Glucosamine conjugates bearing N,N,O-donors: Potential imaging agents utilizing the [M(CO)3]+ core (M = Re, Tc)

Article abstract of DOI:10.1039/b914310f

The design rationale, synthesis and radiolabeling evaluation of four glucosamine conjugated ligands for the [^99mTc(CO)₃] ⁺ core is described. The capability to bind the tricarbonyl core is initially demonstrated using the cold surrogate [Re(CO)₃] ⁺. The four compounds are competent chelates in binding [ ^99mTc(CO)₃]⁺ as labeling studies show, with yields ranging from 79 to 96% and the resulting complexes showing stability in the presence of competing chelates for 24 h at 37 °C. The rhenium complexes were tested for hexokinase-catalysed phosphorylation, and the technetium complexes were tested for GLUT-1 mediated cell uptake - they showed a small amount of uptake but it was not glucose dependent, suggesting that it was not via the GLUT-1 transporters.

Full text of DOI:10.1039/b914310f

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PAPER
www.rsc.org/dalton | Dalton Transactions
Glucosamine conjugates bearing N,N,O-donors: potential imaging agents
utilizing the [M(CO)3]+ core (M = Re, Tc)†
Meryn L. Bowen,a Nathaniel C. Lim,a,b Charles B. Ewart,a,b Ripen Misri,c Cara L. Ferreira,a,b Urs Ha¨feli,c
Michael J. Adam*b and Chris Orvig*a
Received 16th July 2009, Accepted 4th September 2009
First published as an Advance Article on the web 25th September 2009
DOI: 10.1039/b914310f
The design rationale, synthesis and radiolabeling evaluation of four glucosamine conjugated ligands for
the [99mTc(CO)3]+ core is described. The capability to bind the tricarbonyl core is initially demonstrated
using the cold surrogate [Re(CO)3]+. The four compounds are competent chelates in binding
[
99mTc(CO)3]+ as labeling studies show, with yields ranging from 79 to 96% and the resulting complexes
showing stability in the presence of competing chelates for 24 h at 37 ◦C. The rhenium complexes were
tested for hexokinase-catalysed phosphorylation, and the technetium complexes were tested for
GLUT-1 mediated cell uptake - they showed a small amount of uptake but it was not glucose
dependent, suggesting that it was not via the GLUT-1 transporters.
is generator produced and has near ideal physical properties (t1/2
=
6 h, g = 143 keV), making it both readily available and suitable
for imaging. Additionally, the similarities between technetium’s
chemistry and bulk properties, and those of its third row congener
rhenium provide both an analogue for macroscopic chemistry
Introduction
Carbohydrates are the principal energy source of the body’s cells,
tissues and organs. Since carbohydrates are broken down extra-
cellularly into monosaccharides which are too polar to passively
diffuse through cell membranes, monosaccharide transporters
(collectively called glucose transporters or GLUTs) facilitate
monosaccharide membrane crossing.1,2 This family of transporters
is also important in the success of FDG (2-deoxy-2-[18F]-D-
glucose) as a positron emission tomography (PET) agent in
oncology. FDG is taken up into cells by way of the GLUTs,
and is then phosphorylated by the enzyme hexokinase.3,4 Once
phosphorylated, FDG becomes trapped in cells because it is not
a good substrate for any other cellular enzyme, and is negatively
charged, meaning it cannot diffuse out of the cell. Phosphorylated
FDG levels build up in cells with high concentrations of GLUTs
and hexokinase, such as cancer cells. This, combined with the
elevated glucose metabolism of many tumor cells,5 leads to a larger
amount of FDG being trapped in tumor cells compared to normal
cells, giving a good tumor : background ratio and therefore good
contrast in the PET images obtained.
FDG, however, has logistic and economic drawbacks associated
with its radioisotope, 18F. 18F is cyclotron produced and has a
relatively short half-life (t1/2 = 110 min), significantly limiting its
availability. Because of this there is interest in developing “FDG-
like” radiopharmaceuticals that are more economical and read-
ily accessible. Single Photon Emission Computed Tomography
(SPECT) imaging, utilizing 99mTc in various chemical forms, is
used in over 90% of nuclear medicine scans.6 This is because 99mTc
(
185/187Re) and the possibility of a therapeutic radiopharmaceutical
based on the same molecular scaffold (186Re t1/2 = 3.68 d, b =
1.07 MeV, g = 137 keV; 188Re t1/2 = 16.98 h, b = 2.12 MeV, g =
155 keV). Unlike 123I or 18F which can be directly substituted for
a hydrogen, carbon, nitrogen or oxygen atom in a biomolecule,
technetium and rhenium are metals, and as such normally need
to be chelated and “masked” before being utilized. The discovery
of an easy synthesis of tricarbonyl technetium(I) by Alberto and
coworkers7 introduced a particularly useful radionuclide core for
this type of work. This core is small, stable and inert and the three
aqua ligands present following the kit preparation can easily be
substituted for the desired chelate. Once formed these technetium
tricarbonyl complexes are inert and stable, and the metal binding
sphere relatively small, which is advantageous when appending
biomolecules, for them to have the best chance of being recognized
as the parent molecule in vivo, as is often the goal in this kind of
work.
The general design for technetium-tagged tracers normally
comprises a biomolecule responsible for the compound’s efficacy
and targeting, a spacer group between the biomolecule and the
metal binding moiety, a chemical tether which will allow simple
conjugation to the biomolecule and a strong chelator as the metal
binding pocket. Many biomolecules have been labeled in this
general way,8 including proteins9 and small molecules10,11 as well
as carbohydrates.12-16 In this work, we have investigated the use of
glucosamine as the biomolecule, as there is evidence describing its
in vivo role in the transport and accumulation of compounds in
tumors, as glucosamine conjugates have been reported to be both
transported by GLUT transporters17,18 and phosphorylated by
hexokinase.17,19 The primary amine of glucosamine lends itself to
functionalization, and we have chosen to do this by conjugation to
tridentate monoprotic proligands. We are particularly interested in
assessing the system compatibility of a donor system we reported
aMedicinal Inorganic Chemistry Group, Department of Chemistry, The
University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1,
Canada. E-mail: orvig@chem.ubc.ca; Fax: +01 604 822 2847; Tel: +01 604
822 4449
bTRIUMF, 4004 Wesbrook Mall, Vancouver, BC, V6T 2A3, Canada.
E-mail: adam@triumf.ca
cFaculty of Pharmaceutical Sciences, The University of British Columbia,
2146 East Mall, Vancouver, BC, V6T 1Z3, Canada
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of selected compounds. See DOI: 10.1039/b914310f
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earlier,20 which bears a pyridyl-tert-nitrogen-phenol moiety, and is
capable of forming stable neutral complexes with [99mTc(CO)3]+.20
The versatility of our approach renders itself useful for further
evaluation of these types of systems. Our synthetic results are
presented here, along with hexokinase catalysed phosphorylation
and GLUT-mediated cell uptake studies.
Reductive amination of 1 with 14 resulted in the fully protected
compound 15. Selective acetate deprotection with hydrazine
acetate yielded the free C1-OH compound 16. Silyl protection
of 16 with TBDMS-Cl resulted in protected compound 17. The
changing of the protecting group at C1 of glucosamine was
necessary as initial investigations using the C1-acetyl protected
compound resulted in side reactions in the succeeding step
and failure to furnish the desired product. Deprotection of the
remaining acetyl protected alcohols was achieved by addition
of NaOMe, yielding 18 which was further deprotected by H2
using Pd(OH)2/C as a catalyst to yield amine derivative 19.
Sequential reductive aminations with 2-pyridinecarboxaldehyde
and salicylaldehyde yielded C1 protected proligand 21. Final
silyl deprotection of 21 with benzyltrimethylammonium fluoride
afforded proligand 22.
Results and discussion
Tridentate proligands have been reported to form more stable
complexes with the [99mTc(CO)3]+ core than bidentate proligands
with equivalent donor groups, and they also possess higher
stability in vitro and better in vivo clearance as compared to their
bidentate counterparts.21,22 Combining these findings with our in-
terest in utilizing glucosamine as a biomolecule, we have designed
and synthesized a series of monoanionic tridentate proligands
conjugated to glucosamine for binding to the tricarbonyl core.
Reaction of proligands 6, 8, 11 and 22 with [Re(H2O)3(CO)3][Br]
in refluxing ethanol afforded macroscopic quantities the non-
radioactive rhenium surrogate compounds 6-Re(CO)3, 8-Re(CO)3,
11-Re(CO)3 and 22-Re(CO)3 (Scheme 3). The “cold” complexes
have spectroscopic properties that confirm their expected struc-
tures.
Synthesis
Amide coupling of 1 with N,N-dibenzylglycine using the water
soluble carbodiimide, EDC, afforded the fully protected com-
pound 2 (Scheme 1). Hydrogenolysis of the benzyl groups using
hydrogen and palladium hydroxide on carbon as a catalyst with
acetic acid as a solvent gave the acetate salt 3. The ratio of
acetic acid in the product differed from batch to batch such that
before utilizing a particular batch for the succeeding step, the
As a representative example, 6-Re(CO)3 showed significant
1
shifts in both its H and 13C NMR spectra upon metal binding,
compared to the spectra of proligand 6 (see Experimental section
and ESI†). The 1H NMR shifts due to the carbohydrate resonances
were unchanged or showed very small shifts, confirming the
1
pendant nature of the glucosamine moiety in solution. The H
1
amount of acetic acid was quantified by H NMR spectroscopy.
NMR shifts of the aromatic resonances are indicative of metal
binding, with the proton resonances all shifting significantly.
In the spectra of 6-Re(CO)3 the shifts of the aromatic protons
upon metal binding were: H13 (8.49 to 8.84 ppm), H11 (7.77 to
8.07 ppm), H2 (7.50 to 7.70 ppm), H10/12 (7.13 to 7.53 ppm),
H4 (7.28 to 7.35 ppm), and H3/5 (6.79 to 6.99 ppm). The same
qualitative trend was also observed for 19-Re(CO)3, whereas for
8-Re(CO)3 two of the aromatic protons shift upfield and two shift
downfield, and for 11-Re(CO)3 all the aromatic protons bar one
shift downfield, while the proton on the imidazole ring adjacent
to the coordinating nitrogen shifted upfield.
Sufficient base (sodium carbonate) to neutralize this acid was
added prior to addition of the appropriate aldehyde. 2-Pyridine
carboxaldehyde or 1-methylimidazolecarboxaldehyde was added,
and the corresponding imine formed and subsequently reduced to
give the secondary amines 4 and 9. Compound 4 was then used
to prepare protected proligands 5 and 7 by reductive amination
with salicylaldehyde and alkylation with methylbromoacetate,
respectively. Compound 9 was used to prepare protected proligand
10 by reductive amination with salicylaldehyde. Proligands 6
and 11 were prepared in one step each by deacetylation of the
acetyl protecting groups of 5 and 10 respectively with sodium
methoxide. Proligand 8 was prepared in one step by addition
of sodium hydroxide to 7 which deprotected both the methyl
and the acetyl groups. These compounds displayed the expected
spectroscopic properties, as analysed by infrared, 1H and 13C NMR
spectroscopies and mass spectrometry, confirming their assigned
structure. The building block approach to making these conjugates
is manifested by easy modification of the binding motif donor sets
as well as capability to change the length of the spacer between
the amide functionality and the binding motif, and lends itself to
extension to making more derivatives for SAR studies.
The 13C NMR spectra (ESI†) are indicative of the expected
coordination mode as well. The carbon resonances of the sugar
regions shift very little, confirming that the glucosamine moiety
remains pendant. The 13C NMR spectrum of the ligand and the
rhenium complex resulted in two sets of similar, yet distinct,
resonances for the carbohydrate and spacer moieties, due to
the presence of two anomers. As there is splitting in both the
proligands and the complexes, it is likely that the observed splitting
is due to the presence of anomers in both cases, rather than from
the presence of D and K metal complex isomers. As the donor set
is composed of three distinct donors, the complexes formed can
be in either an A-B-C or a C-B-A fac arrangement, and although
it is probable that both of these isomers form, it seems that they
In the course of our study, we were also interested in investigat-
ing the effect of different connectivity between the glucosamine
and the binding motif on their possible “glucose-like” uptake
mechanism. There are amide linkages in proligands 6, 8, and 11,
so a less bulky secondary amine was investigated in proligand 19.
Reductive amination of ethanolamine with salicylaldehyde
yielded 13 (Scheme 2). 2-(Dibenzylamino)ethanol 13 was then
oxidized to the corresponding aldehyde 14 according to a previ-
ously reported procedure.23 The aldehyde was unstable, so was used
1
produce the same signals as each other in both the H and 13C
NMR spectra.
The appearance of multiple carbon signals between 197.5 and
196.8 ppm in the 13C NMR spectrum of 6-Re(CO)3 indicates
an arrangement of three carbonyl groups in a fac orientation
around the metal centre. For the aromatic resonances, C9 and
C6 shift upfield (158.0 from 159.8 ppm and 119.8 from 124.6 ppm,
respectively) upon binding, while the other nine carbon resonances
1
immediately following quantification by H NMR spectroscopy.
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Scheme 1 Reaction conditions: (i) 1 equiv dibenzylglycine,24 1.1 equiv EDC.HCl, 1.1 equiv HOBT.H2O, 0.1 equiv DMAP, DMF, 25 ◦C, 18 h;
(ii) Pd(OH)2/C, H2, AcOH, 25 ◦C, 18 h; (iii) ~6 equiv Na2CO3, 1 equiv 2-pyridine carboxaldehyde, ClCH2CH2Cl, 25 ◦C, 18 h, 2.5 equiv NaBH(OAc)3,
25 ◦C, 4 h; (iv) 3.1 equiv salicylaldehyde, 5.1 equiv NaBH(OAc)3, ClCH2CH2Cl, 25 ◦C, 20 h; (v) 5 equiv NaOMe, MeOH, 25 ◦C, 2 h; (vi) ~6 equiv
Na2CO3, 2 equiv methylbromoacetate, CH2Cl2, 25 ◦C, 48 h; (vii) 1 M NaOH, 25 ◦C, 1 h; (viii) ~6 equiv Na2CO3, 1-methylimidazole, EtOH, 25 ◦C, 0.5 h,
2.5 equiv NaBH4, 25 ◦C, 10 min; (ix) 2 equiv salicylaldehyde, 5.1 equiv NaBH(OAc)3, ClCH2CH2Cl, 25 ◦C, 20 h; (x) 5 equiv NaOMe, MeOH, 25 ◦C, 2 h.
shift downfield upon binding: C15 (174.5 to 182.8 ppm), C1
(158.0 to 160.7 ppm), C13 (149.7 to 154.0 ppm), C11 (139.0 to
142.5 ppm), C5 (132.6 to 134.9 ppm), C3 (130.3 to 132.7 ppm),
C10 (124.9 to 127.4 ppm), C12 (124.1 to 125.7 ppm), C4 (120.6
to 121.2 ppm), and C2 (117.1 to 117.3 ppm). Diagnostic shifts
occur for the methylene carbon resonances as well: C8 (60.5 to
69.2 ppm), C7 (56.7 to 68.2 ppm), C14 (58.1 to 62.6 ppm), whereas
for C20, the resonance remains constant at 63.0 ppm, indicating
the pendant nature of the glucosamine moiety, while providing
evidence of binding of the tridentate unit. Further, the other
9218 | Dalton Trans., 2009, 9216–9227
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Table 1 Summary of labeling and stability results for 99mTc complex formation
Ligand
RT (Re compound)a
RT (99mTc compound)a
Radiochemical yield
Stability in cysteine–24 hb
Stability in histidine–24 hb
6
18.7 min
12.9 min
19.6 min
16.2 min
18.8 min
13.2 min
19.4 min
16.3 min
94%
94%
96%
79%
99 2%
94 2%
100 4%
97 3%
98 1%
85 3%
94 3%
92 3%
8
11
22
a HPLC conditions: 100% H2O (with 0.1% TFA) linear gradient to 100% ACN at 30 min. b 37 ◦C.
Scheme 2 Reaction conditions: (i) 2 equiv salicylaldehyde, ClCH2CH2Cl, 25 ◦C, 10 min, 4.3 equiv NaBH(OAc)3, 25 ◦C, 16 h; (ii) 5.7 equiv Et3N, 4.9 equiv
SO3-py complex, DMSO, 25 ◦C, 1 h;23 (iii) 1.2 equiv 1, ClCH2CH2Cl, 25 ◦C, 0.5 h, 2 equiv NaBH(OAc)3, 25 ◦C, 16 h; (iv) 1.1 equiv NH2NH2-AcOH,
DMF, 25 ◦C, 2.5 h; (v) 6.9 equiv imidazole, 4 equiv TBDMSCl, CH2Cl2, 25 ◦C, 2 h; (vi) 1.3 equiv NaOMe, MeOH, 25 ◦C, 2.5 h; (vii) Pd(OH)2/C, H2,
MeOH, 25 ◦C, 24 h; (viii) 10 equiv Na2CO3, 1.05 equiv 2-pyridinecarboxaldehyde, MeOH, N2, 25 ◦C, 24 h, 2.4 equiv NaBH4, 25 ◦C, 24 h; (ix) 1 equiv
salicylaldehyde, ClCH2CH2Cl, 25 ◦C, 1 h, 2 equiv NaBH(OAc)3, 25 ◦C, 24 h; (x) 1.8 equiv benzyltrimethylammonium fluoride, THF, MeOH, 25 ◦C, 24 h.
glucosamine resonances C21, C17, C19, C18, and C16 shift little,
if at all, upon binding: 92.7 to 91.9 ppm, 73.2 to 73.1 ppm, 73.0
to 72.0 ppm, 72.3 to 71.9 ppm, and 56.0 to 57.0 ppm, respectively.
This qualitative trend of resonances near the predicted metal
binding atoms shifting a significant amount and the carbohydrate
resonances shifting much less so is also observed for 8-Re(CO)3,
11-Re(CO)3 and 22-Re(CO)3.
isotopes, 185Re and 187Re (37:63) and thus gives rise to diagnostic
isotope peak patterns in the mass spectra. ESI-MS+ of 6-Re(CO)3,
showed m/z values for [MH]+ of 702 (M185ReH+), 704 (M187ReH+),
8-Re(CO)3 showed m/z values for [MH]+ of 676 (M185ReH+), 678
(M187ReH+), 11-Re(CO)3 showed m/z values for [MH]+ of 705
(M185ReH+), 707 (M187ReH+) while 22-Re(CO)3 showed m/z values
for [MH]+ 688 (M185ReH+), 690 (M187ReH+).
Further evidence for complex formation is provided by IR spec-
troscopy and mass spectrometry of dilute MeOH solutions. For
6-Re(CO)3, 8-Re(CO)3, 11-Re(CO)3 and 22-Re(CO)3, two car-
bonyl bands are present at 2032 and 1905 cm-1, 2027 and 1910 cm-1,
2028 and 1884 cm-1, and 2029 and 1906 cm-1, respectively, which
are significantly higher than the rhenium precursor carbonyl
stretches at 2000 and 1868 cm-1. Rhenium exists as a mixture of
Labeling and stability
The 99mTc complexes formed in high radiochemical yields after
reacting for 30 min (Table 1). The phenolate-based ligands
required a base to give these short reaction times, whereas the
acid-based ligand coordinated readily in PBS buffer at pH 7.4.
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complex. Agreement of their retention times (RTs) supports
the expected outcome of the technetium analogue having the
same chemical structure as the rhenium complex. There is a
slight difference between the retention times of the rhenium and
technetium complexes because the UV (rhenium) and radiation
(
99mTc) detectors are connected in series. Percentage radiochemical
yield was calculated by integration of all the peaks that appear in
the radiation trace. The other major species seen in these HPLC
traces was the unreacted starting material [99mTc(H2O)3(CO)3]+
(RT =12 min).
All 99mTc complexes were tested for stability in excess concen-
trations of cysteine and histidine for 24 h at 37 ◦C (Table 1).
Cysteine and histidine are strong, potentially tridentate ligands for
the tricarbonyl core, and are found ubiquitously in vivo. There was
no major degradation product seen, but rather several small peaks
slowly grew in, suggesting the presence of several minor species.
The compounds in this work all exhibit satisfactory stability to
justify further investigation as molecular imaging agents, as they
have been shown to be stable enough under relevant conditions.
The phenolates showed greater stability under these challenge
conditions than did the equivalent acid-based ligand, showing
one potential benefit of using this ligating group.
A recent study found that a [Tc(CO)3]+ complex with two
phenolate donors was not as stable to similar challenge conditions
as were ligands made up of other binding group combinations.25
It may be that the phenolate is best used in conjunction with
two nitrogenous donors, as this provides a neutral binding sphere
as well as a large nitrogen content, known to be for optimal
[Tc(CO)3]+ binding.26
As the pKa of the phenol is around 10, the reaction mixture
needs to be made basic to enable sufficient ligand deprotonation
for complex formation in a timely manner. In our systems this
proceeded well and with no sign of degradation products, but these
conditions may limit its general applicability. This potential draw-
back may be outweighed, however, by the increased lipophilicity of
a phenol compared to an acid group, which in certain cases may be
able to improve the biodistribution and solubilities of the resulting
compound. As these biological properties can be very sensitive to
small changes, this is a useful addition to the expanding knowledge
on the coordination chemistry of technetium.
GLUT-1 cell uptake assay
Cell uptake of several 99mTc complexes was examined in LCC6-
HER2 cells—a human breast cancer cell line with high surface
concentrations of GLUT-1 glucose transporters.27 Fig. 1 shows
the results of this assay. The cell uptake of each of the 99mTc
compounds examined was independent of glucose, meaning that
the uptake was not due to transport via the GLUT-1 transporters.
The percentage uptake for each of the compounds is quite
low. For comparison, FDG, the positive control, showed uptake
of around 3.4% of the total activity when in a glucose free
environment, and 0.1% when in 5 mM glucose (results not
shown). The trend of lipophilic compounds having higher uptake,
suggests that passive diffusion or membrane association was
responsible for the observed results. There was no significant dif-
ference between the uptake of the amine-linked and amide-linked
compounds.
Scheme
3
Reaction conditions: (i) [Re(H2O)3(CO)3][Br], EtOH, D,
6–24 h; (ii) [99mTc(H2O)3(CO)3]+, 3 equiv NaOEt, EtOH, 0.5 h, 80 ◦C;
(iii) [99mTc(H2O)3(CO)3]+, PBS, 0.5 h, 80 ◦C.
The phenolate ligands did coordinate to the metal without base,
but not on a radiochemistry-appropriate timeframe.
The 99mTc complexes were analyzed using HPLC performed with
coinjection of the thoroughly characterized analogous rhenium
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Experimental
Instruments and materials
All solvents and reagents were used as received from Sigma-
Aldrich or Acros unless otherwise noted. Re(CO)5Br is commer-
cially available (STREM). [Re(H2O)3(CO)3]Br,29 dibenzylglycine,24
1,3,4,6-tetra-O-acetyl-b-D-glucosamine 1,30 were prepared as de-
scribed previously.
The analytical TLC plates were aluminium backed ultra pure
silica gel 60, 250 mm, the glass backed preparative TLC plates
˚
1000 mm, and the flash column silica gel (standard grade, 60 A,
32-63 mm) used were all provided by Silicycle. 1H and 13C
NMR, 13C APT, H-1H COSY and H-13C HMQC spectra were
recorded on a Bruker AV300, AV400 or DRX400 spectrometer.
The NMR spectra are expressed on the d scale and referenced to
residual solvent peaks. Infrared spectra were recorded between
400 and 4000 cm-1 in transmission mode on a Nicolet 4700
FT-IR spectrophotometer as KBr discs at a resolution of 4 cm-1
or on a Nicolet 6700 FT-IR spectrophotometer at a resolution of
0.09 cm-1. Melting points were measured using a Mel-Temp by
Laboratory Devices, Cambridge, Massachusetts. ESI mass spectra
were recorded on a Micromass LCT instrument at the University
of British Columbia, Department of Chemistry. High resolution
mass spectra (Micromass LCT TOF-MS)) and elemental analysis
(Carlo Erba analytical instrument) were provided by the Ana-
lytical Services Facility in the Department of Chemistry. HPLC
analyses of cold compounds were done on a Phenomenex Synergi
4 mm C18 Hydro-RP column (4.6 ¥ 250 mm) using a Waters
WE 600 HPLC system equipped with a 2478 dual wavelength
absorbance UV detector run using an Empower software package.
HPLC analyses of radiolabeled complexes were performed on
a Knauer Wellchrom K-1001 HPLC equipped with a K-2501
absorption detector and a Capintec radiometric well counter. A
Phenomenex Synergi 4 mm C-18 Hydro-RP analytical column with
dimensions of 4.6 ¥ 250 mm was used. The identities of the novel
1
1
Fig. 1 Results of cell uptake studies – each compound’s uptake was
examined in a glucose free environment and in 5 mM glucose solution
(average % of total activity in the cells after a 30 min incubation for four
independent experiments are shown – error bars are standard deviations).
Hexokinase assay
Hexokinase catalyses the phosphorylation of glucose in the
C-6 position, and is also known to phosphorylate certain glu-
cose analogues.3,17,19 We assessed whether our compounds were
phosphorylated by hexokinase, an important factor when looking
for a glucose-based imaging agent, as without phosphorylation
to trap the compound, it can diffuse out of a cell just as quickly
as it diffused in. Unfortunately none of the compounds tested
were found to be phosphorylated by hexokinase, and therefore no
differences could be identified between activities of the different
compounds. This lack of activity may be because the metal-
binding portions of the molecules are too bulky to allow the cleft of
hexokinase to narrow as is required for phosphorylation to occur.
1
compounds were confirmed by H and 13C NMR spectroscopy,
Conclusions
high resolution mass spectrometry and infrared spectroscopy.
To our knowledge we were the first group to use the phenolate-
based ligand system for the tricarbonyl core,20,28 though others
have shown interest in this donor system more recently.25 The
phenolate was investigated here as an anionic binding group
to provide neutral complexes when bound to the monocationic
tricarbonyl core. It has been demonstrated again here that the
phenolate group is a suitable donor for the tricarbonyl core, as
it binds to both the technetium and rhenium centers within a
radiochemistry-appropriate timeframe and gives complexes that
are stable to cysteine and histidine challenges. It is interesting
to note that the phenolates are more stable in our hands under
these physiologically relevant conditions than the equivalent acid
compounds.
The assays performed on the compounds in this work, whether
based on phenolates or other ligand types, showed the compounds
not to be as biologically active as we had initially hoped. These
compounds are likely too different from glucose for the enzymes to
recognize and process them as they would their native substrates.
In this case, that perturbation is too large to allow for a functional
imaging agent to be formed. Work on compounds with longer
linkers and different binding groups that is ongoing in our lab
may circumvent these issues.
Synthesis
2-((Bis(phenylmethyl)amino)acetamido)-2-deoxy-1,3,4,6-tetra-
O-acetyl-b-D-glucopyranose, (2). N,N-Dibenzylglycine24 (5.00 g,
0.0196 mol) was dissolved in hot DMF (125 mL). The resulting
clear colorless solution was cooled in an ice bath. EDC·HCl (N-
(3-dimethylaminopropyl)-N¢-ethylcarbodiimide hydrochloride)
(4.14 g, 0.0216 mol), HOBt·H2O (1-hydroxybenzotriazole hydrate)
(2.92 g, 0.0216 mol), and DMAP (4-dimethylaminopyridine)
(0.239 g, 0.00196 mol) were added sequentially to the DMF
solution. The reaction mixture was stirred at 0 ◦C for 30 minutes
and to this solution was added 1,3,4,6-tetra-O-acetyl-b-D-
glucosamine 130 (6.81 g, 0.0177 mol). The ice-bath was removed
and the clear colorless solution was stirred at room temperature.
After 18 hours, the solvent was removed under reduced pressure,
leaving behind a pale white residue. The residue was dissolved
in CH2Cl2 (125 mL), washed with saturated aqueous Na2CO3
solution (3 ¥ 40 mL), 1M HCl (3 ¥ 40 mL) and then brine (2 ¥
30 mL). The organic layer was dried over anhydrous MgSO4,
and evaporated to dryness under reduced pressure. Column
chromatography (silica-CH2Cl2/5% MeOH) was used to isolate 2
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as a white solid (9.25 g, 80% yield). Rf = 0.56 (silica-CH2Cl2/5%
MeOH:); 1H NMR (DMSO-d6, 400 MHz, 25 ◦C, d): 7.97 (d, J =
9.6 Hz, 1H), 7.37-7.21 (m, 10H), 5.99 (d, J = 8.4 Hz, 1H), 5.50
(t, J = 9.6 Hz, 1H), 4.91 (t, J = 10.0 Hz, 1H), 4.21 (dd, J =
12.4, 4.4 Hz, 1H), 4.10-3.97 (m, 3H), 3.53 (s, 4H), 2.93 (d, J =
16.0 Hz, 1H), 2.83 (d, J = 15.6 Hz, 1H), 2.01 (s, 3H), 1.98 (s, 3H),
1.82 (s, 3H), 1.78 (s, 3H) ppm. 13C NMR (DMSO-d6, 100 MHz,
25 ◦C, d): 170.2, 170.0, 169.4, 169.3, 168.6, 137.7, 128.8, 128.2,
127.1, 91.7, 72.0, 71.5, 68.3, 61.5, 57.2, 55.8, 51.7, 20.5, 20.4, 20.3,
20.2 ppm; IR (KBr) nmax (cm-1): 1742 (s), 1663 (m), 1514 (m),
1371 (w), 1261 (m), 1225 (s), 1079 (m), 1039 (m), 740 (w); MS
(ES+, 100% MeOH, 30V): m/z = 607 (MNa+); HR-MS (ES+ of
MNa+) m/z calcd for C30H36N2O10Na: 607.2268, found 607.2267;
Anal. Calcd. for C30H36N2O10: C, 61.63; H, 6.21; N, 4.79. Found:
C, 61.90; H, 6.59; N, 5.11.
d): 171.4, 170.1, 169.7, 169.3, 168.9, 159.4, 148.8, 136.5, 122.1,
121.9, 91.9, 72.2, 71.5, 68.2, 61.5, 53.9, 51.7, 51.5, 20.6, 20.5, 20.4,
20.4 ppm; IR (KBr) nmax (cm-1): 1747 (s), 1668 (m), 1522 (m),
1371 (w), 1229 (s), 1074 (m), 1041 (m), 598 (w); MS (ES+, 100%
MeOH, 30 V): m/z = 496 (MH+); HR-MS (ES+ of MH+) m/z
calcd for C22H30N3O10: 496.1931, found: 496.1930; Anal. Calcd for
C22H29N3O10: C, 53.33; H, 5.90; N, 8.48. Found: C, 53.41; H, 6.20;
N, 8.65.
2-((N-(2-Hydroxybenzyl)-N-(pyridin-2-ylmethyl)amino)acet-
amido)-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glucopyranose,
(5).
Compound 4 (700 mg, 1.4 mmol) was dissolved in ClCH2CH2Cl
(15 mL). Salicylaldehyde (470 mL, 4.4 mmol) and NaBH(OAc)3
(1.5 g, 7.2 mmol) were added sequentially and the reaction
mixture was stirred at room temperature for 20 h. The reaction
was quenched by addition of saturated aqueous Na2CO3 solution
(10 mL). The mixture was then partitioned and the aqueous
layer further extracted with CH2Cl2 (2 ¥ 8 mL). The combined
organic extract was washed with brine (2 ¥ 10 mL), then dried
over anhydrous MgSO4. After filtration of the drying agent, the
filtrate was taken to dryness by rotary evaporation to yield crude
5. Column chromatography (silica-CH2Cl2/5% MeOH) was used
to isolate and purify 5 as a yellow solid (0.670 g, 79% yield);
Rf = 0.74 (silica-CH2Cl2/10% MeOH); 1H NMR (DMSO-d6,
2-(2-Aminoacetamido)-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glu-
copyranosyl acetate, (3). Compound 2 (7.64 g, 0.0131 mol) was
dissolved in glacial acetic acid (100 mL). Pd(OH)2/C (0.800 g,
0.0057 mmol) was added in one portion, the flask was capped with
a rubber septum and purged with H2 from a balloon. The mixture
was kept under a positive pressure of H2 at room temperature for
18 h. The suspension was then filtered through a pad of celite and
the solvent was removed under reduced pressure to yield yellow oil
3 (quantitative). Rf = 0.05 (silica-CH2Cl2/5% MeOH:); 1H NMR
◦
400 MHz, 25 C, d): 9.95 (s, 1H), 8.50 (d, J = 2.4 Hz, 2H), 7.72
◦
(DMSO-d6, 400 MHz, 25 C, d): 5.76 (d, J = 8.8 Hz, 1H), 5.22
(td, J = 7.8 and 1.8 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.27 (t,
J = 6.6 Hz, 1H), 7.16-7.09 (m, 2H), 6.81-6.73 (m, 2H), 5.86 (d,
J = 8.8 Hz, 1H), 5.35 (t, J = 9.6 Hz, 1H), 4.92 (t, J = 9.8 Hz,
1H), 4.21 (dd, J = 12.4 and 4.6 Hz, 1H), 4.09-4.00 (m, 3H), 3.66
(s, 2H), 3.59 (s, 2H), 3.09-2.98 (m, 2H), 2.01 (s, 3H), 1.98 (s, 3H),
1.92 (s, 3H), 1.83 (s, 3H) ppm. 13C NMR (DMSO-d6, 100 MHz,
25 ◦C, d): 170.5, 170.0, 169.5, 169.3, 168.7, 158.2, 156.2, 148.8,
136.7, 130.8, 128.6, 123.1, 122.5, 122.3, 118.8, 115.4, 91.6, 72.0,
71.5, 68.1, 61.5, 58.2, 56.0, 53.7, 51.8, 20.5, 20.4, 20.4, 20.2 ppm;
IR (KBr) nmax (cm-1): 3314 (m), 1754 (s), 1685 (m), 1522 (w),
1368 (m), 1221 (s), 1078 (m), 1040 (m), 760 (m); MS (ES+, 100%
MeOH, 30 V): m/z = 602 (MH+); HR-MS (ES+ of MH+) m/z
calcd for C29H36N3O11: 602.2350, found: 602.2348; Anal. Calcd.
for C29H35N3O11: C, 57.90; H, 5.86; N, 6.98. Found: C, 58.04; H,
6.28; N, 7.29.
(t, J = 10.0 Hz, 1H), 4.90 (t, J = 10.0 Hz, 1H), 4.19 (dd, J =
12.4 and 4.4 Hz, 1H), 4.06-3.97 (m, 3H), 3.39 (s, 2H), 2.04 (s,
3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.92 (s, 3H), 1.87 (s, 30H, Acetic
acid) ppm. 13C NMR (DMSO-d6, 100 MHz, 25 ◦C, d) 172.6, 170.2,
169.9, 169.8, 169.4, 169.0, 91.7, 72.1, 71.6, 68.2, 61.6, 52.2, 41.1,
21.5, 20.6, 20.6, 20.5, 20.4 ppm. MS (ES+, 100% CH3CN, 30 V):
m/z = 427 (M+Na+, 100%); HR-MS (ES- of M-) m/z calcd for
C16H23N2O10 403.1353, found 403.1354. Note: The IR spectrum
was not obtained due to decomposition and EA was not obtained
due to varying amounts of acetic acid present in the oily sample.
2-(((N-Pyridin-2-ylmethyl)amino)acetamido)-2-deoxy-1,3,4,6-
tetra-O-acetyl-b-D-glucopyranose, (4). Compound 3 (6.0 g) was
dissolved in ClCH2CH2Cl (100 mL). Na2CO3 (34.2 g, 0.323 mol)
and an equimolar amount of 2-pyridinecarboxaldehyde were
added sequentially. The cloudy red solution was stirred for 18 h
at room temperature. NaBH(OAc)3 (6.85 g, 0.0323 mol) was
then added in one portion and the resulting reaction mixture
further stirred for an additional 4 h. The reaction was quenched
by addition of saturated aqueous Na2CO3 solution (50 mL).
The mixture was then partitioned and the aqueous layer further
extracted with CH2Cl2 (2 ¥ 25 mL). The combined organic extract
was washed with brine (2 ¥ 25 mL), then dried over anhydrous
MgSO4. After filtration of the drying agent, the filtrate was
taken to dryness by rotary evaporation to yield crude 4. Column
chromatography (silica-CH2Cl2/5% MeOH) was used to isolate
and purify 4 as a pale yellow solid (2.19 g, 43% yield). Rf = 0.51
(silica-CH2Cl2/10% MeOH); 1H NMR (DMSO-d6, 400 MHz,
25 ◦C, d): 8.49 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 9.7 Hz, 1H),
7.75 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.25 (t, J =
5.8 Hz, 1H), 5.89 (d, J = 8.8 Hz, 1H), 5.37 (t, J = 9.6 Hz, 1H), 4.90
(t, J = 9.6 Hz, 1H), 4.19 (dd, J = 12.4 and 4.4 Hz, 1H), 4.08-3.94
(m, 3H), 3.65 (s, 2H), 3.09 (s, 2H), 2.03 (s, 3H), 2.01 (s, 3H), 1.97
(s, 3H), 1.92 (s, 3H) ppm. 13C NMR (DMSO-d6, 100 MHz, 25 ◦C,
2-((N-(2-Hydroxybenzyl)-N-(pyridin-2-ylmethyl)amino)acet-
amido)-2-deoxy-D-glucopyranose, (6). Compound 5 (670 mg,
1.11 mmol) was dissolved in MeOH (5 mL) and NaOMe (310 mg,
5.7 mmol) was added in one portion. After stirring for 2 h at room
temperature, the solvent was removed under reduced pressure
to yield a pale white solid. Compound 6 (300 mg, 62% yield)
was isolated and purified using column chromatography (silica-
CH2Cl2/20% MeOH): Rf = 0.34 (silica-CH2Cl2/20% MeOH); 1H
◦
NMR (MeOH-d4, 400 MHz, 25 C, d): 8.55-8.45 (m, 1H), 7.81-
7.75 (m, 1H), 7.51-7.49 (m, 1H), 7.30-7.27 (m, 1H), 7.15-7.11 (m,
2H), 6.83-6.76 (m, 2H), 5.06 (d, J = 3.5 Hz, 1H-b), 4.64 (d, 8.4 Hz,
1H-a), 3.90-3.62 (m, 8H), 3.35-3.27 (m, 2H), 3.23 (s, 2H) ppm. 13
C
NMR (MeOH-d4, 100 MHz, 25 ◦C, d): 174.5, 159.7, 158.0, 149.6,
139.0, 132.3, 130.3, 124.9, 124.5, 124.0, 120.5, 117.1, 97.1 (a), 92.7
(b), 73.2, 73.0, 72.6, 63.0, 60.2, 58.0, 56.7, 55.8 ppm; IR (KBr) nmax
(cm-1): 3386 (br, s), 1655 (s), 1597 (w), 1541 (w), 1489 (w), 1247 (m),
1104 (w), 1039 (m), 758 (s); MS (ES+, 100% MeOH, 30 V): m/z
434 (MH+); HR-MS (ES+ of MH+): m/z calcd for C21H28N3O7:
9222 | Dalton Trans., 2009, 9216–9227
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∑
434.1927, found: 434.1925; Anal. Calcd. for C21H27N3O7 (H2O)0.5:
2-((N-(1-Methylimidazol-2-ylmethyl)amino)acetamido)-2-deoxy-
C, 56.77; H, 6.40; N, 9.46. Found: C, 56.52; H, 6.23; N, 9.21.
1,3,4,6-tetra-O-acetyl-b-D-glucopyranose,
(9). 2-(2-Amino-
acetamido)-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glucopyranose
(3) (6.52 mmol) was dissolved in dry ethanol (20 mL), and
an appropriate amount of sodium carbonate was added to
2-((N-(Pyridin-2-ylmethyl)-N-(methoxyethanoic acid)amino)-
acetamido)-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glucopyranose,
(7). 2-((N-(Pyridin-2-ylmethyl)amino)acetamido)-2-deoxy-1,3,
4,6-tetra-O-acetyl-b-D-glucopyranose (4) (0.950 g, 1.92 mmol)
was dissolved in dry dichloromethane (15 mL). Na2CO3 (0.406 g,
3.84 mmol) and methyl bromoacetate (0.241 mL, 2.88 mmol)
were added. The flask was evacuated and filled with argon,
and the reaction mixture stirred at room temperature for 48 h.
Water was added, the two layers were separated, and the organic
layer was washed again with water, and once with brine, before
being dried over MgSO4. The drying agent was filtered off; the
filtrate was evaporated on a rotary evaporator, and then purified
by column chromatography on silica gel with 5% methanol in
dichloromethane as eluent. The solvents were removed in vacuo
to give an off white solid (0.790 g, 72% yield). Rf = 0.47
(silica-CH2Cl2/10% MeOH). 1H NMR (CDCl3, 400 MHz, 25 ◦C,
d): 8.98 (d, J = 9.4 Hz, 1H), 8.60 (d, J = 4.5 Hz, 1H), 7.67 (ddd,
J = 7.7 Hz, 7.6 Hz, 1.7 Hz, 1H), 7.23 (m, 2H), 5.84 (d, J =
8.8 Hz, 1H), 5.31 (dd, J = 10.2 Hz, 9.5 Hz, 1H), 5.13 (dd, J =
9.6 Hz, 9.8 Hz, 1H), 4.30 (m, 2H), 4.13 (dd, J = 2.0 Hz, 12.4 Hz,
1H), 3.88 (m, 3H), 3.71 (s, 3H), 3.40 (d, J = 3.1 Hz, 2H), 3.31 (s,
2H◦), 2.09, 2.02, 1.99, 1.94 (s, 12H). 13C NMR (CDCl3, 100 MHz,
25 C, d): 171.90, 171.87, 170.89, 170.55, 169.59, 169.34, 157.77,
149.87, 137.08, 123.34, 123.00, 92.62, 73.12, 72.93, 68.43, 61.91,
59.60, 58.64, 54.76, 52.76, 51.99, 20.96, 20.93, 20.80, 20.78. IR
1
neutralise the associated acetic acid, as determined by H NMR
spectroscopy of the starting material (4.60 g, 43.4 mmol).
After ten minutes stirring to ensure all the acid was quenched,
1-methylimidazole-2-carboxaldehyde (0.770 g, 6.99 mmol) was
added to the reaction. A nearly immediate colour change to green
was observed, and within ten minutes the reaction was complete,
as tested by TLC. Sodium borohydride (0.780 g, 21.1 mmol) was
added and the reaction mixture stirred for ten minutes longer, as
the green colour faded. The reaction mixture was quenched with
water (30 mL), which also dissolved the excess sodium carbonate.
The solution was extracted three times with dichloromethane
(3 ¥ 30 mL), the organic fractions were combined and washed
once with water (30 mL), once with brine (30 mL), then dried
over magnesium sulfate before being filtered and reduced to an
oil on the rotary evaporator. This oil was purified using column
chromatography (5% methanol in dichloromethane) and the
solvents evaporated in vacuo to give an off white solid (0.81 g,
25% yield). Rf = 0.40 (silica-CH2Cl2/10% MeOH). 1H NMR
(MeOH-d4, 400 MHz, 25 ◦C, d): 7.67 (d, J = 9.0 Hz, 1H),6.95
(s, 2H), 6.84 (s, 2H), 5.80 (d, J = 8.7 Hz, 1H), 5.24 (dd, J =
9.4 Hz, 10.2 Hz, 1H), 5.13 (dd, J = 9.4 Hz, 9.7 Hz, 1H), 4.27
(m, 2H), 4.13 (dd, J = 2.2 Hz, 12.4 Hz, 1H), 3.83 (ddd, J =
9.8 Hz, 2.2 Hz, 4.7 Hz, 1H), 3.77 (d, J = 6.4 Hz, 2H, 3.63 (s, 3H),
3.29 (d, J = 4.6 Hz, 2H). 13C NMR (CDCl3, 100 MHz, 25 ◦C,
d): 171.76, 170.66, 169.32, 144.21, 127.29, 121.31, 92.46, 72.81,
72.70, 67.89, 61.66, 52.73, 52.12, 44.98, 32.46, 20.90, 20.69, 20.56.
IR nmax (cm-1): 3324 (w), 2953 (w), 2360 (m), 2341 (m), 1743 (s),
1673 (m), 1519 (m), 1367 (m), 1214 (s), 1034 (s), 728 (m). HR-MS
(ES+ of MNa+): m/z calcd for C21H30N4O10Na : 521.1860, found:
521.1863.
n
max (cm-1): 3321 (w), 2958 (w), 1744 (s), 1663 (m), 1508 (m), 1390
(m), 1218 (s), 1038 (m). HR-MS (ES+ of MNa+): m/z calcd for
C25H33N3O12Na : 590.1962, found: 590.1948.
2-((N-(Pyridin-2-ylmethyl)-N-(ethanoic acid)amino)acetamido)-
2-deoxy-D-glucopyranose, (8). 2-((N-(Pyridin-2-ylmethyl)-N-
(methoxyethanoic acid) amino) acetamido)-1,3,4,6-tetraacetyl-2-
deoxy-D-glucopyranose (7) (40 mg, 0.071 mmol) was suspended
in 1 M NaOH (2 mL) and stirred vigorously. After about
5 min the solution became homogenous and slightly yellow.
The reaction was complete according to ESI-MS after about
60 min. Amberlite CG-50 ion exchange resin was added and the
resulting slurry stirred vigorously for 15 min, before the resin was
filtered off. The aqueous filtrate was reduced to a yellow solid
on the rotary evaporator before being taken up in a minimum
amount of methanol and filtered to remove most of the NaOH.
The methanolic solution was then purified by reverse phase
HPLC (isocratic, H2O with 0.1% TFA), and the solvents removed
in vacuo to give a pale orange oil (15 mg, 55% yield). Rf = 0.33
2-((N -(1-Methylimidazol-2-ylmethyl)-N -(2-hydroxybenzyl)-
amino)acetamido)-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glucopy-
ranose, (10). 2-((N-(1-Methylimidazol-2-ylmethyl)amino)acet-
amido)-2-deoxy-D-glucopyranose (9) (0.800 g, 1.62 mmol) was
dissolved in 1,2-dichloroethane (15 mL) and sodium carbonate
(0.343 g, 3.24 mmol) was added. The reaction flask was purged
for ten minutes with argon, salicylaldehyde (337 mL, 3.21 mmol)
was added, and the reaction mixture stirred at room temperature
for 30 min. Sodium triacetoxyborohydride (1.02 g, 4.83 mmol)
was added and the reaction mixture stirred for 24 h. Upon
completion of the reaction, as determined from loss of starting
materials by TLC, the solvent was removed by rotary evaporator
and the off white solid partitioned between dichloromethane
and water (20 mL each). The layers were separated and the
aqueous layer washed twice with dichloromethane (20 mL), the
two fractions of which were then combined. These were dried
with brine (30 mL) then MgSO4, filtered and reduced in volume
on the rotary evaporator. The yellow solution was purified by
column chromatography on silica gel using 4% methanol in
dichloromethane as an eluent, and the solvent was removed
in vacuo to give an off white solid (0.626 g, 64% yield). Rf =
(silica-MeOH). H NMR (MeOH-d4, 400 MHz, 25 ◦C, d): 8.79
1
(s, 1H), 8.41 (dd, J = 7.4 Hz, 7.0 Hz, 1H), 7.93 (m, 1H), 7.86 (m,
1H), 5.10 (d, J = 3.1 Hz, 0.75H - a), 4.66 (d, J = 8.2 Hz, 0.25H
- b), 4.41 (d, J = 4.7 Hz, 2H), 4.30, 4.02, 3.66–3.90, 3.50–3.64,
3.34–3.42 (m). 13C NMR (MeOH-d4, 100 MHz, 25 ◦C, d): 173.79,
172.72, 162.63, 162.29, 156.53, 145.78, 145.66, 143.35, 126.71,
126.22, 96.40, 94.27, 92.10, 77.58, 75.38, 73.16, 72.76, 72.26,
71.95, 71.71, 68.21, 62.28, 61.88, 58.31, 58.23, 55.48, 57.31, 57.17.
IR nmax (cm-1): 3272 (s, br), 2918 (m), 1667 (s, br), 1538 (m), 1417
(w), 1188 (s), 1031 (s). Purity assessed by HPLC: 96.5%. HR-MS
(ES+ of MNa+): m/z calcd for C16H23N3O8Na: 408.1383, found:
408.1388.
1
0.55 (silica-CH2Cl2/10% MeOH). H NMR (CDCl3, 400 MHz,
25 ◦C, d): 7.97 (d, J = 9.0 Hz, 1H), 7.14 (ddd, J = 7.2 Hz, 8.2 Hz,
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1.5 Hz, 1H), 7.07 (d, J = 1.1 Hz, 1H), 6.96 (dd, J = 7.4 Hz, 1.3 Hz,
1H), 6.87 (s, 1H), 6.85 (s, 1H), 6.71 (dd, J = 7.4 Hz, 7.3 Hz, 1H),
5.72 (d, J = 8.4 Hz, 1H), 5.10 (dd, J = 9.6 Hz, 9.9 Hz, 1H), 5.00
(dd, J = 9.8 Hz, 9.4 Hz, 1H), 4.23 (dd, J = 12.4 Hz, 4.5 Hz, 1H),
4.02 (dd, J = 2.1 Hz, 12.6 Hz, 1H), 3.96 (dd, J = 9.4 Hz, 9.6 Hz,
1H), 3.84 (d, J = 13.1 Hz, 1H), 3.78 (ddd, J = 4.5 Hz, 2.0 Hz,
9.9 Hz, 1H), 3.67 (dd, J = 15.6 Hz, 14.1 Hz, 2H), 3.62 (d, J =
13.1 Hz, 1H), 3.52 (s, 3H), 3.18 (dd, J = 16.7 Hz, 10.6 Hz, 2H),
2.03, 1.94, 1.85, 1.76 (s, 12H). 13C NMR (CDCl3, 100 MHz, 25 ◦C,
d): 171.71, 170.85, 170.26, 169.55, 169.04, 157.42, 145.28, 131.14,
129.88, 125.93, 122.35, 121.82, 119.06, 117.85, 92.04, 72.84, 72.56,
68.27, 61.85, 56.57, 56.49, 53.24, 50.27, 32.70, 20.95, 20.87, 20.76,
20.68. IR nmax (cm-1): 3274 (w), 1744 (s), 1674 (m), 1388 (m), 1211
(s), 1072 (m), 1035 (s), 760 (s). HR-MS (ES+ of MNa+): m/z calcd
for C28H36N4O11Na : 627.2278, found: 627.2288.
13 is commercially available from TCI America as 95% pure
by GC.
2-(Dibenzylamino)acetaldehyde, (14)23. To a solution of 13
(3.5 g, 14.5 mmol) in DMSO (70 mL) was added triethylamine
(11.5 mL, 82.5 mmol). Sulfur trioxide-pyridine complex (11.2 g,
70.4 mmol) in DMSO (70 mL) was added dropwise and the
reaction mixture was stirred at ambient temperature. After 1 hour,
diethyl ether (200 mL), saturated ammonium chloride solution
(100 mL) and water (100 mL) was added. The mixture was parti-
tioned and the aqueous layer further extracted with diethylether
(3 ¥ 100 mL). The combined organic extract was washed with
brine (100 mL) and dried over MgSO4. After the drying agent was
filtered, the filtrate was taken to dryness by rotary evaporation. The
target compound was not purified further as it is highly unstable. A
DMSO-d6 NMR sample was however taken to verify the presence
of the aldehyde moiety (9.5 (s) ppm).
2-((N -(1-Methylimidazol-2-ylmethyl)-N -(2-hydroxybenzyl)-
amino)acetamido)-2-deoxy-D-glucopyranose,
Methylimidazol-2-ylmethyl)-N-(2-hydroxybenzyl) amino) acet-
amido)-2-deoxy-1,3,4,6-tetraacetyl-D-glucopyranose (10)
(11). 2-((N-(1-
2-(2-Bis(phenylmethyl)-1,2-diaminoethyl)-2-deoxy-1,3,4,6-tetra-
O-acetyl-b-D-glucopyranose, (15). Compound 1 was free-based
by dissolving 1·HCl (5 g, 13.0 mmol) in CH2Cl2 (100 mL)
and saturated aqueous Na2CO3 solution (150 mL), and stirring
vigorously for 0.5 h. The resulting mixture was then partitioned
and separated, then the aqueous phase was further extracted with
CH2Cl2 (3 ¥ 60 mL). The organic layers were combined and
reduced to dryness by rotary evaporation under reduced pressure
yielding 4.20 g, 12.1 mmol. This was dissolved in ClCH2CH2Cl
(80 mL), and added to a solution of 14 (~10.35 mmol, quantified
(0.225 g, 0.372 mmol) was dissolved in methanol (10 mL).
An excess of NaOMe (0.110 g, 20.4 mmol) was added and the
reaction mixture stirred at room temperature for two hours.
Amberlite CG-50 ion exchange resin was added, and after ten
minutes of rapid stirring, was removed by filtration. The filtrate
was reduced in volume on a rotary evaporator, and the resulting
yellow-orange oil purified by reverse phase HPLC (H2O with
0.1% TFA to 100% ACN over 30 min). The solvents were removed
in vacuo to give a pale yellow oil (0.040 g, 25% yield). Rf = 0.24
(silica-CH2Cl2/20% MeOH). 1H NMR (MeOD, 400 MHz, 25 ◦C,
d): 7.28–7.32 (m, 2H), 7.18–7.20 (m, 1H), 7.09–7.14 (m, 1H),
6.76–6.82 (m, 2H), 5.09 (d, J = 3.4 Hz, 0.63H - a), 4.67 (d, J =
8.3 Hz, 0.37H - b), 4.00–4.10 (m, 2H), 3.79–3.95 (m, 4H) 3.74
(s, 3H), 3.72 (s, 2H), 3.44 (s, 2H)3.35–3.43 (m, 2H). 13C NMR
(MeOD, 100 MHz, 25 ◦C, d): 173.64, 173.24, 157.74, 157.59,
146.42, 133.31, 133.15, 131.20, 131.12, 125.48, 123.97, 121.25,
121.09, 119.65, 116.92, 97.23, 92.98, 78.49, 76.31, 73.54, 73.01,
72.93, 72.65, 63.20, 63.12, 59.96, 59.89, 58.88, 56.00, 57.61, 57.55,
49.50, 35.26, 35.18. IR nmax (cm-1): 3293 (s, br), 2942 (m, br), 1671
(s), 1609 (w), 1531 (m), 1459 (m), 1210 (s), 1133 (s), 757 (m).
Purity assessed by HPLC: 97.8%. HR-MS (ES+ of MNa+) m/z
calcd for C20H28N4O7Na: 459.1856, found 459.1848.
1
by H NMR) in ClCH2CH2Cl (40 mL). The resulting mixture
was stirred at ambient temperature for 0.5 h before addition of
NaBH(OAc)3 (4.4 g, 20.8 mmol). After 16 h, the reaction was
quenched with saturated aqueous Na2CO3 solution (100 mL).
The layers were then partitioned and the aqueous phase extracted
with CH2Cl2 (2 ¥ 100 mL). The combined organic extracts was
washed with brine (100 mL) and dried over anhydrous MgSO4.
The solution was then taken to dryness by rotary evaporation
yielding crude 15 as dark brown oil. Column chromatography
(silica-CH2Cl2/10% CH3CN) was used to isolate and purify 15 as
light yellow oil (2.52 g, 44% yield): Rf = 0.60 (silica-CH2Cl2/10%
CH3CN); 1H NMR (DMSO-d6, 400 MHz, 25 ◦C, d): 7.31-7.21 (m,
10H), 5.65 (d, J = 8.4 Hz, 1H), 5.15 (t, J = 9.6 Hz, 1H), 4.81 (t,
J = 9.7 Hz, 1H), 4.15-4.11 (m, 1H), 4.01-3.91 (m, 2H), 3.50 (q,
J = 10.7 Hz, 4H), 2.75-2.60 (m, 3H), 2.36 (t, J = 6.2 Hz, 2H),
2.00 (s, 3H), 1.98 (s, 3H), 1.95 (s, 3H), 1.89 (s, 3H) ppm; 13C NMR
(DMSO-d6, 100 MHz, D1 = 2 s, 25 ◦C, d): 170.5, 170.3, 169.8,
169.3, 139.6, 129.0, 128.6, 127.3, 94.3, 73.5, 71.6, 69.0, 62.1, 61.1,
57.9, 53.7, 44.8, 21.2, 21.1, 21.0, 20.9 ppm; IR (NaCl) nmax (cm-1):
1752 (s), 1367 (w), 1222 (s), 1069 (m), 1040 (m), 751 (w); MS (ES+,
100% CH3CN, 30V): m/z = 571 (MH+); HR-MS, (ES+ of MH+)
m/z calcd for C30H39N2O9: 571.2656, found: 571.2657.
2-(Dibenzylamino)ethanol, (13). To a solution of ethanolamine
(12) (1.007 g, 16.49 mmol) in ClCH2CH2Cl (100 mL) was added
salicylaldehyde (3.4 mL, 33.46 mmol) and the resulting mixture
stirred at ambient temperature. After 10 minutes, NaBH(OAc)3
(15 g, 70.77 mmol) was added. After 16 hours, the reaction was
quenched by addition of saturated aqueous Na2CO3 solution
(100 mL). The mixture was then partitioned and the aqueous
layer further extracted with CH2Cl2 (2 ¥ 100 mL). The combined
organic extract was washed with brine (150 mL) and dried over
MgSO4. After filtration of the drying agent, the filtrate was taken
to dryness by rotary evaporation. 13 was purified and isolated as
a light yellow oil by column chromatography (silica-hexanes/10%
EtOAc to hexanes/20% EtOAc) in 83% yield: Rf = 0.15 (silica-
2-(2-Bis(phenylmethyl)-1,2-diaminoethyl)-2-deoxy-3,4,6-tri-O-
acetyl-b-D-glucopyranose, (16). To a solution of 15 (1.65 g,
2.89 mmol) in DMF (5 mL) was added NH2NH2·AcOH (0.30 g,
3.3 mmol). The resulting solution was stirred at ambient tempera-
ture for 2.5 h followed by addition of EtOAc (17 mL). The mixture
was then washed with ice-cold saturated aqueous Na2CO3 solution
(4 ¥ 40 ml), brine (20 mL) and dried over anhydrous MgSO4. The
solvent was removed by rotary evaporation to yield crude 16 as
hexanes/10% EtOAc); H NMR (DMSO-d6, 400 MHz, 25 ◦C,
1
d): 7.36-7.20 (m, 10H), 4.39 (t, J = 5.3 Hz, 1H), 3.57 (s, 4H),
1
3.49 (m, 2H), 2.47 (t, J = 6.6 Hz, 2H) ppm; Alternatively,
white hygroscopic solid (~80% yield, quantified by H NMR):
9224 | Dalton Trans., 2009, 9216–9227
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
1
Rf = 0.22 (silica-CH2Cl2/10% CH3CN); H NMR (DMSO-d6,
2-(1,2-Diaminoethyl)-2-deoxy-1-tert-butyldimethylsilyl-b-D-
glucopyranose, (19). To a solution of 18 (0.58 g, 1.12 mmol) in
MeOH (10 mL) was added Pd(OH)2/C (0.15 g). The reaction
was then stirred at ambient temperature under a positive H2
atmosphere using an H2-filled balloon. After 24 hours, the mixture
was filtered through a MeOH pre-washed pad of celite. The
resulting filtrate was reduced to dryness by rotary evaporation
yielding a light yellow solid (◦0.375 g, 99% yield). 1H NMR
(DMSO-d6, D2O 400 MHz, 25 C, d): 4.35 (d, J = 7.6 Hz, 1H),
3.43-3.39 (m, 2H), 3.15-3.01 (m, 3H), 2.86-2.78 (m, 1H), 2.70-2.61
(m, 2H), 2.56-2.52 (m, 1H), 2.06 (t, J = 8.4 Hz, 1H), 0.85 (s, 9H),
0.07 (s, 6H) ppm; IR (KBr) nmax (cm-1): 3406 (br), 2954 (m), 2929
(m), 2882 (m), 2957 (m), 1655 (w), 1637 (w), 1618 (w), 1473 (m),
1390 (w), 1255 (m), 1172 (m), 1079 (s), 840 (s), 782 (s); MS (ES+,
100% ACN, 30V): m/z = 337 (MH+); HR-MS, (ES+ of MH+) m/z
calcd for C14H33N2O5Si: 337.2159, found: 337.2158.
◦
300 MHz, 25 C, d): 7.32-7.21 (m, 10H), 5.18 (br s, 1H), 5.06 (t,
J = 9.8 Hz, 1H), 4.79 (t, J = 9.5 Hz, 1H), 4.15-3.95 (m, 3H),
3.60 (d, J = 13.7 Hz, 2H), 3.41 (d, J = 13.7 Hz, 2H), 2.83-2.80
(m, 1H), 2.66-2.52 (m, 2H), 2.46-2.35 (m, 2H), 2.00 (s, 3H), 1.96
(s, 3H), 1.89 (s, 3H) ppm; MS (ES+, 100% CH3CN, 30V): m/z =
529 (MH+); HR-MS, (ES+ of MH+) m/z calcd for C28H37N2O8:
529.2550, found: 529.2551.
2-(2-Bis(phenylmethyl)-1,2-diaminoethyl)-2-deoxy-1-tert-butyl-
dimethylsilyl-3,4,6-tri-O-acetyl-b-D-glucopyranose, (17). To
a
solution of 16 (7.88 mmol) in CH2Cl2 (25 mL) was added imidazole
(3.7 g, 54.3 mmol) and tert-butyldimethylsilyl chloride (4.81 g,
31.9 mmol). After stirring for 2 h at ambient temperature, the
reaction was quenched by addition of water (50 mL) and the
mixture partitioned. The aqueous layer was further extracted with
CH2Cl2 (3 ¥ 25 mL) and the combined organic extract was washed
with brine (20 mL), and dried over anhydrous MgSO4. After the
drying agent was filtered, the filtrate was taken to dryness by rotary
evaporation yielding crude 17 which was isolated and purified by
column chromatography (silica-CH2Cl2 to CH2Cl2/5% CH3CN)
as a yellow oil in 55% yield; Rf = 0.25 (silica-CH2Cl2/5% CH3CN);
1H NMR (DMSO-d6, 400 MHz, 25 ◦C, d): 7.31-7.21 (m, 10H), 4.99
(t, J = 9.6 Hz, 1H), 4.74-4.68 (m, 2H), 4.11-4.06 (m, 1H), 3.98-3.95
(m, 1H), 3.84-3.78 (m, 1H), 3.50 (q, J = 13.6 Hz, 4H), 2.89-2.85
(m, 1H), 2.72-2.64 (m, 1H), 2.43-2.37 (m, 3H), 1.99 (s, 3H), 1.96
(s, 3H), 1.89 (s, 3H), 0.84 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H) ppm;
13C NMR (DMSO-d6, 100 MHz, D1 = 1.5 s, 25 ◦C, d): 170.0,
169.9, 169.5, 139.1, 128.5, 128.1, 126.8, 98.0, 73.4, 70.5, 69.4, 63.4,
62.3, 57.6, 53.2, 45.0, 25.6, 20.6, 20.5, 20.4, 17.6, -4.2, -5.2 ppm;
IR (NaCl) nmax (cm-1): 3477 (m), 3319 (m), 2900 (br), 2118 (m),
1951 (m), 1770 (s), 1674 (m), 1651 (m), 1603 (m), 1585 (m), 1513
(s), 1495 (s), 1471 (s), 1455 (s), 1092 (m); MS (ES+, 100% MeOH,
30V): m/z = 643 (MH+), 665 (MNa+); HR-MS, (ES+ of MH+)
m/z calcd for C34H51N2O8Si: 643.3415, found: 643.3411.
2-((2-(N-Pyridin-2-ylmethyl))diaminoethyl)-2-deoxy-1-tert-but-
yldimethylsilyl-b-D-glucopyranose, (20). To a solution of 19
(0.37 g, 1.1 mmol) in MeOH (10 mL) was added 2-
pyridinecarboxaldehyde (0.11 mL, 1.15 mmol) and Na2CO3
(1.17 g, 11.04 mmol). The mixture was then stirred at ambient
temperature under a N2 atmosphere. After 24 hours, NaBH4 (0.1 g,
2.64 mmol) was added and the mixture further stirred at ambient
temperature. After another 24 hours, the solid was filtered and the
filtrate reduced to dryness by rotary evaporation. Compound 20
was isolated and purified using column chromatography (silica-
CH2Cl2/20% MeOH to CH2Cl2/19% MeOH/1% Et3N) as light
yellow solid (0.21 g, 45% yield): Rf = 0.23 (silica-CH2Cl2/20%
MeOH); 1H NMR (DMSO-d6, 400 MHz, 25 ◦C, d): 8.47 (d, J =
4.3 Hz, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H),
7.24-7.21 (m, 1H), 4.89 (d, J = 4.1 Hz, 1H), 4.34-4.32 (m, 2H),
3.76 (s, 2H), 3.64-3.63 (m, 1H), 3.44-3.33 (m, 1H), 3.07-3.03 (m,
3H), 2.95-2.85 (m, 1H), 2.75-2.65 (m, 1H), 2.65-2.52 (m, 3H), 2.09
(t, J = 8.7 Hz, 1H), 0.86 (s, 9H), 0.76 (s, 6H) ppm; IR (KBr) nmax
(cm-1): 3404 (br), 2930 (m), 2852 (m), 2604 (m), 2498 (m), 1474
(m), 1443 (m), 1453 (m), 1398 (m), 1074 (s), 1078 (s), 1038 (s), 839
(s), 783 (m); MS (ES+, 100% MeOH, 30V): m/z = 428 (MH+),
450 (MNa+); HR-MS, (ES+ of MH+) m/z calcd for C20H38N3O5Si:
428.2581, found: 428.2579.
2-(2-Bis(phenylmethyl)-1,2-diaminoethyl)-2-deoxy-1-tert-butyl-
dimethylsilyl-b-D-glucopyranose, (18). Compound 17 (2.8 g,
4.36 mmol) was dissolved in MeOH (40 mL). NaOMe (0.3 g,
5.56 mmol) was then added and the reaction mixture stirred at
ambient temperature. After 2.5 hours, the reaction was neutralized
by the addition of Sigma-Aldrich Mixed Bed Resin TMD-8
hydrogen and hydroxide form until no color change was observed
upon addition of further resin. The used up resin was then
filtered out and the filtrate taken to dryness by rotary evaporation.
Compound 18 was isolated as a white solid (1.75 g, 78%
yield) by column chromatography (silica-CH2Cl2/1% MeOH to
CH2Cl2/5% MeOH): Rf = 0.20 (silica-CH2Cl2/10% MeOH); 1H
NMR (DMSO-d6, D2O, 400 MHz, 25 ◦C, d): 7.33-7.19 (m, 10H),
4.32 (d, J = 7.6 Hz, 1H), 3.65-3.53 (m, 3H), 3.43-3.37 (m, 3H),
3.09-3.02 (m, 3H), 2.95-2.89 (m, 1H), 2.75-2.69 (m, 1H), 2.48-2.36
(m, 2H), 2.07 (t, J = 8.0 Hz, 1H), 0.82 (s, 9H), 0.03 (s, 3H), 0.02 (s,
3H) ppm; 13C NMR (DMSO-d6, 100 MHz, D1 = 1.5 s, 25 ◦C, d):
139.3, 128.6, 128.1, 126.7, 99.6, 77.2, 75.1, 70.4, 65.8, 61.2, 57.5,
53.4, 46.8, 25.7, 17.6, -3.8, -5.2 ppm; IR (KBr) nmax (cm-1): 3422
(br), 2928 (m), 2856 (m), 1496 (m), 1453 (m), 1257 (m), 1174 (m),
1078 (s), 1029 (m), 839 (s); MS (ES+, 100% MeOH, 30V): m/z =
517 (MH+), 539 (MNa+); HR-MS, (ES+ of MNa+) m/z calcd for
C28H44N2NaO5Si: 539.2917, found: 539.2914.
2-((2-(2-Hydroxybenzyl)-2-(pyridin-2-ylmethyl)diaminoethyl)-
2-deoxy-1-tert-butyldimethylsilyl -b-D-glucopyranose, (21). To
20 (0.21 g, 0.49 mmol) in ClCH2CH2Cl (5 mL) was added
salicylaldehyde (0.05 mL, 0.49 mmol). The resulting solution was
then stirred at ambient temperature. After 1 hour, NaBH(OAc)3
(0.21 g, 0.99 mmol) was added and the mixture further stirred at
ambient temperature. After 24 hours, the reaction was quenched
by addition of saturated aqueous Na2CO3 solution (2 mL). The
mixture was then partitioned and the aqueous layer further
extracted with CH2Cl2 (3 ¥ 5 mL). The combined organic phase
was taken to dryness by rotary evaporation affording crude 21.
Purification was achieved by column chromatography (silica-
CH2Cl2/5% MeOH to CH2Cl2/10% MeOH), yielding 21 as light
yellow solid (0.12 g, 44% yield): Rf = 0.53 (silica-CH2Cl2/10%
MeOH); 1H NMR (DMSO-d6, D2O, 400 MHz, 25 ◦C, d): 8.46 (d,
J = 4.8 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.38 (d, J = 7.6 Hz,
1H), 7.26 (t, J = 6.0 Hz, 1H), 7.15 (d, J = 7.2 Hz, 1H), 7.05 (t, J =
7.6 Hz, 1H), 6.72 (m, 2 H), 4.36 (d, J = 7.6 Hz, 1H), 3.67-3.56 (m,
5H), 3.40 (d, J = 8.8 Hz, 1H), 3.06-2.94 (m, 4H), 2.78-2.72 (m, 1H),
This journal is
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Dalton Trans., 2009, 9216–9227 | 9225
©

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2.53-2.51 (m, 2H), 2.10 (m, 1H), 0.79 (s, 9H), 0.02 (s, 3H), ◦-0.003
(s, 3H) ppm; 13C NMR (DMSO-d6, 100 MHz, D1 = 2 s, 25 C, d):
158.8, 156.8, 149.1, 137.4, 130.3, 128.7, 123.9, 123.4, 122.8, 119.3,
115.9, 99.4, 77.3, 75.1, 70.6, 65.8, 61.4, 59.3, 54.5, 53.6, 46.5, 26.1,
18.0, -3.4, -4.8 ppm; IR (KBr) nmax (cm-1): 3422 (br), 2921 (m),
2856 (m), 1591 (m), 1489 (m), 1473 (m), 1460 (m), 1254 (m), 1078
(s), 1029 (m), 839 (s), 783 (m), 755 (s); MS (ES+, 100% MeOH,
30V): m/z = 534 (MH+), 556 (MNa+); HR-MS, (ES+ of MH+)
m/z calcd for C27H44N3O6Si: 534.2999, found: 534.2997.
TFA to 100% ACN over 30 min) ((66% yield). Rf = 0.12 (silica-
CH2Cl2/20% MeOH). 1H NMR (MeOH-d4, 400 MHz, 25 ◦C, d):
8.81 (d, J = 5.4 Hz, 1H), 8.09 (dd, J = 7.8 Hz, 7.8 Hz, 1H), 7.73
(d, J = 7.6 Hz, 1H, 7.55 (dd, J = 7.40 Hz, 5.8 Hz, 1H), 5.16–
5.33, 4.34–4.70, 4.06–4.12, 3.65–3.91, 3.25–3.53 (m). 13C NMR
(MeOH-d4, 100 MHz, 25 ◦C, d): 197.8, 197.0, 196.4, 183.6, 180.5,
168.4, 159.8, 159.6, 152.2, 140.4, 125.8, 123.9, 95.7, 93.7, 91.3,
76.9, 74.8, 72.1, 71.9, 71.3, 70.9, 69.4, 69.2, 69.1, 69.0, 68.8, 68.7,
61.5, 60.9, 57.1, 54.4. IR nmax (cm-1): 3292 (w, br), 2360 (m), 2341
(m), 2027 (m), 2015 (s), 1910 (s), 1863 (s), 1635 (m, br), 774 (m).
Purity assessed by HPLC: 97.1%. HR-MS (ES+ of MNa+): m/z
calcd for C19H22N3O11Na187Re : 678.0710, found: 678.0704.
2-((2-(2-Hydroxybenzyl)-2-(pyridin-2-ylmethyl)diaminoethyl)-
2-deoxy-b-D-glucopyranose, (22). To 21 (0.21 g, 0.39 mmol) in
THF (5 mL) and MeOH (1 mL) was added benzyltrimethyl-
ammonium fluoride (0.12 g, 0.71 mmol). The resulting mixture was
then stirred at ambient temperature. After 24 hours, the solvent
was removed by rotary evaporation and 22 was isolated by col-
umn chromatography (silica-CH2Cl2/10% MeOH to CH2Cl2/20%
MeOH) as a white solid (0.10 g, 60% yield): Rf = 0.20 (silica-
CH2Cl2/20% MeOH); 1H NMR (DMSO-d6, D2O, 400 MHz,
25 ◦C, d): 8.50 (m 1H), 7.76 (m, 1H), 7.40 (m, 1H), 7.27 (m,
1H), 7.15 (m, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.74 (m, 2 H), 5.02
(d, J = 3.3 Hz, 0.73 H), 4.31 (d, J = 7.8 Hz, 0.26H), 3.79-3.45 (m,
7H), 3.07-3.02 (m, 2H), 2.89-2.71 (m, 2H), 2.56-2.52 (m, 2H), 2.26-
2.23 (m, 0.73H), 2.15-2.05 (m, 0.25H) ppm; 13C NMR (DMSO-d6,
Preparation of 11-Re(CO)3. Refluxed for 8 h then purified
to a pale yellow oil by semi-preparative HPLC (H2O with 0.1%
TFA to 100% ACN over 30 min) (43% yield). Rf = 0.30 (silica-
CH2Cl2/20% MeOH). 1H NMR (MeOD, 400 MHz, 25 ◦C, d): 7.53
(d, J = 7.4 Hz, 1H), 7.35 (dd, J = 7.1 Hz, J = 7.1 Hz, 1H), 7.20
(d, J = 5.8 Hz, 1H), 7.14 (s, 1H), 6.98 (m, 2H), 5.10 (d, J = 3.3 Hz
0.45H - a), 4.40 (s, 0.55H - b), 4.66–4.95, 4.08–4.14, 3.64–3.81,
3.01–3.55, (m, 13H), 3.64 (s, 3H). 13C NMR (MeOD, 100 MHz,
◦
25 C, d): 197.94, 197.44, 197.35, 196.55, 196.35, 181.29, 181.23,
158.24, 158.20, 152.17, 152.04, 135.03, 135.00, 133.00, 129.61,
125.88, 125.80, 121.49, 120.15, 120.01, 117.58, 96.19, 92.53, 91.99,
78.46, 75.69, 75.18, 73.51, 73.44, 72.36, 72.27, 72.13, 72.05, 68.18,
67.92, 65.29, 64.81, 64.43, 62.92, 62.74, 59.14, 58.95, 57.47, 57.42,
35.40, 35.28, 35.21. IR nmax (cm-1): 3232 (m, br), 2360 (w), 2028
(s), 1884 (s), 1672 (m), 1620 (s), 1198 (s), 1134 (s), 757 (m), 527
(s). Purity assessed by HPLC: 95.9%. HR-MS (ES+ of MH+) m/z
calcd for C23H28N4O10187Re: 707.1363, found 707.1365.
◦
100 MHz, D1 = 2 s, 25 C, d): 158.6, 158.4, 156.8, 156.7, 148.8,
148.7, 136.8, 136.7, 130.0, 129.8, 128.2, 128.1, 123.7, 123.5, 123.1,
123.0, 122.3, 122.2, 118.7, 118.6, 115.6, 97.8, 90.3, 76.9, 75.3, 72.2,
72.0, 70.8, 70.5, 65.0, 62.3, 61.2, 58.7, 54.4, 53.8, 53.1, 52.9, 45.8,
44.1 ppm; IR (KBr) nmax (cm-1): 3421 (br), 1596 (s), 1571 (s),
1458 (s), 1409 (s), 1252 (m), 1041 (s), 755 (s); MS (ES+, 100%
MeOH, 30V): m/z = 420 (MH+); HR-MS, (ES+ of MH+) m/z
calcd for C21H30N3O6: 420.2135, found: 420.2133; Anal. Calcd.
Preparation of 22-Re(CO)3. Refluxed for 24 h then isolated
∑
for C21H29N3O6 (CH3)2CO∑(CH3OH)0.3: C, 59.91; H, 7.49; N, 8.63.
by preparative TLC (silica-CH2Cl2/20% MeOH) as white solid
1
(87% yield). Rf = 0.31 (silica -CH2Cl2/20% MeOH). H NMR
Found: C, 59.68; H, 7.46; N, 8.95.
(DMSO-d6, D2O, 400 MHz, 25 ◦C, d): 8.97-8.74 (m, 1H), 8.15 (t,
J = 7.6 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.65-7.48 (m, 2H), 7.32
(t, J = 7.6 Hz, 1H), 7.00-6.98 (m, 1H), 6.93 (t, J = 7.6 Hz, 1H),
4.96-4.91 (m, 0.71H), 4.83-4.75 (m, 2H), 4.65-4.57 (m, 0.23H),
4.53-4.33 (m, 2H), 4.11-3.50 (m, 4H), 3.26-3.17 (m, 2H), 3.11-
2.85 (m, 2H), 2.62-2.51 (m, 1H), 2.28-2.04 (m, 1H); 13C NMR
(DMSO-d6, 100 MHz, 25 ◦C, d): 196.2, 196.0, 195.9, 160.0, 159.8,
156.8, 154.2, 153.8, 141.1, 141.0, 140.9, 140.8, 134.2, 131.2, 126.4,
126.2, 119.5, 118.3, 115.9, 97.3, 94.2, 90.9, 77.6, 77.5, 76.4, 73.3,
72.4, 71.4, 71.2, 70.5, 68.3, 68.0, 65.8, 60.8, 60.7, 60.0, 59.9, 55.0,
54.7, 53.3, 53.0, 52.8,; IR (KBr) nmax (cm-1): 3386 (br, m), 2029 (s),
1906 (s), 1609 (m), 1458 (m), 1033 (m), 762 (m); MS (ES+, 100%
MeOH, 30V): m/z = 688 (M185ReH+), 690 (M187ReH+); HR-MS
(ES+ of MH+): m/z calcd for C24H29N3O9187Re: 690.1461, found:
Synthesis of L-Re(CO)3 complexes. Equivalent amounts of
ligand and [Re(H2O)3(CO)3]Br in EtOH was stirred under reflux
conditions for between 6 and 24 h. The solvent was then removed
by rotary evaporation and the pure complex was isolated using
either column chromatography, HPLC or preparative TLC.
Preparation of 6-Re(CO)3. Refluxed for 24 h then solated as
a pale white solid (72% yield) by column chromatography (silica-
CH2Cl2/20% MeOH). Rf = 0.27 (silica-CH2Cl2/20% MeOH). 1H
NMR (MeOH-d4, 400 MHz, 25 ◦C, d): 8.85 (m, 1H), 8.07 (m,
1H), 7.70 (m, 1H), 7.53 (m, 2H), 7.35 (t, J = 8.3 Hz, 1H), 7.00
(m, 2H), 5.06-4.95 (m, 4H), 4.91-4.83 (m, 1H), 4.49-4.39 (m, 1H),
3.82-3.55 (m, 5H), 3.50-3.33 (m, 1H), 3.23-3.15 (m, 1H); 13C NMR
(MeOH-d4, 100 MHz, 25 ◦C, d): 197.5, 196.8, 182.8, 182.3, 182.1,
160.7, 160.3, 158.0, 154.0, 153.8, 142.5, 142.2, 134.9, 132.7, 127.4,
125.7, 125.4, 121.2, 119.8, 119.6, 117.3, 95.8, 91.9, 91.8, 78.2, 78.0,
75.3, 74.9, 73.1, 72.1, 72.0, 71.9, 71.8, 69.2, 69.1, 68.2, 68.1, 63.0,
62.7, 62.6, 62.5, 59.4, 57.1, 57.0. IR (KBr) nmax (cm-1): 3386 (br,
m), 2032 (s), 1905 (s), 1618 (m), 1458 (w), 764 (w). MS (ES+, 100%
MeOH, 30V): m/z = 702 (M185ReH+), 704 (M187ReH+); HR-MS
(ES+ of MH+): m/z calcd for C24H27N3O10187Re: 704.1254, found:
704.1256; Anal. Calcd. for C24H26N3O10Re∑(HBr)1.5: C, 34.98; H,
3.36; N, 5.10 Found: C, 34.75; H, 3.51; N, 4.88.
∑
690.1460; Anal. Calcd. for C24H28N3O9 (HBr)0.1∑H2O: C, 36.60; H,
3.97; N, 5.34 Found: C, 36.70; H, 3.92; N, 5.17.
General procedure for radiolabeling with [99mTc(H2O)3(CO)3]+.
The organometallic precursor [99mTc(H2O)3(CO)3]+ was prepared
from a saline solution of Na[99mTcO4] (1 mL, 100 MBq) using
an Isolink(tm) kit generously provided by Mallinckrodt (now
Covidien). Briefly, a 1 mL saline solution of Na[99mTcO4] was added
to an Isolink(tm) kit, and the vial was heated to reflux for 20 min.
Upon cooling, ~0.12 mL of a 1 M HCl solution was added to
adjust the pH between 9-10. To 0.50 mL of compound 6, 8, or 22
Preparation of 8-Re(CO)3. Refluxed for 6 h, then purified as
an off-white solid by semi-preparative HPLC (H2O with 0.1%
(10-3 M) in EtOH, was added 3 equiv of NaOEt (0.15 mL, 10-2
M
9226 | Dalton Trans., 2009, 9216–9227
This journal is The Royal Society of Chemistry 2009
©

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in EtOH) followed by 0.10 mL of [99mTc(H2O)3(CO)3]+ solution.
Alternatively, to 0.5 mL of 11 in PBS solution at pH 7.4 was added
0.10 mL of [99mTc(H2O)3(CO)3]+ solution. The resulting solutions
were heated to 80 ◦C for 0.5 h and cooled to ambient temperature
prior to HPLC injection.
(C.L.F.) also supported by MDS Nordion. We acknowledge
support from Mallinckrodt Inc. (now Covidien) for supply of
IsolinkTM kits, and UBC Hospital Nuclear Medicine Department
for supply of Na[99mTcO4].
Notes and references
Cysteine and histidine challenge experiments. To either cysteine
or histidine (1 mM, 0.9 mL) in PBS buffer solution at pH 7.4
was added a solution of 99mTc complex (final ligand concentration
10-5 M). The samples were incubated at 37 ◦C for 24 h and were
then analyzed by HPLC.
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GLUT-1 cell uptake studies
These experiments were performed in a manner similar to that
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˚
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that of ADP was 12.5 min.
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Acknowledgements
Acknowledgement is made to NSERC for funding the research
via a Strategic Grant, and an Industrial Postgraduate Scholarship
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 9216–9227 | 9227
©