DOTMA-based amides (DOTMAMs) as a platform for the development of PARACEST MRI contrast agents

Article abstract of DOI:10.1039/c6ra11741d

A synthetic methodology leading to previously unknown DOTMA-based secondary amides (DOTMAMs) has been developed. Alkylation of cyclen with l-lactic acid-derived pseudohalides was used as a key step affording the alkyl- and aryl-decorated DOTMAMs. Amino acid-decorated DOTMAMs were obtained via peptide coupling between DOTMA and protected amino acids. Metallation of the DOTMAMs ligand with Tm³⁺ gave complexes exhibiting proximal amide proton based paramagnetic CEST effects at <-50 ppm relative to water.

Full text of DOI:10.1039/c6ra11741d

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DOTMA-based amides (DOTMAMs) as a platform for
the development of PARACEST MRI contrast agents
Received 00th January 20xx,
Accepted 00th January 20xx
M. Suchý,a,c A.X. Li,c M. Milne,a R. Barthac,d and R.H.E. Hudsona,b*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A synthetic methodology leading to previously unknown DOTMA-based secondary amides (DOTMAMs) has been
developed. Alkylation of cyclen with L-lactic acid-derived pseudohalides was used as a key step affording the alkyl- and
aryl-decorated DOTMAMs. Amino acid-decorated DOTMAMs were obtained via peptide coupling between DOTMA and
protected amino acids. Metallation of the DOTMAMs ligand with Tm3+ gave complexes exhibiting proximal amide proton
based paramagnetic CEST effects at < -50 ppm relative to water.
to magnetization transfer (MT) from endogenous
macromolecules.11,12 The MT effect is a competing mechanism
capable of saturating the bulk water spins and consequently
Introduction
Nowadays magnetic resonance imaging (MRI) represents an
indispensable tool in clinical diagnostics. MRI is primarily
based on the detection of water protons in the body and
oftentimes is used in combination with an exogenous agent
that enhances the image contrast.1 Such agents are termed
‘contrast agents’ (CAs) and the development of CAs suitable
for MRI is an active area of research. Several types of CAs are
currently available,2 among them paramagnetic chemical
exchange saturation transfer (PARACEST) MRI CAs3 represent a
relatively new group characterized by exceptional sensitivity to
various parameters relevant to clinical diagnostics, such as
temperature,4 pH,5 redox state,6 concentration of primary
metabolites7 or enzymatic activity.8 Despite the potential
usefulness of PARACEST CAs, their low in vivo sensitivity
requires the administration of high concentrations (20-100
mM) of CAs;9 thus, it is important to employ ligands that stably
bind the potentially toxic lanthanide metal, such as those
based on the macrocyclic chelator DOTA, and employ
strategies to increase the observable signals.10
lowers the contrast efficiency of PARACEST MRI CAs within the
MT frequency range (ca. -100 to 100 ppm). Development of
PARACEST MRI CAs exhibiting highly shifted CEST signals lying
toward or beyond the outer limit of the MT frequency range
represents a way to address the low sensitivity associated with
these agents. In this regard, we recently demonstrated Tm3+
,‡
DOTAM-t-butyl (
1
, Figure 1),13 possessed an amide proton-
based CEST effect clearly lying outside of the MT frequency
range. The presence of the highly shifted CEST effect (δ = -102
ppm) was attributed to the predominance of the twisted
square antiprism (TSAP) isomer of CA
favoured by the sterically demanding t-butylamides.14
1
in solution13 which was
Unlike the Tm3+ complexes derived from DOTA (
and unsubstituted DOTAM ( , Figure 1) which exist in solution
predominantly (ca. 90%) in the square antiprism isomer (SAP),
2, Figure 1)
3
,
§
the Tm3+ complex derived from DOTMA15
(4, Figure 1) favours
the TSAP isomer in solution (ca. 90%).16 This conformational
bias is attributed to the steric effect due to the presence of the
additional four methyl groups on the side arms of DOTMA (4).
Upon administration into a biological system PARACEST MRI
CAs experience a significant decrease in sensitivity attributable
We hypothesized tetraamides derived from DOTMA, herein
referred to as DOTMAMs,§§ may possess highly shifted CEST
signals similar to that observed for
1, and have a greater
a.Department of Chemistry The University of Western Ontario, London ON N6A 5B7
Canada
preference for the TSAP isomer as observed for the carboxylic
acid DOTMA, thus potentially resulting in a more intense,
highly-shifted CEST effect.
b.Centre for Advanced Materials and Biomaterials Research, The University of
Western Ontario
c. Center for Functional and Metabolic Mapping, Robarts Research Institute, The
University of Western Ontario, London ON N6A 5K8 Canada
d.Department of Medical Biophysics, The University of Western Ontario
While the syntheses of various ligands based on the DOTA (2)
*
or DOTAM (3) scaffold via peralkylation of cyclen (5, Figure 1)
Robert H. E. Hudson, phone +519-661-2111, ext. 86349; e-mail: rhhudson@uwo.ca
† Electronic Supplementary Information (ESI) available: Full experimental details, are well documented in the literature,17 only a small number
of DOTMA-based ligands are presently known.16,18
NMR and MS spectra associated with synthetic intermediates and final products.
CEST spectra and ꢀ-plots associated with CAs 6-8, 10 and 11. See
DOI: 10.1039/x0xx00000x
Furthermore, only the primary amide derived from DOTMA
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(DOTMAM)19 has been described. This ligand was accessed by
the peralkylation of cyclen with racemic 2-propanionamide
resulting in a mixture of stereoisomeric ligands of which only
the Eu3+ chelate was studied.19
Results and discussion
Our studies began with the synthesis of corresponding
electrophiles, which we envisioned using for the peralkylation
of cyclen (5). A DCC-mediated peptide coupling between (S)-2-
chloropropionic acid (12, Scheme 1) and benzylamine afforded
corresponding secondary chloroderivative 13 (Scheme 1) in
good yield using a modified literature procedure.20 Despite
the use of 2-chloropropionic acid derivatives previously for the
preparation of DOTMA,15 the reactivity of electrophile 13 was
found to be insufficient to achieve the peralkylation of cyclen.
Therefore, we turned our attention to the use of (S)-lactic acid
derived pseudohalides as potential electrophiles. Coupling of
(S)-lactic acid (14) with a small variety of amines,21 namely
benzylamine, t-butylamine, aniline, and (L)-Lys(Boc)-OMe•HCl,
Our interest in Tm3+ chelates of DOTMAMs (with a secondary
amide) required the development of
methodology. Herein, we report the preparation of the
hitherto unreported Tm3+ complex of known ligand
and well
as the synthesis of five new ligands and the Tm3+ complexes
thereof ( 11, Figure 1) as well as evaluation of their CEST
properties.
a new synthetic
6
7
-
proceeded smoothly and afforded the lactamides 15-18
(Scheme 1) in good yields (60-93%).
Firstly, conversion of the secondary OH group to the corresponding
triflate (OTf-lactamide) was attempted. Among the few known
preparations of DOTMA-based ligands, peralkylation of cyclen with
OTf-(S)-lactic acid esters is used as a key step almost exclusively.16,18
Treatment of lactamide 15 with trifluoroacetic acid anhydride
(Tf2O)18a afforded the unstable OTf-lactamide 15b (two signals
observed in 19F NMR spectrum). However, treatment of lactamide
16 with Tf2O produced the more stable OTf-lactamide 16b (one
signal at δ -74.9 ppm in 19F NMR spectrum after storage for 24
hours at room temperature). Unfortunately, OTf-lactamide 16b
was not stable under the forcing conditions required for
peralkylation of cyclen (heat, excess of base) resulting in the
formation of complex mixture devoid of the desired product of
peralkylation.
Figure 1. Chemical structures of Tm3+ DOTAM-t-Bu (
), DOTAM ( ), DOTMA ( ), cyclen ( ) and the DOTMA-derived
complexes 6-11
1), DOTA
(
2
3
4
5
.
Scheme 1. Preparation of electrophiles 15a-15c, 16a, 16b, 17a-19a
Allen’s group has recently reported an alkylation of N,O- Scheme 1) proceeded smoothly (82% yield).23 Unfortunately, the
macrocycles related to cyclen by OTs-(S)-lactamide.22
The alkylation of cyclen with 15c resulted in a complex mixture
conversion of lactamide 15 to OTs-(S)-benzyl lactamide (15c,
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containing only small amount (< 10%) of the desired product of Scheme 3. Preparation of CAs 10 and 11
peralkylation, obtained after careful HPLC purification.
The problem was solved by using OMs-(S)-lactamides 15a-18a
(Scheme 1) obtained in excellent yields (> 90%) after treatment of
lactamides 15-18 with MsCl under basic conditions (Scheme 1).24
With electrophiles 15a-18a in hand we investigated the alkylation of
cyclen. Mixtures of di-, tri- and tetrasubstituted cyclens have been
obtained, when electrophiles 15a-17a were used. The desired
ligands were purified by HPLC, affording DOTMAM-Bn, DOTMAM-t-
Bu and DOTMAM-Ph in low to moderate yields (14-42%) as
described in the Supporting Information and shown in Scheme 2.
Scheme 2. Preparation of CAs 6-9
With complexes 6-8, 10 and 11 prepared, their potential as
PARACEST MRI CAs was investigated. The CEST spectra associated
with CAs 6-8, 10 and 11 were acquired as described in the
Supporting Information. Temperature and pH sensitivity of the CAs
6-8, 10 and 11 was also investigated. When possible, the results
were compared with those obtained previously for related DOTAM-
based complexes.13,26 The results of these studies are summarized
in Table 1 and discussed below.
This methodology also proved successful for the preparation of the
DOTMAM ligand with a primary amide group (Scheme 2). Thus 6
was obtained in 51% yield (see the Supporting Information for
details) by peralkylation of cyclen with OMs-(S)-lactamide (19a,
Scheme 1), followed by purification by flash column
chromatography (FCC) on Al2O3.† Prolonged heating (48 h at 90 °C)
of above mentioned ligands with TmCl3•H2O (in dioxane/water
mixture, pH ~ 7) afforded the desired complexes 6-8 (Scheme 3),
interestingly no metalation (complex 9) was observed when
DOTMAM-Ph was treated with TmCl3•H2O under identical
conditions (Scheme 2).
Table 1. CEST properties for CAs 6-8, 10 and 11.*
CA
CEST effect (ppm)
signal intensity (%)
Related DOTAM complex; CEST
effect (ppm)/signal intensity
(%)
Peralkylation of cyclen with OMs-lactamide 18a did not lead to the
formation of the desired DOTMAM-Lys(Boc)-OMe; therefore, we
explored an alternative synthetic route to obtain the amino acid-
6
-54 (6); -63 (7)
-65 (6)
-46 (32); -51 (33)13b$
-51 (27)13b$
7
decorated DOTMAM derived ligands.
An HBTU-mediated
-68 (10); -102 (21)13a†
-50 (15)26#
coupling∫,25 between DOTMA tetrasodium salt (4, Scheme 3)
prepared according to the literature procedures16,18a and (L)-
Lys(Boc)-OMe•HCl afforded the desired DOTMAM-(L)-Lys(Boc)-
OMe (in 41% yield after the HPLC purification). Subsequent
8
-115 (3)
10
11
-53 (20); -62 (10)
-51 (1)
not available
removal of protecting groups and metallation with TmCl3•H2O * CEST spectra for 6-8, 10 and 11 were collected on a 9.4 T clinical
o
furnished the complex 10 in 27% overall yield (based on DOTMA MRI scanner at 15 mM of complex, 37 C, pH 7.5 using a saturation
tetrasodium salt) as shown in Scheme 3 and described in the pulse of 14 μT for 3.95 s. $ CEST spectra collected at: 14 T, 10 mM of
o
†
Supporting Information. Conjugation of 4 with (L)-Phe-OEt•HCl, complex, 37 C, pH 7.0 using a saturation pulse of 650 Hz for 2 s.
followed by removal of ester protecting groups by saponification CEST spectra collected at: 14 T, 10 mM complex (in 90% D2O), 37 oC,
#
and metalation with TmCl3•H2O gave the complex 11 in 25% overall pH 7.2 using a saturation pulse of 20 μT for 2 s. CEST spectra
o
yield (Scheme 3; see the Supporting Information for experimental collected at: 14 T, 10 mM complex, 37 C, pH 7.0 using a saturation
details).
pulse of 20 μT for 10 s.
CA 6, derived from the unsubstituted DOTMAM ligand, and CAs 7
and 8, decorated with alkyl groups, all featured amide-proton based
CEST effect with intensities that are 4-7 fold smaller (Table 1) than
signals produced by the corresponding DOTAM-based counterparts
(Tm3+ DOTAM, Tm3+ DOTAM-Bn and Tm3+ DOTAM-t-Bu).13 A CEST
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signal at lower chemical shift was not observed for CA 8 (Table 1), Curiously, only a very weak CEST effect (Table 1) was observed for
presumably it was too weak to be detected. On the other hand, the Tm3+ DOTMAM-(L)-Phe-OH (11). The intensity of the signal was
CEST signals associated with CAs 6-8 were shifted farther from bulk found to increase with increasing pH (see the Supporting
water signal (by 8-14 ppm, Table 1) as compared to the analogous Information), implying that the exchange kinetics associated with
DOTAM CAs. The signal reduction of DOTMAM-based CAs is caused CA 11 is out of the optimal range (too slow)¤ for the observation of
by slower exchange rates (by ca. 2 fold) compared to related the CEST effect at pH 7; presence of base increases the exchange
DOTAM-based complexes (244 μs for 7 versus 125 μs for Tm3+ rate of the side chain amide protons resulting in a somewhat
DOTAM-Bn; 579 μs for 8 versus 273 μs for Tm3+ DOTAM-t-Bu).27 stronger signal at pH 8 (see Supporting Information).29 Our findings
Should these findings with respect to pendant arm amide proton indicate that further variation of the amino acid residues30 present
exchange rates extend to a slowing of water exchange at the metal in DOTMAM-derived CAs might lead to complexes endowed with
center, examination of Eu3+ DOTMAM-based complexes (requiring interesting CEST properties.
slow exchange rates for bound water)3 may lead to the formation of
Examination of the 1H NMR spectra for Yb3+ DOTMAM, Yb3+
DOTMAM-Bn and Yb3+ DOTMAM-t-Bu (supporting information)
PARACEST MRI CAs. These studies are beyond the scope of the
present work.
indicate these complexes exist predominantly in as a single isomer.
Tm3+ DOTMAM-(L)-Lys-OH (10) possessed two CEST effects (Table Comparison of the mostly highly shifted signal, the axial cyclen
1), both showed a dependence on pH and temperature (Figure 2). proton designated H4, is commonly done discriminate coordination
The origin of the two peaks is possibly due to the presence of s-cis complex geometry (i.e. SAP versus TSAP),31 and estimate population
and s-trans amide conformations as suggested by the similarity in distributions of the two isomers, but is unenlightening in the
chemical shifts of the CEST signals to those present in the current case. Comparison of Eu3+ to Yb3+ metallated ligands show
unsubstituted DOTMAM (6).28
the following trends. The chemical shift of H4 for Eu3+DOTAM (SAP
isomer ~40 ppm; TSAP isomer ~ 5 ppm)19 less highly shifted than
the Eu3+DOTMAM complex (SAP isomer ~44-59 ppm; TSAP isomer
~ 15-20 ppm). The Yb3+DOTAM complex exists as a single isomer
(SAP) displaying a chemical shift of H4 = 95 ppm. The Yb3+DOTMAM
complex prepared during this work shows H4 = 124 ppm, which
follows the trend of increasing chemical shift in going from the
DOTAM to DOTMAM ligand observed for the Eu3+ complexes. The
Yb3+DOTMAM-Bn shows a similar chemical shift for H4 (127 ppm). It
is notable that both the Tm3+DOTMAM and Tm3+DOTMAM-Bn
complexes both give rise to relatively low-shifted amide-proton
100
pH: 5.5
80
5.5
6.0
6.0
60
6.5
6.5
7.0
7.0
40
20
7.5
CEST signals.
This is in contrast to Yb3+DOTMAM-t-Bu which
7.5
possesses a lower shifted H4 (104 ppm) yet the Tm3+ complex
displays the CEST signal with the greatest hyperfine shift. It is
tempting to suggest that the lower H4 chemical shift for
8.0
0
8.0
-20
100
50
0
-50
-100
Tm3+DOTMAM-t-Bu indicates that it exists in
a different
Frequency offset (ppm)
coordination geometry, possibly TSAP, which is responsible for the
high shifting CEST signal and would be consistent with our previous
work.13
Experimental
General Experimental Procedures
Reagents were commercially available and all solvents were
HPLC grade except for water (18.2 MΩ cm millipore water),
CH2Cl2 and DMF (dried over Al2O3, in a solvent purification
system). Solvents were removed under reduced pressure in a
rotary evaporator, aqueous solutions were lyophilized and
organic extracts were dried over Na2SO4. Flash column
chromatography (FCC) was carried out using silica gel (SiO2),
mesh size 230 - 400 Å and basic alumina (Al2O3), pH 9.5-10.5,
mesh size 10 – 100 Å. Thin-layer chromatography (TLC) was
carried out on Al backed silica gel or alumina plates with
compounds visualised by I2 vapours, anisaldehyde stain, 5%
ninhydrin stain, phosphomolybdic acid stain, and UV light.
Melting points (mp) were obtained on Fisher–Johns apparatus
Figure 2. CEST spectra associated with Tm3+ DOTMAM-(L)-Lys-OH
(10), measured at 9.4 T, 15 mM of complex, using a saturation pulse
of 14 μT for 3.95 s. The CEST effects are modulated by both pH
(top) and temperature (bottom).
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and are uncorrected. Specific rotations [α]D were determined 3H); 1.79 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3) δ 169.4, 137.4,
by polarimeter at ambient temperature using a 1 ml, 10 cm 128.6, 127.5 (2 × C), 55.6, 43.7, 22.5. HRMS (EI) m/z; found
path length cell; the units are 10-1 deg cm2 g-1 and the 197.0602 [M]+ (calcd 197.0607 for C10H12ClNO); LRMS (EI) m/z
concentrations are reported in g/100 mL. Ultra performance (rel. abundance): 197 [M+] (23), 162 (85), 106 (14).
liquid chromatography (UPLC) was performed using a BEH C18
Coupling of (S)-lactic acid with aniline, benzylamine and t-
column (particle size 1.7μm; 1.0 id × 100 mm) and HR-ESI-MS
detector. Mobile phase: Method A: 100% H2O – 100% MeCN
(both solvents containing 0.1% HCOOH) over 5 min, then 100%
MeCN for 2 min, linear gradient, flow rate 0.1 mL/min. HPLC
purification was performed using a Delta-Pak C18 300 Ǻ column
(particle size 15 μm; 8 × 100 mm Radial-Pak cartridge). Mobile
phase for Method B (DOTMAM-Bn) was 0 min, 90% H2O – 10%
MeCN (for each method both solvents containing 0.1% TFA) to
butylamine
Separate mixtures containing (S)-lactic acid (14, 450 mg, 5
mmol for benzylamine or 180 mg, 2 mmol), HOBt (1.35 g, 10
mmol for benzylamine or 540 mg, 4 mmol), DCC (1.55 g, 7.5
mmol for benzylamine or 619 mg, 3 mmol) and benzylamine
(660 μL, 6 mmol), aniline (220 μL, 2.4 mmol) or t-butylamine
(220 μL, 2.4 mmol) in dry CH2Cl2 (15 mL for benzylamine or 6
mL) were stirred for 24 h at room temperature (rt). The solids
were filtered off, the filters were washed with ice cold CH2Cl2.
Resulting solutions were washed with saturated NaHCO3
solution (30 mL for benzylamine or 20 mL), the aqueous layers
were extracted with CH2Cl2 (2 × 30 mL for benzylamine or 2 ×
13 min, 20% H2O – 80% MeCN,; Method C (DOTMAM-t-Bu, 7):
0 min, 90% H2O – 10% MeCN to 11 min, 13% H2O – 87% MeCN;
Method D (DOTMAM-Ph): 0 min, 90% H2O – 10% MeCN to 13
min, 100% MeCN; Method E [DOTMAM-(L)-Lys(Boc)-OMe,
DOTMAM-(L)-Phe-OEt]: 0 min, 90% H2O – 10% MeCN to 10
min, 100% MeCN; Method F (8): 0 min, 90% H2O – 10% MeCN
20 mL).
Combined organic extracts were dried, were
to 11 min, 20% H2O – 80% MeCN; linear gradient and 3
mL/min flowrate were used for each method. Size exclusion
chromatography (SEC) was carried out on Bio-Gel P2, 45-90
μm mesh resin (8 g, per 0.1 mmol of compound). Ten fractions
(10 ml each) were collected and identified with I2 vapours and
UV light. Absence of free Tm3+ was verified by xylenol orange
concentrated, the residues were dissolved in small amount of
cold acetone (ca. 5 – 10 mL) and precipitated dicyclohexylurea
was filtered off using a Pasteur pipette with a cotton plug. The
volatiles were evaporated and the residues were subjected to
FCC on 40 g SiO2, eluting with hexanes/EtOAc (3:1, reaction
with benzylamine); 30 g SiO2, eluting with CH2Cl2/MeOH (98:2,
reaction with aniline) or 25 g SiO2, eluting with CH2Cl2/MeOH
(95:5). Evaporation of the eluates afforded (S)-N-benzyl
lactamide (15, 727 mg, 81%), (S)-N-t-butyl lactamide (16, 175
mg, 60%) or (S)-N-phenyl lactamide (17, 266 mg, 81%).
test.32 NMR spectra were recorded on
a 400 MHz
1
spectrometer for H NMR spectra δ values were recorded as
follows: CDCl3 (7.27 ppm); CD3OD (3.31; 4.87 ppm), D2O (4.75
ppm) for 13C (125 MHz) δ CDCl3 (77.0 ppm); CD3OD (49.2 ppm).
Mass spectra (MS) were obtained using electron impact (EI) or
electrospray ionisation (ESI). Chemical exchange saturation
transfer (CEST) spectra were acquired using a 9.4 T small
animal clinical MRI scanner as follows: NMR tubes with
solutions of the complexes (15 mM, pH 7.5) were imaged at 37
°C [the temperature was monitored and controlled by blowing
hot air using a Model 1025 Small Animal Monitoring and
Gating System (SA Instruments, Inc., Stony Brook, NY)] using a
(S)-N-benzyl lactamide (15), colorless oil. [α]D -6.4 (c 1,
MeOH); lit.35 [α]D -6.8 (c 6.9, CHCl3). 1H NMR (CDCl3) δ 7.29 (m,
5H); 7.11 (br s, D2O exch, 1H); 4.41 (d, J = 6.0 Hz, 2H); 4.24 (q, J
= 7.0 Hz, 1H); 1.42 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3) δ 174.7,
137.8, 128.7, 127.6, 127.5, 68.3, 43.0, 21.2. HRMS (EI) m/z;
found 179.0952 [M]+ (calcd 179.0946 for C10H13NO2); LRMS (EI)
m/z (rel. abundance): 179 [M+] (33), 161 (15), 134 (10), 91
(100), 65 (12).
fast spin echo pulse sequence (FOV: 12.8
32 32, TR = 4000 ms, 4 echoes, and TE = 10 ms), preceded by
×
12.8 mm2, matrix:
×
a frequency selective saturation pulse (B1 = 14 μT, saturation
range = -150 to 50 ppm in steps of 1 ppm, saturation time =
3.95 s). CEST spectra were generated using the average signal
intensity from each tube. Similar methodology was used to
evaluate the temperature (15 mM, pH 7.5, temperature range
33-39 °C) and pH (15 mM, 37 °C, pH range 6.0-8.0) sensitivity
of selected agents. The exchange rates of the amide protons
with bulk water were measured by Ω-plot method as
described previously.33
(S)-N-t-butyl lactamide (16), colorless solid. [α]D -28.0 (c 1,
MeOH). 1H NMR (CDCl3) δ 6.47 (br s, D2O exch, 1H); 4.09 (q, J
= 6.5 Hz, 1H); 3.52 (br s, D2O exch., 1H); 1.38 (d, J = 6.5 Hz, 3H);
1.36 (s, 9H). 13C NMR (CDCl3) δ 174.5, 68.3, 50.7, 28.6, 21.1.
HRMS (EI) m/z; found 145.1107 [M]+ (calcd 145.1103 for
C7H15NO2); LRMS (EI) m/z (rel. abundance): 145 [M+] (95), 130
(100), 101 (67).
(S)-N-phenyl lactamide (17), colorless oil. [α]D -41.0 (c 0.5,
MeOH); lit.36 [α]D -23.5 (c 0.48, CHCl3). 1H NMR (CDCl3) δ 8.67
(br s, D2O exch., 1H); 7.52 (m, 2H); 7.30 (m, 2H); 7.12 (m, 1H);
4.31 (q, J = 6.5 Hz, 1H); 4.14 (br s, D2O exch, 1H); 1.48 (d, J =
6.5 Hz, 3H). 13C NMR (CDCl3) δ 173.2, 137.0, 129.0, 124.6,
119.9, 68.7, 21.0. HRMS (EI) m/z; found 165.0797 [M]+ (calcd
165.0790 for C9H11NO2); LRMS (EI) m/z (rel. abundance): 165
[M+] (50), 121 (21), 93 (100), 65 (13).
Preparation of (S)-N-benzyl-2-chloropropanamide
The reaction was carried out as described in the literature,34
starting from 430 μL (5 mmol) of S-chloropropionic acid (12).
The product was isolated by FCC on 70 g SiO2, eluting with
hexanes/EtOAc
(3:1),
obtained
(S)-N-benzyl-2-
chloropropanamide (13, 813 mg, 82%), colorless solid. [α]D
-
1.3 (c 1, MeOH); lit.34 [α]D -3.8 (c 1, MeOH). 1H NMR (CDCl3) δ
7.37 (m, 2H); 7.31 (m, 3H); 6.88 (br s, D2O exch, 1H); 4.49 (m,
Coupling of (S)-lactic acid with H-(L)-Lys(Boc)-OMe•HCl
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A stirred mixture containing (S)-lactic acid (14, 180 mg, 2 HRMS (EI) m/z; found 179.0952 [M]+ (calcd 179.0946 for
mmol), H-(L)-Lys(Boc)-OMe · HCl (594 mg, 2 mmol), HOBt (270 C10H13NO2); LRMS (EI) m/z (rel. abundance): 179 [M+] (33), 161
mg, 2 mmol) and Et3N (280 μL, 2 mmol) in dry CH2Cl2 (15 mL) (15), 134 (10), 91 (100), 65 (12). HRMS (ESI) m/z: found
was cooled to 0 ºC, followed by an addition of DCC (825 mg, 4 334.1124 [M + H]+ (calcd. 334.1113 for C17H20NO4S).
mmol). The stirring continued for further 30 min at 0 °C, the
Reaction of (S)-lactamides 15-19 with MsCl
cooling bath was removed and the stirring was continued for
further 48 h at rt. The solids were filtered off, the filter was
washed with ice cold CH2Cl2. Resulting solution was washed
with saturated NaHCO3 solution (30 mL), the aqueous layer
was extracted with CH2Cl2 (30 mL). Combined organic extract
was dried, was concentrated, the residue was dissolved in
small amount of cold acetone (ca. 5 – 10 mL) and precipitated
dicyclohexylurea was filtered off using a Pasteur pipette with a
cotton plug. The volatiles were evaporated and the residue
was subjected to FCC on 50 g SiO2, eluting with CH2Cl2/MeOH
(95:5). Evaporation of the eluate afforded (S)-N-(L)-Lys(Boc)-
OMe lactamide (18, 621 mg, 93%) as colorless oil. [α]D -11.9 (c
1.4, MeOH). 1H NMR (CDCl3) δ 7.20 (d, D2O exch., J = 8.5 Hz,
1H); 4.71 (br s, D2O exch, 1H); 4.57 (m, 1H); 4.24 (q, J = 6.5 Hz,
1H); 3.73 (s, 3H); 3.08 (m, 2H); 1.86 (m, 1H); 1.71 (m, 1H); 1.49
(m, 2H); 1.43 (m, 12H); 1.35 (m, 2H). 13C NMR (CDCl3) δ 174.8,
172.8, 156.1, 79.2, 68.3, 52.4, 51.5, 40.1, 31.9, 29.4, 28.4, 22.4,
21.0. HRMS (ESI) m/z: found 333.2025 [M + H]+ (calcd.
333.2026 for C15H29N2O6).
Separate suspensions or solutions of (S)-N-benzyl lactamide
(
15, 788 mg, 4.4 mmol), (S)-N-t-butyl lactamide (16, 175 mg,
1.21 mmol), (S)-N-phenyl lactamide (17, 266 mg, 1.61 mmol),
(S)-N-(L)-Lys(Boc)-OMe lactamide (18, 279 mg, 0.84 mmol) or
(S)-lactamide (19, 891 mg, 10 mmol) in dry CH2Cl2 [20 mL in
the case of (S)-N-benzyl lactamide (15); 6 mL in the case of (S)-
N-t-butyl lactamide (16); 8 mL in the case of (S)-N-phenyl
lactamide (17); 4 mL in the case of (S)-N-(L)-Lys(Boc)-OMe
lactamide (18); 15 mL in the case of (S)-lactamide (19)] and
Et3N [1.12 mL, 8 mmol in the case of (S)-N-benzyl lactamide
(
(
(
15); 310 μL, 2.19 mmol in the case of (S)-N-t-butyl lactamide
16); 410 μL, 2.93 mmol in the case of (S)-N-phenyl lactamide
17); 580 μL, 4.2 mmol in the case of (S)-N-(L)-Lys(Boc)-OMe
lactamide (18); 2.54 mL, 18.2 mmol in the case of (S)-lactamide
19)] were cooled to 0 °C, followed by a dropwise addition
(
(over ca. 1 min) of MsCl [430 μL, 5.58 mmol in the case of (S)-
N-benzyl lactamide (15); 120 μL, 1.53 mmol in the case of (S)-
N-t-butyl lactamide (16); 160 μL, 2.04 mmol in the case of (S)-
N-phenyl lactamide (17); 90 μL, 1.07 mmol in the case of (S)-N-
(L)-Lys(Boc)-OMe lactamide (18); 980 μL, 12.7 mmol in the
case of (S)-lactamide (19)]. The cooling baths were removed
Reaction of (S)-N-t-butyl lactamide with Tf2O
A round bottom flask containing (S)-N-t-butyl lactamide (16
,
202 mg, 1.39 mmol) was flushed with N2, followed by the and the mixtures were stirred at rt as follows: 2 h in the case
addition of dry CH2Cl2 (2 mL) and dry pyridine (120 μL, 1.46 of (S)-N-benzyl lactamide (15) and (S)-N-(L)-Lys(Boc)-OMe
mmol). The solution was cooled to 0 °C, followed by a lactamide (18); 4 h in the case of (S)-N-t-butyl lactamide (16
)
dropwise addition (over ca. 1 min) of Tf2O (230 μL, 1.39 mmol). and (S)-N-phenyl lactamide (17); 24 h in the case of (S)-
The stirring continued for further 1 h at 0 °C, the solvent was lactamide (19). The volatiles were evaporated and the
evaporated and the residue was subjected to FCC on 25 g SiO2, residues were subjected to FCC as follows: 40 g SiO2, eluting
eluting with hexanes/EtOAc (1:1). Evaporation of the eluate with hexanes/EtOAc (1:1) in the case of (S)-N-benzyl lactamide
afforded (S)-O-Tf-N-t-butyl lactamide (16b, 223 mg, 34%) as
(15) and (S)-N-phenyl lactamide (17); 30 g SiO2, eluting with
colorless solid. [α]D -16.8 (c 0.5, MeOH). 1H NMR (CDCl3) δ hexanes/EtOAc (1:1) in the case of (S)-N-t-butyl lactamide (16);
5.94 (br s, D2O exch, 1H); 5.14 (q, J = 7.0 Hz, 1H); 1.70 (d, J = 30 g SiO2, eluting with CH2Cl2/MeOH (95:5) in the case of (S)-N-
7.0 Hz, 3H); 1.38 (s, 9H). 13C NMR (CDCl3) δ 166.0, 123.1, (L)-Lys(Boc)-OMe lactamide (18); 60 g SiO2, eluting with
120.0, 116.8, 113.6, 83.4, 52.1, 28.4, 19.1. 19F NMR (CDCl3) δ - CH2Cl2/MeOH (95:5) in the case of (S)-lactamide
(19).
74.9. HRMS (EI) m/z; found 277.0591 [M]+ (calcd 277.0596 for Evaporation of the eluates afforded (S)-O-Ms-N-benzyl
C8H14F3NO4S); LRMS (EI) m/z (rel. abundance): 277 [M+] (16), lactamide (15a, 1.05 g, 93%), (S)-O-Ms-N-t-butyl lactamide
262 (100), 222 (44), 177 (20), 112 (42), 69 (90).
(
16a, 253 mg, 94%), (S)-O-Ms-N-phenyl lactamide (17a, 373
mg, 95%) and (S)-O-Ms-N-(L)-Lys(Boc)-OMe lactamide (18a
,
Reaction of (S)-N-benzyl lactamide with TsCl
322 mg, 93%). In the case of (S)-lactamide (19) was the eluate
concentrated to ca. one quarter of its original volume (a
precipitate started to form), hexanes were added and the
mixture was set aside for 2 h at -10 °C. Separated precipitate
was filtered off, was washed with hexanes and was dried to
afford (S)-O-Ms-lactamide (19a, 1.34 g, 80%).
A solution of (S)-N-benzyl lactamide (15, 295 mg, 1.65 mmol)
in dry CH2Cl2 (6 mL) was cooled to 0 °C, followed by the
addition of Et3N (460 μL, 3.29 mmol), DMAP (24 mg, 0.2 mmol)
and TsCl (471 mg, 2.47 mmol). The mixture was stirred for 1 h
at 0 °C, then for 24 h at rt. The solvent was evaporated and
the residue was subjected to FCC on 30 g SiO2, eluting with
hexanes/EtOAc (3:1). Evaporation of the eluate afforded (S)-
O-Ts-N-benzyl lactamide (15c, 450 mg, 82%) as colorless solid.
[α]D -53.9 (c 1, MeOH). 1H NMR (CDCl3) δ 7.77 (d, J = 8.0 Hz,
2H); 7.33 (m, 5H); 7.20 (d, J = 6.5 Hz, 2H); 6.62 (br s, D2O exch,
1H); 4.92 (q, J = 7.0 Hz, 1H); 4.41 (d, J = 6.0 Hz, 2H); 2.46 (s,
3H); 1.47 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3) δ 168.6, 145.4,
137.3, 132.6, 130.0, 128.6, 127.7, 127.4, 77.3, 43.1, 21.6, 18.7.
(S)-O-Ms-N-benzyl lactamide (15a), colorless solid. [α]D -39.8
(c 1, MeOH). 1H NMR (CDCl3) δ 7.34 (m, 5H); 6.64 (br s, D2O
exch, 1H); 5.14 (q, J = 7.0 Hz, 1H); 4.53 (dd, J = 15.0, 6.0 Hz,
1H); 4.47 (dd, J = 15.0, 6.0 Hz, 1H); 3.08 (s, 3H); 1.67 (d, J = 7.0
Hz, 3H). 13C NMR (CDCl3) δ 168.7, 137.4, 128.6, 127.5, 127.4,
76.5, 43.2, 38.4, 18.9. HRMS (EI) m/z; found 258.0806 [M + H]+
(calcd 258.0800 for C11H16NO4S); LRMS (EI) m/z (rel.
6 | J. Name., 2012, 00, 1-3
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abundance): 258 [M+] (10), 161 (62), 133 (23), 106 (44), 91 42%), DOTMAM-t-Bu × 4CF3COO- (67 mg, 24%) or DOTMAM-
(100).
Ph × 4CF3COO- (43 mg, 14%) The residue obtained in the case
of (S)-O-Ms-lactamide (19a) was subjected to FCC on 25 g
(S)-O-Ms-N-t-butyl lactamide (16a), colorless solid. [α]D -68.8 Al2O3, eluting first with CH2Cl2/MeOH/NH4OH (80:19:1, 3
(c 0.5, MeOH). 1H NMR (CDCl3) δ 6.09 (br s, D2O exch, 1H); fractions ca. 30 mL each) later with MeOH (8-10 fractions, ca.
4.95 (q, J = 7.0 Hz, 1H); 3.10 (s, 3H); 1.60 (d, J = 7.0 Hz, 3H); 30 mL each). The later fractions were concentrated to give
1.38 (s, 9H). 13C NMR (CDCl3) δ 167.7, 51.6, 38.8, 28.5, 18.8. DOTMAM (141 mg, 51%).
HRMS (EI) m/z; found 223.0874 [M]+ (calcd 223.0878 for
C8H17NO4S); LRMS (EI) m/z (rel. abundance): 223 [M+] (29), 208 DOTMAM-Bn × 4CF3COO-, colorless hygroscopic solid. HPLC,
(100), 180 (16), 136 (15), 123 (50), 84 (79).
Method B, tR 9.1 min. 1H NMR (D2O) δ 6.82-6.71 (br m, 20H);
4.17-2.47 (br m, 28H); 1.48 (br s, 6H); 0.92 (br s, 6H). HRMS
(S)-O-Ms-N-phenyl lactamide (17a), colorless solid. [α]D -64.6 (ESI) m/z: found 817.5093 [M + H]+ (calcd. 817.5129 for
(c 1, MeOH). 1H NMR (CDCl3) δ 8.13 (br s, D2O exch., 1H); 7.55 C48H65N8O4).
(m, 2H); 7.35 (m, 2H); 7.17 (m, 1H); 5.21 (q, J = 7.0 Hz, 1H);
3.16 (s, 3H); 1.72 (d, J = 7.0 Hz, 3H). 13C NMR (CDCl3) δ 166.6, DOTMAM-t-Bu × 4CF3COO-, colorless hygroscopic solid. HPLC,
136.6, 129.1, 125.2, 120.2, 76.6, 38.9, 18.8. HRMS (EI) m/z; Method C, tR 7.9 min. 1H NMR (D2O) δ 4.12-2.60 (br m, 20H);
found 243.0562 [M]+ (calcd 243.0565 for C10H13NO4S); LRMS 1.49 (br s, 6H); 1.22 (br s, 36H); 1.08 (br s, 6H). HRMS (ESI)
(EI) m/z (rel. abundance): 243 [M+] (83), 120 (100), 92 (43).
m/z: found 681.5723 [M +
H]+ (calcd. 681.5755 for
C36H73N8O4).
(S)-O-Ms-N-(L)-Lys(Boc)-OMe lactamide (18a), pale yellow oil.
[α]D -36.5 (c 1, MeOH). 1H NMR (CDCl3) δ 6.89 (d, D2O exch., J DOTMAM-Ph × 4CF3COO-, colorless hygroscopic solid. HPLC,
= 8.0 Hz, 1H); 5.08 (q, J = 7.0 Hz, 1H); 4.63 (br s, D2O exch., 1H) Method D, tR 7.8 min. 1H NMR (D2O) δ 7.32-6.88 (br m, 20H);
4.58 (m, 1H); 3.74 (s, 3H); 3.14 (s, 3H); 3.10 (m, 2H); 1.90 (m, 4.74-2.66 (br m, 20H); 1.68-1.17 (br m, 12H). HRMS (ESI) m/z:
1H); 1.72 (m, 1H); 1.63 (d, J = 7.0 Hz, 3H); 1.49 (m, 2H); 1.42 (s, found 761.4512 [M + H]+ (calcd. 761.4503 for C44H57N8O4).
9H); 1.35 (m, 2H). 13C NMR (CDCl3) δ 172.2, 168.6, 156.0, 79.1,
76.3, 52.5, 51.8, 40.0, 38.7, 31.8, 29.4, 28.4, 22.3, 19.2. HRMS DOTMAM, colorless solid. 1H NMR (CD3OD) δ 3.63 (q, J = 7.0
(ESI) m/z: found 411.1786 [M + H]+ (calcd. 411.1801 for Hz, 4H); 3.00 (t, J = 13.5 Hz, 4H); 2.82 (t, J = 12.5 Hz, 4H); 2.38
C16H31N2O8S).
(d, J = 14.0 Hz, 4H); 2.15 (d, J = 13.5 Hz, 4H); 1.19 (d, J = 7.0 Hz,
12H). 13C NMR (CD3OD) δ 179.5, 57.3, 47.7, 46.2, 7.6. HRMS
(S)-O-Ms-lactamide (19a), colorless crystals; mp 110-112 ºC. (ESI) m/z: found 457.3230 [M + H]+ (calcd. 457.3251 for
[α]D -50.9 (c 1, MeOH). 1H NMR (CD3OD) δ 5.02 (q, J = 7.0 Hz, C20H41N8O4).
1H); 3.16 (s, 3H); 1.57 (d, J = 7.0 Hz, 3H). 13C NMR (CD3OD) δ
174.6, 77.0, 38.6, 19.6. HRMS (EI) m/z; found 167.0247 [M]+ Coupling of DOTMA · 4Na+ with H-(L)-Lys(Boc)-OMe · HCl and
(calcd 167.0252 for C4H9NO4S); LRMS (EI) m/z (rel. abundance): H-(L)-Phe-OEt•HCl
167 [M+] (10), 123 (56), 109 (12), 65 (29).
A stirred separate suspensions of DOTMA×4Na+ (
4, 82 mg, 0.15
mmol), prepared according to previously described
,
Alkylation of cyclen with (S)-O-Ms-lactamides 15a-19a
procedures37 38 and H-(L)-Lys(Boc)-OMe•HCl (178 mg, 0.6
K2CO3 [320 mg, 2.31 mmol or 767 mg, 5.55 mmol in the case of mmol) or H-(L)-Phe-OEt•HCl (138 mg, 0.6 mmol) in DIPEA (210
(S)-O-Ms-lactamide (19a)] was added to separate solutions of μL, 1.2 mmol) and dry DMF (1 mL) were cooled to 0 °C,
cyclen [
of (S)-O-Ms-lactamide (19a)] and (S)-O-Ms-N-benzyl lactamide mixtures were stirred for 10 min at 0 °C, then for 24 h at 60 °C,
15a, 257 mg, 1 mmol), (S)-O-Ms-N-t-butyl lactamide (16a, 223 then they were cooled to rt and were diluted with brine (50
5, 43 mg, 0.25 mmol or 103 mg, 0.6 mmol in the case followed by the addition of HBTU (228 mg, 0.6 mmol). The
(
mg, 1 mmol), (S)-O-Ms-N-phenyl lactamide (17a, 243 mg, 1 mL), followed by the extraction with EtOAc (2 × 25 mL).
mmol) or (S)-O-Ms-lactamide (19a, 401 mg, 2.4 mmol) in Combined organic extracts were washed with brine (3 × 50
MeCN [1.5 mL or 5 mL in the case of (S)-O-Ms-lactamide mL), were dried and were concentrated. The residues were
(
19a)]. The mixtures were stirred for 48 h at 60 °C, were dissolved in MeOH (3 mL) and were subjected to semi-
cooled to rt, were diluted with brine (40 mL) and were preparative HPLC purification as described in General
extracted with EtOAc (2 × 20 mL). In the case of (S)-O-Ms- experimental considerations. The fractions containing the
lactamide (19a) the solids were filtered off with suction and desired product were combined and concentrated to leave
the filtrate was washed with MeCN. Combined organic DOTMAM-(L)-Lys(Boc)-OMe × 4CF3COO- (117 mg, 41%) or
extracts were dried and were concentrated along with the DOTMAM-(L)-Phe-OEt × 4CF3COO- (195 mg, 81%).
filtrate obtained in the case of (S)-O-Ms-lactamide (19). The
residues obtained after extraction were dissolved in MeOH (3 DOTMAM-(L)-Lys(Boc)-OMe × 4CF3COO-, colorless solid. HPLC,
mL) and were subjected to semi-preparative HPLC purification Method E, tR 7.5 min. 1H NMR (CD3OD) δ 4.78-3.94 (br m, 4H);
as described in General experimental procedures. The 3.76 (br s, 12H); 3.47-2.55 (br m, 24H); 1.86-1.34 (br m, 72H).
fractions containing the desired product were combined and HRMS (ESI) m/z: found 1429.9065 [M + H]+ (calcd. 1429.9133
concentrated to leave DOTMAM-Bn × 4CF3COO- (115 mg, for C68H125N12O20).
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DOTMAM-t-Bu were dissolved in MeOH/water (1.5 mL each)
DOTMAM-(L)-Phe-OEt × 4CF3COO-, pale brown solid. HPLC, and were subjected to semipreparative HPLC purification as
Method E, tR 7.8 min. 1H NMR (CD3OD) δ 7.31-7.02 (br m, 20 described in General experimental procedures. The fractions
H); 5.06 (br m, 4H); 4.46-4.12 (br m, 12H); 3.41-2.31 (br m, containing the desired product were combined, were
24H); 1.48 (br s, 6H); 1.35 (m, 12H); 1.08 (br s, 6H). HRMS (ESI) concentrated, were dissolved in water (1 mL) and were
m/z: found 1161.6594 [M
+
H]+ (calcd. 1161.6600 for neutralized (pH ca. 7.0, 1 M HaOH solution). Resulting
solutions were subjected to SEC as described in General
C64H89N8O12).
experimental procedures. The fractions containing the desired
product were combined and concentrated to leave Tm3+
Deprotection of DOTMAM-(L)-Lys(Boc)-OMe
DOTMAM-Bn (7 8, 7
, 21 mg, 50%) and Tm3+ DOTMAM-t-Bu (
A solution of DOTMAM-(L)-Lys(Boc)-OMe × 4CF3COO- (117 mg,
0.062 mmol) in TFA (1 mL) was stirred for 20 min at rt. The
volatiles were evaporated, the residue was dissolved in MeOH
(600 μL), followed by the addition of NaOH solution (2.5 M, 1
mL, 2.5 mmol). Resulting mixture was stirred for 2 h at 60 ºC,
MeOH was evaporated, the mixture was cooled to 0 °C and the
pH was adjusted to ca. 7 (1 M HCl). Resulting aqueous solution
was subjected to SEC as described in General experimental
procedures. The fractions containing the product were
combined and concentrated to leave DOTMAM-(L)-Lys-OH (50
mg, 83%) as colorless solid. 1H NMR (D2O) δ 4.09 (m, 4H); 3.78
(m, 4H); 3.09-2.88 (br m, 24H); 1.76-1.30 (br m, 36H). HRMS
(ESI) m/z: found 973.6386 [M + H]+ (calcd. 973.6410 for
C44H85N12O12).
mg, 40%). The residues obtained from DOTMAM, DOTMAM-
(L)-Lys-OH and DOTMAM-(L)-Phe-OH were dissolved in water
(2 mL) and were subjected to SEC as described in General
experimental procedures. The fractions containing the desired
product were combined and concentrated to leave Tm3+
DOTMAM (
mg, 78%) and Tm3+ DOTMAM-(L)-Phe-OH (11, 38 mg, 58%).
6
, 32 mg, 49%), Tm3+ DOTMAM-(L)-Lys-OH (10, 49
Tm3+ DOTMAM (
623.2344 [M - 2H]+ (calcd. 623.2358 for C20H38N8O4Tm).
6), colorless solid. HRMS (ESI) m/z: found
Tm3+ DOTMAM-Bn (
7), colorless solid. HPLC, Method C, tR 6.1
min. HRMS (ESI) m/z: found 983.4254 [M - 2H]+ (calcd.
983.4236 for C48H62N8O4Tm).
Saponification of DOTMAM-(L)-Phe-OEt
Tm3+ DOTMAM-t-Bu (
8), colorless solid. HPLC, Method F, tR 6.6
A solution of NaOH (64 mg, 1.6 mmol) in H2O (1 mL) was added
to a solution of DOTMAM-(L)-Phe-OEt × 4CF3COO- (117 mg, 0.1
mmol) in THF (1 mL). The mixture was vigorously stirred for 24
h at 60 °C, was diluted with small amount of H2O (ca. 3 mL)
and was cooled to 0 °C. The pH was adjusted to ca. 4-5 (1 M
HCl), the mixture was set aside for 4 h at 3 °C, the precipitate
was filtered off with suction, was washed with water and was
dried to leave DOTMAM-(L)-Phe-OH (57 mg, 54%) as pale
brown solid. 1H NMR (CD3OD) δ 7.19 (m, 20 H); 4.71 (br m,
4H); 3.58-2.82 (br m, 28H); 1.14 (br m, 12H). HRMS (ESI) m/z:
found 1049.5399 [M + H]+ (calcd. 1049.5348 for C56H73N8O12).
min. HRMS (ESI) m/z: found 847.4824 [M - 2H]+ (calcd.
847.4862 for C36H70N8O4Tm).
Tm3+ DOTMAM-(L)-Lys-OH (10), colorless solid. HRMS (ESI)
m/z: found 1139.5552 [M
-
2H]+ (calcd. 1139.5523 for
C44H82N12O12Tm).
Tm3+ DOTMAM-(L)-Phe-OH (11), colorless solid. HRMS (ESI)
m/z: found 1237.4275 [M - 3H + Na]+ (calcd. 1237.4239 for
C56H69N8O12NaTm).
Metallation of DOTMAM, DOTMAM-Bn, DOTMAM-t-Bu,
Conclusions
DOTMAM-(L)-Lys-OH and DOTMAM-(L)-Phe-OH with TmCl3•H2O
Separate solutions of TmCl3•H2O [34 mg, 0.13 mmol in the In summary, we have developed a new synthetic methodology
case of DOTMAM; 13 mg, 0.047 mmol in the case of and prepared the first examples of DOTMAM ligands
DOTMAM-Bn; 6 mg, 0.021 mmol in the case of DOTMAM-t-Bu; possessing secondary amides including amino acids. This was
18 mg, 0.066 mmol in the case of DOTMAM-(L)-Lys-OH and achieved by tetraalkylation of cyclen with OMs-lactamides as
DOTMAM-(L)-Phe-OH] in water [2 mL in the case of DOTMAM, a key step for the preparation of simple alkyl- and aryl-
DOTMAM-(L)-Lys-OH and DOTMAM-(L)-Phe-OH; 2.5 mL in the decorated ligands, while peptide coupling of DOTMA
case of DOTMAM-Bn; 1 mL in the case of DOTMAM-t-Bu] were tetrasodium salt with protected amino acids furnished amino
added to separate solutions of DOTMAM (46 mg, 0.1 mmol), acid-decorated DOTMAM ligands. Furthermore, the Tm3+
DOTMAM-Bn × 4CF3COO- (54 mg, 0.043 mmol), DOTMAM-t-Bu complexes of these ligands were prepared and their CEST
× 4CF3COO- (22 mg, 0.019 mmol), DOTMAM-(L)-Lys-OH × properties were investigated. Although the CEST effects were
4CF3COO- (54 mg, 0.055 mmol) and DOTMAM-(L)-Phe-OH × more highly shifted than comparable Tm3+DOTAM-based
4CF3COO- (57 mg, 0.055 mmol) in dioxane [2 mL in the case of complexes, the intensity of the CEST effects were observed to
DOTMAM, DOTMAM-(L)-Lys-OH and DOTMAM-(L)-Phe-OH; 2.5 be weaker. This has been ascribed to less favourable (slower)
mL in the case of DOTMAM-Bn; 1 mL in the case of DOTMAM- amide proton exchange rates. The L-lysine conjugated agent
t-Bu]. The mixtures were stirred for 48 h at 90 °C, while the pH 10 displayed temperature- and pH-dependent CEST effects in
was maintained at ca. 6.5-7.0 (1 M NaOH solution). Reaction the physiological range. Based on similarity to the analogous
mixtures were transferred into centrifuge tubes and were DOTAM-based agent, it is expected to have reasonable
lyophilized. The residues obtained from DOTMAM-Bn and biocompatibility39 and may be suitable for future in vivo
8 | J. Name., 2012, 00, 1-3
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studies.9,39 Future work should investigate Eu3+ DOTMAM-alkyl
ARTICLE
decorated complexes as well as a wider selection of Tm3+ 5Suchý, M.; Li, A. X.; Milne, M.; Bartha, R.; Hudson, R. H. E. Contrast
Med. Mol. Imaging 2012, 7, 441ꢁ449.
DOTMAM-amino acid decorated complexes for useful CEST
properties.
6Ratnakar, S. J.; Viswanathan, S.; Kovacs, Z.; Jindal, A. K.; Green, K.
N.; Sherry, A. D. J. Am. Chem. Soc. 2012, 134, 5798ꢁ5800.
Examination of the Yb3+ complexes for DOTMAM, DOTMAM-
Bn and DOTMAM-t-Bu indicated an overwhelming
predominance of a single coordination isomer in solution
although the t-butyl amide appeared different than the other
two. The Tm3+ complexes of these ligands also show the
greatest difference in the chemical shift of the amide-proton
CEST signal. On the basis of the cyclen H4 proton chemical
shift we speculate that the TSAP isomer is most favoured in
this series by the combination of the t-butyl amide substituent
and DOTMAM ligand, although further studies are needed to
elucidate the geometry unambiguously.
7Zhang, S.; Trokowski, R.; Sherry, A. D. J. Am. Chem. Soc. 2003, 125,
15288ꢁ15289.
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Acknowledgements
11Li, A. X.; Hudson, R. H. E.; Barret, J. W.; Jones, C. K.; Pasternak, S.
Financial support from the Canadian Institutes of Health
Research (CIHR) and the Natural Sciences and Engineering
Research Council of Canada (NSERC) is gratefully
acknowledged. We thank Prof. J. M. Chong (University of
Waterloo) for the assistance with optical rotation
measurements.
H.; Bartha, R. Magn. Reson. Med. 2008, 60, 1197ꢁ1206.
12
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‡ DOTAM refers to the tetraamide derivative of DOTA (1,4,7,10- Chem. 2009
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tetraazacyclododecane-1,4,7,10-tetraacetic acid).
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§ The term DOTMA is used to refer to the RRRR-enantiomer of
', ", '''-tetramethyl-1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetate in this manuscript.
α
,
α
α
α
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G.; Woods, M. Inorg. Chem. 2011, 50, 7955ꢁ7965.
§§ The term DOTMAM is used to denote DOTMA-based amides
in analogy to DOTAM which is used to denote DOTA-based
amides. This abbreviation was first used by Merbach and 17 Reviewed by: Suchý, M.; Hudson, R. H. E. Eur. J. Org. Chem. 2008,
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†
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∫ The coupling between DOTMA•4Na+ and benzylamine (7%
yield) was found to be inferior to that obtained by
tetraalkylation of cyclen with OMs-(S)-benzyl lactamide.
19
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¤ The CEST effect associated with CA 11 was too weak to measure
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