Chemical Approach to Positional Isomers of Glucose-Platinum Conjugates Reveals Specific Cancer Targeting through Glucose-Transporter-Mediated Uptake in Vitro and in Vivo

Article abstract of DOI:10.1021/jacs.6b06937

Glycoconjugation is a promising strategy for specific targeting of cancer. In this study, we investigated the effect of d-glucose substitution position on the biological activity of glucose-platinum conjugates (Glc-Pts). We synthesized and characterized all possible positional isomers (C1α, C1β, C2, C3, C4, and C6) of a Glc-Pt. The synthetic routes presented here could, in principle, be extended to prepare glucose conjugates with different active ingredients, other than platinum. The biological activities of the compounds were evaluated both in vitro and in vivo. We discovered that varying the position of substitution of d-glucose alters not only the cellular uptake and cytotoxicity profile but also the GLUT1 specificity of resulting glycoconjugates, where GLUT1 is glucose transporter 1. The C1α- and C2-substituted Glc-Pts (1α and 2) accumulate in cancer cells most efficiently compared to the others, whereas the C3-Glc-Pt (3) is taken up least efficiently. Compounds 1α and 2 are more potent compared to 3 in DU145 cells. The α- and β-anomers of the C1-Glc-Pt also differ significantly in their cellular uptake and activity profiles. No significant differences in uptake of the Glc-Pts were observed in non-cancerous RWPE2 cells. The GLUT1 specificity of the Glc-Pts was evaluated by determining the cellular uptake in the absence and in the presence of the GLUT1 inhibitor cytochalasin B, and by comparing their anticancer activity in DU145 cells and a GLUT1 knockdown cell line. The results reveal that C2-substituted Glc-Pt 2 has the highest GLUT1-specific internalization, which also reflects the best cancer-targeting ability. In a syngeneic breast cancer mouse model overexpressing GLUT1, compound 2 showed antitumor efficacy and selective uptake in tumors with no observable toxicity. This study thus reveals the synthesis of all positional isomers of d-glucose substitution for platinum warheads with detailed glycotargeting characterization in cancer.

Full text of DOI:10.1021/jacs.6b06937

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Article
Chemical approach to positional isomers of glucose-
platinum conjugates reveals specific cancer targeting through
glucose-transporter mediated uptake in vitro and in vivo
Malay Patra, Samuel G. Awuah, and Stephen J. Lippard
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b06937 • Publication Date (Web): 27 Aug 2016
Downloaded from http://pubs.acs.org on August 28, 2016
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Chemical approach to positional isomers of glucoseꢀplatinum conjugates reveals specific
cancer targeting through glucoseꢀtransporter mediated uptake in vitro and in vivo
Malay Patra, Samuel G. Awuah and Stephen J. Lippard*
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Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts
02139, United States
ABSTRACT: Glycoconjugation is a promising strategy for specific targeting of cancer. In this
study, we investigated the effect of Dꢀglucose substitution position on the biological activity of
glucoseꢀplatinum conjugates (GlcꢀPts). We synthesized and characterized all possible positional
isomers (C1α, C1β, C2, C3, C4 and C6) of a GlcꢀPt. The synthetic routes presented here could in
principle be extended to prepare glucoseꢀconjugates with different active ingredients than
platinum. The biological activities of the compounds were evaluated both in vitro and in vivo.
We discovered that variation in position of substitution of Dꢀglucose not only alters the cellular
uptake and cytotoxicity profile but also the GLUT1 specificity of resulting glycoconjugates,
where GLUT1 is glucose transporter 1. The C1αꢀ and C2ꢀsubstituted GlcꢀPts (1α and 2)
accumulate in cancer cells most efficiently compared to the others, whereas the C3ꢀGlcꢀPt (3) is
taken up least efficiently. Compounds 1α and 2 are more potent compared to 3 in DU145 cells.
The αꢀ and βꢀanomer of the C1ꢀGlcꢀPt also differ significantly in their cellular uptake and
activity profiles. No significant differences in uptake of the GlcꢀPts were observed in
noncancerous RWPE2 cells. The GLUT1 specificity of the GlcꢀPts was evaluated by
determining the cellular uptake in the absence and presence of the GLUT1 inhibitor cytochalasin
B, and by comparing their anticancer activity in DU145 cells and a GLUT1 knockdown cell line.
The results reveal that C2ꢀsubstituted GlcꢀPt 2 has the highest GLUT1 specific internalization,
which also reflects the best cancer targeting ability. In a syngeneic breast cancer mouse model
overexpressing GLUT1, compound 2 showed antitumor efficacy and selective uptake in tumors
with no observable toxicity. This study thus reveals the synthesis of all positional isomers of Dꢀ
glucose substitution for platinum warhead with detailed glycotargeting characterization in
cancer.
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INTRODUCTION
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The essential nutrient Dꢀglucose is the main carbon and energy source for cells. Cellular uptake
of Dꢀglucose is mediated by membrane bound glucose transporters which are classified into two
main families: facilitative (GLUTs) and sodium dependent (SGLTs).1 GLUTs transport Dꢀ
glucose by an energy independent facilitative diffusion process whereas SGLTs mediated Dꢀ
glucose uptake is an active and energy dependent process. There are fourteen human GLUTs
discovered to date, among which only GLUTs1ꢀ4 have been extensively studied.1,2 The
molecular structures of human GLUT1 and GLUT3 have been reported very recently.2,3 GLUT1
is broadly overexpressed in various cancers, including hepatic, pancreatic, breast, esophageal,
brain, renal, lung, cutaneous, colorectal, endometrial, ovarian, and cervical.1,4 GLUT1 expression
levels in patientꢀderived tumor samples correlate well with poor prognosis.1,4ꢀ6 Moreover,
overexpression of other glucose transporters, such as GLUT2 (gastric, breast, colon, and liver
carcinomas), GLUT3 (Bꢀcell nonꢀHodgkin's lymphoma), GLUT12 (prostate adenocarcinomas)
and SGLT1ꢀ2 (colorectal and metastatic lung carcinomas) has also been reported for certain type
of cancers.1,4,7,8
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Dꢀglucose is metabolized oxidatively in the mitochondria of healthy nonꢀcancerous cells and
produces 36 moles ATP per mole of glucose. In contrast, cancer cells metabolize glucose by
aerobic glycolysis, which is energetically very inefficient and provides only 4 moles ATP from
one mole of glucose.9 Therefore, the rapid growth and proliferation of cancer cells demand
drastic increase in glucose uptake and metabolite flux; this phenomenon is known as the
“Warburg effect”.10 The altered glucose intake and glucose transporter overexpression are
common in cancers and provide clinically validated targets for cancer treatment.11ꢀ13 Over the
years, the design of glycoconjugates to exploit the overexpressed glucose transporters for
specific delivery of various organic anticancer drugs, fluorophores, and radiotracers to cancer
tissues, has been a field of active research.14ꢀ20 The potential of glycoconjugation as a strategy in
diagnosis and therapy is supported by GLUTsꢀmediated uptake and selective accumulation of the
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radiolabeled glucose derivative FꢀFDG in cancer tissues. The tracer is frequently employed in
the clinic for diagnosis and staging of various types of cancers.12,21 As an alternative to the
radioisotopeꢀlabeled glucose bioꢀprobes, various fluorescentꢀtagged glucose molecules were also
developed.15 Glycoconjugates of various anticancer drugs such as paclitaxel, adriamycin, and
azomycin have been synthesized for specific delivery to cancer cells.14 In order to introduce
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tumorꢀtargeting properties to platinum and other metalꢀbased anticancer compounds, others and
we have reported the synthesis and biological activities of glucoseꢀconjugates of metal
complexes.22ꢀ27
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Figure 1. Representative examples of positional isomers of Dꢀglucose conjugates having
different biological properties.
Since the introduction of glufosfamide in 1995,28 the first explicitly designed glucoseꢀ
conjugate of a DNA alkylating agent ifosfamide, which exploits the overexpressed glucose
transporters in the membrane of cancer cells, there has been a significant body of work on
glycoconjugates in the literature.14,15,26,29ꢀ31 However, in the last two decades, there has not been
even a single report describing the synthesis of a complete set of positional isomers (i.e. C1ꢀC6
with the same linker length) of a given glycoconjugate to investigate the influence of position of
substitution in Dꢀglucose on its biological activity. There are one primary and four secondary
hydroxyl groups in Dꢀglucose, which offer the possibility to attach a warhead or a sensor, such as
a fluorophore. During the transporterꢀmediated translocation, these “hydroxyl” groups play
important roles in hydrogen bonding interactions with various amino acid residues of GLUTs, as
revealed recently in crystal structures of Dꢀglucose bound GLUT1 and GLUT3.2,3 Therefore, the
recognition and translocation efficiencies through the glucose transporters are expected to vary
for different positional isomers of a glycoconjugate.14 In fact, analyzing the results from a few
earlier studies revealed that the position of substitution of Dꢀglucose influences the biological
properties of glucoseꢀconjugates.14,15,30,32,33 The C6ꢀsubstituted glucoseꢀconjugate of 4ꢀ
nitrobenzofurazan (6ꢀNBDG, Figure 1) binds to GLUT1 transporters with a 300ꢀfold greater
affinity than Dꢀglucose, and that is why the rate of cellular uptake of 6ꢀNBDG is relatively slow
and not reduced efficiently by Dꢀglucose in a competition assay.30,34 On the other hand, its C2ꢀ
positional isomer (2ꢀNBDG, Figure 1) accumulates readily in cells and its uptake can be
inhibited efficiently when coꢀincubated with Dꢀglucose.15,35 Moreover, 2ꢀNBDG, but not 6ꢀ
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NBDG, enters the glycolytic pathway in cells and is converted to its nonꢀfluorescent 6ꢀphosphate
derivative, thereby offering the ability to study the cellular metabolic events.15 The C6 positional
isomer of a glucoseꢀconjugate of 7ꢀchlorokynurenic acid showed superior protective activity,
compared to its C3ꢀpositional isomer, against seizures induced by NꢀmethylꢀDꢀaspartate
(NMDA) in mice (Figure 1).33 Thus syntheses and a systematic study of biological activities of
various positional isomers of a given glycoconjugate are highly desirable for they might shed
light on identifying the appropriate “hydroxyl” group of Dꢀglucose at which to attach a warhead,
thereby contributing to the design of next generation glycoconjugates.14 Towards this goal, we
synthesized positional isomers of glucoseꢀplatinum conjugates using [(transꢀ1,2ꢀdiaminoꢀ
cyclohexane)(2,2ꢀdimethyl malonato)Pt(II)] (7) as the warhead (Figure 2). Biological studies
revealed that the position of substitution is an important determinant of cellular uptake,
cytotoxicity, and GLUT1 specificity of a glucoseꢀdrug conjugate.
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HO
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HO
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OH
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O
C2-Glc-Pt (2)
O
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Pt
N
H2
O
[(DACH)(DMM)Pt] (7)
Figure 2. Structures of C1ꢀC6ꢀsubstituted positional isomers (1ꢀ6) of a glucoseꢀplatinum
conjugate and aglycone 7.
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RESULTS AND DISCUSSION
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Synthesis of positional isomers
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Glucoseꢀconjugates were designed in which attachment to the sugar moiety via a spacer
preserved key structural features of the entity, a prerequisite for optimal glucoseꢀtransporter
recognition. Dꢀglucose offers five hydroxyl groups to which the spacer can be appended. Here
we attached a biologically active platinum anticancer drug candidate 7 (Figure 2) to the different
positions of Dꢀglucose iteratively via a two carbon spacer using an ether glycosidic linkage.27
The rigorous syntheses of the complete set of positional isomers with the same linker length of
the glucoseꢀplatinum conjugates involve multiple steps with several selective protection and
deprotection steps.
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OH
OAc
O
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OAc
O
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(a)
(b)
HO
HO
AcO
AcO
AcO
AcO
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13 (α-anomer)
OH
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HO
HO
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OH
OH
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19%
OH
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O
Scheme 1. Synthesis of C1ꢀGlcꢀPts 1β and 1α. Reagents and conditions: (a)36,37 3ꢀ
Bromopropanol, BF3ꢀOEt2, 20 h; b) i) 2ꢀmethylꢀdiꢀtertꢀbutyl malonate, NaH, DMF, 24 h, ii)
NaOMe, MeOH, 2 h; (c) TFA, DCM, 5 h; (d) i) 3ꢀBromopropanol, BF3ꢀOEt2, 20 h, ii) anhydrous
FeCl3, DCM, 36 h; (e) i) (DACH)PtCl2, Ag2SO4, H2O, 20 h, ii) Ba(OH)2, H2O, 24 h.
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The synthesis of the C1 derivatives is relatively facile and summarized in Scheme 1. The
special reactivity profile offered by the anomeric “OAc” group of βꢀDꢀglucose pentaꢀacetate (8)
allows Lewis acid catalyzed regioselective introduction of 3ꢀbromoꢀpropanol at the C1 position.
Both anomers of the desired C1ꢀsubstituted glucoseꢀmalonic acid derivatives 11 and 14 could be
synthesized from the starting material 8. Treatment of 8 with 3ꢀbromoꢀpropanol in the presence
of BF3ꢀOEt2 provided compound 9 exclusively as the βꢀanomer. Formation of only a small
amount (10ꢀ15%) of the αꢀanomer (12) was observed, which was removed during
chromatographic purification. Compound 9 was then subjected to a nucleophilic substitution
reaction with 2ꢀmethylꢀdiꢀtertꢀbutyl malonate followed by deprotection of the acetylꢀprotecting
groups using NaOMe. The resulting intermediate 10 was treated with trifluoroacetic acid (TFA)
to obtain the desired glucoseꢀmalonic acid derivative 11. For the synthesis of 14, which is the αꢀ
anomer of 11, FeCl3 mediated anomerization was performed after installation of the 3ꢀbromoꢀ
propanol unit at the C1 position of 8 in the first step.38 The αꢀanomer 12 was isolated in 15%
overall yield after two steps. Subsequently, a similar reaction sequence as that described for the
synthesis of 11 yielded ligand 14. The last synthetic step involved platination of ligands 11 or 14
with [dichloro(1R,2R)ꢀcyclohexaneꢀ1,2ꢀamine)platinum(II)], [(DACH)PtCl2],39 which gave the
C1ꢀsubstituted GlcꢀPts 1β and 1α, respectively. The anomeric purity of the C1ꢀGlcꢀPts was
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confirmed by their respective H NMR spectra (Figure S6). The C1 anomeric proton signals of
1β and 1α appear at 4.45 ppm (d, J = 7.9 Hz) and 4.97 ppm (d, J = 3.7 Hz) in their respective
NMR spectra. Furthermore, the coupling constants (J = 7.9 Hz for β and 3.7 Hz for α) confirmed
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the nature of the anomers of 1.40 As expected, significant differences in the C NMR signals of
1β and 1α was also noticed. The C1 anomeric carbon resonances appeared at 102.3 ppm and 98.2
ppm for 1β and 1α, respectively. These spectroscopic data confirm the distinct molecular
configurations of 1β and 1α.
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Scheme 2. Synthesis of C2ꢀGlcꢀPt 2. Reagents and conditions: (a) BnBr, NaH, DMF; (b) BnOH,
HCl in Et2O or BF3ꢀOEt2, (c) TFA, H2O, DCM; (d) i) diꢀbutyl tin (IV) oxide, ii) BnBr, K2CO3;
(e) allyl bromide, NaH; (f) i) NaBH4, BF3ꢀOEt2, ii) NaOH, H2O2; (g) PPh3, CBr4; (h) 2ꢀmethylꢀ
diꢀtertꢀbutyl malonate, NaH, DMF; (i) Pd/C, H2; (j) TFA, DCM; (k) i) (DACH)PtCl2, Ag2SO4,
H2O, 20 h, ii) Ba(OH)2, H2O, 24 h.
In contrast to the C1ꢀGlcꢀPts, the synthesis of C2ꢀGlcꢀPt 2 involves a twelveꢀstep route,
which is presented in Scheme 2. The synthesis was initiated from commercially available,
partially protected 1,2ꢀOꢀisopropylideneꢀαꢀDꢀglucofuranose (15) followed by subsequent
conversion to the fully protected derivative, 16.41 Efforts to synthesize intermediate 1842 from 16
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in a oneꢀstep procedure using benzyl alcohol and either HCl or BF3ꢀOEt2 were unsuccessful.43
Although formation of 18 was traced by thin layer chromatography (TLC) and ESIꢀmass
spectrometry, the purification was tedious, requiring the removal of undesired impurities present
in the reaction mixture. Changing the ratio of reactants (benzyl alcohol and acid) and/or reaction
conditions (temperature and/or time) also did not help. Finally, 18 was synthesized in twoꢀsteps
from 16 in good yield. Compound 18 was then subjected to nucleophilic substitution with allyl
bromide in the presence of NaH to insert the alkyl chain at the C2 position. Hydroborationꢀ
oxidation of 19 afforded compound 20. The hydroxyl group of 20 was then transformed into
bromide (21) using an Appel reaction, and subsequent nucleophilic substitution with 2ꢀmethylꢀ
diꢀtertꢀbutyl malonate in the presence NaH provided 22. The protecting groups of 22 were
removed in a twoꢀstep procedure to give the desired ligand 24. Subsequently, C2ꢀGlcꢀPt 2 was
prepared following a procedure similar to that described for the C1ꢀGlcꢀPts.
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Scheme 3. Synthesis of C3ꢀGlcꢀPt 3. Reagents and conditions: (a) 1ꢀchloroꢀ3ꢀbromoꢀpropane,
50% NaOH, DMSO; (b) NaI, acetone, reflux; (c) 2ꢀmethylꢀdiꢀtertꢀbutyl malonate, NaH, DMF;
(d) TFA, H2O, DCM (e) i) (DACH)PtCl2, Ag2SO4, H2O, 20 h, ii) Ba(OH)2, H2O, 24 h.
Commercial availability of the partially protected 1,2:5,6ꢀdiꢀOꢀisopropylideneꢀαꢀDꢀ
glucofuranose (25, Scheme 3), where all the hydroxyl groups, except that on C3, are protected
makes the synthesis of C3ꢀGlcꢀPt (3) plausible. As shown in Scheme 3, 25 was treated with 1ꢀ
chloroꢀ3ꢀbromoꢀpropane in the presence of NaOH to append the alkyl spacer to the C3 position
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of the sugar unit. The reaction yielded a 1:1 mixture of the desired 26B and an undesired
byproduct 26A. The separation of 26B from the mixture was impossible because both 26A and
26B eluted together during chromatographic purification. We therefore used the mixture for
subsequent steps. Treatment of the mixture with NaI followed by a nucleophilic substitution
reaction with 2ꢀmethylꢀdiꢀtertꢀbutyl malonate yielded 28. The unreacted 26A could be easily
separated from the mixture in this step. Treatment of 28 with trifluoroacetic acid resulted in the
required ligand 29, which was then platinated to give the desired C3ꢀGlcꢀPt 3.
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Scheme 4. Synthesis of C4ꢀGlcꢀPt 4. Reagents and conditions: (a) allyl bromide, NaH; (b) i)
NaBH4, BF3ꢀOEt2, ii) NaOH, H2O2; (c) PPh3, CBr4; (d) 2ꢀmethylꢀdiꢀtertꢀbutyl malonate, NaH,
DMF; (e) Pd/C, H2; (f) TFA, DCM; (g) i) (DACH)PtCl2, Ag2SO4, H2O, 20 h, ii) Ba(OH)2, H2O,
24 h.
For the synthesis of C4ꢀGlcꢀPt (4), we required a glucose derivative where all the hydroxyl
groups, with exception of the C4 hydroxyl, was protected (34 in Scheme 4). The synthetic route
is depicted in Scheme 4. Similar to C1ꢀGlcꢀPts, synthesis of 4 was initiated from βꢀDꢀglucose
pentaꢀacetate (8) which was converted to 34 by a fiveꢀstep procedure (Scheme S1 in SI).44 The
desired ligand 40 was then obtained using the exact same reaction sequence as used for the
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synthesis of 24 from 18 (Scheme 2). The last synthetic step involves platination of 40 to give 4.
The C6ꢀGlcꢀPt 6 and aglycone 7 were synthesized following recently reported synthetic routes.27
All new compounds were characterized by NMR (1H, 13C, 195Pt) spectroscopy and
electrospray ionization (ESI) mass spectrometry. Purities of the platinum compounds were
confirmed to be ≥95% by elemental microꢀanalysis and analytical HPLC (see SI for details).
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Cytotoxicity profile of GlcꢀPts 1ꢀ4, and 6
Having obtained all the positional isomers of the GlcꢀPts, we first investigated whether the
position of substitution influences their antiꢀproliferation properties. The cytotoxicity was
evaluated against a panel of cancer cell lines and a noncancerous one by the MTT (3ꢀ(4,5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) assay. The potencies were expressed as
IC50 values, which represent the concentration of the compounds that confer 50% growth
inhibition. DU145 (prostate), A2780 (ovarian), A549 (lung) cancers, and MRC5 (nonꢀcancerous
lung fibroblast) cells were treated with GlcꢀPts 1ꢀ4 and 6 for 72 h, after which the cell viabilities
were evaluated. As shown in Table S1, all the GlcꢀPts exhibit high potency and inhibited cancer
cell growth at IC50 values of 0.07ꢀ0.32 µM (A2780), 1.7ꢀ4.2 µM (A549), and 2.4ꢀ3.7 µM
(DU145). This tight distribution of IC50 values for the GlcꢀPts against all the three cancer cell
lines in the assay indicates that the position of substitution does not have a significant influence
on cytotoxicity when cells were incubated for 72 h with the compounds. From our recent work
on the C6ꢀglucoseꢀplatinum conjugates we learned that the incubation time plays an important
role in determining the cytotoxicity of this class of compounds.27 We therefore treated DU145
cells with the compounds for 7.5 instead of 72 h, followed by 64 h incubation in fresh media. As
shown in Table 1, significant differences in the IC50 values among the GlcꢀPts emerged under
these conditions. Glucoseꢀconjugate 2 (IC50 = 9 µM) displayed approximately 3ꢀtimes greater
potency compared to 3 (IC50 = 26 µM), with the overall order of potency being 2 ≥ 1α = 1β ≥ 4 =
6 ≥ 3. These results indicate that C1 and C2 substitution of Dꢀglucose is preferred over the C3,
C4, and C6 for conveying cytotoxicity of the glycoconjugates in cancer cells.
Table 1. IC50 values for GlcꢀPts 1ꢀ4, 6 and aglycone 7 in DU145 prostate cancer (7.5 and 72 h
assays) and MRC5 nonꢀcancerous lung epithelial (72 h assay) cells. Data reflect the mean±SD of
results from three or more independent experiments, each performed in triplicate.
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Cell lines
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2.6±0.08 3.7±0.07 2.8±0.27 3.4±0.21 2.4±0.14 2.4±0.35 1.4±0.51
>100 >100 93.6±15 72.3±5.8 95.5±19 34.7±8.8
13±6.2
As shown in Table 1 and S1, in contrast to the cancerous cells, where low micromolar IC50
values were recorded for all GlcꢀPts, several fold higher IC50 values were observed for
noncancerous MRC5 lung fibroblast cells for all GlcꢀPts in the 72 h MTT assay, the exception
being 1β. Notably, 1α and 1β are C1 positional isomers, but the IC50 value of 1α is 100 µM
>
whereas the same for 1β is <20 µM in MRC5 cells (Table S1 and Figure S7a), suggesting that
the configuration at the anomeric center has an important role in determining the cytotoxicity of
glucose conjugates.
Whole cell uptake studies
In order to investigate whether the position of substitution has an influence on the cellular uptake
of glucoseꢀconjugates, we studied the whole cell uptake of GlcꢀPts using DU145, A2780,
SKOV3, and A549 cell lines. All express high levels of the GLUT1 transporter.27,45 Cells were
treated with compounds for 7.5 h and the uptake of the GlcꢀPts was determined by measuring the
platinum content by atomic absorption spectroscopy.
It is expected that the distinctive structural and molecular conformations of the GlcꢀPts under
investigation will influence their function as substrates for glucose transporters. As shown in
Figure 3, the cellular uptake of GlcꢀPts may vary with the position of substitution. In addition,
the extent of variation in uptake is also cell line dependent. Irrespective of the cancer cell type,
the uptake of 2 is significantly higher than that of 3. Strikingly, of the two anomers of 1, uptake
of 1α is higher than that of 1β in A2780 and DU145 cancer cells. These results are consistent
with previously reported C1ꢀsubstituted Cy3ꢀglucose conjugates, where a relatively higher
accumulation of the αꢀanomer was noticed compared to its βꢀanomer.31 The preferential
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Figure 3. Whole cell uptake of GlcꢀPts 1ꢀ4 and 6 in DU145, A2780, SKOV3 and A549 cancer
cells (compound concentration 20 µM for A2780, DU145 and SKOV3 cells and 40 µM for A549
cells, incubation time 7.5 h). Data represent the mean ± SD of at least three replicates. The
asterisks denote differences that statistically significant (*P<0.01, **P<0.001).
uptake of Cꢀ1 substituted αꢀanomer of a glycoconjugate in cancer cells is attributed to its
relatively high recognition by facilitative glucose transporters.2 In a recently published crystal
structure of Dꢀglucose bound to human GLUT3, which shares 66% sequence homology with
human GLUT1, the occupancy of the αꢀanomer in the substrate binding site was dominant (69%)
over that of the βꢀanomer, despite the relative prevalence of the βꢀanomer in aqueous solution.2
In A549 cells, 2 was taken up most efficiently, and 3 and 1β were taken up least efficiently.
These results confirm that the cellular uptake of glucoseꢀconjugates in cancer cells is dependent
on the position of substitution of Dꢀglucose. From their structural similarities, GlcꢀPts 1ꢀ4 are
expected to have octanolꢀwater partition coefficients similar to that of GlcꢀPt 6 (log P = –2.1).27
The highly hydrophilic nature of GlcꢀPts indicate that their cellular internalization via passive
diffusion through the cellular lipid membrane is unlikely. Therefore, differences in uptake
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between different positional isomers in cancer cells most likely arise from differential glucoseꢀ
transporter mediated cellular internalization efficiencies.
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The cellular uptake of GlcꢀPts 1α, 2, and 3 could be correlated with IC50 values obtained from
MTT assays performed over a 7.5 but not 72 h incubation period. For example, as shown in
Figures 3, despite significantly higher uptake of 1α and 2 compared to that of 3 in A2780 or
DU145 cells, all displayed similar cytotoxicity (Tables 1 and S1). Determination of cytotoxicity
using a short incubation time (7.5 h with compounds followed by 64 h with fresh media) in
DU145 cells revealed 1α and 2 to be more cytotoxic compared to 3. This observation parallels
the observed differences in the cellular uptake of these compounds. However, despite being
taken up less efficiently, 1β had a similar IC50 value to those of 1α and 2 in DU145 cells.
As discussed earlier, despite similar activities in various cancer cells, 1α and1β largely differ
in their activities in MRC5 cells in a 72 h MTT assay (Table1). In order to obtain additional
insight, cellular uptake studies of 1α and 1β in MRC5 cells were performed. As shown in Figure
S7b, accumulation of the 1β anomer was 43% greater than that of the 1α anomer. This difference
in uptake at least in part explains the differential cytotoxicity profile of the two anomers of 1.
The results indicate that both the position of substitution as well as the anomeric configuration
influence the biological properties of a glucoseꢀconjugate.
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Cellular uptake in cancer cells versus matched normal cells
We next investigated whether the ability of GlcꢀPts to accumulate selectively in cancer cells
varies with the position of Dꢀglucose substitution. The cellular uptake in prostate epithelial
RWPE2 cells was investigated and compared with uptake in the matched prostate cancer DU145
cell line. Cancer cells, but not the normal healthy cells, upregulate the expression of glucose
transporters and, consequently, glucose influx and utilization.11ꢀ13 We recently showed that
DU145 has an elevated level of GLUT1 expression, whereas the expression of GLUT1 was not
even detectable for RWPE2 cells under a similar experimental condition.27 Therefore,
glycoconjugates capable of exploiting the membraneꢀassociated glucose transporters are
expected to accumulate in the DU145 cancer cells preferentially. Moreover, the position of
substitution can modulate the uptake in DU145 but not RWPE2 cells. As shown in Figures S8a
& b, the accumulation of all the GlcꢀPts in DU145 cells is 3.7ꢀ5.5 fold higher than in RWPE
cells. Importantly, as shown in Figure S8b, accumulation of all the GlcꢀPts is similar in RWPE2
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cells (25ꢀ30 pmole/mg proteins). However, statistically significant variations between different
GlcꢀPts in DU145 cells suggest position of substitution of Dꢀglucose at the platinum warhead
potentiates uptake in DU145 cells. The cellular uptake data together with differences in the level
of glucoseꢀtransporter expression in DU145 and RWPE2 cells indicate that the variable
accumulation of GlcꢀPts arises, at least partly, from differential glucoseꢀtransporter mediated
translocation efficiencies of GlcꢀPts.
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Control
50 ꢀM CytochalasinB
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Figure 4. Effect of GLUT1 inhibitor cytochasinB on the cellular uptake of GlcꢀPts 1ꢀ4, 6 and
aglycone 7 in DU145 cells (compound concentration 80 µM, exposure time 2 h).
Dependence of GLUT1 specificity on position of substitution
The specific cancer targeting ability of a glycoconjugate depends on how well it is recognized by
or translocated through glucose transporters overexpressed in cancer cells. We therefore
investigated how the position of substitution in Dꢀglucose affects specific uptake of the resulting
glycoconjugates. As discussed above, comparison of cellular uptake data in cancerous vs nonꢀ
cancerous cells has already implicated glucose transporters in the uptake of GlcꢀPts. Among the
facilitative glucose transporters overexpressed in cancers, GLUT1 is the most common and best
studied. It is broadly overexpressed in many different cancers and its expression level in biopsy
samples from cancer patients strongly correlates with tumor development, progression, and poor
prognosis.4ꢀ6 Here we screened the GlcꢀPts for their ability to translocate specifically through
GLUT1. Subsequently, the cellular uptake of the GlcꢀPts was determined in the absence and
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presence of a specific GLUT1 inhibitor, cytochalasin B, in DU145 cells. The results are
presented in Figure 4. As expected, uptake of aglycone 7 was not inhibited significantly, but
different degrees of uptake inhibition were observed for the GlcꢀPts. This result confirms our
hypothesis that the position of substitution is an important parameter in determining the GLUT1
specificity of glycoconjugates. Cellular uptake of C2ꢀGlcꢀPt 2 was most significantly inhibited,
by 57%, indicating substitution at C2 to be highly favorable for GLUT1 specificity of this class
of glycoconjugates. The order of uptake inhibition by the GLUT1 inhibitor, which parallels the
degree of GLUT1ꢀspecific uptake, is 2 > 1α ≥ 1β > 3 = 4 = 6. We recently reported the synthesis
and glucoseꢀtransporter mediated uptake of C6ꢀglucose platinum conjugates including 6. The
compounds were designed based on the Dꢀglucose bound crystal structure of XylE, a bacterial
GLUT homolog. Importantly, this current study confirmed that the GLUT1 specificity of 2 is
superior to 6. However, we note that, together with GLUT1, expression of different glucoseꢀ
transporter isoforms such as GLUT2, GLUT3, GLUT4, SGLT1 and SGLT2 are present in
tumors and cancer cells.4 Owing to structural and functional variations, the order of specificity of
GlcꢀPts may not be the same for all glucose transporters. Moreover, owing to the presence of
(1R,2R)ꢀcyclohexaneꢀ1,2ꢀdiamine (DACH) as part of their structures, solute transporters such as
organic cation transporter 2 (OCT2) other than GLUTs might also contribute to the cellular
uptake of these class of compounds.27 In addition to glucose transporters, various enzymes such
as hexokinase II and cell proliferation associated antigen Kiꢀ67 play important roles in
intracellular processing of Dꢀglucose or glucoseꢀconjugates, thereby contributing indirectly to
the cellular accumulation in cancer cells.46ꢀ48 After gaining entry to the cells, 18FꢀFDG is
metabolized to 18FꢀFDGꢀ6ꢀphosphate by hexokinase II that is overexpressed in cancers and
effectively trapped inside the cells. Positive correlations between 18FꢀFDG uptake and
hexokinase II expression and activity in certain type of cancers and cancer cells have been
reported.47ꢀ49 βꢀGlucosidases comprise another class enzymes that play important roles,
especially in the activation of glycoconjugated prodrugs.50,51 Recently, glycosylated Pt(IV)
complexes bearing different glycuronic acids and linkers were synthesized to study antitumor
activity. Despite good in vitro activity owing to improved uptake when compared to their Pt(II)
counterparts, the complexes did not show specific uptake by GLUTs.52 The use of protected
glycuronic acids explains the lack of GLUT mediated uptake. Our glycoconjugation chemical
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approach circumvents this problem because we provide evidence for GLUT1 specificity based
on the position of substitution on glucose.
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Figure 5. a) Western blot analysis of GLUT1 protein in DU145 and its knockdown clone. b) &
c) Representative doseꢀresponse curves for DU145 and DU145ꢀhGLUT1ꢀshRNA cells treated
with different concentrations of the aglycone 7 and GlcꢀPt 2. Cells were treated with compounds
for 7.5 h followed by incubation in fresh media for 64 h before the viability was evaluated by the
MTT assay. d) Comparison of IC50 values of GlcꢀPts 1ꢀ4, 6, and 7 in DU145 and DU145ꢀ
hGLUT1ꢀshRNA cells.
In order to further ascertain whether the position of substitution is an important determinant
of GLUT1 specificity of glycoconjugates, we determined and compared the cytotoxicity of Glcꢀ
Pts in DU145 and its GLUT1 knockdown clone (hereafter designated DU145ꢀhGLUT1ꢀshRNA).
As shown in Figure 5a, approximately 50% knockdown of GLUT1 transporter expression was
achieved by transfecting DU145 cells with a GLUT1ꢀshRNA (see Experimental Section for
details). The anticancer activity determined by MTT assays in DU145 and DU145ꢀhGLUT1ꢀ
shRNA cells revealed that the observed differences in cytotoxicity of GlcꢀPts between wild type
and GLUT1 knockdown cells are highly dependent on the position of substitution of Dꢀglucose
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(Figure 5d). As expected, no significant differences in IC50 values of aglycone 7 were observed
for DU145 and its GLUT1 knockdown clone (Figure 5b,d). However, the IC50 value of GlcꢀPt 2
in DU145ꢀhGLUT1ꢀshRNA was determined to be 26 µM, which is 2.9ꢀfold higher than the IC50
value for parental DU145 cells (9 µM) (Figure 5c,d). The potencies of the 1β and 1α decreased
by 1.8ꢀ and 1.6ꢀfold, respectively, upon knockdown of GLUT1 transporters, but there was no
effect on the efficacy of 3, 4 and 6. These results confirm that the highest GLUT1 specificity is
displayed by C2ꢀsubstituted GlcꢀPt 2, which is consistent with the highest cellular uptake
inhibition of 2 by cytochalasin B.
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In vivo efficacy and biodistribution of GlcꢀPt 2.
The in vivo anticancer activity of our best GLUT1 targeting molecule, GlcꢀPt 2, was assessed in
vivo using a mouse model of GLUT1 expressing breast cancer (G1fpt),53 a syngeneic model
induced in Balb/c mice. Intravenous bolus injections of 5 mg/kg of GlcꢀPt 2 or oxaliplatin
commenced when tumors reached ~50 mm3 for four successive treatments, once every other day.
Treatment groups included GlcꢀPt 2, oxaliplatin, and a control group receiving PBS. Platinum
complexes were freshly prepared by dissolution in PBS prior to injection. Antitumor efficacy
was evaluated by measuring the tumor volume over a period of 30 days. Toxic side effects of the
platinum agents under investigation were assessed daily by thorough examination of physical
condition including, body weight, lethargy, loss of appetite, and general behavior of the animals.
The tumor treatment revealed that GlcꢀPt 2 effectively reduces disease burden by significantly
delaying tumor growth at a dose of 5 mg/kg (Figure 6a). Moreover, oxaliplatin treatment
showed no statistically significant difference compared to the GlcꢀPt 2 treated group. Notably,
tumors treated with GlcꢀPt, 2, or oxaliplatin were 10 times smaller compared to tumors in the
PBS treated control group, indicating excellent efficacy.
The potential toxicity of the platinum constructs 2 and oxaliplatin, compared to PBS
treated controls, was assessed by monitoring the body weight and general behavior of the
animals. Balb/c mice were treated with PBS, 2, or oxaliplatin at the same dosing schedule used
for the efficacy studies (5 mg/kg, 4X in 8 days). As shown in Figure 6b, the body weight of mice
treated with 2 or oxaliplatin was reduced after the second dose but recovered to normal values,
the apparent toxicity of PBS treated mice, throughout the rest of the study, without further
weight changes. Conceivably, despite minor changes in body weight, a more detailed toxicology
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studies would more fully characterize the effects of 2. Also, 2 or 7 accumulated in the kidney and
spleen but minimally in the liver at similar concentrations after 24 h, indicative of clearance.
Renal filtration appears to be a major clearance pathway, which might be expected considering
the hydrophilic nature of 2 and 7.
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Figure 6. Treatment of GLUT1 breast cancer in a Balb/c syngeneic mouse model. Treatment
began when tumors reached ~50 mm3 and was given by an intravenous bolus injection of 5.0
mg/kg of oxaliplatin, GlcꢀPt 2, or PBS. Tumor volumes were monitored daily, and their total
volume was normalized to initial tumor size at time of treatment. Each treatment group consisted
of 5 animals (n = 5). a) Antitumor efficacy studies. b) Toxicity analysis by monitoring body
weight after candidate drug administration.
The biodistribution of GlcꢀPt 2 was assessed by graphite furnace atomic absorption
spectroscopy (GFAAS) at 3 h, 9 h, and 24 h post administration (i.v.) in a mouse model bearing
murine breast cancer overexpressing GLUT1; the results are shown in Figure 7. Given that Glcꢀ
Pt 2 displays selective GLUT1 mediated uptake in vitro, we were interested to learn whether this
phenomenon persists in vivo. To investigate the GLUT1 mediated uptake of 2, we used
compound 7, a nonꢀtargeted platinum complex, as control. It became evident after 24 h (Figure
7c) that 2 accumulates in tumor tissue to approximately 3ꢀfold better than 7, which lacks a Dꢀ
glucose targeting ligand. This result is consistent with the in vitro uptake studies described
above. The encouraging selectivity has the potential to overcome toxic side effects associated
with platinum anticancer agents and subsequently to improve drug efficacy with the possibility
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of increasing drug dose without limiting toxicities. Overall, the biodistribution result confirms
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Figure 7. Biodistribution of GlcꢀPt 2 or aglycone 7 in GLUT1 breast cancer tumor bearing
animals. (a) Quantification of platinum concentration in organ tissue as measured by atomic
absorption spectroscopy 3 h, 9 h, and 24 h following administration of GlcꢀPt 2 after nitric acid
digestion. (b) Quantification of platinum concentration in organ tissue as measured by atomic
absorption spectroscopy 3 h, 9 h, and 24 h postꢀadministration of aglycone 7 after nitric acid
digestion. (c) Comparative plot of platinum concentration in organ tissue of 2 and 7 at 24 h.
SUMMARY AND CONCLUSION.
Feasible synthetic routes were established to all the positional isomers of GlcꢀPt conjugates by
means of etherꢀglycosidic linkages. To our knowledge, this is the first time that a platinum drug
candidate has been attached via a spacer to each of the individual oxygen atoms of Dꢀglucose.
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The synthetic strategies presented here provide a potential platform for the synthesis of any
desired positional isomer of a glucoseꢀconjugate. The current investigation experimentally
establishes that the position of substitution of Dꢀglucose is an important parameter for the
biological activity profile of the resulting glucoseꢀconjugate. GlcꢀPt 1α and 2 accumulate most
efficiently in cancer cells compared to the other positional isomers. No significant differences in
uptake of GlcꢀPts were observed in noncancerous cells that do not express GLUT1 transporter,
suggesting that differential uptake in cancer cells arises mainly from the differential glucoseꢀ
transporter mediated uptake of the GlcꢀPts. Furthermore, the site of substitution modulates the
cytotoxicity profile of the glucoseꢀplatinum conjugates, with the C2ꢀGlcꢀPt derivative 2 being
most potent and C3ꢀGlcꢀPt, 3 the least potent of the positionalꢀisomers in DU145 cells. Cellular
uptake studies in the absence and presence of the GLUT1 inhibitor cytochalasin B revealed that
GlcꢀPt 2 has the highest GLUT1 specific internalization ability. The GLUT1 specificity of 2 was
further confirmed by comparing the potencies of all GlcꢀPts in DU145 cells and in its GLUT1
knockdown clone. The results of this investigation again point to C2 as the most preferable
position on Dꢀglucose for attaching a warhead for GLUT1 mediated delivery of an anticancer
agent. Additional experimental data on other C2ꢀglucose conjugates are desired to confirm this
initial observation. Compound 2 has antitumor efficacy in mice, comparable to that of
oxaliplatin. Importantly, tumor uptake of 2 is significantly higher than that of its nonꢀtargeted
analog 7 in vivo. Although at this stage we do not know the specificity of GlcꢀPts for other
glucoseꢀtransporter isoforms, such as GLUT2ꢀ4 and SGLT1ꢀ2, our findings identify the position
of substitution on Dꢀglucose as an important descriptor of glucose transporter specificity and
biological activity of glycoconjugates in vitro and in vivo.
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EXPERIMENTAL SECTION
General procedure for the synthesis of GlcꢀPts. A suspension of dichlorido[(1R,2R)ꢀ
cyclohexaneꢀ1,2ꢀdiamine]platinum(II)39 ([DACH]PtCl2) and Ag2SO4 in water was stirred
overnight at room temperature in the dark. The precipitate was filtered off using a 0.2 µm
syringe filter and the filtrate containing [DACH]PtSO4 was added in a dropwise manner to an
aqueous solution of the corresponding ligand and Ba(OH)2·8H2O in water, which had been
initially stirred for 30 min at room temperature. The reaction mixture was then stirred for 20 h in
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the dark before removal of insoluble BaSO4 by filtration. The filtrate was lyophilized and the
milky white solid obtained was purified using preparative HPLC with a linear gradients of
millipore water (A) and methanol (B; SigmaꢀAldrich HPLCꢀgrade): t = 0ꢀ5 min, 15% B; t = 8
min, 13% B; t = 30 min, 55% B; t = 35 min, 95% B. The flow rate was 15 mL minꢀ1 and UV
absorption was measured at 220 nm.
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Animal facility and standard of care. Mice were placed in a facility accredited by the
Institutional Animal Care and Use Committee. Treatments were administered by retroꢀorbital
injection of the venous sinus. Amount of anticancer agents was calculated based on the average
animal body weight. The MIT Committee for Animal Care approved all animal protocols.
GlcꢀPt 1β. Reagents used: [DACH]PtCl2 (123 mg, 0.32 mmol) and Ag2SO4 (99 mg, 0.32 mmol)
in water (3 mL), 11 (110 mg, 0.32 mmol) and Ba(OH)2·8H2O (103 mg, 0.32 mmol) in water (4
mL). GlcꢀPt 1β was obtained as a white powder (yield: 31 mg, 15%). tR (RPꢀHPLC) = 13.1 min.
1H NMR (400 MHz, D2O): δ (ppm) 0.99ꢀ1.08 (m, 2H, CH2 DACH),1.13ꢀ1.29 (m, 5H, CH2 DACH
and CH3), 1.42ꢀ1.66 (m, 4H, CH2 DACH and OCH2CH2CH2), 1.89ꢀ2.01 (m, 2H, CH2 DACH), 2.26ꢀ
2.36 (m, 2H, 2×CH DACH), 2.75ꢀ2.97 (m, 2H, OCH2CH2CH2), 3.15ꢀ3.46 (m, 4H, OCH2CH2CH2,
and 2×CH), 3.58ꢀ4.01 (m, 4H, OHCH2 and 2×CH) 4.45 (d, J = 7.9 Hz, 1H, CHanomeric).
13C{1H} NMR (100 MHz, D2O): δ (ppm) 181.76, 181.66, 102.21, 75.92, 75.79, 73.23, 70.22,
69.66, 62.51, 62.27, 60.71, 56.90, 35.76, 31.79, 31.69, 24.88, 23.85, 20.85. 195Pt{1H} NMR (86
MHz, D2O): δ (ppm) −1891. ESIꢀMS (pos. detection mode): m/z (%): 668.3 (100) [M+Na]+,
calculated m/z for [M+Na]+ 668.2. Anal. Calcd for C19H34N2O10Ptꢁ(H2O)2: C, 33.48; H, 5.62; N,
4.11. Found: C, 33.07; H, 5.15; N, 3.99.
GlcꢀPt 1α. Reagents used: [DACH]PtCl2 (137 mg, 0.35 mmol) and Ag2SO4 (110 mg, 0.35
mmol) in water (4 mL), 14 (120 mg, 0.35 mmol) and Ba(OH)2·8H2O (114 mg, 0.35 mmol) in
water (4 mL). GlcꢀPt 1α was isolated as a white powder (yield: 45 mg, 19%). tR (RPꢀHPLC) =
1
13.3 min. H NMR (400 MHz, D2O): δ (ppm) 0.97ꢀ1.08 (m, 2H, CH2 DACH),1.13ꢀ1.31 (m, 5H,
CH2 DACH and CH3), 1.42ꢀ1.64 (m, 4H, CH2 DACH and OCH2CH2CH2), 1.90ꢀ1.99 (m, 2H, CH2
DACH), 2.30ꢀ2.38 (m, 2H, 2×CH DACH), 2.60ꢀ2.67 (m, 1H, OCH2CH2CH2), 3.10ꢀ3.11 (m, 1H,
OCH2CH2CH2), 3.33ꢀ3.37 (m, 1H, OCH2CH2CH2), 3.47ꢀ3.52 (m, 1H, OCH2CH2CH2), 3.57ꢀ3.79
13
(m, 6H, HOCH2 and 4×CH), 4.97 (d, J = 3.7 Hz, 1H, CHanomeric). C{1H} NMR (100 MHz,
D2O): δ (ppm) 181.87, 181.61, 98.12, 73.14, 71.98, 71.33, 69.55, 67.90, 62.43, 62.35, 60.55,
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56.93, 36.28, 31.81, 31.62, 24.82, 23.83, 20.65. 195Pt{1H} NMR (86 MHz, D2O): δ (ppm) −1886.
ESIꢀMS (pos. detection mode): m/z (%): 646.2 (100) [M+H]+, calculated m/z for [M+H]+ 646.5.
Anal. Calcd for C19H34N2O10Ptꢁ2(H2O): C, 33.48; H, 5.62; N, 4.11. Found: C, 33.37; H, 5.31; N,
4.41.
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GlcꢀPt 2. Reagents used: [DACH]PtCl2 (171 mg, 0.45 mmol) and Ag2SO4 (137 mg, 0.45 mmol)
in water (6 mL), 24 (152 mg, 0.45 mmol) and Ba(OH)2·8H2O (141 mg, 0.45 mmol) in water (5
mL). GlcꢀPt 2 was isolated as a white powder (yield: 75 mg, 26%). tR (RPꢀHPLC) = 12.5 and
1
13.0 min. (α and β anomers). H NMR (400 MHz, D2O): δ (ppm) 1.03ꢀ1.13 (m, 2H, CH2 DACH),
1.14ꢀ1.29 (m, 5H, CH2 DACH and CH3), 1.46ꢀ1.62 (m, 4H, 2×CH2 DACH), 1.94ꢀ2.01 (m, 2H,
OCH2CH2CH2), 2.28ꢀ2.37 (m, 2H, 2×CH DACH), 2.72ꢀ3.01 (m, 2H, OCH2CH2CH2), 3.27ꢀ3.49
(m, 3H, OCH2CH2CH2 and CH), 3.71ꢀ3.81 (m, 5H, HOCH2 and 3×CH), 4.62 (d, J =7.9 Hz,
0.5H, CHanomeric), 5.39 (d, J =3.4 Hz, 0.5H, CHanomeric). 13C{1H} NMR (100 MHz, D2O): δ
(ppm) 181.69, 181.56, 126.66, 95.88, 90.11, 82.71, 79.44, 75.84, 75.44, 73.03, 72.19, 71.20,
70.66, 69.75, 69.66, 62.83, 62.73, 62.58, 62.47, 62.31, 60.69, 60.54, 57.03, 57.02, 35.98, 35.89,
31.96, 31.79, 25.36, 25.24, 23.93, 23.88, 20.83, 20.75. 195Pt{1H} NMR (86 MHz, D2O): δ (ppm)
−1876. ESIꢀMS (pos. detection mode): m/z (%): 646.2 (100) [M+H]+, calculated m/z for [M+H]+
646.5. Anal. Calcd for C19H34N2O10Ptꢁ(H2O)2ꢁ2(CF3COOH): C, 30.37; H, 4.43; N, 3.08. Found:
C, 30.55; H, 4.24; N, 3.37. 19F{1H} NMR (376 MHz, D2O): δ (ppm) −75.6 (residual
trifluoroacetic acid).
GlcꢀPt 3. Reagents used: [DACH]PtCl2 (137 mg, 0.35 mmol) and Ag2SO4 (110 mg, 0.35 mmol)
in water (4 mL), 29 (120 mg, 0.35 mmol) and Ba(OH)2·8H2O (114 mg, 0.35 mmol) in water (4
mL). GlcꢀPt 3 was isolated as a white powder (yield: 48 mg, 21%). tR (RPꢀHPLC) = 13.0 and
1
13.3 min. (α and β anomers). H NMR (400 MHz, D2O): δ (ppm) 0.96ꢀ1.11 (m, 2H, CH2 DACH),
1.12ꢀ1.30 (m, 5H, CH2 DACH and CH3), 1.42ꢀ1.64 (m, 4H, 2×CH2 DACH), 1.88ꢀ2.02 (m, 2H,
OCH2CH2CH2), 2.25ꢀ2.37 (m, 2H, 2×CH DACH), 2.74ꢀ3.05 (m, 2H, OCH2CH2CH2), 3.16ꢀ3.45
(m, 3H, OCH2CH2CH2 and CH), 3.48ꢀ3.92 (m, 5H, HOCH2 and 3×CH), 4.57 (d, J =7.8 Hz,
0.6H, CHanomeric), 5.13 (d, J =6.5 Hz, 0.4H, CHanomeric). 13C{1H} NMR (100 MHz, D2O): δ
(ppm) 181.80, 181.66, 95.92, 92.17, 84.46, 81.74, 75.89, 73.90, 73.26, 73.10, 71.56, 71.32,
69.26, 69.22, 62.56, 62.27, 60.63, 60.46, 56.97, 36.02, 31.81, 31.70, 25.43, 23.88, 23.84, 20.73.
195Pt{1H} NMR (86 MHz, D2O): δ (ppm) −1888. ESIꢀMS (pos. detection mode): m/z (%): 646.2
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(100) [M+H]+, calculated m/z for [M+H]+ 646.5. Anal. Calcd for C19H34N2O10Ptꢁ(H2O)3: C,
32.62; H, 5.76; N, 4.00. Found: C, 32.71; H, 5.73; N, 4.01.
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GlcꢀPt 4. Reagents used: [DACH]PtCl2 (171 mg, 0.45 mmol) and Ag2SO4 (137 mg, 0.45 mmol)
in water (6 mL), 40 (152 mg, 0.45 mmol) and Ba(OH)2·8H2O (141 mg, 0.45 mmol) in water (5
mL). GlcꢀPt 4 was isolated as a white powder (yield: 85 mg, 29%). tR (RPꢀHPLC) = 12.6 and
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12.7 min. (α and β anomers). H NMR (400 MHz, D2O): δ (ppm) 1.01ꢀ1.14 (m, 2H, CH2 DACH),
1.17ꢀ1.29 (m, 5H, CH2 DACH and CH3), 1.45ꢀ1.58 (m, 4H, 2×CH2 DACH), 1.92ꢀ2.02 (m, 2H,
OCH2CH2CH2), 2.25ꢀ2.37 (m, 2H, 2×CH DACH), 2.77ꢀ2.98 (m, 2H, OCH2CH2CH2), 3.16ꢀ3.31
(m, 2H, OCH2CH2CH2), 3.39ꢀ3.56 (m, 2H, 2×CH), 3.68ꢀ3.95 (m, 4H, HOCH2 and 2×CH), 4.55
(d, J =8 Hz, 0.6H, CHanomeric), 5.13 (d, J =7.2 Hz, 0.4H, CHanomeric). C{1H} NMR (100
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MHz, D2O): δ (ppm) 181.66, 181.61, 95.87, 91.96, 78.28, 78.20, 75.67, 75.12, 74.25, 73.13,
72.72, 71.59, 70.59, 62.72, 62.61, 62.26, 60.60, 60.51, 56.95, 36.11, 36.01, 31.85, 31.71, 25.38,
23.91, 20.75. 195Pt{1H} NMR (86 MHz, D2O): δ (ppm) −1883. ESIꢀMS (pos. detection mode):
m/z (%): 646.2 (100) [M+H]+, calculated m/z for [M+H]+ 646.5. Anal. Calcd for
C19H34N2O10Ptꢁ(CF3COOH)3: C, 30.40; H, 3.78; N, 2.84. Found: C, 30.12; H, 3.45; N, 3.30.
GlcꢀPt 6. GlcꢀPt 6 was prepared following a literature reported procedure.27
ASSOCIATED CONTENT
Supporting Information
Materials and methods, experimental details, characterization data, NMR spectra of compounds,
analytical HPLC traces of GlcꢀPts, cellular and animal studies. This material is available free of
charge via the internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
lippard@mit.edu
ACKNOWLEDGMENTS
This work was supported by NCI Grant CA034992.
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