).
stirring continued for 2 h at RT. The mixture was then cooled to 0 °C
13
22
2
6
6
527
DOI: 10.1021/acs.jmedchem.5b00621
J. Med. Chem. 2015, 58, 6516−6532

Journal of Medicinal Chemistry
Article
and was slowly quenched by addition of water (30 mL). The organic
concentrated. The residue was subjected to FCC on 30 g of SiO2
eluting 22 with hexanes/acetone (2:1). N-Succinyl-N′-Boc 1,4-
diaminobut-2-yne dimethyl ester (22, 152 mg, 46%): Colorless oil.
layer was separated, and the aqueous layer was extracted with CH Cl2
2
(
10 mL). Combined organic extract was successively washed with 10%
1
H SO solution, saturated NaHCO solution, and water (30 mL each),
H NMR (CDCl ) δ 4.76 (s, D O exch, 1H), 3.92 (m, 2H), 3.78 (m,
2
4
3
3
2
dried, and concentrated to leave 1,4-dichlorobut-2-yne (18, 5.23 g,
6%) as a pale-brown oil of sufficient purity to be used for the next
step.
1H), 3.76 (s, 3H), 3.70 (s, 3H), 3.47 (m, 2H), 2.77 (dd, J = 16.0, 5.5
5
Hz, 1H), 2.72 (dd, J = 16.0, 6.5 Hz, 1H), 2.02 (s, D O exch, 1H), 1.44
2
(s, 9H). 13C NMR (CDCl ) δ 173.6, 171.2, 155.2, 80.3, 80.0, 79.9,
3
A solution of NaN (517 mg, 7.95 mmol) in water (10 mL) was
56.2, 52.3, 51.9, 37.5, 37.1, 30.6, 28.3. HRMS (ESI) m/z: found
3
+
added to a solution of 1,4-dichlorobut-2-yne (18, 468 mg, 3.81 mmol)
329.1723 [M + H] (calcd 329.1713 for C H N O ).
15
25
2
6
38
in THF (10 mL). The mixture was stirred for 24 h at RT. Pulverized
pestle and mortar) PPh (2.1 g, 7.96 mmol) was added, and the
Tetraalkylation of Cyclen with Electrophiles 9a−9c and
(
Subsequent Metalation with TmCl ·H O. Ethyldiisopropylamine
3
3
2
38
stirring continued for further 24 h at RT. The pH of the reaction
(DIPEA, 90 μL, 0.5 mmol) was added to separate solutions of cyclen
(22 mg, 0.125 mmol) in MeCN (2 mL). Electrophiles (0.5 mmol
each) 9a (157 mg), 9b (150 mg), and 9c (165 mg) were added, and
the mixtures were allowed to stir for 18 h at 50 °C (electrophiles 9a
and 9b) or 60 °C (electrophile 9c). The mixtures were cooled to RT,
diluted with water (30 mL), and extracted with EtOAc (30 + 20 mL).
Combined organic extracts were dried, concentrated, and residues
triturated in hexanes to provide the ligands of sufficient purity for the
subsequent steps in nearly quantitative yields.
mixture was adjusted to ca. 10 (2.5 M NaOH solution), and a solution
of Boc O (277 mg, 1.27 mmol) in MeOH (3 mL) was added dropwise
2
39
over 15 min. The pH 10 was maintained during the addition and
subsequent stirring for 30 min at RT. THF was evaporated; the residue
was diluted with brine (30 mL) and was extracted with CH Cl (3 ×
2
2
2
0 mL). Combined organic extract was dried and was concentrated;
the residue was subjected to FCC on 40 g of SiO , eluting with
2
CH Cl /MeOH/NH OH (90:10:1). Evaporation of the eluate
2
2
4
Methyl DOTAM-ω-Aminocaproate. Pale-yellow solid. 1H NMR
(CDCl ) δ 7.47 (m, D O exch, 4H), 3.61 (m, 20H), 3.23−2.88 (br m,
afforded N-Boc-1,4-diaminobut-2-yne (19, 42 mg, 18% based on 18
and 0.33 eq of Boc O used). Na CO (48 mg, 0.46 mmol) was added
2
2
3
3
2
to a solution of N-Boc-1,4-diaminobut-2-yne (19, 42 mg, 0.23 mmol)
in dry DMF (1 mL). Chloroacetyl chloride (24 μL, 0.3 mmol) was
added, and the mixture was stirred for 3 h at RT. It was then diluted
with brine (30 mL) and was extracted with EtOAc (20 mL + 2 × 10
mL). Combined organic extract was washed with brine (2 × 40 mL),
dried, and concentrated. The residue was subjected to FCC on 15 g of
24 H), 2.30 (m, 8H), 1.61 (m, 8H), 1.53 (m, 8H), 1.33 (m, 8H).
+
HRMS (ESI) m/z: found 913.5982 [M + H] (calcd 913.5974 for
C H N O ).
44
81
8
12
1
Methyl DOTAM-3-amino-3-methylbutyrate. Orange oil. H NMR
(DMSO-d ) δ 6.94 (m, D O exch, 3H), 3.67 (m, 12H), 3.42−2.72 (br
6
2
m, 32 H), 1.44 (m, 24H). HRMS (ESI) m/z: found 857.5380 [M +
+
SiO , eluting 20 with hexanes/acetone (2:1). Evaporation of the eluate
H] (calcd 857.5348 for C H N O ).
2
40 73
8
12
Methyl DOTAM-p-aminomethylbenzoate. Colorless solid. 1H
NMR (CDCl ) δ 7.90 (m, 8H), 7.54 (m, D O exch, 4H), 7.20 (m,
afforded N-chloroacetyl-N′-Boc 1,4-diaminobut-2-yne (20, 38 mg,
1
6
4%). Colorless oil. H NMR (DMSO-d ) δ 8.65 (t, D O exch, J = 4.5
6
2
3
2
Hz, 1H), 7.25 (t, D O exch, J = 5.5 Hz, 1H), 4.07 (s, 2H), 3.90 (m,
8H), 4.29 (m, 8H), 3.88 (s, 12 H), 2.97 (m, 8 H), 2.53−2.34 (br m,
2
13
+
2
1
2
H), 3.73 (m, 2H), 1.37 (s, 9H). C NMR (DMSO-d ) δ 165.6,
16H). HRMS (ESI) m/z: found 993.4716 [M + H] (calcd 993.4722
6
55.2, 79.9, 78.2, 78.0, 42.4, 29.4, 28.5, 28.2. HRMS (ESI) m/z: found
for C H N O ).
52
65
8
12
+
83.0818 [M + Na] (calcd 283.0825 for C H ClN O Na).
TmCl ·H O (40 mg, 0.15 mmol) was added to separate suspensions
11
17
2
3
3 2
N-Succinyl-N′-Boc 1,4-Diaminobut-2-yne Dimethyl Ester
22). A solution of 1,4-dichlorobut-2-yne (18, 2.98 g, 24.2 mmol)
and hexamethylenetetramine (3.39 g, 24.2 mmol) in CHCl (25 mL)
of above-mentioned ligands (0.12 mmol each) in water (2 mL). The
pH was adjusted and maintained at ca. 10 (1 M NaOH), while the
mixtures were stirred for 18 h at RT. The complete hydrolysis of ester
functionalities was confirmed by HR-ESI-MS. In the case of methyl
DOTAM-p-aminomethylphenyl tetracarboxylate, the hydrolysis of
ester functionalities was found incomplete, therefore the mixture was
stirred for additional 6 h at 60 °C.
(
3
40
was stirred under reflux for 6 h. The mixture was cooled to RT,
separated precipitate was filtered off with suction, washed with CHCl3,
and dried to leave 6.04 g (95%) of the corresponding quaternary
ammonium salt. The salt (6.01 g, 22.85 mmol) was suspened in EtOH
(
46 mL), conc HCl (8 mL) was added, and mixture was stirred under
DOTAM-ω-aminocaproic Acid. HRMS (ESI) m/z: found 857.5380
40
reflux for 30 min and at RT for 18 h. Separated precipitate (NH Cl)
+
4
[M + H] (calcd 857.5348 for C H N O ).
40
73
8
12
was filtered off with suction and washed with EtOH, and the filtrate
was concentrated and redissolved in 20 mL of EtOH. The solution was
DOTAM-3-amino-3-methylbutyric Acid. HRMS (ESI) m/z: found
+
8
01.4745 [M + H] (calcd 801.4722 for C H N O ).
36
65
8
12
set aside for 2 h at 3 °C; an additional amount of NH Cl was filtered
4
DOTAM-p-aminomethylbenzoic Acid. HRMS (ESI) m/z: found
off with suction and was washed with EtOH. The filtrate was
concentrated, redissolved in water, and lyophilized to leave 1-chloro-4-
aminobut-2-yne hydrochloride (2.93 g, 86% based on 18) as a yellow
solid. A 10 M solution of NaOH in water (380 μL, 3.82 mmol) was
added to a solution of 1-chloro-4-aminobut-2-yne hydrochloride (535
+
9
37.4078 [M + H] (calcd 937.4096 for C H N O ).
48
57
8
12
The pH was then adjusted to ca. 5.5 (1 M HCl), and the stirring of
the mixtures continued for further 18 h at RT or at 60 °C (DOTAM-
p-aminomethylphenyl tetracarboxylate). The mixtures containing the
complexes derived from DOTAM-n-hexyl tetracarboxylate and
DOTAM-t-butyl teracarboxylate were subjected to SEC as described
in General Experimental Procedures. The fractions containing the
products were lyophilized to leave the complexes 5a and 5b. The
mixture containing the complex derived from DOTAM-p-amino-
methylphenyl tetracarboxylate was cooled to 0 °C and was left at 0 °C
for 1 h. Resulting solid (complex 5c) was filtered off with suction,
washed with water, and dried.
mg, 3.82 mmol) in water (6 mL). A solution of Boc O (917 mg, 4.2
2
mmol) in dioxane (12 mL) was added, and the mixture was stirred for
41
2
h at RT. Dioxane was evaporated; the residue was diluted with
water (20 mL) and extracted with EtOAc (2 × 20 mL). Combined
organic extract was dried and concentrated. The residue was subjected
to FCC on 25 g of SiO2 eluting with hexanes/acetone (5:1).
Evaporation of the eluate afforded the product containing a small
amount (ca. 10% of Boc O); this was removed by repeating the FCC
3+
2
Tm DOTAM-ω-aminocaproic Acid (5a, 68 mg, 53%, Based on
purification under identical conditions. 1-Chloro-4-N-Boc-aminobut-2-
1
0). Yellow waxy solid. HRMS (ESI) m/z: found 1023.4425 [M −
1
+
yne (21, 426 mg, 55%). Colorless solid. H NMR (CDCl ) δ 4.78 (s,
2
3
2H] (calcd 1023.4455 for C H N O Tm).
40 70 8 12
13
3+
D O exch, 1H), 4.13 (m, 2H), 3.97 (m, 2H), 1.44 (s, 9H). C NMR
Tm DOTAM-3-amino-3-methylbutyric Acid (5b, 45 mg, 37%,
Based on 10. Yellow waxy solid. HRMS (ESI) m/z: found 967.3815
[M − 2H] (calcd 967.3829 for C H N O Tm).
Tm DOTAM-p-aminomethylbenzoic Acid. (5c, 69 mg, 51%,
based on 10). Colorless solid. HRMS (ESI) m/z: found 1103.3232 [M
− 2H] (calcd 1103.3203 for C H N O Tm).
Trialkylation of Tri-Boc-cyclen (14) with Electrophiles 13a,
(
CDCl ) δ 155.2, 82.9, 80.1, 77.7, 30.6, 30.4, 28.3.
LiOH (53 mg, 2.2 mmol) was added to a solution of 1-chloro-4-N-
3
+
36
62
8
12
3+
Boc-aminobut-2-yne (21, 204 mg, 1 mmol) and L-Asp-(OMe) ·HCl
2
(
1
198 mg, 1 mmol) in dry DMF (2.5 mL). The mixture was stirred for
8 h at 50 °C, cooled to room temperature, diluted with brine (40
+
48
54
8
12
mL), and extracted with EtOAc (20 mL + 2 × 10 mL). Combined
organic extract was washed with brine (3 × 40 mL), dried, and
12b, and 13c. DIPEA (90 μL, 0.5 mmol) was added to separate
6
528
DOI: 10.1021/acs.jmedchem.5b00621
J. Med. Chem. 2015, 58, 6516−6532

Journal of Medicinal Chemistry
Article
3
6
solutions of tri-Boc-cyclen (14, 118 mg, 0.25 mmol) and 0.25 mmol
of electrophiles 13a (111 mg), 12b (84 mg), and 13c (116 mg) in
DMF (2 mL). The mixtures were stirred for 18 h at 90 °C
DOTAM-triacetyl-benzyl-monoacetyl-p-aminomethylbenzoyl-
1
Asp-(OMe)
δ 7.68−7.01 (br m, D
8H), 3.75−3.67 (br m, 6 H), 3.44−2.18 (br m, 26H). HRMS (ESI)
(16c, 68 mg, 36%). Pale-brown solid. H NMR (CDCl
)
2
3
O exch, 24H), 5.02 (m, 1H), 4.53−4.12 (br m,
2
(
electrophiles 13a and 13c) or 24 h at 110 °C (electrophile 12b). The
+
mixtures were cooled to RT, diluted with brine (40 mL), and extracted
with EtOAc (20 mL+ 2 × 10 mL). Combined organic extracts were
washed with brine (2 × 40 mL), dried, and concentrated. The residues
were triturated with hexanes, affording the intermediates 15a−15c of
sufficient purity to be used in the next step.
m/z: found 948.4946 [M + H] (calcd 948.4984 for C51H N O ).
66 9 9
Alkylation of t-butyl cyclen-N-monoacetate (23) with N-chlor-
oacetyl-propargylamine and subsequent conjugation with N-succinyl-
N′-Boc 1,4-diaminobut-2-yne dimethyl ester (22).
9
DIPEA (700 μL, 4 mmol) and N-chloroacetyl-propargylamine
Tri-Boc-mono-N-acetyl-ω-aminocaproyl-Asp-(OMe)2 Cyclen
15a, 186 mg, 94%). Pale-yellow oil. H NMR (CDCl ) δ 6.75 (m,
(530 mg, 4 mmol) were added to a solution of t-butyl cyclen-N-
1
43
(
monoacetate (23, 372 mg, 1.3 mmol) in MeCN (10 mL). The
3
D O exch, 2H), 4.87 (m, 1H), 3.75 (s, 3 H), 3.69 (s, 3H), 3.53−2.55
mixture was stirred for 18 h at 80 °C, the solvent was evaporated, and
the residue was partitioned between water (30 mL) and EtOAc (30
mL + 2 × 20 mL). Combined organic extract was dried and
concentrated. The residue was triturated with hexanes, affording the
intermediate 24.
2
(
br m, 22H), 2.22 (m, 2H), 1.65 (m, 2H), 1.51 (m, 2H), 1.47 (s, 9H),
.45 (s, 9H), 1.34 (m, 2H). HRMS (ESI) m/z: found 787.4794 [M +
1
+
H] (calcd 787.4817 for C H N O ).
37
67
6
12
Tri-Boc-mono-N-acetyl-3-amino-3-methylbutyryl-Asp-(OMe) Cy-
2
1
clen (15b, 120 mg, 70%). Pale-brown oil. H NMR (CDCl ) δ 6.76
t-Butyl 2-{4,7,10-Tris[2-oxo-2-(prop-2-yn-1-ylamino)ethyl]-cy-
3
(
3
m, D O exch, 2H), 4.87 (m, 1H), 3.74−2.78 (br m, 28H), 1.46 (m,
clen-1-yl Acetate (24, 359 mg, 48%). Pale-orange oil. Spectral data
2
+
44
3H). HRMS (ESI) m/z: found 773.4626 [M + H] (calcd 773.4660
were in agreement with those previously reported.
for C H N O ).
TFA (1 mL) and TES (200 μL) were added to separate solutions of
3
6
65
6
12
Tri-Boc-mono-N-acetyl-p-aminomethylbenzoyl-Asp-(OMe)2 Cy-
0.61 mmol of intermediates 22 (350 mg) and 24 (201 mg) in CH Cl .
2
2
1
clen (15c, 187 mg, 93%). Pale-brown oil. H NMR (CDCl ) δ 7.76
(
5
3
8
The mixtures were stirred for 1 h (intermediate 22) or 18 h
(intermediate 24) at RT, the volatiles were evaporated, the excess TFA
was removed by successive coevaporation with CH Cl and toluene
3
d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.25 (m, D O exch, 2H),
2
.06 (m, 1H), 4.46 (d, J = 6.5 Hz, 2H), 3.79 (s, 3H), 3.70 (s, 3H),
2
2
.46−2.63 (br m, 20H), 1.47 (m, 27H). HRMS (ESI) m/z: found
(30 mL each), and the residues (intermediates 25 and 26) were used
for the subsequent step without further purification. A solution of
intermediate 25 in dry DMF (1 mL) was cooled to 0 °C, followed by
the addition of HOBt (25 mg, 0.18 mmol) and HBTU (232 mg, 0.61
mmol). The mixture was stirred for 5 min at 0 °C, followed by the
addition of the solution of intermediate 26 in dry DMF (2 mL). The
cooling bath was removed and the stirring continued for 18 h at 50 °C.
The mixture was cooled to RT, diluted with brine (50 mL), and
extracted with EtOAc (30 mL + 2 × 20 mL). Combined organic
extract was washed with brine (3 × 50 mL), dried, and concentrated to
leave a pale-brown oily residue (470 mg), which was dissolved in
MeOH (3 mL) and subjected to semipreparative HPLC purification as
described in the General Experimental Procedures. The fractions
containing the product were combined and concentrated to leave
ligand 27 as trifluoroacetate salt.
+
07.4472 [M + H] (calcd 807.4504 for C H N O ).
39
63
6
12
Removal of Boc groups from intermediates 15a−15c and
peralkylation with N-ioodoacetyl-n-hexylamine, N-ioodoacetyl-t-butyl-
amine, and N-ioodoacetyl-benzylamine. TES (200 μL) and TFA (500
μL) were added to separate solutions of 0.2 mmol of intermediates
1
5a (157 mg), 15b (155 mg), and 15c (161 mg) in CH Cl (1.5 mL).
2 2
The mixtures were stirred for 18 h at RT, the volatiles were
evaporated, the excess TFA was removed by successive coevaporation
with CH Cl and toluene (30 mL each), and the residues were used
2
2
for subsequent alkylation without further purification. Complete
deprotection was confirmed by HR-ESI-MS.
N-Acetyl-ω-aminocaproyl-Asp-(OMe)2 Cyclen. Pale-brown oil.
+
HRMS (ESI) m/z: found 487.3223 [M + H] (calcd 487.3244 for
C H N O ).
2
2
43
6
6
N-Acetyl-3-amino-3-methylbutyryl-Asp-(OMe)2 Cyclen. Pale-
DOTAM-triacetyl-propargyl-monoacetyl-1,4-diamonobuty-2-
+
−
brown oil. HRMS (ESI) m/z: found 473.3104 [M + H] (calcd
ynyl-Asp(OMe) ·4CF COO (27, 110 mg, 14%). Colorless solid.
2
3
1
4
73.3088 for C H N O ).
HPLC, method B, t 4.6 min. H NMR (D O) δ 4.23 (m, 1H), 3.76
21
41
6
6
R
2
N-Acetyl-p-aminomethylbenzoyl-Asp-(OMe) Cyclen. Pale-brown
(m, 4H), 3.67 (m, 6H), 3.50 (s, 3H), 3.39 (s, 3H), 3.31−2.35 (br m,
26H), 2.30 (m, 3H). HRMS (ESI) m/z: found 726.3925 [M + H]+
(calcd 726.3939 for C H N O ).
2
+
oil. HRMS (ESI) m/z: found 507.2910 [M + H] (calcd 507.2918 for
C H N O ).
2
4
39
6
6
35 52
9
8
DIPEA (870 μL, 5 mmol) was added to separate solutions of Boc-
Metalation of Ligands 16a−16c and 27 with TmCl ·H O.
3 2
deprotected intermediates described above in MeCN (6 mL), followed
by the addition of 0.66 mmol of the corresponding electrophiles as
TmCl ·H O (30 mg, 0.11 mmol) was added to separate solutions of
3 2
ligands 16a−16c and 27 (0.1 mmol each) in MeOH/water as follows:
ligands 16a and 16c (2 mL MeOH/1 mL water), ligand 16b (1 mL
MeOH/2 mL water), and ligand 27 (3 mL water). The pH was
adjusted and maintained at ca. 10 (1 M NaOH), while the mixtures
were stirred for 18 h at RT. The pH was then adjusted to ca. 5.5 (1 M
HCl), and the stirring of the mixtures continued for further 18 h at
RT, MeOH was evaporated, and remaining aqueous solutions were
subjected to SEC as described in General Experimental Procedures.
The fractions containing the products were lyophilized to leave the
complexes 6a−6d.
26b
follows: N-ioodoacetyl-n-hexylamine
(178 mg), N-ioodoacetyl-t-
26b
26b
butylamine (159 mg), and N-ioodoacetyl-benzylamine (182 mg).
The mixtures were stirred for 18 h at 50 °C, the solvent was
evaporated, and the residues were partitioned between water (30 mL)
and EtOAc (2 × 30 mL). Combined organic extracts were washed
with brine (2 × 30 mL), dried, and concentrated. The residues were
triturated with hexanes, affording the desired ligands 16a−16c of
sufficient purity for metalation.
DOTAM-triacetyl-n-hexyl-monoacetyl-ω-aminocaproyl-Asp-
1
Tm3 DOTAM-triacetyl-n-hexyl-monoacetyl-ω-aminocaproyl-
+
(
8
OMe) (16a, 180 mg, 99%). Pale-brown solid. H NMR (CDCl ) δ
2
3
.35 (br m, D O exch, 1H), 7.60 (br m, D O exch, 2H), 7.23−6.62 (br
Asp-(OH) (6a, 50 mg, 48%). Pale-yellow waxy solid. HRMS (ESI)
2
2
2
+
m, D O exch, 2H), 4.85 (m, 1H), 3.75 (s, 3 H), 3.70 (s, 3H), 3.58−
m/z: found 1048.5483 [M − 2H] (calcd 1048.5500 for
2
2
.24 (br m, 36H), 1.51 (m, 10H), 1.28 (m, 20H), 0.88 (m, 9H).
C H N O Tm).
4
4
81
9
9
+
3+
HRMS (ESI) m/z: found 910.6678 [M + H] (calcd 910.6705 for
Tm DOTAM-triacetyl-t-butyl-monoacetyl-3-amino-3-methyl-
butyryl-Asp-(OH)2 (6b, 15 mg, 16%). Pale-brown solid. HRMS
C H N O ).
4
6
88
9
9
+
DOTAM-triacetyl-t-butyl-monoacetyl-3-amino-3-methylbutyryl-
(ESI) m/z: found 950.4406 [M − 2H] (calcd 950.4404 for
1
Asp-(OMe) (16b, 143 mg, 88%). Pale-brown oil. H NMR (CDCl )
C H N O Tm).
2
3
37 67
9
9
3+
δ 6.94 (m, D O exch, 3H), 6.55 (m, D O exch, 2H), 4.84 (m, 1H),
Tm DOTAM-triacetyl-benzyl-monoacetyl- p-aminomethylben-
zoyl-Asp-(OH) (6c, 40 mg, 37%). Pale-yellow solid. HRMS (ESI) m/
2
2
3
.74 (s, 3 H), 3.69 (s, 3H), 3.56−3.09 (br m, 28H), 1.36 (m, 43H).
2
+
+
HRMS (ESI) m/z: found 812.5625 [M + H] (calcd 812.5610 for
z: found 1086.3760 [M − 2H] (calcd 1086.3778 for
C H N O ).
C H N O Tm).
3
9
74
9
9
49 59
9
9
6
529
DOI: 10.1021/acs.jmedchem.5b00621
J. Med. Chem. 2015, 58, 6516−6532

Journal of Medicinal Chemistry
Article
Tm3+ DOTAM-triacetyl-propargyl-monoacetyl-1,4-diamonobuty-
TSAP, twisted square antiprism; UPLC, ultrahigh pressure
liquid chromatography; UV, ultraviolet
2
-ynyl-Asp(OH) (6d, 41 mg, 47%). Colorless solid. HRMS (ESI) m/
2
+
z: found 864.2704 [M − 2H] (calcd 864.2733 for C H N O Tm).
33
45
9
8
REFERENCES
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00621.
■
■
*
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AUTHOR INFORMATION
Corresponding Author
Phone: +1 519-661-2111 ext 86349. E-mail: rhhudson@uwo.
■
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ACKNOWLEDGMENTS
■
The financial support of this work was provided by the Ontario
Institute for Cancer Research (OICR), Canadian Health
Research Institute (CIHR), and National Science and
Engineering Research Council of Canada (NSERC). We
thank Elyse Hudson for assistance with graphic designs.
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ABBREVIATIONS USED
■
(8) Li, A. X.; Wojciechowski, F.; Suchy,
H. E.; Menon, R. S.; Bartha, R. A sensitive PARACEST contrast agent
́
M.; Jones, C. K.; Hudson, R.
Boc, tert-butyloxycarbonyl; BSA, bovine serum albumin; CA,
contrast agent; CEST, chemical exchange saturation transfer;
DCC, dicyclohexylcarbodiimide; DMF, dimethylformamide;
DOTAM, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid amide; EI, electron impact; ESI, electron spray; FCC,
flash column chromatography; FISP, fast imaging with steady
state precession; FLASH, fast low angle shot; FLEX, frequency
labeled exchange transfer; HBTU, 1-benzotriazolyl-N,N,N′,N′-
tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxy-
benzotriazole; HPDO3A, 10-(2-hydroxypropyl)-1,4,7-triscar-
boxymethyl-1,4,7,10-tetraazacyclododecane; HPLC, high pres-
sure liquid chromatography; HRMS, high resolution mass
spectrometry; MRI, magnetic resonance imaging; MRS,
magnetic resonance spectroscopy; MT, magnetization transfer;
NHS, N-hydroxysuccinimide; NMR, nuclear magnetic reso-
nance; OPARACHEE, on resonance paramagnetic chemical
exchange effects; PARACEST, paramagnetic chemical exchange
saturation transfer; RT, room temperature; SAP, square
antiprism; SEC, size exclusion chromatography; SPIO, super
paramagnetic iron oxide; T3P, propanephosphonic acid
anhydride; TES, triethylsilane; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; TLC, thin layer chromatography;
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