Synthesis of DTPA analogues derived from piperidine and azepane: Potential contrast enhancement agents for magnetic resonance imaging

Article abstract of DOI:10.1021/jo0106344

Two DTPA derivatives (PIP-DTPA and AZEP-DTPA) as potential contrast enhancement agents in MRI are synthesized. The T1 and T2 relaxivities of their corresponding Gd(III) complexes are reported. At clinically relevant field strengths, the relaxivities of the complexes are comparable to that of the contrast agent, Gd(DTPA) which is in clinical use. The serum stability of the ¹⁵³Gd-labeled complexes is assessed by measuring the release of ¹⁵³Gd from the ligands. The radiolabeled Gd chelates are found to be kinetically stable in human serum for up to at least 14 days without any measurable loss of radioactivity.

Full text of DOI:10.1021/jo0106344

J . Org. Chem. 2001, 66, 7745-7750
7745
Syn th esis of DTP A An a logu es Der ived fr om P ip er id in e a n d
Azep a n e: P oten tia l Con tr a st En h a n cem en t Agen ts for Ma gn etic
Reson a n ce Im a gin g
Hyun-Soon Chong,† Kayhan Garmestani,† L. Henry Bryant, J r.,‡ and Martin W. Brechbiel*,†
Chemistry Section, National Cancer Institute, and Diagnostic Radiology Department,
Clinical Center, NIH, Bethesda, Maryland
martinwb@mail.nih.gov
Received J une 21, 2001
Two DTPA derivatives (PIP-DTPA and AZEP-DTPA) as potential contrast enhancement agents in
MRI are synthesized. The T1 and T2 relaxivities of their corresponding Gd(III) complexes are
reported. At clinically relevant field strengths, the relaxivities of the complexes are comparable to
that of the contrast agent, Gd(DTPA) which is in clinical use. The serum stability of the 153Gd-
labeled complexes is assessed by measuring the release of 153Gd from the ligands. The radiolabeled
Gd chelates are found to be kinetically stable in human serum for up to at least 14 days without
any measurable loss of radioactivity.
In tr od u ction
derivatives having a rigid cyclohexyl ring have shown
considerable improvement of in vivo stability.6 We have
also found that the stereochemistry of cyclohexyl substi-
tuted DTPA derivatives can provide significant increases
on the in vivo stability of their metal complexes.6e High
lipophilicity is known to result in greater hepatobiliary
clearance. Some DTPA chelates with suitable liphophi-
licity are found to provide significantly enhanced hepa-
tobiliary clearance as compared to the parent DTPA.7
In our continuing effort to develop Gd(III) contrast
agents for MRI,8 DTPA chelates (AZEP-DTPA, 1, and
PIP-DTPA, 2, vide infra) based on either a piperidine ring
or an azepane ring were designed and synthesized. The
liphophilicity and rigidity of these chelates are greater
than those of the parent DTPA. Substitution of either
the piperidine ring or an azepane ring with an amino
group of the DPTA is expected to potentially increase
complex stability and hepatobiliary clearance of the
system in terms of liphophilicity and rigidity.
DTPA (diethylenetriaminepentaacetic acid) and its
derivatives have been used in MRI,1 radiotherapy,2 and
radiodiagnostics.3 In particular, its gadolinium complex,
Gd(DTPA), has been successful as a contrast agent in
MRI and is currently in clinical use along with several
derivatives.1 Although Gd(III) is an optimal paramag-
netic metal for MRI due to high electronic spin and high
relaxation rate, its toxicity produces severe side effects.
Thus, it is required that Gd(III) complexes be stable in
vivo and excreted completely after administration. Re-
search has been focused on the synthesis and physical
characteristics of new Gd(III) complexes to minimize
toxicity while maintaining high relaxation enhancement.4
In general, conformational constraint of chelating
agents, i.e., inclusion of a rigid structure to control the
geometry of the metal binding donor groups may have a
significant effect on the stability of the formed metal
complexes.5 For example, we have reported that DTPA
Herein, we report the synthesis of two DTPA deriva-
tives and relaxometric studies of the corresponding Gd
* To whom correspondence should be addressed. Fax: (301) 402-
1923.
† Chemistry Section, National Cancer Institute.
(5) Fossheim, R.; Dugstad, H.; Dahl, S. G. J . Med. Chem. 1991, 34,
819.
‡ Diagnostic Radiology Department, Clinical Center.
(1) (a) Caravan, P.; Ellison, J . J .; McMurry, T. J .; Lauffer, R. B.
Chem. Rev. 1999, 99, 2293. (b) Lauffer, R. B.; Parmelee, D. J .; Dunham,
S. U.; Ouellet, H. S.; Dolan, R. P.; Witte, S.; McMurry, T. J .; Walovitch,
R. C. Radiology 1998, 207, 529. (c) Aime, S.; Botta, M.; Fasano, M.;
Terreno, E. Chem. Soc. Rev. 1998, 27, 19. (d) Lauffer, R. B. Chem.
Rev. 1987, 87, 901.
(6) (a) McMurry, T. J .; Pippin, C. G.; Wu, C.; Deal, K. A.; Brechbiel,
M. W.; Mirzadeh, S.; Gansow, O. A. J . Med. Chem. 1998, 41, 3546. (b)
Camera, L.; Kinuya, S.; Garmestani, K.; Wu, C.; Brechbiel, M. W.; Pai,
L. H.; McMurry, T. J .; Gansow, O. A.; Pastan, I.; Paik, C. H.;
Carrasquillo, J . A. J . Nucl. Med. 1994, 35, 882. (c) Brechbiel, M. W.;
Gansow, O. A. J . Chem. Soc., Perkin Trans. 1 1992, 1173. (d) Brechbiel,
M. W.; Gansow, O. A. Bioconjugate Chem. 1991, 2, 187. (e) Wu, C.;
Kobayashi, H.; Sun, B.; Yoo, T. M.; Paik, C. H.; Gansow, O. A.;
Carrasquillo, J . A.; Pastan, I.; Brechbiel, M. W. Bioorg. Med. Chem.
1997, 5, 1925.
(2) Volkert, W. A.; Hoffmann, T. J . Chem. Rev. 1999, 99, 2269.
(3) Anderson, C. J .; Welch, M. J . Chem. Rev. 1999, 99, 2219.
(4) (a) Pickersgil, I. F.; Rapoport, H. J . Org. Chem. 2000, 65, 4048.
(b) Aime, S.; Botta, M.; Frullano, L.; Crich, S. G.; Giovenzana, G.;
Pagliarin, R.; Palmisano, G.; Sirtori, F. R.; Sisti, M. J . Med. Chem.
2000, 43, 4017. (c) Lemieux, G. A.; Yarema, K. J .; J acobs, C. L.;
Bertozzi, C. R. J . Am. Chem. Soc. 1999, 121, 4278. (d) Sajiki, H.; Ong,
K. Y.; Nadler, S. T.; Wages, H. E.; McMurry, T. J . Synth. Commun.
1996, 16, 2511. (e) Caufield, T. J .; Guo, P.; Illig, C. R.; Kellar, E.;
Liversidge, J .; Shen, J .; Wellons, J .; Ladd, D.; Peltier, N.; Toner, L.
Bioorg. Med. Chem. Lett. 1995, 5, 1657. (f) Williams, M. A.; Rapport,
H. J . Org. Chem. 1994, 59, 3616. (g) Williams, M. A.; Rapoport, H. J .
Org. Chem. 1993, 58, 1151. (h) Aime, S.; Anelli, P. L.; Botta, M.; Fedeli,
F.; Grandi, M.; Paoli, P.; Uggeri, F. Inorg. Chem. 1992, 31, 2422. (i)
Brucher, E.; Cortes, S.; Chavez, F.; Sherry, A. D. Inorg. Chem. 1991,
30, 2092. (j) Sherry, A. D.; Brown, R. D., III; Geraldes, C. F. C.; Koenig,
S. H.; Kuan, K.-T.; Spiller, M. Inorg. Chem. 1989, 28, 620.
(7) Schumann-Giampieri, G.; Schitt-Willich, H.; Frenzel, T. J . Phar-
macol. Sci. 1993, 799.
(8) (a) Magersta¨dt. M.; Gansow, O. A.; Brechbiel, M. W.; Colcher,
D.; Baltzer, L.; Knop, R. H.; Girton, M. E.; Naegele, M. Magn. Reson.
Med. 1986, 3, 808. (b) Wiener, E. C.; Auteri, F. P.; Chen, J . W.;
Brechbiel, M. W.; Gansow, O. A.; Schneider, D. S.; Belford, R. L.;
Clarkson, R. B.; Lauterbur, P. C. J . Am. Chem. Soc. 1996, 118, 7774.
(c) Bulte J . W. M.; Wu, C.; Brechbiel, M. W.; Brooks, R. A.; Vymazal,
J .; Holla, M.; Frank J . A. Invest. Radiol. 1998, 33, 841. (d) Bryant, L.
H., J r.; Brechbiel, M. W.; Wu, C.; Bulte, J . W. M.; Berynek, V.; Frank,
J . A. J . Magn. Reson. Imag. 1999, 9, 348. (e) Bryant, L. H., J r.;
Yordanov, A. T.; Linnoila, J . J .; Brechibel, M. W.; Frank, J . A. Angew.
Chem., Int. Ed. 2000, 39, 1641.
10.1021/jo0106344 This article not subject to U.S. Copyright. Published 2001 by the American Chemical Society
Published on Web 10/18/2001

7746 J . Org. Chem., Vol. 66, No. 23, 2001
Chong et al.
Sch em e 1
Sch em e 2
complexes. The serum stability of the 153Gd-labeled
complexes was investigated for an understanding of the
in vitro dissociation of the complexes.
strongly indicates that the piperidine moiety in the
starting material 7 has disappeared and the diazide 9
1
does not possess the plane of symmetry as in 7. The H
NMR spectrum of 9 shows a multiplet at δ 3.4 ppm for
the C-6 methine proton. In addition, a doublet at 3.31
ppm for the methylene group at the side chain and a
multiplet at δ 2.89 ppm for the C-7 methylene proton and
the C-2 methine proton were observed, supporting the
structure of the ring-expanded 9. The HRMS produced
a molecular ion peak at m/z 286, which corresponds to
the expected diazide 8, but also supports the structure
of 9 (see the Experimental Section). Ultimately, viscous
diazide 9 was converted to tosylate 11 to confirm struc-
ture and stereochemistry via X-ray analysis (Scheme 2).
Thus, diazide 9 was hydrogenated and subsequently
reacted with TsCl to afford compound 11. The structure
and stereochemistry of the derivative 11 was confirmed
via application of X-ray diffraction methods.12
It seems likely that ring expansion leading to diazide
9 proceeds via an aziridinum intermediate, which further
undergoes regioselective nucleophilic attack of azide
anion at the methine carbon.12 In fact, several reports
on synthesis of a few interesting piperidine or azepane
derivatives via similar ring expansion were made in the
literature.13 However, these reports showed that the ring-
expanded products were routinely obtained along with
the expected products, which were then inseparable by
chromatographic techniques. The result of rearrange-
ment shown herein, to the best of our knowledge, is a
Resu lt a n d Discu ssion
Syn th esis of DTP A Der iva tives 1 a n d 2. The
azepane-backboned DTPA 1 was in fact prepared by a
fortuitous discovery of a rearrangement leading to diazide
9 while in route to prepare triamine 18. The synthetic
route for diazide 9 is summarized in Scheme 1. Com-
mercially available 2,6-pyridinedicarboxylic acid 3 was
used as starting material and converted into cis-2,6-bis-
(methoxycarbonyl)piperidine (4) in 73% yield by following
the reported procedure of Cheˆnevert and Dickman.9
Reaction of 4 with benzyl bromide in the presence of K2-
CO3 produced compound 5 in 94% yield. The base-
promoted reaction of 5 with LiBH4 in THF produced the
N-benzylated diol 610 in 73% yield. The subsequent
reaction of 6 with SOCl2 provided 7,11 which was reacted
with NaN3 in DMSO. However, the latter reaction
provided only the ring-expanded diazide 9 rather than
the expected compound 8. As an initial effort to solve the
1
structure of diazide 9, mass spectra, H, and 13C NMR
spectra were analyzed. The NMR spectra reveal loss of
molecular symmetry, which was expected for desired
diazide 8. In its 13C NMR spectrum, diazide 9 displays
four peaks between δ ) 127.3-138.3 ppm as expected
from the aromatic unit and eight peaks between δ )
22.0-61.2 ppm, two of which are the additional peaks
based on symmetry. A triplet signal at δ 34.8 ppm
(12) The X-ray structure of compound 11 along with extensive
studies of the rearrangement leading to the diazide 9 will be published
elsewhere.
(9) Cheˆnevert, R.; Dickman, M. J . Org. Chem. 1996, 61, 3332.
(10) Schipper, E.; Boehme, W.; Graeme, E. S.; Chinery, E. J . Med.
Pharm. Chem. 1961, 4, 79.
(11) Fehnel, E. A.; Oppenlander, G. C. J . Am. Chem. Soc. 1953, 75,
4660.
(13) (a) Morie, T.; Kato, S.; Harada, H.; Fujiwara, I.; Watanabe, K.;
Matsumoto, J .-I. J . Chem. Soc., Perkin Trans. 1 1994, 2565. (b) Kim,
D.-K.; Kim, G.; Kim, Y.-W. J . Chem. Soc., Perkin Trans. 1 1996, 803.
(c) Brown, G. R.; Foubister, A. J .; Wright, B. J . Chem. Soc., Perkin
Trans. 1 1987, 553.

DTPA Analogues Derived from Piperidine and Azepane
Sch em e 3
J . Org. Chem., Vol. 66, No. 23, 2001 7747
Sch em e 4
unique example affording only the ring-expanded product
with exclusive regioselectivity. Furthermore, this rear-
rangement is of particular value in that the rearranged
product was obtained only as diastereomerically pure cis
isomer and in high yield (92%). This example might
provide a useful entry for the synthesis of precursor
molecules of azepane or piperidine-backboned natural
products14 such as (-)-balanol,14a a fungal metabolite
having potent protein kinase C inhibitory properties.
Our continuing interest in the synthesis of chelating
agents prompted us to prepare the DTPA derivative 1
(AZEP-DTPA) from the rearranged diazide 9, which
contains a DTPA moiety. The procedure used to prepare
1 is shown in Scheme 3. Thus, triamine 12 was obtained
by de-N-benzylation of compound 10, and subsequent
alkylation with tert-butyl bromoacetate provided 13 in
68% yield. The penta-acid 1 as a HCl salt was obtained
after deprotection of the tert-butyl ester (HCl(g)/dioxane).
The synthetic route for DTPA derivative 2 (PIP-DTPA)-
having a piperdine backbone15 is outlined in Scheme 4.
The starting material, cis-2,6-bis(methoxycarbonyl)pip-
eridine (4),9 was directly reduced to 2,6-dimethanolpip-
eridine (14)16 under similar reaction conditions used for
the preparation of 6. To eliminate the possibility of
rearrangement, the free amine of 14 was protected with
a tosyl group. Thus, 14 was treated with TsCl to afford
compound 15, which on reaction with sodium azide
provided the expected diazide 16 in 87% yield. Interest-
ingly, the tosyl-protected compound 15 did not afford any
rearranged diazide contrary to 7 protected by a benzyl
group (see the Experimental Section).
Reductive hydrogenation of diazide 16 afforded 17 in
93% yield and subsequent deprotection of the tosyl group
with concentrated sulfuric acid provided 2,6-bis(aminom-
ethyl)piperidine (18) in high yield. The triamine 18 was
alkylated with tert-butyl bromoacetate to afford 19 in 61%
yield. The tert-butyl group in 19 was removed (HCl(g)/
dioxane) to provide pentaacid 2 in 86% yield.
Ser u m Sta bility. The serum stability of the Gd(III)
complexes was assessed by measuring the release of 153
-
Gd from the chelates (1 and 2). Serum stability of the
153Gd-labeled complexes was evaluated for up to 14 days.
The results thereby obtained are shown in Figure 1. The
two Gd(III) complexes are found to be stable in serum at
least for 14 days without any noticeable loss of radio-
activity. Previously, we reported that, in serum, Gd-
(DTPA) lost 20% of the radioactivity after 5 days (Figure
1).8a Although serum stability is not necessarily an
absolute indicator of in vivo stability, it is noteworthy
that the two novel DTPA derivatives studied herein
display superior stability in serum as compared to the
parent DTPA when coordinated to Gd(III).
Rela xivity. T1 and T2 NMRD profiles for the Gd(III)
complexes are depicted in Figures 2 and 3, respectively.
For comparison, the T1 and T2 relaxivities of the com-
mercially available Gd(DTPA) (Magnevist) are measured.
Qualitatively and quantitatively, the T1 and T2 NMRD
profiles for the Gd(III) complexes Gd(PIP-DTPA) and
Gd(AZEP-DTPA) are similar to those of the clinically
used MR contrast agent GdDTPA (Magnevist) (Figures
2 and 3) and to those of other previously reported Gd
acylic and macrocyclic complexes where the Gd(III) is
(14) (a) Nicolaou, K. C.; Bunnage, M. E.; Koide, K. J . Am. Chem.
Soc. 1994, 116, 8402. (b) Lampe, J . W.; Hughes, P. F.; Biggers, C. K.;
Smith, S. H.; Hu, H. J . Org. Chem. 1994, 59, 5147. (c) Ma, D.; Sun, H.
J . Org. Chem. 2000, 65, 6009.
(15) During the manuscript preparation for this article, we found
that previously, a piperidine-backboned DTPA was synthesized as a
mixture of cis and trans isomers. For synthesis of 2 as a racemic
mixture, see: Gries, H.; Renneke, F.-J .; Weinmann, H.-J . Eur. Pat. 0
250 358 A2, J une 15, 1987.
(16) Kasuga, S.; Taguchi, T. Chem. Pharm. Bull. 1965, 13, 233.

7748 J . Org. Chem., Vol. 66, No. 23, 2001
Chong et al.
to the Gd(III), which could have occurred with dissocia-
tion of the Gd(III) from the chelate in solution, would
have resulted in a significant increase in the measured
relaxivities. No indication of Gd dissociation is evident
from the NMRD profiles, which is in agreement with the
serum stability studies.
Con clu sion
The azepane- and piperidine-backboned DTPA deriva-
tives (1 and 2) for gadolinium contrast agents in MRI
have been synthesized. The azepane-backboned DTPA 1
was prepared by a fortuitous discovery of a rearrange-
ment leading to diazide 9, which was isolated as a single
regioselective isomer and in high yield (92%). The result
of the rearrangement might be applied to synthesis of
precursor molecules for azepane or piperidine-backboned
natural products. The T1 and T2 relaxivities and the
serum stability of the corresponding Gd(III) complexes
are reported. Serum stability studies show that the novel
Gd(III)-labeled complexes are stable in serum for up to
at least 14 days without any measurable loss of radio-
activity, a very promising result in that the clinically used
Gd(DTPA) loses 20% of 153Gd after 5 days. The relaxivites
of two Gd(III) complexes are comparable to that of the
contrast agent, Gd(DTPA) which is in clinical use. The
similar relaxivity to the clinically used MR contrast
agent, Gd(DTPA) (Magnevist) and the exceptional serum
stability indicate that the chelates studied herein may
definitely be effective in vivo MR contrast agents. Further
toxicological evaluations such as biodistribution and
blood clearance studies of these promising MR contrast
agents will be disclosed in the due course.
F igu r e 1. Serum stability of Gd(AZEP-DTPA) (0), Gd(PIP-
a
DTPA) ((), and Gd(DTPA) (2)a at pH 7 and 37 °C. Previous
result in ref 8a.
Additionally, these chelates might be modified for the
covalent attachment to biomolecules (bifunctional chelat-
ing agents) or may be derivatized for noncovalent inter-
action with in-vivo serum proteins for use in MR angiog-
raphy and perfusion. Studies on chelate modifications are
currently being pursued.
F igu r e 2. Plots of T1 relaxivity for Gd(AZEP-DTPA) (0), Gd-
(PIP-DTPA) ((), and Gd(DTPA) (2) at pH 7 and 23 °C.
Exp er im en ta l Section
Gen er a l Meth od s. 1H, 13C, and APT NMR spectra were
obtained using a Varian Gemini 300 instrument, and chemical
shifts are reported in ppm on the δ scale relative to TMS, TSP,
or solvent. Proton chemical shifts are annotated as follows:
ppm (multiplicity, integral, coupling constant (Hz)). Fast atom
bombardment mass spectra (FAB-MS) were obtained on an
Extrel 4000 in the positive ion detection mode. Chromatograms
(SE-HPLC) were obtained on a Dionex isocratic system with
a Waters 717 autosampler, a Gilson 112 UV detector and an
in-line IN/US γ-Ram Model 2 radiodetector. Elemental mi-
croanalyses were performed by Galbraith Laboratories, Knox-
ville, TN. Gd(DTPA) was used as its N-methylglucamine salt,
also known as gadopentate dimeglumine (Magnevist), and was
obtained from Berlex Laboratories, Wayne, NJ . The 153Gd was
obtained from Isotope Products, Valencia, CA. Phosphate
buffered saline (PBS), 0.1 M of pH 7.4 consisted of 0.08 M Na2-
HPO4, 0.02 M KH2PO4, 0.01 M KCl, and 0.14 M NaCl.
Ca u tion : 153Gd (t1/2 ) 241.6 days) is a γ-emitting radionu-
clide. Appropriate shielding and handling protocols should be
in place when using this isotope. Caution: Perchlorate salts
can be explosive and should be handled with care.
F igu r e 3. Plots of T2 relaxivity for Gd(AZEP-DTPA) (0), Gd-
(PIP-DTPA) ((), and Gd(DTPA) (2) at pH 7 and 23 °C.
coordinated to the eight donor atoms of the ligands with
one vacant coordination site available to bind a water
molecule.1a The relaxation rates for the Gd(III) chelates
are dominated by the inner-sphere dipolar coupling
between the coordinated water molecule and the para-
magnetic Gd(III). The measured relaxivities arise from
the exchange between the coordinated water molecule
and the surrounding water molecules in solution. An
increase in the number of coordinated water molecules
N-Ben zyl-cis-2,6-bis(m eth oxyca r bon yl)p ip er id in e (5).
To a solution of 4 (6 g, 29.8 mmol) and K2CO3 (20.6 g, 149
mmol) in CH3CN (100 mL) under argon was added benzyl
bromide (5.1 g, 29.8 mmol), and the resulting mixture was
refluxed for 48 h. The reaction mixture was allowed to cool
gradually to ambient temperature and then filtered and the
filtrate concentrated in vacuo. The residue was purified via

DTPA Analogues Derived from Piperidine and Azepane
J . Org. Chem., Vol. 66, No. 23, 2001 7749
column chromatography on silica gel eluting with 15% EtOAc-
hexane. Pure 5 (8.2 g, 94%) was thereby obtained as a colorless
oil: 1H NMR (CDCl3) δ 1.22-1.41 (m, 2 H), 1.70-1.95 (m, 4
H), 3.20-3.28 (m, 2 H), 3.60 (s, 6 H), 3.87 (s, 2 H), 7.24-7.38
(m, 5 H); 13C NMR (CDCl3) δ 20.4 (t), 28.7 (t), 51.4 (d), 59.0
(t), 62.0 (q), 127.2 (d), 128.0 (d), 128.4 (d), 137.4 (s), 173.4 (s).
Anal. Calcd for C16H21NO4: C, 65.96; H, 7.26. Found: C, 66.01;
H, 7.42.
CH2Cl2-hexane. Pure 16 (1.0 g, 87.0%) was thereby obtained
as a colorless viscous oil: 1H NMR (CDCl3) δ 1.35-1.80 (m, 6
H), 2.53 (s, 3 H). 3.42-3.49 (m, 2 H), 3.59-3.65 (m, 2 H), 4.10-
4.23 (m, 2 H), 7.41 (AB, J AB ) 10.3 Hz, 2 H), 7,81 (AB, J AB
)
10.3 Hz, 2 H); 13C NMR (CDCl3) δ 13.5 (t), 21.4 (q), 24.3 (t),
51.0 (d), 54.3 (t), 126.6 (d), 129.8 (d), 137.3 (s), 145.6 (s). Anal.
Calcd for C14H19N7SO2: C, 48.13; H, 5.48. Found: C, 48.42;
H, 5.63.
Gen er a l P r oced u r e for Red u ction of Ca r ba m a te Di-
ester s 4 a n d 5. A solution of 4 or 5 (28 mmol) in anhydrous
THF (150 mL) under N2 was cooled to 0 °C via application of
an external ice-water bath. To this cooled solution was added
LiBH4 (2.44 g, 111 mmol) portionwise with stirring. The
resulting mixture was stirred at 0 °C for 1 h. The reaction
mixture was allowed to warm gradually to ambient temper-
ature with stirring during 12 h. The reaction mixture was
poured into a stirred mixture of EtOAC (300 mL), 5% NaHCO3
solution (100 mL), and NaHCO3 (10 g) and stirred for 30 min.
After saturation of the aqueous phase with NaCl, the resulting
mixture was extracted with EtOAc (4 × 150 mL). The
combined organic layers were dried (MgSO4) and filtered, and
the filtrate was concentrated in vacuo.
Gen er a l P r oced u r e for Red u ction s of Dia zid es 9 a n d
16. To a solution of 9 or 16 (3.85 mmol) in CH3OH (20 mL)
was added 10% Pd/C catalyst (100 mg). The resulting mixture
was subjected to hydrogenolysis by agitation with excess H2-
(g) at 25 psi in a Parr hydrogenator appartus at ambient
temperature for 3 h. The reaction mixture was filtered through
Celite, and the filtrate was concentrated in vacuo.
cis-7-Am in om eth yl-1-ben zyla zep a n -3-yla m in e (10). The
residue was purified via column chromatography on neutral
alumina eluting with 15% CH3OH-EtOAc. Pure 10 (830 mg,
98%) was thereby obtained as a colorless oil: 1H NMR (CDCl3)
δ 1.20-2.15 (m, 8 H), 2,60-3.25 (m, 8 H), 3.95 (AB, J AB ) 15.2
Hz, 1 H), 4.10 (AB, J AB ) 15.2 Hz, 1 H), 7.35-7.52 (m, 5 H);
13C NMR (CDCl3) δ 22.6 (t), 32.0 (t), 39.4 (t), 45.8 (t), 48.4 (d),
55.9 (t), 59.5 (t), 65.0 (d), 126.8 (d), 128.2 (d), 128.5 (d), 140.5
N-Ben zyl-cis-2,6-bis(h ydr oxym eth yl)piper idin e (6). The
residue was purified via column chromatography on silica gel
by eluting with 50% EtOAc-hexane. Pure 6 (4.8 g, 73%) was
(s). Anal. Calcd for
C14H23N3(H2O)0.5: C, 70.69; H, 9.96.
Found: C, 70.45, H, 9.90.
1
thereby obtained as a colorless viscous oil. The IR, H NMR,
N-Tosyl-cis-2,6-bis(a m in om eth yl)p ip er id in e (17). The
residue was purified via column chromatography on neutral
alumina eluting with 15% CH3OH-EtOAc. Pure 17 (630 mg,
93%) was thereby obtained as a colorless oil: 1H NMR (CDCl3)
δ 1.10-1.62 (m, 6 H), 2.28 (s, 4 H), 2.44 (s, 3 H), 2.80-3.10
(m, 4 H), 4.00-4.14 (m, 2 H), 7.13 (AB, J AB ) 7.8 Hz, 2 H),
7.25 (AB, J AB ) 7.8 Hz, 2 H); 13C NMR (CDCl3) δ 13.9 (t), 21.1
(q), 24.7 (t), 45.0 (t), 54.2 (d), 126.2 (d), 129.5 (d), 138.1 (s),
142.8 (s). Anal. Calcd for C14H23N3SO2(H2O)0.5: C, 54.88; H,
7.89. Found: C, 54.52; H, 7.82.
and 13C NMR spectra of the material thereby obtained were
essentially identical to data reported previously for authentic
6.10
cis-2,6-Bis(h yd r oxym eth yl)p ip er id in e (14). The residue
was purified via column chromatography on silica gel by
eluting with 50% MeOH-CH2Cl2. Pure 14 (1.87 g, 46%) was
1
thereby obtained as a colorless viscous oil. The IR, H NMR,
and 13C NMR spectra of the material thereby obtained were
essentially identical to data reported previously for authentic
14.16
cis-N-(1-Ben zyl-6-t olu en esu lfon yla m in oa zep a n -2-yl-
m eth yl)tolu en esu lfon a m id e (11). To a solution of 10 (260
mg, 1.11 mmol) and sodium hydroxide (140 mg, 3.5 mmol) in
H2O (2 mL) was added dropwise a solution of TsCl (637 mg,
3.34 mmol) in diethyl ether (5 mL). The reaction mixture was
stirred for 3 h at ambient temperature, at which time the
reaction mixture was diluted with diethyl ether (20 mL) and
washed with H2O (10 mL). The organic layer was dried
(MgSO4) and filtered, and the filtrate was concentrated in
vacuo. The residue was purified via column chromatography
on silica gel by eluting with 30% EtOAc-hexane. Pure 11 (518
mg, 86%) was thereby obtained as a colorless solid and
recrystallized from CH2Cl2/hexane: 1H NMR (CDCl3) δ 1.50-
1.80 (m, 6 H), 2.45 (s, 3 H), 2.53 (s, 3 H), 2.78-3.37 (m, 6 H),
3.56 (AB, J AB ) 12.4 Hz, 1 H), 3.75 (AB, J AB ) 12.4 Hz, 2 H),
5.34 (d, J ) 8.3 Hz, 2 H), 7.16-7.46 (m, 11 H), 8.75 (AB, J AB
) 10.3 Hz, 1 H); 13C NMR (CDCl3) δ 19.5 (t), 21.36 (q), 21.41
(q), 30.0 (t), 36.6 (t), 45.3 (t), 51.0 (d), 52.7 (t), 58.4 (t), 61.4
(d), 126.8 (d), 126.9 (d), 127.3 (d), 128.7 (d), 128.8 (d), 129.4
(d), 129.6 (d), 136.7 (s), 137.3 (s), 139.3 (s), 142.8 (s), 143.2 (s).
Anal. Calcd for C28H35N3S2O4: C, 62.08; H, 6.51. Found: C,
62.00, H, 6.70.
cis-7-Am in om eth yla zep a n -3-yla m in e (12). To a solution
of 10 (900 mg, 3.9 mmol) in CH3OH (10 mL) was added 10%
Pd/C catalyst (150 mg). The resulting mixture was subjected
to hydrogenated by agitation with excess H2(g) at 60 psi in a
Parr hydrogenation apparatus at ambient temperature for 72
h. The reaction mixture was filtered through Celite, and the
filtrate was concentrated in vacuo. The residue was purified
via column chromatography on neutral alumina eluting with
30% CH3OH-EtOAc. Pure 12 (528 mg, 94%) was thereby
obtained as a colorless oil: 1H NMR (CD3OD) δ 1.21-1.53 (m,
4 H), 1.67-2.04 (m, 4 H), 2.44-2.90 (m, 6 H), 3.31-3.38 (m, 3
H); 13C NMR (CD3OD) δ 23.4 (t), 35.4 (t), 38.2 (t), 48.2 (t), 53.8
(t), 54.0 (d), 61.8 (t); HRMS (positive ion FAB) calcd for
C7H15N3 Mr+ m/z 144.1501, found Mr+ m/z 144.1490.
Gen er a l P r oced u r e for Alk yla tion of Tr ia m in es 12 a n d
18. To a suspension of 12 or 18 (2.1 mmol) and K2CO3 (1.45 g,
10.5 mmol) in CH3CN (25 mL) under argon was added
dropwise tert-butyl bromoacetate (4.08 g, 20.95 mmol), and the
N-Ben zyl-cis-2,6-bis(ch lor om eth yl)p ip er id in e (7). A so-
lution of 6 (2.8 g, 11.9 mmol) in dry benzene (30 mL) was
saturated with HCl(g) at 0 °C. After addition of thionyl chloride
(5 mL), the mixture was heated at 60 °C for 3 h. The cooled
reaction mixture was concentrated and neutralized with 5%
Na2CO3 solution. The resulting mixture was extracted with
CH2Cl2 (3 × 50 mL), the combined organic layers were dried
(MgSO4) and filtered, and the filtrate was concentrated in
vacuo to afford crude 7 (2.62 g, 81%): 1H NMR (CDCl3) δ 1.45-
2.03 (m, 6 H), 3.00-3.12 (m, 2 H), 3.38 (t, J AB ) 11.3 Hz, 2 H),
3.63 (d, J AB ) 9.4 Hz, 2 H), 3.98 (s, 2 H), 7.34-7.50 (m, 5 H);
13C NMR (CDCl3) δ 18.2 (t), 26.5 (t), 45.7 (t), 56.4 (d), 61.2 (t),
126.6 (d), 127.0 (d), 128.0 (d), 140.0 (s). The crude product was
used directly in the next step. The IR, 1H NMR, and 13C NMR
spectra of 7 as a HCl salt were essentially identical to data
reported previously.10
Gen er a l P r oced u r e for th e Rea ction w ith Sod iu m
Azid e of 7 a n d 15. A mixture of 7 or 15 (3.3 mmol) and NaN3
(7.6 mmol) in DMSO (5 mL) was heated to 90 °C for 4 h. The
resulting mixture was poured into ice-water and extracted
with Et2O (2 × 30 mL). The combined organic layers were
washed with H2O (3 × 15 mL), dried (MgSO4), and filtered.
The filtrate was concentrated in vacuo.
cis-6-Azid o-2-a zid om et h yl-1-b en zyla zep a n e (9). The
crude product could be used for the next step or could be
purified via column chromatography on basic alumina eluting
with 10% EtOAc-hexane. Pure 9 was thereby obtained as a
colorless viscous oil (866 mg, 92%): 1H NMR (CDCl3) δ 1.38-
1.76 (m, 3 H), 1.84-2.15 (m, 3 H), 2.89-3.18 (m, 3 H), 3.31 (d,
2 H), 3.4-3.53 (m, 1 H), 3.98 (AB, J AB ) 15.5 Hz, 1 H), 4.11
(AB, J AB ) 15.5 Hz, 1 H), 7.38-7.51 (m, 5 H); 13C NMR (CDCl3)
δ 22.0 (t), 31.8 (t), 34.8 (t), 51.6 (t), 54.3 (t), 59.2 (t), 59.3 (d),
61.2 (d), 127.3 (d), 128.4 (d), 128.6 (d), 139.3 (s); IR (film) ν
3066, 3032, 2943, 2862, 2094, 1735, 1453, 1265, 739, 701 cm-1
;
MS (CI/NH3) m/z 286 [Mr + H]. Anal. Calcd for C14H19N7: C,
58.93; H, 6.71. Found: C, 59.30; H, 6.96.
N-Tosyl-cis-2,6-bis(a zid om eth yl)p ip er id in e (16). The
crude product could be used directly for thenext step or purified
via column chromatography on basic alumina eluting with 30%

7750 J . Org. Chem., Vol. 66, No. 23, 2001
Chong et al.
resulting mixture was heated at 65 °C for 24 h. The reaction
mixture was allowed to cool gradually to ambient temperature
and after being filtered, and the filtrate was concentrated in
vacuo.
J AB ) 8.6 Hz, 2 H), 7.44 (AB, J AB ) 8.6 Hz, 2 H), 7.65 (AB, J AB
) 8.6 Hz, 2 H), 7.86 (AB, J AB ) 8.6 Hz, 2 H); 13C NMR (CDCl3)
δ 13.3 (t), 21.3 (q), 21.4 (q), 24.3 (t), 49.6 (d), 70.2 (t), 126.4
(d), 127.6 (d), 129.7 (d), 129.8 (d), 132.1 (s), 136.5 (s), 143.7
(s), 145.0 (s). Anal. Calcd for C28H33NS3O8: C, 55.34; H, 5.47.
Found: C, 55.43; H, 5.80.
cis-[6-(Bis-ter t-b u t oxyca r b on ylm et h yla m in o)-2-[(b is-
ter t-bu toxyca r bon ylm eth yla m in o)m eth yl]a zep a n -1-yl]-
a cetic Acid ter t-Bu tyl Ester (13). The residue was purified
via column chromatography on silica gel eluting with 10%
EtOAc-hexane. Pure 13 (1.02 g, 68%) was thereby obtained
as a colorless oil: 1H NMR (CDCl3) δ 1.29-1.97 (m, 45 H),
2.03-2.30 (m, 4 H), 2.52-2.60 (m, 2 H), 2.50-3.10 (m, 6 H),
3.35-3.75 (m, 10 H); 13C NMR (CDCl3) δ 23.0 (t), 27.8 (q), 27.9
(q), 27.9 (q), 32.0 (t), 33.1 (t), 52.3 (t), 53.2 (t), 56.6 (t), 57.7 (t),
58.2 (t), 60.9 (d), 61.0 (d), 80.0 (s), 80.2 (s), 80.4 (s), 170.6 (s),
171.4 (s), 171.8 (s). Anal. Calcd for C37H67N3O10: C, 62.25; H,
9.46. Found: C, 62.04; H, 9.62.
cis-2,6-Bis[N,N-b is(ter t-b u t oxyca r b on ylm et h yl)a m i-
n om eth yl]-1-p ip er id in ea cetic Acid ter t-Bu tyl Ester (19).
The residue was purified via column chromatography on silica
gel eluting with 20% EtOAc-hexane. Pure 19 (913 mg, 61%)
was thereby obtained as a colorless oil: 1H NMR (CDCl3) δ
1.20-1.95 (m, 45 H), 2.52-2.65 (m, 4 H), 2.76-3.07 (m, 8 H),
3.45 (s, 8 H), 3.65 (s, 2 H); 13C NMR (CDCl3) δ 23.2 (t), 27.8
(q), 30.0 (t), 51.3 (t), 56.2 (t), 58.9 (t), 59.0 (d), 79.7 (s), 80.3
(s), 170.2 (s), 171.9 (s). Anal. Calcd for C37H67N3O10: C, 62.25;
H, 9.46. Found: C, 61.87; H, 9.27.
Gen er a l P r oced u r e for Dep r otection of ter t-Bu tyl
Ester in 13 a n d 19. A solution of 13 or 19 (0.21 mmol) in dry
1,4-dioxane (10 mL) in an ice-water bath was saturated with
HCl for 4 h, at which time the mixture was allowed to ambient
temperature and then was stirred for 18 h. The precipitate
was collected and washed with ethyl ether. The collected solid
was dissolved in water immediately and lyophilized.
cis-[2,7-Bis[bisca r boxym eth yla m in o)m eth yl]a zep a n -1-
yl]a cetic Acid (1). Pure 1 (90 mg, 79%) was obtained as a
salt: 1H NMR (D2O, pD 1, CD3OD as reference) δ 1.05-1.75
(m, 3H), 1.86-2.22 (m, 3 H), 2.90-3.76 (m, 8 H), 4.02-4.18
(m, 8 H), 4.75-5.03 (m, 5 H); 13C NMR (D2O, pD 1, CD3OD as
reference) δ 27.9 (t), 30.4 (t), 45.8 (t), 52.7 (t), 53.9 (t), 54.8 (t),
55.3 (t), 58.7 (d) 63.0 (d), 65.9 (t), 171.5 (s), 171.4 (s), 179.6 (s).
Anal. Calcd for C17H30N3O10Cl3(H2O)2: C, 35.28; H, 5.92; N,
7.26. Found: C, 35.66; H, 6.03; N, 7.08.
cis-2,6-Bis[N,N-bis(ca r boxym eth yl)a m in om eth yl]-1-p i-
p er id in ea cetic Acid (2). Pure 2 (83 mg, 86%) was obtained
as a salt: 1H NMR (D2O, pD 1, CD3OD as reference) δ 1.15-
1.57 (m, 6 H), 3.03-3.42 (m, 8 H), 3.50-3.84 (m, 13 H); 13C
NMR (D2O, pD 1, CD3OD as reference) δ 21.0 (t), 21.5 (t), 42.7
(t), 55.4 (t), 55.5 (t), 61.1 (d), 171.0 (s), 171.5 (s). Anal. Calcd
for C17H30N3O10Cl3(H2O)2: C, 35.28; H, 5.92; N, 7.26. Found:
C, 35.36; H, 6.33; N, 7.15.
cis-2,6-Bis(a m in om eth yl)p ip er id in e (18). N-Tosyl-2,6-
diaminomethylpiperidine (17, 700 mg, 2.35 mmol) was dis-
solved in concentrated H2SO4 (10 mL) and heated to 115 °C
for 72 h under argon. The resulting solution was cooled to
ambient temperature and added in portions to Et2O (150 mL)
at -60 °C. The resulting precipitate was collected, washed with
Et2O (20 mL), and immediately dissolved in H2O (25 mL). The
aqueous solution was extracted with Et2O (10 mL), concen-
trated to 5 mL, and neutralized with 50% NaOH. The resulting
mixture was extracted with CH2Cl2 (3 × 50 mL), the combined
organic layers were dried (MgSO4) and filtered, and the filtrate
was concentrated in vacuo to afford 18 (292 mg, 87.0%): 1H
NMR (CDCl3) δ 1.08-1.75 (m, 6 H), 2.21-2.62 (m, 11 H);13C
NMR (CDCl3) δ 24.0 (t), 30.0 (t), 47.8 (t), 58.6 (d). HRMS
(Positive ion FAB) calcd for C7H17N3 Mr+ m/z 144.1501, found
+
Mr m/z 144.1498.
Mea su r em en ts of Ser u m Sta bility. The 153Gd complexes
of 1 and 2 were prepared by the addition of 350 µCi of 153Gd
(0.1 M HCl adjusting the pH to 4.5 with 5 M NH4OAc) to 20
µL of 0.2 M ligand solution in 0.15 M NH4OAC of pH 4.5. The
reactions went to completion after the reaction mixture was
heated at 80 °C for 18 h and were then loaded onto a column
of Chelex-100 resin (1 mL volume bed, equilibrated with 0.15
M NH4OAc). The complexes were eluted from the resin with
0.15 M NH4OAc while the resin retained free 153Gd. The pH
of 153Gd complex solutions was adjusted to 7 with PBS buffer,
and 250 µCi of the complex was added to 1 mL of human serum
which was incubated at 37 °C. A aliquot of the serum (5-10
µL) was taken at selected times (Figure 1) and analyzed by
SE-HPLC. The serum stability of the 153Gd complexes was
assessed by measuring the release of 153Gd radionuclide from
the complexes to serum proteins using SE-HPLC with TSK
3000 column. The column was eluted with PBS at 1 mL/min
flow rate.
Mea su r em en ts of Rela xivity. NMR dispersion measure-
ments were made on a custom-designed variable field T1-T2
analyzer (Southwest Research Institute, San Antonio, TX) at
23 °C. The magnetic field was varied from 0.02 to 1.5 T
(corresponding to a proton larmor frequency of 1-64 MHz).
T1 was measured by using a saturation recovery pulse
sequence with 32 incremental recovery times. The relaxivities
(relaxation rates per mM Gd(III) concentration) were obtained
after subtracting the water contribution. The commercial Gd-
(DTPA) (Magnevist) was used without further modifications.
The other Gd chelates were prepared by dissolving the
appropriate ligand and GdCl3 in water with stirring at a 1:0.9
mole ratio with the pH adjusted to 6.5 with 1M NaOH. The
total Gd concentration for each sample was determined by ICP-
AAS (Desert Analytics, Tuscon, AZ).
N-Tosyl-cis-2,6-bis(m eth yltosyl)p ip er id in e (15). A solu-
tion of 14 (900 mg. 6.2 mmol) in CH2Cl2 (3 mL) under nitrogen
was cooled to 0 °C via application of an external ice-water
bath. To this cooled solution were added sequentially Et3N
(1.88 g, 18.6 mmol) and a solution of TsCl (3.55 g, 18.6 mmol)
in CH2Cl2 (4 mL). The resulting mixture was allowed to warm
gradually to ambient temperature with stirring during 18 h.
The reaction mixture was diluted with CH2Cl2 and washed
with H2O and with saturated NaCl solution. The organic layer
was dried (MgSO4) and filtered and the filtrate concentrated
in vacuo. The residue was purified via column chromatography
on silica gel eluting with 20% EtOAc-hexane. Pure 15 (1.36
g, 36%) was thereby obtained as a colorless viscous oil: 1H
NMR (CDCl3) δ 1.18-1.35 (m, 6 H), 2.70 (d, J ) 13.2 Hz, 2
H). 2.46 (s, 3 H), 2.51 (s, 6 H), 4.01-4.18 (m, 8 H), 7.32 (AB,
Ack n ow led gm en t. We thank the structural Mass
Spectra Group (Dr. L. Pannell, NIDDK, Bethesda, MD)
for obtaining the mass spectra of 12 and 18.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for compound 9. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O0106344