Synthesis and biological evaluation of non-glucose glycoconjugated N-hydroyxindole class LDH inhibitors as anticancer agents

Article abstract of DOI:10.1039/c5ra00946d

Inhibitors of human lactate dehydrogenase A (LDH-A) are promising therapeutic agents against cancer. The development of LDH-A inhibitors that possess cellular activities has so far proved to be particularly challenging, since the enzyme's active site is narrow and highly polar. In the recent past, we were able to develop a glucose-conjugated N-hydroxyindole-based LDH-A inhibitor designed to exploit the sugar avidity expressed by cancer cells (the Warburg effect). Herein we describe a structural modulation of the sugar moiety of this class of inhibitors, with the insertion of α-d-mannose, β-d-gulose, or β-N-acetyl-d-glucosamine portions in their structures. Their stereospecific chemical synthesis, which involves a substrate-dependent stereospecific glycosylation step, and their biological activity in reducing lactate production and proliferation in cancer cells are reported. Interestingly, the α-d-mannose conjugate displayed the best properties in the cellular assays, demonstrating an efficient antiglycolytic and antiproliferative activity in cancer cells. This journal is

Full text of DOI:10.1039/c5ra00946d

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PAPER
Synthesis and biological evaluation of non-glucose
glycoconjugated N-hydroyxindole class LDH
inhibitors as anticancer agents†
Cite this: RSC Adv., 2015, 5, 19944
Valeria Di Bussolo,a Emilia C. Calvaresi,‡b Carlotta Granchi,a Linda Del Bino,a
Ileana Frau,a Maria Chiara Dasso Lang,§a Tiziano Tuccinardi,a Marco Macchia,a
a
b
a
*
*
Adriano Martinelli, Paul J. Hergenrother and Filippo Minutolo
Inhibitors of human lactate dehydrogenase A (LDH-A) are promising therapeutic agents against cancer. The
development of LDH-A inhibitors that possess cellular activities has so far proved to be particularly
challenging, since the enzyme's active site is narrow and highly polar. In the recent past, we were able to
develop a glucose-conjugated N-hydroxyindole-based LDH-A inhibitor designed to exploit the sugar
avidity expressed by cancer cells (the Warburg effect). Herein we describe a structural modulation of the
sugar moiety of this class of inhibitors, with the insertion of a-D-mannose, b-D-gulose, or b-N-acetyl-D-
glucosamine portions in their structures. Their stereospecific chemical synthesis, which involves a
substrate-dependent stereospecific glycosylation step, and their biological activity in reducing lactate
production and proliferation in cancer cells are reported. Interestingly, the a-D-mannose conjugate
displayed the best properties in the cellular assays, demonstrating an efficient antiglycolytic and
antiproliferative activity in cancer cells.
Received 16th January 2015
Accepted 10th February 2015
DOI: 10.1039/c5ra00946d
www.rsc.org/advances
peculiar metabolism occurring in cancer by acting as anti-
glycolytic agents, as reviewed recently.3,4
Introduction
Lactate dehydrogenase (LDH), the enzyme that catalyzes the
interconversion of pyruvate and lactate, establishes a key
checkpoint for the switch from aerobic to anaerobic glycolysis.
LDH is a tetrameric enzyme that may exist in ve isoforms
(hLDH1-5), which results from the possible combinations of the
two subunits: LDH-A and LDH-B. Subunit LDH-A (and, conse-
quently, its tetrameric functional form hLDH5 or LDH-A4) is
very frequently found to be overexpressed in invasive cancer; its
genetic silencing has been shown to reduce proliferation and
invasiveness of tumor cells, especially under hypoxic condi-
tions.5 The validity of LDH-A as an anticancer target is further
strengthened by the fact that individuals homozygous for LDH-
A deciency do not show any particular clinical symptoms,
except for myoglobinuria upon intense physical exertion.6
Therefore, several recent research efforts have been dedicated to
the development of new LDH-A inhibitors with therapeutic
potential as anticancer drugs, although problems related to
poor cellular activities of these inhibitors have been oen
reported.7,8
Tumors generally follow unusual metabolic pathways to obtain
the energy and anabolites required for their persistent growth.
Growing cancer cells rewire their metabolism to meet their
bioenergetic and biosynthetic needs for rapid proliferation by
increasing their consumption of glucose and glutamine,
enhancing glycolysis, and increasing the excretion of lactate. In
particular, glycolysis, rather than oxidative phosphorylation
(OXPHOS), is predominantly used even in the presence of
oxygen, as described by the well-known “Warburg effect”.1,2 In
fact, in normal cells, most of the pyruvate produced by glycol-
ysis goes into the tricarboxylic acid (TCA) cycle and is eventually
oxidized to CO2 via OXPHOS. On the contrary, tumor cells
mostly convert pyruvate to lactate anaerobically. Several
prospective drugs have been developed to take advantage of this
a
`
Dipartimento di Farmacia, Universita di Pisa, Via Bonanno 33, 56126 Pisa, Italy.
E-mail: lippo.minutolo@farm.unipi.it
bDepartment of Chemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL
The high rate of glycolysis in cancer cells is associated with a
striking glucose avidity. This feature has been exploited by
glycoconjugation of anticancer drugs, in order to improve their
targeting to tumor cells versus normal tissues.9 We have previ-
ously developed a novel class of N-hydroxyindole (NHI)-based
LDH-A inhibitors,10,11 and we recently reported that gluco-
conjugated NHIs, in particular NHI-Glc-2 (1, Fig. 1), showed a
61801, USA. E-mail: hergenro@illinois.edu
† Electronic supplementary information (ESI) available: NMR spectra of 2–4, 9,
12–14, 16–19 and 21; HRMS of 2–4; calibration curves of 1–3. See DOI:
10.1039/c5ra00946d
‡ Present address: College of Medicine, University of Illinois, 506 S. Mathews
Avenue, Urbana, IL 61801, USA.
`
§ Present address: Dipartimento di Biotecnologia, Chimica e Farmacia, Universita
degli Studi di Siena, via Aldo Moro 2, 53019 Siena, Italy.
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glycoconjugates 2–4 (Fig. 1) and their biological evaluations as
prospective anticancer agents interfering with the cellular
production of lactic acid.
Results and discussion
Synthesis of the glycoconjugates
Recently, we reported a new glycosylation process, using glycal
derived vinyl epoxides 6a and 6b as new glycosyl donors
(Scheme 1). These systems, in the presence of alcohols or alkyl
lithium reagents as nucleophiles, afford only 6-O-benzyl-
protected O- and C-glycosides with the same conguration of
the starting epoxide, in an uncatalyzed substrate-dependent
stereospecic process.17–19 The driving force of this regio- and
stereoselective 1,4-addition process is the occurrence of a
coordination between the nucleophile and the oxirane oxygen
in the form of hydrogen bond, in the case of O-nucleophile, or
through the metal lithium cation, in the case of alkyl lithium
reagents. Vinyl epoxides 6a and 6b cannot be isolated, because
they are unstable and, therefore, they must be prepared only in
situ by cyclization under basic conditions with t-BuOK of their
ultimate precursor, the corresponding trans-hydroxymesylates 5
and 7.
Fig. 1 Chemical structures of the NHI-glycoconjugates: b-D-Glc-(1),
a-D-Man-(2), b-D-Gul-(3), and b-D-Glc(NAc)-(4) NHIs.
remarkably improved potency in reducing the cellular produc-
tion of lactic acid in cancer cells.12 Anticancer glycoconjugates
are not limited to D-glucose; other sugar and aminosugar
moieties have been previously introduced in anticancer agents
with improved selectivity. This is due to the fact that glucose
transporters overexpressed by tumor tissues, such as GLUT1,
are able to transport a number of sugar substrates besides
glucose.9 Therefore, the ability of glucose analogues, such as a-
manno-2 and b-gulo-conjugate 3, to be efficiently taken up by
cancer cells, results in an attractive strategy of extending our
“dual targeting” of the Warburg effect.12 to a wider class of sugar
conjugates. Compound 2 is a conjugate of D-mannose, a C2
epimer of D-glucose; D-mannose is found at concentrations of
around 50 mM in human serum, and it is primarily used in the
glycosylation of proteins (specically, mannose-6-phosphate is
a localization tag applied to proteins destined for lysosomal
import). Mannose is known to be transported into mammalian
cells primarily by mannose-specic transporters normally
expressed in intestinal cells; however, it has also been reported
to be a good substrate for GLUT1 (ref. 13) with a 13-fold
reduction of its affinity for this transporter (Km ¼ 20 mM), when
compared to that of glucose (Km ¼ 1.5 mM).14 Compound 3
contains D-gulose, which is a stereoisomer of glucose differing
in stereochemistry at the C3 and C4 positions; it is used by some
archaea but it is not known to be found in human serum or used
in human metabolism. So far, the ability of D-gulose to be
transported by GLUTs has not been reported. In addition, since
cancer cells have been found to utilize O-GlcN-acylation as a
post-translational modication,15,16 we also synthesized
compound 4, in which the NHI pharmacophoric unit is conju-
gated with GlcNAc.
Scheme 1 Substrate-dependent stereospecific glycosylation: reac-
tion conditions and mechanism of the stereochemical outcome [O-
nucleophiles (alcohols), X ¼ OH; C-nucleophiles (lithium alkyls), X ¼ Li;
R ¼ Me, Et, i-Pr, t-Bu, Ph, monosaccharides].
Herein, we describe the application of our originally-
developed glycosylation protocols to the synthesis of
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In the synthesis of glycoconjugates 2 and 3, due to the
instability the N–O single bond under the reductive conditions
utilized for the nal removal of the benzyl protective group
which was present in our original methodology (see 6a and 6b,
Scheme 1), we decided to use synthetic intermediates in which
the primary hydroxy functionality is protected as a tetrahy-
dropyranyl ether (–OTHP), a protective group which can be
easily removed by acid hydrolysis. The key intermediates are
represented by THP-protected hydroxymesylate 8, which was
synthesized in six steps starting from tri-O-acetyl-D-glucal as
previously reported20 and 9, which was instead specically
synthesized from 8 (Scheme 2). The conversion of 8 to 9 started
with a cyclization with t-BuOK in CH3CN, leading to the in situ
formation of epoxide 10b, which was followed by a ring opening
reaction with a non-coordinating hydroxy ion equivalent, such
as Me3SiOꢀ present in Bu4N+Me3SiOꢀ, a salt prepared by
addition of potassium trimethyl silanolate (Me3SiOK) to a
solution of tetrabutyl ammonium bromide (TBAB) in THF.19 In
this way, aer exposure to water, a clean 1,2-addition process is
obtained, affording the desired trans diol 12, which was treated
with TBSCl to give the mono allyl C(3)-O-TBS derivative 13.
Subsequent mesylation of 13 at C(4) produced the all-protected
glycal derivative 14, which was nally deprotected with TBAF/
THF to give trans hydroxymesylate 9.
The synthesis of b-gulo-derivative 3 started from trans-
hydroxymesylate 8 (Scheme 3), with the in situ formation of vinyl
epoxide 10b by cyclization with t-BuOK in CH3CN. This reactive
intermediate was not isolated. It was immediately treated with
the NHI-based glycosyl acceptor 15 (NHI-2, a LDH-A inhibitor
previously reported by us11) to give, aer only 30 minutes at
room temperature, the glycosylation product 16 with complete
1,4-regio- and b-stereoselectivity. Functionalization of the
double bond present in 16 by OsO4/NMO protocol afforded the
corresponding syn-dihydroxylated product 18, in accordance
with a complete sterically-favored a-facial stereoselective elec-
trophilic addition (70% yield). The deprotection of the
Scheme 3 Synthesis of glycoconjugates 2 and 3 by stereospecific
glycosylation of NHI-derivative 15.
C(6)-OTHP functionality of 18 by using catalytic PPTS in abso-
lute EtOH under controlled temperature at 40 ꢁC for 46 h,
afforded the desired b-O-D-gulo-conjugate 3 in good yield aer
recrystallization (75% yield). A similar protocol was utilized for
the preparation of the a-manno-derivative 2 (Scheme 3). In this
case, the reaction sequence started from trans-hydroxymesylate
9 with a cyclization reaction with t-BuOK in CH3CN to produce
highly reactive vinyl epoxide 10a, which was treated in situ with
glycosyl acceptor 15. Here again, the glycosylation step proved
to be very efficient aer only 30 minutes at room temperature,
Scheme 2 Synthesis of THP-protected trans-hydroxymesylates 8 and
9 from tri-O-acetyl-D-glucal.
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Table 1 In vitro kinetic inhibition of sugar conjugates 1–4 against
with the production of a-glycoconjugate 17 with a complete 1,4-
regio- and a-stereoselectivity (58% yield aer purication). Once
obtained, the unsaturated a-glycoconjugate 17 was submitted to
cis-dihydroxylation with OsO4/NMO and, in this case, the b-
stereoselective attack of the electrophile is directed by the allyl
substituents at C(4) and C(1), now located on the a-face.
Therefore the corresponding b-dihydroxylated derivative 19 was
the only stereoisomer obtained (65% yield). The nal depro-
tection of the THP moiety present at C(6) of 19, carried out in
absolute EtOH for 20 h at 40 ꢁC in the presence of PPTS,
provided the a-O-D-manno-conjugate 2 in 80% yield aer
recrystallization.
LDH-Aa
Compound
LDH-A – Ki (mM)
1
2
3
4
37.8 ꢂ 0.9b
92.4 ꢂ 9.0
68.1 ꢂ 17.5
98.4 ꢂ 15.6
a
Ki values were obtained by non-linear regression analysis with
GraphPad Prism soware (GraphPad, La Jolla, CA) using a second
order polynomial regression analysis by applying the mixed-model
inhibition t (mean values
ꢂ
SD calculated from at least 2
experiments, see Experimental section). b From ref. 12.
The synthesis of GlcNAc-conjugate 4 (Scheme 4) utilized as
the glycosyl donor oxazoline 20, which was prepared as previ-
ously reported.21 Glycosyl acceptor 15 displayed a low reactivity potent than glucose-conjugate 1 (ref. 12) in these in vitro assays
as the nucleophile with oxazoline 20 and, therefore, we needed on the isolated enzyme.
to test a wide array of reaction conditions. The best results were
obtained by using TMSOTf as the catalyst.22 This way, the ring
Molecular modeling
opening of oxazoline 20 was realized in anhydrous dichloro-
Docking studies followed by molecular dynamic (MD) simula-
ꢁ
˚
ethane at 80 C for 24 h in the presence of molecular sieves 4 A
using 0.5 equiv of TMSOTf and 1.5 equiv of NHI 15 to afford
peracetylated Glc-NAc-conjugate 21 in 58% yield aer purica-
tion. Deacetylation of 21 with MeONa in CH2Cl2/MeOH afforded
Glc-NAc-conjugate 4 in quantitative yields.
tions were carried out to examine the putative modes of inter-
action of sugar conjugates 2–4 with LDH-A and the results of
these studies are shown in Fig. 2.
As highlighted in Fig. 2, all three compounds show a very
similar positioning into the LDH-A binding site. In all cases, the
C]O portion of the ester group of these compounds forms an
H-bond with R169, and the methyl group of the ester establishes
lipophilic interactions with the isopropyl side chain of V235.
The 4-(triuoromethyl)indole central scaffold is placed in a cle
mainly delimited by H193, G194, A238, V241, and I242, whereas
the 6-phenyl group is directed toward the entrance of the
binding site cavity. The sugar moiety is always placed in the
NADH-binding pocket but, depending on the type of mono-
saccharide attached, it shows different H-bonds with the
protein. Specically, the a-mannose ring of 2 (Fig. 2A) forms two
H-bonds with the hydroxyl group of T248 through its 2- and 3-
hydroxyl groups. The b-gulose ring of 3 (Fig. 2B) shows a slightly
different interaction mode with the protein, with a H-bond
occurring between its 4-OH group and T248, and a peculiar H-
bond that is formed by its 6-OH group and the side chain of
N138. Finally, the GlcNAc portion of 4 (Fig. 2C) forms
completely different interactions with the enzyme active site,
the most important of which seem to be the interaction of the
acetamido-group with T248, through its N–H portion, and with
R169 through its C]O moiety.
Enzyme inhibition assays
The inhibitory activities of the sugar conjugates 2–4 were
measured by standard enzyme kinetic experiments on puried
human enzyme isoform hLDH5. The Ki values of each
compound (Table 1) were measured in NADH-competition
experiments as previously described.12
All the newly synthesized sugar conjugates (2–4) proved to be
moderately active in these assays, with Ki values ranging from
68.1 mM with the manno-derivative 3 to 98.4 mM with the
amino–glucose-derivative 4. None of them proved to be more
Cellular lactate production inhibition assays
The ability of the glycol-conjugated NHIs 2–4 to inhibit lactate
production in cells was evaluated using the same previously
reported technique developed for glucose-conjugate 1,12 and the
results are displayed in Fig. 3.
In testing these glyco-conjugates at 50–200 mM concentra-
tions for their ability to reduce lactate production in HeLa cells
following an 8 h incubation, it was found that the a-manno-
derivative 2 led to a potent, dose-dependent reduction in
lactate production, which is comparable to that obtained with
glucose-conjugate 1, with a remarkable 55% inhibition at the
Scheme 4 Synthesis of GlcNAc conjugate 4 via the formation of
oxazoline 20 and subsequent stereospecific glycosylation of NHI-
derivative 15.
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Fig. 2 MD simulation results for the complex of LDH-A with 2 (A), 3 (B), and 4 (C).
lowest concentration used (50 mM).
A noticeable dose- A549 cells were treated with 100 mM concentrations of each
dependent activity was also found with b-gulo derivative 3, compound for 4 h. Aer washing and lysing the cells, the
although it was lower than those observed with 1 and 2. On the cellular fraction of each compound was assessed by LC-MS and
contrary, GlcNAc-conjugate 4 lacked any signicant activity in correlated to concentration using calibration standards (Fig. S1,
this assay, and, therefore, was not considered in our subsequent ESI†), and the intracellular concentrations are displayed in
experiments. Under these conditions, a very modest effect is Fig. 4.
observed with very high concentrations (10 mM) of hexokinase
As we had previously observed with glucose-conjugate 1,12
inhibitor 2-deoxyglucose, whereas negligible effects are mannose-derivative 2 displays a signicantly enhanced cell uptake
observed upon incubation with 10 mM of the topoisomerase II compared to the aglycone NHI-2. While 1 and 2 had similar levels
inhibitor etoposide, a cytotoxic compound that does not affect of cell uptake, the gulose-derivative 3 had slightly lower levels of
glucose metabolism.
cell uptake. These results are consistent with those obtained in the
cellular lactate production inhibition assays (Fig. 3), where the
most potent cellular inhibitors were found to be 1 and 2, thus
positively correlating cellular activity and cell uptake. It is impor-
tant to note that no cleavage of any of these compounds was
observed in either the UV trace or TIC of the resultant lysate
samples from 4 h of incubation in A549 cells, so all the effects
should be ascribed to the parent compounds.
Cellular uptake assays
The relative cell uptake levels of glyco-conjugates 2 and 3, and of
reference glucose-conjugate 1,12 versus their aglycone counter-
part NHI-2 (15, Scheme 3), and the potential cleavage of the
mannose-(2) and gulo-portions (3) in cell culture, was assessed.
Fig. 3 Lactate production inhibition of glycoconjugates 1–4. HeLa human cervical carcinoma cells were treated under normoxic conditions with
indicated compound concentrations or 1% DMSO vehicle, in DMEM supplemented with 10 mM unlabeled glucose, 1 mM pyruvate, and 4 mM
glutamine. Following 8 h incubation, medium aliquots were collected, concentrated, derivatized using MTBSTFA + 1% TBDMCS catalyst, and
assessed by GC-MS. Lactate was normalized in each sample using the 1 mM chlorophenylalanine (CPA) internal standard, and lactate relative to
vehicle is depicted. Averages from three or more independent experiments are depicted, with error bars depicting standard error (n $ 3).
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the highest potency levels were found with the new mannose-
derivative 2, which is even more potent than glucose-
derivative 1, and shows IC50 values of 5.4 mM against HeLa
cells and 15.2 mM against A549 cells.
Conclusions
We have successfully planned and implemented the stereo-
specic synthesis of three new glycoconjugates (2–4), which
were intended to extend the chemical class of sugar-
conjugates of NHIs. These glycoconjugates were designed
to be LDH-A inhibitors with efficient cell uptake. The bio-
logical assays in cancer cells displayed a remarkable corre-
lation between the cell uptake, reduction of cellular
production of lactate, and inhibition cellular proliferation.
Overall, the a-manno-derivative 2 proved to be the most
efficient compound in cell-based assays. These results
conrm the validity of the dual targeting strategy of the
Warburg effect and pave the way to a more expansive explo-
ration of glycoconjugates, both as molecular tools and as
potential therapeutic agents that block tumor glycolysis.
Fig. 4 Results of the intracellular concentration comparison study
comparing relative uptake of NHI-2 (15) to its three glycoconjugates
1–3. A549 non-small cell lung carcinoma cells were treated with 100
mM concentrations of each compound for 4 h; cells were then lysed,
sonicated in methanol, and assessed for intracellular compound
concentration by LC-MS. Triplicate data is shown, with error bars
denoting standard error. Statistical analysis was performed using an
unpaired Student's t test to compare glycoconjugate intracellular
concentrations with NHI-2 intracellular concentration, with * denoting
p < 0.05, ** denoting p < 0.01, and *** denoting p < 0.0005.
Cancer cell antiproliferative potency assays
Aer assessing the ability of glycol-conjugates 2 and 3 to
penetrate cells and inhibit cellular lactate production, their
potency in killing HeLa and A549 cancer cells was evaluated and
compared to that of their glucose analogue 1 and of their
aglycone NHI-2 (15). The growth inhibitory effect of these
compounds was evaluated by treating the cancer cells for 72 h
with varying concentrations of the compounds, aer which cell
death was assessed by the Sulforhodamine B (SRB) assay.23 The
antiproliferative potencies are expressed as IC50 values (the
concentration of compound required to kill 50% of cells, where
lower concentrations indicate increased potency) and are pre-
sented in Table 2.
Glycoconjugated NHI compounds 1–3 were all more potent
in killing cancer cells than their respective aglycone (NHI-2, 15),
in agreement with their increased cell uptake (Fig. 4) and
increased cellular lactate production inhibition (Fig. 3)
compared to the aglycone. In particular, the newly-synthesized
compounds have 3-6-fold (compound 2) and 2-3-fold
(compound 3) enhanced potencies compared to NHI-2. Finally,
Experimental
Materials and methods
All solvents and chemicals were used as purchased without
further purication. Chromatographic separations were
performed on silica gel columns by ash (Kieselgel 40, 0.040–
0.063 mm; Merck) or gravity column (Kieselgel 60, 0.063–
0.200 mm; Merck) chromatography. Reactions were followed
by thin-layer chromatography (TLC) on Merck aluminum
silica gel (60 F254) sheets that were visualized under a UV
lamp. Evaporation was performed in vacuo (rotating evapo-
rator). Sodium sulfate was always used as the drying agent.
Proton (1H) and carbon (13C) NMR spectra were obtained with
a Bruker Avance III 400 MHz or Bruker Avance 250 MHz
spectrometer using the indicated deuterated solvents.
Chemical shis are given in parts per million (ppm) (d rela-
tive to residual solvent peak for H and 13C). Yields refer to
isolated and puried products. LC-MS characterization and
high-resolution mass spectrometry (HRMS) analysis were
performed using a Waters Quattro II quadrupole–hexapole–
1
Table 2 Antiproliferative effect (72 h IC50 values, mM ꢂ standard error, quadrupole liquid chromatography/mass spectrometry
n ¼ 3) of glycoconjugates 1–3 in HeLa and A549 human cancer cell
apparatus (Waters, Milford, MA) equipped with an electro-
spray ionization source. LC separation was achieved using a
C18 Waters Xbridge column (2.1 ꢃ 20 mm, Waters) at 25 C
linesa
ꢁ
HeLa
using a linear gradient of mobile phases: 95% H2O, 5%
acetonitrile, and 0.1% formic acid (A) and 95% acetonitrile,
5% H2O, and 0.1% formic acid (B). Solution A was initially
passed through the column but decreased linearly to 50% of
the mobile phase at 10 minutes and 0% of the mobile phase
at 25 minutes. The ow rate was 200 mL minꢀ1, and the
injection volume was 10 mL. The ultraviolet (UV) detector was
programmed to monitor absorbance at 254 nm, to detect the
phenyl ring present in all compounds.
Compound
(cervical carcinoma)
A549 (NSCLC)
NHI-2 (15)
33.4 ꢂ 1.0b
7.2 ꢂ 0.2c
5.4 ꢂ 1.3
44.1 ꢂ 6.2b
17.2 ꢂ 3.0c
15.2 ꢂ 0.7
24.7 ꢂ 0.9
1
2
3
11.8 ꢂ 0.1
a
Three independent experiments were performed under normoxic
conditions. Remaining biomass aer xing with 10% trichloroacetic
b
acid was quantied by sulforhodamine B staining. Data from ref. 11.
c
Data from ref. 12.
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organic solution afforded a crude solid reaction product which
was puried by trituration in hexane, yielding pure 3 (0.031 g,
75% yield) as a white solid. Rf ¼ 0.25 (9 : 1 hexane–acetone).
[a]2D0: +57.71 (c 0.35, CH3OH). 1H NMR (400 MHz; CD3OD) d
8.31–8.36 (m, 1H; Ar), 7.72–7.79 (m, 3H; Ar), 7.45–7.54 (m, 2H;
Ar), 7.40 (tt, 1H, J ¼ 7.4, 1.2 Hz; Ar), 7.23 (qd, 1H, J ¼ 1.8, 0.9 Hz;
Ar), 5.47 (d, 1H, J ¼ 8.3 Hz; anomeric CHO), 4.11 (t, 1H, J ¼ 3.4
Hz; CHO), 4.03 (dd, 1H, J ¼ 8.3, 3.4 Hz; CHO), 3.98 (s, 3H;
COOMe), 3.93 (td, 1H, J ¼ 6.2, 1.1 Hz; CHO), 3.81–3.85 (m, 1H;
CHO), 3.79 (dd, 1H, J ¼ 10.8, 4.6 Hz; CH2O), 3.66 (dd, 1H, J ¼
10.8, 6.1 Hz; CH2O). 13C NMR (62.5 MHz; CD3OD) d 162.2, 141.2,
140.1, 139.9, 130.1 (2C), 129.8, 129.1, 128.4 (2C), 125.9 (q, J ¼
271.7 Hz), 124.4 (q, J ¼ 32.3 Hz), 120.1 (q, J ¼ 4.6 Hz), 118.4 (q, J
¼ 1.3 Hz), 115.2, 108.8, 106.7, 75.5, 73.2, 70.4, 68.6, 61.9, 52.4.
HRMS: (M + H+) found 498.1370; C23H23F3NO8 requires
498.1376.
Synthetic procedures
Methyl 1-(((2S,5R,6R)-5-hydroxy-6-(((tetrahydro-2H-pyran-2-
yl)oxy)methyl)-5,6-dihydro-2H-pyran-2-yl)oxy)-6-phenyl-4-(tri-
uoromethyl)-1H-indole-2-carboxylate (16). A solution of trans-
hydroxymesylate 8 (ref. 20) (0.150 g, 0.488 mmol) in anhydrous
CH3CN (13 mL) was treated with t-BuOK (0.060 g, 0.54 mmol,
1.1 equiv) at room temperature and, aer the disappearance of
the starting material (TLC), with N-hydroxyindole derivative 15
(0.180 g, 0.537 mmol, 1.1 equiv) and the resulting reaction
mixture was stirred 1 h at the same temperature. Aer dilution
with Et2O, the organic phase was washed with brine, dried
(MgSO4) and concentrated to afford a crude reaction product
(0.272 g), which was subjected to ash chromatography. Elution
with a 1 : 1 hexane–AcOEt (0.1% Et3N) mixture afforded pure
glycoside 16 (0.206 g, 77% yield) as a liquid. Rf ¼ 0.33 (4 : 6
1
hexane–AcOEt). H NMR (250 MHz; CDCl3) d 8.13–8.27 (m cor-
(2R,3R,4S)-2-(((Tetrahydro-2H-pyran-2-yl)oxy)methyl)-3,4-di-
hydro-2H-pyran-3,4-diol (12). A solution of Bu4NBr (2.94 g, 9.12
mmol, 4.0 equiv) in anhydrous THF (21.0 mL) was treated with
Me3SiOK (1.18 g, 9.12 mmol, 4.0 equiv) and the reaction mixture
was stirred at room temperature for 10 min. Filtration through a
short (1 cm) Celite column afforded a clear THF solution which
was concentrated at reduced pressure (rotary evaporator, nal
volume about 10 mL) (Solution B, containing Bu4N+Me3SiOꢀ, 4.0
equiv). A solution of hydroxymesylate 8 (0.702 g, 2.28 mmol) in
anhydrous THF (10.5 mL) was treated with t-BuOK (0.282 g, 2.51
mmol, 1.1 equiv) and the reaction mixture was stirred 15 min at
room temperature (Solution A, containing epoxide 10b). Solution
B was added dropwise to Solution A and the reaction mixture
was stirred 48 h at room temperature. Dilution with Et2O and
evaporation of the washed (brine) organic solution afforded a
crude liquid product that was subjected to ash chromatog-
raphy. Elution with a 3 : 7 hexane–AcOEt (0.1% Et3N) mixture
afforded pure trans diol 12 (0.235 g, 45% yield), as a colorless
liquid. Rf ¼ 0.13 (3 : 7 hexane–AcOEt). 1H NMR (250 MHz;
CDCl3) d 6.58–6.66 (m corresponding to two diastereoisomers,
overall 1H, vinyl CHO), 4.95–5.05 (m corresponding to two
diastereoisomers, overall 1H, vinyl CH), 4.64–4.72 (m corre-
sponding to two diastereoisomers, overall 1H, THP–CHO2),
4.06–4.20 (m corresponding to two diastereoisomers, overall
1H; CHO), 3.77–4.04 (m corresponding to two diastereoisomers,
overall 5H CHO and CH2O), 3.47–3.62 (m corresponding to two
diastereoisomers, overall 1H; CHO), 1.69–1.85 (m correspond-
ing to two diastereoisomers, overall 3H, THP–CH2), 1.48–1.68
(m corresponding to two diastereoisomers, overall 3H; THP–
CH2).
responding to two diastereoisomers, overall 1H; Ar), 7.62–7.96
(m, 3H; Ar), 7.15–7.52 (m, 4H; Ar), 6.23–6.47 (m corresponding
to two diastereoisomers, overall 2H; vinyl CH), 5.96–6.05 (m
corresponding to two diastereoisomers, overall 1H; anomeric
CH), 4.31–4.59 (m corresponding to two diastereoisomers,
overall 1H; THP–CHO2), 3.08–4.15 (m corresponding to two
diastereoisomers, overall 6H; CHO & CH2O), 3.96 (s, 3H;
COOCH3), 1.29–1.74 (m corresponding to two diastereoisomers,
overall 6H; THP–CH2).
Methyl
6-phenyl-4-(triuoromethyl)-1-(((2S,3R,4R,5R,6R)-
3,4,5-trihydroxy-6-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)tetra-
hydro-2H-pyran-2-yl)oxy)-1H-indole-2-carboxylate (18). A solu-
tion of glycoside (16) (0.050 g, 0.091 mmol) in an 1 : 1 t-BuOH–
acetone mixture (0.31 mL) was added to a 50% p/v aqueous
solution of N-methyl morpholine-N-oxide (NMO) (80 mL) and the
resulting reaction mixture was treated with 2.5% p/v OsO4
solution in t-BuOH (80 mL) and stirred for 2 h at the same
temperature. Dilution with AcOEt and evaporation of the
ltered (Celite®) organic solution afforded a crude reaction
product (0.092 g), which was subjected to ash chromatog-
raphy. Elution with a 2 : 8 hexane–AcOEt (0.1% Et3N) mixture
afforded 18 (0.037 g, 70% yield), practically pure as a liquid. Rf ¼
1
0.12 (2 : 8 hexane–AcOEt). H NMR (250 MHz; CDCl3) d 8.25–
8.31 (m corresponding to two diastereoisomers, overall 1H; Ar),
7.58–7.79 (m, 3H; Ar), 7.34–7.54 (m, 4H; Ar), 5.37–5.49 (m cor-
responding to two diastereoisomers, overall 1H; anomeric
CHO), 5.14–5.25 (m corresponding to two diastereoisomers,
overall 1H; THP–CHO2), 3.90–4.46 (m corresponding to two
diastereoisomers, overall 5H; CH2O and CHO), 3.98 (s, 3H;
COOMe), 3.34–3.80 (m corresponding to two diastereoisomers,
overall 3H; CH2O and CHO), 1.40–1.74 (m corresponding to two
diastereoisomers, overall 6H; THP–CH2).
(2R,3S,4S)-4-((tert-Butyldimethylsilyl)oxy)-2-(((tetrahydro-2H-
pyran-2-yl)oxy)methyl)-3,4-dihydro-2H-pyran-3-ol (13). Treat-
ment of trans diol 12 (0.216 g, 0.939 mmol) in anhydrous DMF
(2.5 mL) with imidazole (0.128 g, 1.88 mmol, 2.0 equiv) and
TBSCl (0.170 g, 1.13 mmol) afforded, aer 18 h stirring at room
temperature, a crude liquid product (0.283 g, 88% yield) con-
sisting of mono-silylated 13, pure as a liquid, which was used in
the next step without any purications. Rf ¼ 0.25 (8 : 2 hexane–
AcOEt). 1H NMR (250 MHz; CDCl3) d 6.49–6.58 (m corresponding
to two diastereoisomers, overall 1H; vinyl CHO), 4.81–4.90 (m
corresponding to two diastereoisomers, overall 1H; vinyl CH),
Methyl
6-phenyl-4-(triuoromethyl)-1-(((2S,3R,4R,5R,6R)-
3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-
oxy)-1H-indole-2-carboxylate (3). PPTS (0.002 g, 0.008 mmol, 0.1
equiv) was added to a solution of 6-OTHP-protected precursor
18 (0.048 g, 0.083 mmol) in absolute EtOH (0.3ꢁmL) and the
reaction mixture was carefully stirred 46 h at 40 C. Aer dilu-
tion with CH2Cl2, solid NaHCO3 was added until the solution
turned out to be slightly basic. Evaporation of the ltered
19950 | RSC Adv., 2015, 5, 19944–19954
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4.63–4.71 (m corresponding to two diastereoisomers, overall 1.1 equiv) and, aer the disappearance of the starting material
1H; THP–CHO2), 3.95–4.17 (m corresponding to two diastereo- (TLC), with N-hydroxyindole derivative 15 (0.180 g, 0.537 mmol,
isomers, overall 2H; CHO and CH2O), 3.72–3.94 (m corre- 1.1 equiv) and the resulting reaction mixture was stirred for 1 h
sponding to two diastereoisomers, overall 4H CHO and CH2O), at room temperature. Aer dilution with Et2O, the organic
3.43–3.62 (m corresponding to two diastereoisomers, overall phase was washed with brine, dried (MgSO4) and concentrated
1H, CHO), 1.44–1.86 (m corresponding to two diastereoisomers, to afford a crude reaction product, which was subjected to ash
overall 6H; THP–CH2), 0.87 (s, 9H; (CH3)3CSi), 0.10 (s, 6H; chromatography. Elution with a 1 : 1 hexane–AcOEt (0.1% Et3N)
CH3Si).
mixture afforded pure glycoside 17 (0.155 g, 58% yield) as a
1
(2R,3S,4S)-4-((tert-Butyldimethylsilyl)oxy)-2-(((tetrahydro-2H- white solid. Rf ¼ 0.33 (4 : 6 hexane–AcOEt); mp 50–53 ꢁC; H
pyran-2-yl)oxy)methyl)-3,4-dihydro-2H-pyran-3-yl methanesulfo- NMR (250 MHz; CDCl3) d 7.77–7.89 (m corresponding to two
nate (14). Treatment of 13 (0.283 g, 0.82 mmol) in anhydrous diastereoisomers, overall 1H; Ar), 7.59–7.76 (m corresponding
pyridine (2.5 mL) with MsCl (0.13 mL, 1.6 mmol, 2.0 equiv) was to two diastereoisomers, overall 3H; Ar), 7.34–7.56 (m corre-
stirred at 0 ꢁC for 18 h. Aer concentration under vacuum, the sponding to two diastereoisomers, overall 3H; Ar), 7.27–7.33 (m
crude product was subjected to ash chromatography. Elution corresponding to two diastereoisomers, overall 1H; Ar), 6.12–
with a 8 : 2 hexane–AcOEt (0.1% Et3N) mixture afforded pure 6.36 (m corresponding to two diastereoisomers, overall 2H,
mesylate derivative 14 (0.254 g, 71% yield) as a yellow liquid. Rf vinyl CH), 5.78–5.89 (m corresponding to two diastereoisomers,
¼ 0.19 (8 : 2 hexane–AcOEt). 1H NMR (250 MHz; CDCl3) d 6.45– overall 1H; anomeric CHO), 3.26–4.53 (m corresponding to two
6.54 (m corresponding to two diastereoisomers, overall 1H; diastereoisomers, overall 7H; CH2O and CHO), 3.94 (s, 3H;
vinyl CHO), 4.82–4.91 (m corresponding to two diastereoiso- COOMe), 1.28–1.86 (m corresponding to two diastereoisomers,
mers, overall 1H; vinyl CH), 4.58–4.76 (m corresponding to two overall 6H; THP–CH2).
diastereoisomers, overall 2H; CHOMs + THP–CHO2), 4.22–4.34
Methyl
6-phenyl-4-(triuoromethyl)-1-(((2R,3S,4S,5S,6R)-
(m corresponding to two diastereoisomers, overall 1H; CHO), 3,4,5-trihydroxy-6-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)tetra-
4.15–4.21 (m corresponding to two diastereoisomers, overall hydro-2H-pyran-2-yl)oxy)-1H-indole-2-carboxylate (19). A solu-
1H; CHO), 3.81–4.02 (m corresponding to two diastereoisomers, tion of 17 (0.040 g, 0.073 mmol) in an 1 : 1 t-BuOH–acetone
overall 2H; CH2O), 3.57–3.80 (m corresponding to two diaste- mixture (0.25 mL) was added to 50% p/v aqueous solution of N-
reoisomers, overall 1H; CHO), 3.43–3.56 (m corresponding to methyl morpholine-N-oxide (NMO) (60 mL) and the resulting
two diastereoisomers, overall 1H; CHO), 3.04–3.10 (m corre- reaction mixture was treated with 2.5% p/v OsO4 solution in t-
sponding to two diastereoisomers, overall 3H; CH3SO2), 1.66– BuOH (60 mL) and stirred for 8 h at room temperature. Dilution
1.89 (m corresponding to two diastereoisomers, overall 2H; with AcOEt and evaporation of the ltered (Celite®) organic
THP–CH2), 1.44–1.62 (m corresponding to two diastereoiso- solution afforded a crude reaction product which was puried
mers, overall 4H; THP–CH2), 0.83–0.92 (m corresponding to two by ash chromatography. Elution with a 2 : 8 hexane–AcOEt
diastereoisomers, overall 9H; (CH3)3CSi), 0.09–0.16 (m corre- (0.1% Et3N) mixture afforded pure a-mannopyranoside 19
sponding to two diastereoisomers, overall 6H; CH3Si).
(0.028 g, 65% yield) as a white solid. Rf ¼ 0.17 (2 : 8 hexane–
1
(2R,3R,4S)-4-Hydroxy-2-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)- AcOEt). H NMR (250 MHz; CDCl3) d 7.76–7.88 (m correspond-
3,4-dihydro-2H-pyran-3-yl methanesulfonate (9). Treatment of 14 ing to two diastereoisomers, overall 1H; Ar), 7.55–7.72 (m cor-
(0.249 g, 0.589 mmol) in anhydrous THF (20 mL) with 1 M TBAF responding to two diastereoisomers, overall 3H; Ar), 7.32–7.50
ꢁ
in THF (0.59 mL, 0.59 mmol) at 0 C for 20 min. Aer dilution (m corresponding to two diastereoisomers, overall 4H; Ar),
with Et2O, evaporation of the washed (brine) and dried (MgSO4) 5.66–5.71 (m corresponding to two diastereoisomers, overall
organic solution afforded a crude product, which was subjected 1H; anomeric CHO), 4.66–4.77 (m corresponding to two dia-
to ash chromatography (1 : 1 hexane–AcOEt) to produce pure stereoisomers, overall 1H; THP–CHO2), 4.30–4.47 (m corre-
trans-hydroxymesylate 9 (0.122 g, 67% yield) as a yellow liquid. sponding to two diastereoisomers, overall 2H; CHO), 3.93–4.22
Rf ¼ 0.19 (1 : 1 hexane–AcOEt). 1H NMR (250 MHz; CDCl3) d (m corresponding to two diastereoisomers, overall 3H; CHO and
6.53–6.61 (m corresponding to two diastereoisomers, overall CH2O), 3.90 (s, 3H; COOMe), 3.71–3.86 (m corresponding to two
1H; vinyl CHO), 4.93–5.04 (m corresponding to two diastereo- diastereoisomers, overall 2H; CH2O), 3.29–3.48 (m corre-
isomers, overall 1H; vinyl CH), 4.72–4.87 (m corresponding to sponding to two diastereoisomers, overall 1H; CHO), 1.30–1.71
two diastereoisomers, overall 1H; THP–CHO2), 4.59–4.68 (m (m corresponding to two diastereoisomers, overall 6H; THP–
corresponding to two diastereoisomers, overall 1H; CHOMs), CH2).
4.18–4.30 (m corresponding to two diastereoisomers, overall
Methyl
6-phenyl-4-(triuoromethyl)-1-(((2R,3S,4S,5S,6R)-
2H; CH2O), 3.43–4.05 (m corresponding to two diastereoiso- 3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-
mers, overall 4H CHO & CH2O), 3.04–3.13 (m corresponding to oxy)-1H-indole-2-carboxylate (2). PPTS (0.018 g, 0.007 mmol, 0.1
two diastereoisomers, overall 3H; CH3SO2), 1.36–1.90 (m cor- equiv) was added to a solution of 19 (0.040 g, 0.068 mmol) in
responding to two diastereoisomers, overall 6H; THP–CH2).
absolute EtOH (1.3 mL) and the reaction mixture was stirred for
Methyl 1-(((2R,5S,6R)-5-hydroxy-6-(((tetrahydro-2H-pyran-2- 20 h at 40 ꢁC. Aer dilution with CH2Cl2, solid NaHCO3 was
yl)oxy)methyl)-5,6-dihydro-2H-pyran-2-yl)oxy)-6-phenyl-4-(tri- added until the solution turned out to be slightly basic. Evap-
uoromethyl)-1H-indole-2-carboxylate (17). A solution of trans oration of the ltered organic solution afforded a crude reaction
hydroxymesylate 9 (0.150 g, 0.488 mmol) in anhydrous CH3CN product, which was puried by trituration with hexane, yielding
(12.6 mL) was treated with t-BuOK (0.060 g, 0.54 mmol, pure a-O-D-mannopyranoside 2 (0.014 g, 83% yield) pure as a
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white solid. Rf ¼ 0.08 (9 : 1 AcOEt–acetone). [a]2D0 +18.14 (c 0.95, 7.44 (t, 1H, J ¼ 7.3 Hz; Ar), 7.11 (s, 1H; Ar), 5.18–5.21 (m, 2H, 2 ꢃ
1
CH3OH). H NMR (400 MHz; CD3OD) d 8.04 (s, 1H; Ar), 7.75– OH), 5.11 (d, J ¼ 8.7 Hz, 1H, anomeric CHO), 4.39 (t, J ¼ 4.8 Hz,
7.78 (m, 3H; Ar), 7.48–7.53 (m, 2H; Ar), 7.41 (tt, 1H, J ¼ 7.4, 1.2 1H, OH), 3.92 (s, 3H, COOMe), 3.80 (q, J ¼ 9.0 Hz, 1H; CHO),
Hz; Ar), 7.25–7.29 (m, 1H; Ar), 5.57 (d, 1H, J ¼ 1.9 Hz; anomeric 3.41–3.67 (m, 4H; CH2O, CHO and CHN), 3.11–3.19 (m, 1H;
CHO), 4.57–4.63 (m, 1H; CHO), 4.20–4.28 (m, 1H; CHO), 3.96 (s, CHO), 1.94 (s, 3H; CH3CO); 13C NMR (100 MHz; DMSO-d6): d
3H; COOMe), 3.83–3.89 (m, 4H; CH2O and CHO). 13C NMR (62.5 169.4, 159.4, 139.1, 137.7, 137.5, 129.9, 129.2 (2C), 128.1, 127.4
MHz; CD3OD) d 161.3, 141.1, 140.5, 139.2, 130.2 (2C), 129.3, (2C), 124.7 (q, J ¼ 271.0 Hz), 122.4 (q, J ¼ 32.3 Hz), 119.4 (q, J ¼
129.1, 128.5 (2C), 125.2 (q, J ¼ 271.0 Hz), 124.8 (q, J ¼ 32.9 Hz), 4.7 Hz), 117.2 (q, J ¼ 2.0 Hz), 113.7, 106.5, 104.2, 77.0, 73.5, 69.8,
120.2 (q, J ¼ 4.9 Hz), 118.3 (q, J ¼ 1.7 Hz), 113.6, 111.2, 106.9, 60.8, 54.0, 52.3, 23.2. HRMS: (M + H+) found 539.1639;
77.5, 72.4, 70.5, 67.9, 62.6, 52.8. HRMS: (M + H+) found C25H26F3N2O8 requires 539.1641.
498.1366; C23H23F3NO8 requires 498.1376.
(2R,3S,4R,5R,6S)-5-Acetamido-2-(acetoxymethyl)-6-((2-(methoxy-
carbonyl)-6-phenyl-4-(triuoromethyl)-1H-indol-1-yl)oxy)tetra-
Molecular modeling
hydro-2H-pyran-3,4-diyl diacetate (21). Activated molecular The compounds were built using Maestro 9.0 (ref. 24) and was
˚
sieves 4 A (0.215 g), oxazoline 20 (ref. 21) (0.230 g, 0.69 mmol, subjected to a conformational search (CS) of 1000 steps, using a
1.0 equiv) and N-hydroxyindole derivative 15 (0.250 g, 0.75 water environment model (generalized-Born/surface-area
mmol, 1.1 equiv) were dissolved in 3.5 mL of anhydrous DCE model) by means of Macromodel.25 The algorithm used was
and the obtained mixture was stirred at 80 ꢁC until complete based on the Monte Carlo method with the MMFFs force eld
dissolution of the reagents. Aer cooling to room temperature, and a distance-dependent dielectric constant of 1.0. The ligand
TMSOTf (60 mL, 0.34 mmol, 0.5 equiv) was added. The resulting was then energy minimized using the conjugated gradient (CG)
solution was heated again at 80 ꢁC and stirred overnight at the method until a convergence value of 0.05 kcal (molꢀ1
ꢀ1) was
˚
A
same temperature. Then the mixture was diluted with CH2Cl2 reached, using the same force eld and parameters used for the
and ltered; the ltered solution was neutralized by saturated CS. The hLDH5 chain was extracted from the minimized
aqueous NaHCO3 and washed with brine. Evaporation of the average structure of the complex between LDH and 1J obtained
washed organic solution afforded a crude product, which was by us through molecular dynamic simulations.10 Automated
subjected to ash chromatography. Elution with a 9 : 1 CHCl3/ docking was carried out by means of the GOLD 5.1 program.26
acetone mixture yielded pure compound 21 (0.140 g, yield 40%) The “allow early termination” option was deactivated, the
as a pale yellow solid: mp 222–225 ꢁC. [a]D20 ¼ +26.9 (c 0.79; remaining GOLD default parameters were used, and the ligand
CHCl3). Rf ¼ 0.3 (9 : 1 CHCl3–acetone). 1H NMR (400 MHz; was submitted to 30 genetic algorithm runs by applying the
CDCl3): d 8.10 (s, 1H; Ar), 7.74 (s, 1H; Ar), 7.63 (d, 2H, J ¼ 7.7 Hz; ChemScore tness function. The best docked conformation was
Ar), 7.36–7.49 (m, 3H; Ar), 7.33 (bs, 1H; Ar), 6.70 (d, 1H, J ¼ 8.6 taken into account. The so obtained complexes were energy
Hz; NH), 5.52 (d, 1H, J ¼ 9.05, anomeric CHO), 5.16–5.28 (m, minimized using AMBER 11.27 Each complex was placed in a
2H; CH2OAc), 4.55 (q, 1H, J ¼ 9.0 Hz; CHOAc), 4.26 (dd, 1H, J ¼ rectangular parallelepiped water box, an explicit solvent model
12.4, 4.7 Hz; CHOAc), 3.98–4.08 (m, 1H; CHO), 3.95 (s, 3H; for water (TIP3P) was used, and the complex was solvated with a
˚
COOMe), 3.69–3.75 (m, 1H; CHN), 2.07 (s, 3H; CH3CO), 2.03 (s, 10 A water cap. Chloride ions were added as counterions to
3H; CH3CO), 2.02 (s, 3H; CH3CO), 1.77 (s, 3H; CH3CO). 13C NMR neutralize the system. Two steps of minimization were then
(100 MHz; CDCl3): d 171.0, 170.6 (2C), 169.4, 161.0, 140.2, 140.0, carried out. In the rst stage, we kept the complex xed with a
139.6, 129.2 (2C), 128.3, 127.4 (2C), 124.3 (q, J ¼ 272.4 Hz), 123.8 position restraint of 500 kcal (molꢀ1
A
ꢀ2) and we solely mini-
˚
(q, J ¼ 33.0 Hz), 120.0 (q, J ¼ 4.8 Hz), 119.03 (q, J ¼ 4.7 Hz), 117.4 mized the positions of the water molecules. In the second stage,
(q, J ¼ 2.1 Hz), 114.3, 108.4, 105.8, 73.6, 72.5, 68.0, 61.8, 52.5, we minimized the entire system through 20 000 steps of
52.4, 23.3, 20.8, 20.7, 20.4.
Methyl
steepest descent followed by conjugate gradient until a
1-(((2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6- convergence of 0.05 kcal (molꢀ1
ꢀ1) was attained. All the a
˚
A
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-phenyl-4-(tri- carbons of the protein were blocked with a harmonic force
uoromethyl)-1H-indole-2-carboxylate (4). Compound 21 (56.5 constant of 10 kcal (molꢀ1
ꢀ2). Ten nanoseconds of MD
˚
A
mg, 0.087 mmol, 1.0 equiv) was dissolved in a 2 : 3 mixture of simulation were then carried out. The time step of the simula-
CH2Cl2 and MeOH (4 mL) and cooled at 0 ꢁC. A freshly prepared tions was 2.0 fs with a cutoff of 10 A for the non-bonded
˚
0.33 M solution of MeONa/MeOH (0.05 mL) was added to the interaction, and SHAKE was employed to keep all bonds
resulting solution and the reaction mixture was stirred for 4 h at involving hydrogen atoms rigid. Constant-volume periodic
room temperature. The mixture was then neutralized with an boundary MD was carried out for 400 ps, during which the
acidic Amberlite™ IR 120 H resin The resin was then removed temperature was raised from 0 to 300 K. Then 9.6 ns of constant
by ltration and repeatedly extracted with methanol. The pressure periodic boundary MD was carried out at 300 K using
combined ltrate was concentrated under vacuum to give a the Langevin thermostat to maintain constant the temperature
crude product, which was recrystallized from MeOH to yield of our system. General Amber Force Field (GAFF) parameters
pure glycoside 4 (0.020 g, yield 50%) as a pale yellow solid: mp: were assigned to the ligand, while partial charges were calcu-
190–194 ꢁC. [a]D20 ¼ ꢀ36.9 (c 0,17; MeOH). Rf ¼ 0.15 (9 : 1 AcOEt– lated using the AM1-BCC method as implemented in the
MeOH). 1H NMR (400 MHz; DMSO-d6): d 8.15 (s, 1H; Ar), 8.06 (d, Antechamber suite of AMBER 11. The nal structure of the
J ¼ 9.0 Hz, 1H, NH), 7.82–7.91 (m, 3H; Ar), 7.50–7.55 (m, 2H; Ar), complex was obtained as the average of the last 8 ns of MD
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minimized by the CG method until a convergence of 0.05 kcal
Compounds were identied using AMDIS Chromatogram
(molꢀ1
ꢀ1). The average structure was obtained using the ptraj soware (Amdis, freeware available from amdis.net) and pro-
˚
A
program implemented in AMBER 11.
grammed WIST and Niley commercial libraries. The integration
area of lactate in each sample was divided by the integration
area of CPA in the same sample to achieve a lactate/internal
standard ratio. The ratios were averaged for duplicates, and
Biological assays
Enzyme inhibition assay. The compounds were evaluated in percent lactate production over vehicle was calculated for each
enzymatic assays to assess their inhibitory properties against independent experiment. The mean lactate production/vehicle
commercially available puried human isoform of lactate was then averaged between three or more independent
dehydrogenase hLDH5 (LDH-A4, Lee Biosolution, Inc.). The experiments.
reaction of lactate dehydrogenase was conducted using the
Intracellular concentration assessment. Assessment of
“forward” direction (pyruvate / lactate) and the kinetic intracellular concentration was performed as described previ-
parameters for the substrate (pyruvate) and the cofactor (NADH) ously.12 A549 human non-small cell lung carcinoma cells (ATCC,
were measured by uorescence (emission wavelength at 460 Manassas, VA) were grown in T25 cell culture asks prior to
ꢁ
nm, excitation wavelength at 340 nm), to monitor at 37 C the being treated with 100 mM compound or vehicle control
rate of conversion of NADH to NAD+ and, therefore, the (prepared in DMSO) in 5 mL total volume (0.2% nal concen-
progression of the reaction. Such assays were conducted in tration DMSO in all treatments). At each time point, cells were
wells containing 200 mL of a solution comprising the reagents rapidly collected by scraping, washed twice with sterile PBS, 37
dissolved in 100 mM phosphate buffer at pH 7.4. DMSO stock ꢁC, and disrupted by sonication in methanol, ꢀ80 ꢁC, using an
solution of compounds were prepared (concentration of DMSO XL-2000 Misonix sonicator (Qsonica, Newton, CT). Aer a 30
did not exceed 4% during the measurements). Assays were minute incubation at 4 ꢁC to facilitate precipitation of proteins,
performed in 96-well plates and compounds were assayed in the the sonicates were centrifuged, and a portion of the supernatant
presence of scalar concentrations of NADH. They were added in was analyzed using the protocol described above. Data analysis
scalar amounts (concentration range ¼ 15–60 mM) to a reaction was performed using MassLynx spectrometry soware (Waters).
mix containing phosphate buffer, 1.4 mM pyruvate and a scalar
These LC-MS parameters allowed for the clear resolution of
concentration of NADH (10–150 mM), nally LDH solution was compound 1 (elution time: 12.4 minutes in the UV trace, 12.6
added (0.015 U mLꢀ1). LDH activity was measured by recording minutes in the TIC), compound 2 (elution time: 12.7 minutes in
the decrease in NADH uorescence using Victor X3 Microplates the UV trace, 12.9 minutes in the TIC), compound 3 (elution
Reader (PerkinElmer®).
time: 12.9 minutes in the UV trace, 13.1 minutes in the TIC),
Assessment of lactate production by GC-MS. This assay was and 15 (elution time: 15.8 minutes in the UV trace; 16.0 minutes
performed under normoxic conditions as previously in the TIC). The UV traces of vehicle-treated sonicates from both
described12 Briey, conuent HeLa cervical carcinoma cells the start and end of the experiment time course contained no
(ATCC, Manassas, VA) in a 96 well plate were treated with peaks in this range. Calibration curves of 15 and glyco-
compound or vehicle control (1% DMSO nal concentration in conjugates 1, 2, and 3 demonstrated a linear relationship
all samples) in DMEM minus phenol red + 10% dialyzed FBS + between concentration and UV trace integration area, so a linear
1% Penstrep, supplemented with 10 mM glucose, 1 mM equation was generated for each compound to convert inte-
sodium pyruvate and 4 mM glutamine, in a nal volume of 125 gration area to concentration.
mL per well. Immediately following compound addition, plates
Cell proliferation assay. Determination of cellular IC50
were incubated for 4 or 8 h at 37 ꢁC in a 95% air/5% CO2 values was performed under normoxic conditions as
atmosphere. Duplicate wells were prepared for each treat- described previously.12 Briey, HeLa and A549 cells, grown in
ment. Following treatment, medium was collected, and 100 mL RPMI 1640 medium supplemented with 10% FBS and 1%
were added to 2 mL 50 mM chlorophenylalanine (CPA; internal Penicillin/Streptomycin, were added at a density of 5000 cells
standard for GC-MS analysis). Samples were concentrated, per well to 96 well plates to which 31.6 nM–200 mM
derivatized by a four-hour incubation with MTBSTFA + 1% compound in DMSO was already added (1% nal concen-
TBDMCS (Thermo Scientic, Walthman, MA) in acetonitrile at tration DMSO in all wells; triplicate wells at the same
ꢁ
85 ꢁC, and immediately analyzed using GC-MS (Agilent 6890N concentration per repetition). Plates were incubated at 37 C
GC/5973 MS, equipped with an Agilent DB-5 capillary column, in a 95% air/5% CO2 atmosphere for 72 h. Medium was
30 M ꢃ 320 mM ꢃ 0.25 mM, model number J&W 123-5032, removed and cells were xed by the addition of 50 mL 10%
ꢁ
Agilent Technologies, Santa Clara, CA) and an electron impact trichloroacetic acid in water, 4 C, to each well. Plates were
ionization source. One microliter of each sample was injected incubated at 4 ꢁC for at least one hour, and the sulforhod-
using an automated injector, and a solvent delay of 8.20 amine B colorimetric assay23 was performed to assess
minutes was implemented. The initial oven temperature was remaining biomass in each well. Cells treated with 1% DMSO
120 ꢁC, held for 5 minutꢀes1; then the temperature was increased were used as the 100% live control for biomass, and wells
at a rate of 10 ꢁC min until a temperature of 250 ꢁC was incubated with medium alone were used as the baseline zero
reached. Temperature was then increased by 40 ꢁC minꢀ1 until biomass control. IC50 values were calculated using SoMax
ꢁ
a nal temperature of 310 C was reached. Total run time per Pro soware (Molecular Devices, Sunnyvale, CA).
sample was 22.5 min.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 19944–19954 | 19953

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Paper
A. Martinelli, F. Minutolo and P. J. Hergenrother,
ChemBioChem, 2013, 14, 2263–2267.
Acknowledgements
13 J. E. Barnett, G. D. Holman and K. A. Munday, Biochem. J.,
1973, 131, 211–221.
14 H. Lodish, A. Berk, S. L. Zipursky, P. Matsudaira,
D. Baltimore and J. Darnell, in Molecular Cell Biology, ed.
W. H. Freeman, New York, 4th edn, 2000, http://
www.ncbi.nlm.nih.gov/books/NBK21669/.
15 Z. Ma and K. Vosseller, Amino Acids, 2013, 45, 719–733.
16 Z. Ma and K. Vosseller, J. Biol. Chem., 2014, 289, 34457–
34465.
17 V. Di Bussolo, M. Caselli, M. Pineschi and P. Crotti, Org.
Lett., 2002, 4, 3695–3698.
18 V. Di Bussolo, M. Caselli, M. Pineschi and P. Crotti, Org.
Lett., 2003, 5, 2173–2176.
19 V. Di Bussolo, M. Caselli, M. R. Romano, M. Pineschi and
P. Crotti, J. Org. Chem., 2004, 69, 7383–7386.
20 V. Di Bussolo, L. Checchia, M. R. Romano, M. Pineschi and
P. Crotti, Org. Lett., 2008, 10, 2493–2496.
21 S. Nakabayashi, C. D. Warren and R. W. Jeanlozs, Carbohydr.
Support from the National Institutes of Health (R01GM098453)
is gratefully acknowledged. ECC is supported by an NCI F30
Ruth L. Kirschstein National Research Service Award
(1F30CA168323).
Notes and references
1 O. Warburg, Science, 1956, 124, 269–270.
2 P. F. Oliveira, A. D. Martins, A. C. Moreira, C. Y. Cheng and
M. G. Alves, Med. Res. Rev., 2015, 35, 126–151.
3 C. Granchi and F. Minutolo, ChemMedChem, 2012, 7, 1318–
1350.
4 C. Granchi, D. Fancelli and F. Minutolo, Bioorg. Med. Chem.
Lett., 2014, 24, 4915–4925.
5 (a) V. R. Fantin, J. St-Pierre and P. Leder, Cancer Cell, 2006, 9,
425–434; (b) H. Xie, J. Hanai, J. G. Ren, L. Kats, K. Burgess,
P. Bhargava, S. Signoretti, J. Billiard, K. J. Duffy, A. Grant,
X. Wang, P. K. Lorkiewicz, S. Schatzman, M. Bousamra II,
A. N. Lane, R. M. Higashi, T. W. Fan, P. P. Pandol,
V. P. Sukhatme and P. Seth, Cell Metab., 2014, 19, 795–809.
6 T. Kanno, K. Sudo, M. Maekawa, Y. Nishimura, M. Ukita and
K. Fukutake, Clin. Chim. Acta, 1988, 173, 89–98.
7 C. Granchi, S. Bertini, M. Macchia and F. Minutolo, Curr.
Med. Chem., 2010, 17, 672–697.
Res., 1986, 150, C7–C10.
ˇ
ˇ
´
´
22 K. Krenek, P. Simon, L. Weignerova, B. Fliedrova, M. Kuzma
ˇ
and V. Kren, Beilstein J. Org. Chem., 2012, 8, 428–432.
23 V. Vichai and K. Kirtikara, Nat. Protoc., 2006, 1, 1112–1116.
¨
24 Maestro, version 9.0, Schrodinger Inc, Portland, OR, 2009.
¨
25 Macromodel ver. 9.7, Schrodinger Inc, Portland, OR, 2009.
8 C. Granchi, I. Paterni, R. Rani and F. Minutolo, Future Med.
Chem., 2013, 5, 1967–1991.
26 M. L. Verdonk, J. C. Cole, M. J. Hartshorn, C. W. Murray and
R. D. Taylor, Proteins, 2003, 52, 609–623.
9 E. C. Calvaresi and P. J. Hergenrother, Chem. Sci., 2013, 4,
2319–2333.
10 C. Granchi, S. Roy, C. Giacomelli, M. Macchia, T. Tuccinardi,
A. Martinelli, M. Lanza, L. Betti, G. Giannaccini,
27 D. A. Case, T. A. Darden, T. E. Cheatham III,
C. L. Simmerling, J. Wang, R. E. Duke, R. Luo,
R. C. Walker, W. Zhang, K. M. Merz, B. Roberts, B. Wang,
´
S. Hayik, A. Roitberg, G. Seabra, I. Kolossvary, K. F. Wong,
´
A. Lucacchini, N. Funel, L. G. Leon, E. Giovannetti,
F. Paesani, J. Vanicek, J. Liu, X. Wu, S. R. Brozell,
T. Steinbrecher, H. Gohlke, Q. Cai, X. Ye, J. Wang,
M.-J. Hsieh, G. Cui, D. R. Roe, D. H. Mathews,
M. G. Seetin, C. Sagui, V. Babin, T. Luchko, S. Gusarov,
A. Kovalenko and P. A. Kollman, AMBER 11, University of
California, San Francisco, CA, 2010.
G. J. Peters, R. Palchaudhuri, E. C. Calvaresi,
P. J. Hergenrother and F. Minutolo, J. Med. Chem., 2011,
54, 1599–1612.
11 C. Granchi, E. C. Calvaresi, T. Tuccinardi, I. Paterni,
M. Macchia, A. Martinelli, P. J. Hergenrother and
F. Minutolo, Org. Biomol. Chem., 2013, 11, 6588–6596.
12 E. C. Calvaresi, C. Granchi, T. Tuccinardi, V. Di Bussolo,
R. W. Huigens, H. Y. Lee, R. Palchaudhuri, M. Macchia,
19954 | RSC Adv., 2015, 5, 19944–19954
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