Synthesis and DNA-binding affinity studies of glycosylated intercalators designed as functional mimics of the anthracycline antibiotics

Article abstract of DOI:10.1039/b909153j

Anthracycline antibiotics such as daunomycin (Dauno) and doxorubicin (Dox) are well-known clinically used cancer chemotherapeutics, which, among other mechanisms, bind to DNA, thereby triggering a cascade of biological responses leading to cell death. However, anthracyclines are cardiotoxic, and drug resistance develops rapidly, thus limiting their clinical use. We report here the synthesis and DNA-binding affinity of a novel class of functional anthracycline mimetics consisting of an aromatic moiety linked to a carbohydrate (1-12). In the targets, the aromatic core consists of a 2-phenylbenzo[b]furan- 3-yl, 2-phenylbenzo[b]thiophen-3-yl, 1-tosyl-2-phenylindol-3-yl, or 2-phenylindol-3-yl group that is bound to one of three aminosugars (daunosamine, acosamine, or 4-amino-2,3,4,6-tetradeoxy-α-l-hexopyranoside) via a propargyl linker. The DNA binding affinity of these twelve compounds has been evaluated by using both direct and indirect fluorescence measurements. Compared to Dauno and Dox, the DNA binding affinity of these analogues is weaker. However, both aromatic and aminosugar motifs are critical to DNA binding, with more influence coming from the structural features of the aromatic portion.

Full text of DOI:10.1039/b909153j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and DNA-binding affinity studies of glycosylated intercalators
designed as functional mimics of the anthracycline antibiotics†
Wei Shi,a Robert S. Colemanb and Todd L. Lowary*a
Received 8th May 2009, Accepted 12th June 2009
First published as an Advance Article on the web 17th July 2009
DOI: 10.1039/b909153j
Anthracycline antibiotics such as daunomycin (Dauno) and doxorubicin (Dox) are well-known
clinically used cancer chemotherapeutics, which, among other mechanisms, bind to DNA, thereby
triggering a cascade of biological responses leading to cell death. However, anthracyclines are
cardiotoxic, and drug resistance develops rapidly, thus limiting their clinical use. We report here the
synthesis and DNA-binding affinity of a novel class of functional anthracycline mimetics consisting of
an aromatic moiety linked to a carbohydrate (1–12). In the targets, the aromatic core consists of a
2-phenylbenzo[b]furan-3-yl, 2-phenylbenzo[b]thiophen-3-yl, 1-tosyl-2-phenylindol-3-yl, or
2-phenylindol-3-yl group that is bound to one of three aminosugars (daunosamine, acosamine, or
4-amino-2,3,4,6-tetradeoxy-a-L-hexopyranoside) via a propargyl linker. The DNA binding affinity of
these twelve compounds has been evaluated by using both direct and indirect fluorescence
measurements. Compared to Dauno and Dox, the DNA binding affinity of these analogues is weaker.
However, both aromatic and aminosugar motifs are critical to DNA binding, with more influence
coming from the structural features of the aromatic portion.
Introduction
Deoxyribonucleic acid, DNA, is the most fundamental building
block of life. Any error within the total DNA content of a cell
may affect its proper functioning, thus potentially leading to
cell death. By taking advantage of the uncontrolled proliferation
of cancer cells, drugs that bind to DNA and inhibit DNA-
processing enzymes have long been recognized as effective cancer
chemotherapeutic agents.1 There are two classes of DNA-binding
drugs: covalent and non-covalent. The latter group can be further
divided into two categories: intercalating agents and minor groove
binding agents.2
Anthracycline antibiotics are an important class of DNA-
intercalating drugs, and rank among the most effective anticancer
agents.3 Among the most well-known anthracycline antibiotics are
the naturally occurring compounds daunorubicin (Dauno) and
doxorubicin (Dox) (Chart 1).4,5 Following the discovery of the
Chart 1 Structures of daunorubicin (Dauno) and doxorubicin (Dox).
The anthraquinone domain is composed of rings B, C, and D.
pharmaceutical potential of Dauno and Dox, much research has
been directed towards identifying analogues with better clinical
efficacy. To date, hundreds, if not thousands, of analogues of these
two natural products have been synthesized.6 However, only a few
have been approved for cancer treatment: idarubicin, valrubicin,
epirubicin, pirarubicin, and aclarubicin.7
The mechanism by which the anthracylines act has been sug-
gested to be concentration dependent.8 At the typical concentra-
tions present after the administration of these compounds, DNA
binding is considered to be a key event that triggers cell death.8
Crystal structures of daunorubicin with DNA have established
that these species bind to DNA through intercalation of the
anthraquinone moiety between adjacent nucleoside base pairs,
while the carbohydrate residue and the saturated cyclohexane ring
(ring A) occupy the minor groove.9
Although anthracycline antibiotics are widely used in can-
cer therapy, two major problems exist: drug resistance,10 and
irreversible cardiotoxicity.11 Although drug resistance could be
alleviated by the identification of new anthracycline analogues,6
the cardiotoxicity, which is associated with the anthraquinone
aAlberta Ingenuity Centre for Carbohydrate Science and Department of
Chemistry, The University of Alberta, Gunning-Lemieux Chemistry Centre,
Edmonton, AB T6G 2G2, Canada. E-mail: tlowary@ualberta.ca
bDepartment of Chemistry, The Ohio State University, 100 West 18th Avenue,
Columbus, OH 43210, USA
† Electronic supplementary information (ESI) available: Synthesis of
compounds 15–16, 18–27, 29, 30, 33–37, 40 and 42–47, 1H and 13C NMR
spectra for new compounds or known compounds synthesized by a new
approach (1–12, 16, 20, 21, 24–27, 29, 30, 33–37, 40 and 42–47), typical
HPLC conditions for the purity analysis (examples of 1 and 5), and one
sample figure from the FID(EtBr) asays of compounds 1–4. See DOI:
10.1039/b909153j
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Chart 2 Generation of reactive oxygen species from daunorubicin.
core, remains a significant problem. The mechanism underlying
anthracycline-induced cardiotoxicity is complex, but it is believed
that the major cause is the single-electron reduction of ring C
(Chart 2) leading to the generation of reactive oxygen species
that damage the heart.8,12 There is no specific treatment for
anthracycline-related cardiotoxicity. Thus, new strategies that
afford a good therapeutic response with minimal cardiotoxicity
are of interest.8
The propargyl group was selected as the linker because the
hydroxyl group and the terminal alkyne can be used to connect
carbohydrates and intercalators in a straightforward manner by
glycosylation and Sonogashira coupling, respectively. In addi-
tion, the alkyne can be further modified to provide additional
analogs through, for example, hydrogenation to an alkane or
the formation of triazoles through an azide–alkyne [2 + 3]
cycloaddition.18
Among the strategies used for preparing synthetic anthracycline
analogs, a diversity-oriented synthesis approach is popular.6 In this
approach a limited number of functional groups at certain loci
are modified without changing the basic structural framework of
the drug. An alternate strategy, function-oriented synthesis,13 is
based on the preparation of analogs in which the function of each
structural moiety in a given molecule are mimicked. In targeting
analogs of Dox and Duano for synthesis, we employed this second
approach. Based on available crystal structures of anthracycline–
DNA complexes, we designed compounds including two structural
components: a DNA intercalating moiety and a minor-groove
binding moiety.
As a starting point, we chose to incorporate aminosugars
found in the clinically used anthracycline drugs (daunosamine
in daunorubicin and doxorubicin; acosamine in epirubicin) as the
minor-groove binding carbohydrate domain (1–4, and 5–8, respec-
tively, Chart 3). In addition, because 4-amino-2,3,4,6-tetradeoxy-
a-L-hexopyranosides are found in anthracycline analogues that
exhibit unique biological properties,19 we selected 4-amino-2,3,4,6-
tetradeoxy-a-L-threo-hexopyranoside (4-N-TDTH, 9–12) as the
third carbohydrate moiety.
Results and discussion
Design of synthetic route
Because of the vast number of carbohydrate-modified anthra-
cyclines that have been synthesized,6 our efforts were focused
on novel aglycones. Thus, we chose to replace the tetracyclic
anthracycline core with relatively simple intercalating moieties.
This design feature has two advantages. First, these aromatic struc-
tures are more accessible than complex anthracycline aglycones.14
Moreover, avoiding cardiotoxicity with compounds containing an
anthraquinone or anthraquinone-like portion would be unlikely.8
It should be noted that analogs of Dauno and Dox containing
a truncated anthraquinone core have been prepared;12,15 however,
the cytotoxicity of these simplified analogues is lower than the
clinically-used drugs.12
We envisioned two strategies that could be used to synthesize
targets 1–12. The first (Scheme 1, route A) would involve the
initial coupling of propargyl alcohol with iodinated aromatic
moieties via Sonogashira coupling and subsequent reaction with
an appropriate glycosyl donor. The second strategy (Scheme 1,
route B) would be to glycosylate propargyl alcohol first, followed
by Sonogashira coupling of the terminal alkyne in the product
with iodinated precursors of aromatic intercalators. Both of these
routes were investigated, but route B is the preferred method
because of its higher degree of convergency (see below).
Earlier studies by Denny, et al. showed that aromatic molecules
such as 2-phenylquinolines possess antitumor activity due to their
ability to bind to DNA via “minimal intercalation.”16 It was
hypothesized that the use of minimal intercalators would increase
the effective drug concentration at a remote tumor site because
the loss of the drug due to strong DNA binding during delivery
is decreased.16 More recent work has shown that conjugation
of a carbohydrate moiety to simple intercalators via a linker
functionality improves the binding affinity of these compounds
for DNA.17 Mindful of these considerations, we envisioned
conjugating carbohydrates to a flat, aromatic intercalating system
through a simple linker (Chart 3).
Synthesis of iodinated aromatic moieties
Starting from 2-iodophenol and 2-(methylthio)aniline, 3-iodo-
2-phenyl[b]benzofuran (13)20 and 3-iodo-2-phenyl[b]benzothio-
phene (14)21 (Scheme 1), were synthesized in high yields based on
literature-reported synthetic methodologies. The third required
aromatic precursor was 3-iodo-2-phenylindole (16), which has
been previously synthesized from 2-phenylindole upon treatment
with iodine and potassium hydroxide.22 In our hands, although 16
could be detected in the reaction mixture by TLC, following col-
umn chromatography only starting material (2-phenylindole) was
recovered. The same result was obtained using N-iodosuccinimide
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Chart 3 Designed intercalator and carbohydrate conjugate system.
phenylacetylene was followed by tosylation of the nitrogen to
give 19 in 91% yield over the two steps.23 This compound was
cyclized upon treatment with iodine and potassium carbonate in
acetonitrile to afford the protected indole derivative 15.24 Removal
of tosylate protecting group was achieved in 86% yield by heating
a solution of 16 at 60–70 ◦C in THF in the presence of tetra-
n-butylammonium fluoride. In the absence of iodine or NIS,
compound 16 can be purified by column chromatography and
is sufficiently stable for storage.
Synthesis of aromatic intercalator control compounds
To identify the contribution made by each structural component
(the aromatic intercalating moiety, carbohydrate, and the propar-
gyl linker), it was necessary to synthesize the appropriate control
compounds consisting solely of these structural domains. Thus,
compounds containing only the intercalating moiety, or those that
contain both intercalating moieties and the propargyl linker, were
synthesized (20–27, Chart 4).
Scheme 1 Retrosynthetic analysis of 1–12.
(NIS). Thus, another route to 16 was investigated (Scheme 2).
Starting from 2-iodoaniline (17), Sonogashira coupling with
Chart 4 Structures of control compounds containing aromatic domains.
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Scheme 2 Reagents and conditions: (a) Phenylacetylene, Pd(PPh3)2Cl2, CuI, Et3N, 95%; (b) TsCl, Pyridine, THF, 96%; (c) I2, K2CO3, CH3CN, 98%;
(d) n-Bu4NF, THF, 60–70 ◦C, 86%.
The preparation of 20, 21, 24 and 25 is shown in Scheme 3.
2-Phenyl[b]benzofuran (20) was synthesized from 3-iodo-2-
phenyl[b]benzofuran (13) in 86% yield by lithium–halogen ex-
change, followed by protonation with ammonium chloride.25,26
Similar treatment of the corresponding benzothiophene derivative
14, gave 21 in a comparable (85%) yield. Compounds 24 and
25 were synthesized from 13 and 14, respectively, in high yields
using standard Sonogashira coupling with propargyl alcohol
(Scheme 3).
Use of the same general approaches for preparing 22, 23, 26 and
27, led to problems (Scheme 4). For example, treatment of 15 with
n-butyllithium yielded none of the desired 1-tosyl-2-phenylindole
(22). Instead, the major product, isolated in 74% yield, was 2-
phenylindole (23); a minor product was alkyne 19, which was
produced in 20% yield. The latter compound is undoubtedly
formed by b-elimination of the organolithium species derived
from 15, while the strongly basic conditions of the reaction
appear to promote the deprotection of the tosyl group, thus
leading to 23. Therefore, while this approach gave, unexpectedly,
one the target compounds (23), an alternative approach was
needed for access to 1-tosyl-2-phenylindole, 22. Fortunately, it
was possible to synthesize 22 in modest 52% yield by heating a 1,2-
dichloroethane solution of 19 at reflux with 20 mol% copper (II)
triflate.23b
Difficulties were also encountered in the coupling of 15 to
propargyl alcohol. The conditions used in the preparation of 24
and 25 resulted in no reaction; only unreacted 15 was isolated.
However, when the reaction mixture was heated at 100 ◦C in a
microwave reactor, the desired product 26 was formed, albeit in
modest (52%) yield. Also unexpected was that attempted coupling
of 3-iodo-2-phenylindole (16) with propargyl alcohol afforded
none of 27, but instead 2-phenylindole (23) was isolated as the only
product, in 77% yield. Protection of indole nitrogen thus appears
necessary to favor Sonogashira coupling over protodemetallation
of the organopalladium species derived from 16. Compound 27
could, however, be synthesized in 45% yield from 26 by treatment
with tetra-n-butylammonium fluoride as described above for the
synthesis of 16 from 15 (Scheme 2). It was also found that
compound 27 slowly decomposed during column purification,
even using base-deactivated silica gel at the adsorbent.
Scheme 3 Reagents and conditions: (a) 1. n-BuLi, THF, -78 ◦C; 2. aq. NH4Cl, 86% for 20, 85% for 21; (b) Propargyl alcohol, Pd(PPh3)2Cl2, CuI, Et3N,
73% for 24, 81% for 25.
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Scheme 4 Reagents and conditions: (a) 1. n-BuLi, THF, -78 ◦C; 2. aq. NH4Cl, 20% for 19, 74% for 23; (b) Propargyl alcohol, Pd(PPh3)2Cl2, CuI,
piperidine, 100 ◦C (microwave), 2 h, 52%; (c) Cu(OTf)2, 1,2-dichloroethane, reflux, 52%; (d) n-Bu4NF, THF, 60–70 ◦C, 45%; (e) Propargyl alcohol,
Pd(PPh3)2Cl2, CuI, piperidine, 77% for 23.
Synthesis of propargyl daunosamine and acosamine glycosides
by column chromatography in 55% and 5% yield, respectively.
However, the two b stereoisomers, 31 and 32, were inseparable
and were obtained as a mixture in 24% yield. Assignment of the
stereochemistry at C-1 and C-3 in 29–32 was straightforward based
on the multiplicity of the resonances arising from H-1 and H-3.
For both a-anomers 29 and 30, no large coupling was seen for the
resonance of the equatorial H-1 with 3J1,2a ~ 3–4 Hz and 3J1,2e ~ 1–
2 Hz (a = axial, e = equatorial). For the axially disposed H-3 in 29,
its resonance in the 1H NMR spectrum appeared as a doublet of
Having a route in place to the aromatic domains, we turned our
attention to the preparation of the carbohydrate moieties. The
required building blocks, 36 and 37, were synthesized from a
previously reported precursor, 28 (Scheme 5).27 Compound 28,
prepared as a mixture of four stereoisomers, was converted to
propargyl glycosides 29–32 in 84% combined yield. The two a
stereoisomers, 29 and 30, could be obtained as pure compounds
∑
Scheme 5 Reagents and conditions: (a) Propargyl alcohol, BF3 Et2O, 84% for 29–32; (b) K2CO3, methanol, 99% for 33, 94% for 35; (c) PPh3, H2O, THF,
60 ◦C, 97% for 37, 89% for 36; (d) 1. Tf2O, pyridine; 2. n-Bu4NOAc, CH3CN, 86%.
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doublet of doublets with 3J2a,3 ~ 12.4 Hz, 3J3,4 ~ 9.8 Hz, and 3J2e,3
~ 5.0 Hz. On the other hand, the resonance of the equatorially
oriented H-3 in 30 appeared as an apparent quartet because the
coupling constants for J3,4, J2a,3, and J2e,3 are almost identical
(~ 3.6 Hz) (see ESI† for details). Although the synthesis of
the desired product, 29, via this route is not high yielding, the
preparation of 28 is simple and efficient, and the conversion of 28
to 29 proceeds in reasonable yield.
Glycoside 29 could then be efficiently converted into 33
upon reaction with K2CO3 in methanol. The reduction of the
azide group in 33 using triphenylphosphine–water afforded the
propargyl acosaminoside, 37, in 96% overall yield from 29. The
daunosamine isomer of 37, glycoside 36, was obtained from 33
by inversion of the stereochemistry at C-4 through formation of
the triflate ester, and displacement with acetate. Deacetylation,
followed by the reduction of the azide under the conditions
used for the synthesis of 37, afforded the propargyl glycoside
of daunosamine, 36. Inversion of stereochemistry at C-4 in 33
could also be accomplished via a two-step approach involving
triflate ester formation and reaction with sodium nitrite or tetra-
n-butylammonium nitrite.28 However, the three-step process in
Scheme 5 gave a better yield (81%) than the two-step process
(45%).
With 40 in hand, the deprotection of the acetyl group with
potassium carbonate in methanol afforded 42 in 91% yield.
Introduction of the azido group with inverted stereochemistry was
achieved by mesylate ester formation followed by displacement
with sodium azide, affording compound 43 in 76% overall yield.
Subsequently, the glycosylation reaction was performed before the
reduction of the azido group, which facilitates anomer separation.
Glycoside 44a was then subjected to a Staudinger reaction, which
afforded the desired product 45 in 85% yield.
3
3
3
Synthesis of amino analogues 1–8: Coupling of propargyl
daunosamine and acosamine glycosides with iodinated aromatics
As mentioned previously, we envisioned two possible synthetic
routes for the preparation of final compounds 1–8 (Scheme 1).
However, there are two drawbacks associated with route A. First,
because 28 is a mixture of four inseparable stereoisomers, it cannot
be used as a donor directly, unless the stereoisomeric products
would be separable after coupling. Second, the use of route A
would require the synthesis of phenylindole derivatives 26 and 27,
which, as outlined in Scheme 4, was difficult. Therefore route B
was adopted for the synthesis of 1–8.
As shown in Scheme 7, coupling of benzofuran 13 with the
terminal alkynes proceeds efficiently, requiring only 5 mol%
of palladium catalyst. Slightly increased amounts of catalyst
(7 mol%) were needed to achieve a comparable transformation for
benzothiophene 14. Because the reaction time for benzothiophene
14 was longer than that for benzofuran 13, the load of copper(I)
co-catalyst was decreased to avoid homocoupling of the alkyne,
a common by-product in Sonogashira couplings.33 The reaction
was most difficult with indole 15, which required 10 mol% of the
catalyst and less copper(I) iodide, compared to the reaction with
13. The use of the amine solvents most commonly employed in
Sonagashira couplings (e.g., triethylamine) led to poor product
yields. After screening a number of solvents, piperidine was found
to give the best results, affording the products in 61–74% yields.
Although N-tosylated indoles 3 and 7 were not initially among
the desired structures, because they were easily available along the
synthetic pathway, their DNA-binding abilities were evaluated.
Finally, the protected indoles 4 and 8 were synthesized in 89
and 91% yield, respectively, by reaction of 3 and 7 with tetra-
n-butylammonium fluoride.
Synthesis of 2-propargyl 4-amino-2,3,4,6-tetradeoxy-a-
L-threo-hexopyranoside (4-N-TDTH)
The preparation of the propargyl 4-N-TDTH glycoside started
from glycal 38 (Scheme 6). A Ferrier I-rearrangement29 of this
alkene in the presence of SnCl4 and methanol afforded a 90%
yield of compound 39 as a mixture of a and b glycosides in an
approximately 9:1 ratio.30 The reduction of the alkene 39 to alkane
40 was surprisingly difficult. When 5% palladium on charcoal
was used as the hydrogenation catalyst, we observed a significant
amount of byproduct 41 in which the anomeric methoxy group
was replaced with a hydrogen atom. Although the formation of
this byproduct could be avoided by using Wilkinson’s catalyst31 or
using diimide reagents32 as means of hydrogenation, the reaction
rate was significantly slower (3–5 days). Finally, it was found that
by using ethyl acetate as a solvent, 10% palladium on charcoal
gave the highest yield of 40, with a slight contamination of the
byproduct 41.
Scheme 6 Reagents and conditions: (a) SnCl4, MeOH, 81% for 39a and 9% for39b; (b) H2, 10% Pd/C, ethyl acetate, 90%; (c) K2CO3, methanol, 91%;
∑
(d) 1). MsCl, Et3N, 0 ◦C; 2) NaN3, DMF, 110 ◦C, 76% over two steps; (e) Propargyl alcohol, BF3 Et2O, -40 ◦C, 69% for 44a and 20% for 44b; (e) PPh3,
H2O, THF, 60 ◦C, 85%.
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Scheme 7 Reagents and conditions: (a) 13, 5% Pd(PPh3)2Cl2, 10% CuI, 74% for 1, 77% for 5; (b) 14, 7% Pd(PPh3)2Cl2, 5% CuI, 67% for 2, 71% for 6;
(c) 15, 10% Pd(PPh3)2Cl2, 5% CuI, 61% for 3, 74% for 7; (d) n-Bu4NF, 89% for 4, 94% for 8.
Synthesis of amino analogues 9–12
however, we were unable to couple the propargyl amino glycoside
45 with the tosyl protected indole iodide 15 with acceptable
efficiency. The yield of 11 was less than 5% under the optimal
conditions used for the synthesis of 3 and 7. The application
of microwave heating slightly improve the yield to around 15–
20%, but the decomposition of the catalyst under these conditions
impeded further improvement.
Unlike 28, which has four stereoisomers, compound 40 could
be obtained in the isomeric form at the anomeric position only
(Scheme 6). Therefore, we carried out trial reactions for coupling
24 and 25 with methyl glycoside 40 under the catalysis of several
different Lewis acids. Boron trifluoride diethyl etherate gave the
best results (Scheme 8); however, the yields were generally low, less
than 50%.
Therefore, the preparation of 9–12 adopted the same route
as for the synthesis of 1–8, in which propargyl glycosides were
synthesized first, followed by Sonogashira coupling with aromatic
iodides (Scheme 9). This reaction worked well with 13 and 14;
Fluorescence DNA binding studies
With sufficient quantities of 1–12 available, their DNA binding
ability was examined. Due to the significance of DNA binding
in drug design, a number of approaches have been developed to
∑
Scheme 8 Reagents and conditions: (a) alcohol, BF3 Et2O,–40→0 ◦C, 49% for 46 and 43% for 47.
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Scheme 9 Reagents and conditions: (a) 13, 5% Pd(PPh3)2Cl2, 10% CuI, 85%; (b) 14, 7% Pd(PPh3)2Cl2, 5% CuI, 76%; (c) 15, 10% Pd(PPh3)2Cl2, 5% CuI,
100 ◦C (microwave), 2 h, 17%; (d) n-Bu4NF, 75%.
probe the interactions between DNA and small organic ligands,
including, calorimetry,34 NMR spectroscopy,35 viscometry,36 and
fluorescence.37 Due to the relative low water solubility of our
analogues, we were interested in sensitive assays that could mea-
sure DNA binding affinity at concentrations in the micromolar
range. Therefore, fluorescence measurements were deemed most
appropriate.
The change in fluorescence of a compound upon binding to
DNA can be achieved in two different ways: direct and indirect.
The direct assay measures the changes in the fluorescence of the
ligand of interest after the addition of that ligand into a DNA
solution.36a,37a–d The indirect method, the fluorescent intercalator
displacement (FID) assay, involves a second fluorescent molecule
that is a standard DNA intercalator such as ethidium bromide
(EtBr).37d–f Instead of monitoring the fluorescence of the ligand
being tested, the fluorescence of the standard intercalator is
monitored before and after the addition of the ligand.
believe that fluorescence quenching is most likely due to electron
transfer between guanine nucleotides and the photoactivated
state of compounds 1–12. The changes in the emission curves
illustrated in Figure 1 demonstrated the ability of 1–12 to bind
to DNA, but no more information could be obtained, given the
different flourophores in the compounds. To evaluate the DNA
binding affinity between different compounds, the FID assay was
used.
Fluorescent intercalator displacement (FID) assay
Although the single point direct titration cannot definitively tell
the differences of the binding affinity of different ligands, the
single point FID titration is a well-accepted method for ranking
DNA binding affinity of different ligands using EtBr as a ‘ruler’.39
Multipoint titration, on the other hand, is extensively used to semi-
quantitate DNA binding affinity by calculating relative associate
constants.40
From single point FID titration, the percentage of remaining
EtBr could be calculated and was used to determine the relative
affinity of 1–12 for DNA (Table 1). Based on the data, we can
rank the binding affinity of 1–4 as 3 > 1 > 2 > 4. When obtaining
an overall ranking for 1–12, some interesting trends emerged.
The ranking appears to be determined mainly by the aromatic
moiety, and the affinity decreases in the order of N-tosyl protected
indole > benzo[b]furan > benzo[b]thiophene ≥ free indole. The
carbohydrate moiety, on the other hand, has less influence on the
binding affinity. It is of interest to note that although there is a
bulky substituent present in the N-tosyl protected indole core, it
demonstrated the strongest binding. We propose that results from
the positioning of the N-tosyl group in the minor or major groove,
therefore providing additional interactions with the groove walls.
To elucidate possible contribution from each structural moi-
ety (intercalator, minor groove binder, and the linker) to the
overall binding affinity of the molecule, we assayed compounds
Direct fluorescence measurements
A one point direct fluorescence titration is an efficient way to detect
the binding of a fluorescent ligand to DNA.36a,37a,b Figure 1 shows
the change of the emission curves of compounds 1–4 at 50 mM
upon the addition of sonicated herring sperm DNA (hsDNA) at
20 mg/mL. After the addition of DNA, a fluorescence quenching
effect of different amplitudes was observed for all four compounds
(Figure 1–B). Similar results were also obtained for compounds
5–12.
Although it is well known that for EtBr, the intensity of its
fluorescence increases upon the addition of DNA,37a fluorescence
quenching is also common due to either spectral overlap with
other fluorophores in the system being tested, or electron transfer
from the activated ligand to guanine nucleotides.38 Due to the
lack of spectral overlap between the excited ligand and DNA
nucleotides, the only other fluorophores in our system, we
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Table 1 Percentage of remaining EtBr in FID assays of 1–12, 20–26, 36,
37, and 45
Compound
Structural domains
% Remaining EtBr
1
62.0 1.9
67.5 2.1
47.9 2.7
76.7 0.7
50.5 4.1
73.6 1.1
49.7 4.9
76.5 2.0
68.5 1.9
79.9 1.0
38.6 1.9
78.4 1.9
2
3
4
5
6
Intercalator-linker-carbohydrate
7
8
9
10
11
12
20
21
22
23
98.6 3.3
96.5 4.0
100.5 2.5
96.0 2.2
Intercalator
24
25
26
107.9 0.6
95.0 2.7
110.7 1.3
Intercalator-linker
36
37
45
100.0 3.9
90.5 1.0
93.3 1.6
Carbohydrate-linker
affinity of 11 is approximately 15-fold less than EtBr. Because
Dauno and Dox have similar DNA binding affinity compared
to EtBr,42 the DNA binding affinity of our analogues is ~15-
fold weaker compared to these two clinical anthracycline drugs, a
respectable binding affinity given the simplicity of our systems.
Conclusions
Fig. 1 Emission spectra of 1–4 in the absence and presence of DNA:
50 mM of 1 (black), 2 (blue), 3 (purple), and 4 (red) in the absence (bold
line) and presence (thin line) of 20 mg/mL of hsDNA showing (A) absolute
intensity and (B) normalized intensity based on the emission maxima from
A. Emission (y-axis) was measured as a function of emission wavelength
(x-axis) upon exciting each compound at its excitation maximum.
A panel of novel daunosamine and acosamine-containing gly-
coconjugates (1–12) have been synthesized. These compounds
were designed as functional mimics of natural anthracycline
antibiotics, and contain three functional subunits: a planar
aromatic system as the intercalating moiety, a carbohydrate as
the minor/major groove binding moiety, and a propargyl spacer
that links these two groups. The DNA binding affinity of all
compounds has been evaluated and confirmed using direct and
indirect fluorescent assays. Although the aromatic moiety has
been identified as the key structural feature affecting the DNA
binding affinity, an aminosugar appendage is also indispensible,
but structural changes in this group appear to have a weak effect on
binding. Further studies of the binding mode and more biological
evaluation are currently in progress.
20–23 (containing only the aromatic intercalating moiety), 24–26
(composed of the intercalating moiety and the propargyl linker),
and 36, 37 and 45 (possessing the carbohydrate moiety and the
linker). None showed detectable binding. Thus, there is necessarily
a cooperative effect between these three components to achieve the
observed binding affinity. Based on the proposed binding model
from simulation studies for anthracycline drugs,41 it is possible
that the carbohydrate moiety introduces the molecule to DNA to
form a minor groove-bound state. After this, a binding equilibrium
is established by the interaction between DNA and the aromatic
moiety.
Experimental section
By multipoint titration, the relative binding constant for a
ligand can be calculated based on the equation described in the
experimental section.40 Based on the data provided in Table 1, the
DNA binding affinity of 3 and 7 is ~20-fold less than EtBr. For
compound 11, which exhibited the highest relative DNA binding
affinity, an FID titration was carried out (data not shown). After
linear fitting, the concentration of 11 at which there is 50% loss
of EtBr fluorescence is about 75 mM; therefore, the DNA binding
Chemistry
Reactions were carried out in oven dried glassware. All reagents
were purchased from commercial sources and were used without
further purification unless noted. Before use, reaction solvents
were purified by successive passage through columns of alumina
and copper in a PURESOLV-400 System from Innovative Tech-
nology Inc. under argon atmosphere. Unless stated otherwise,
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The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 3709–3722 | 3717
©

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all reactions were carried out under a positive pressure of argon
and were monitored by TLC on silica gel G-25 UV254 (0.25 mm,
Macherey-Nagel). Spots were detected under UV light and/or
by charring with 10% H2SO4 in ethanol, or in acidified ethanolic
anisaldehyde/vanillin. Solvents were evaporated under reduced
pressure and below 40 ◦C (water bath). Column chromatography
was performed on silica gel 60 (40–60 mM). The ratio between silica
gel and crude product ranged from 100 to 50:1 (w/w). Iatrobeads
refers to a beaded silica gel 6RS-8060, which is manufactured by
Iatron◦Laboratories (Tokyo). Optical rotations were measured at
22 2 C. Melting points are uncorrected. 1H NMR spectra were
recorded on VARIAN INOVA-NMR spectrometers at 400, 500
or 600 MHz, and chemical shifts are referenced to either TMS
(0.0, CDCl3) or CD2HOD (4.78, CD3OD). 13C NMR spectra were
recorded at 100 or 125 MHz, and 13C chemical shifts are referenced
to CDCl3 (77.23 ppm, CD3Cl3) or CD3OD (49.00 ppm, CD3OD).
1H data are reported as though they were first order. The errors
between the coupling constants for two coupled protons were less
than 0.5 Hz, and the average number was reported. Assignment
propargyl glycoside 45 (22 mg, 0.13 mmol) was added CuI (1.5 mg,
10 mol%). The mixture was stirred at 100 ◦C in a microwave reactor
for 2 h, the reaction was then quenched by the addition of a satd
aqueous NH4Cl solution, and the resulting solution was extracted
with EtOAc. The organic fractions were dried (Na2SO4), filtered,
and concentrated under vacuum to yield the crude product, which
was purified by column chromatography.
General procedure for the preparation of compounds 4, 8, and 12
To a solution of N-tosyl protected indole 3, 7, or 11 (31 mg,
0.058 mmol) in THF (3 mL) was added tetra-n-butylammonium
fluoride (TBAF) solution in THF (1.0 M, 0.35 mL, 0.35 mmol)
at room temperature, and the mixture was stirred for 24 h. A satd
aqueous solution of NaHCO3 (30 mL) was then added and the
mixture was extracted with CH2Cl2. The organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated to a
residue that was purified by column chromatography.
3-(2-Phenyl-benzo[b]furan-3-yl)-prop-2-ynyl 3-amino- 2,3,6-
1
of NMR resonances was done based on H–1H COSY, HMQC,
trideoxy-a-L-lyxo-hexopyranoside (1). Pure
1 was obtained
and in some cases HMBC experiments. In the interpretation of
the NMR data for methylene protons on the carbohydrate ring,
‘a’ and ‘e’ refer to axial and equatorial orientation, respectively.
In the cases where no clear assignment of these hydrogens could
be made based on all NMR data, assignments were made taking
into consideration the anisotropy effect of a ring s bond, which
results in equatorial hydrogens resonating more downfield than
axial hydrogens.43 Electrospray mass spectra were recorded on
samples suspended in mixtures of THF with CH3OH and added
NaCl. Optical rotations were measured on a Perkin-Elmer 241
Polarimeter at the sodium D line (589 nm). Optical rotations
are in units of deg mL(dm·g)-1. IR spectra were recorded on
the Nicolet Magna 750 FTIR spectrometer. The reported purity
values were obtained with a Varian HPLC system, using an
evaporative light scattering detector (ELSD) 2000ES from Alltech,
and a Varian Microsorb-MV 100–5 C18 column. The eluant
consisted of acetonitrile and water, the ratio of which depends
on the compound. For all basic amino compounds, 0.1% of
trifluoroacetic acid was added to facilitate elution and avoid
aggregation. When the purity derived from HPLC analysis is
greater than 99%, it is reported as > 99%.
by column chromatography on Iatrobeads (1% Et3N in 7:1
CH2Cl2–CH3OH) in 74% yield: white foamy solid, Rf 0.39
(1% Et3N in 7:1 CH2Cl2–CH3OH); IR n 3319 (N–H), 3048
-1
23
(O–H), 2010 (C C) cm ; [a] D)–98.9 (c 0.3, CH3OH); 1H NMR
(400 MHz, CD3OD, dH) 8.22–8.26 (m, 2H, Ar), 7.60–7.63 (m, 1H,
Ar), 7.48–7.56 (m, 3H, Ar), 7.41–7.45 (m, 1H, Ar), 7.35–7.39 (m,
1H, Ar), 7.29–7.33 (m, 1H, Ar), 5.27 (br d, 1H, J1,2a = 3.3 Hz,
≡
≡
H-1), 4.65 (s, 2H, OCH2C C), 4.05 (br q, 1H, J5,6 = 6.5 Hz,
¯
H-5), 3.63–3.69 (m, 2H, H-3, H-4), 2.08 (ddd, 1H, J2a,2e = J2a,3
=
12.7 Hz, J1,2a = 3.3 Hz, H-2a), 1.91 (ddd, 1H, J2a,2e = 12.7 Hz,
J2e,3 = 4.5 Hz, J1,2e = 1.0 Hz, H-2e), 1.22 (d, 3H, J5,6 = 6.5 Hz,
H-6); 13C NMR (100 MHz, CD3OD, dC) 158.0 (Ar), 154.8 (Ar),
131.1 (Ar), 130.9 (Ar), 130.6 (Ar), 129.9 (2, Ar), 126.9 (2, Ar),
126.8 (Ar), 124.8 (Ar), 121.0 (Ar), 112.2 (Ar), 99.4 (Ar), 96.6
≡
≡
(C-1), 94.0 ( C), 78.5 ( C), 68.0 (C-4), 67.8 (C-5), 56.2 (OCH2),
48.6 (C-3), 29.4 (C-2), 16.9 (C-6). HRMS (ESI) calcd for (M + H)
C23H24NO4: 378.1700. Found: 378.1702. Purity: >99%.
3-(2-Phenyl-benzo[b]thiophene-3-yl)-prop-2-ynyl 3-amino-2,3,6-
trideoxy-a-L-lyxo-hexopyranoside (2). Pure 2 was obtained by
column chromatography (1% Et3N in 9:1 CH2Cl2–CH3OH) in
67% yield: solid yellow paste, Rf 0.43 (1% Et3N in 7:1 CH2Cl2–
-1
≡
CH3OH); IR n 3361 (N–H), 3013 (O–H), 2010 (C C) cm ;
General procedure for the preparation of compounds 1–3, 5–7 and
9–10 via Sonogashira coupling
[a]23D)–86.8 (c 0.3, CH3OH); H NMR (400 MHz, CD3OD, dH)
1
7.94–7.97 (m, 2H, Ar), 7.84–7.89 (m, 2H, Ar), 7.38–7.50 (m, 5H,
To a degassed solution of sugar alkyne (1.3 mmol), iodi-
nated aromatic compound (1.0 mmol), and PdCl2(PPh3)2 (see
Scheme 2.8/2.9 for amount) in piperidine (6 mL) was added
CuI (see Scheme 2.8/2.9 for amount). The reaction mixture was
stirred under Ar at room temperature and followed by TLC. Once
complete, the reaction was quenched by the addition of a satd
aqueous solution of NH4Cl (10 mL). The aqueous solution was
extracted with Et2O (3 ¥ 10 mL) and the combined organic
layers were washed with brine, dried (Na2SO4), filtered, and
concentrated to yield the crude product, which was purified by
column chromatography.
Ar), 5.21 (br d, 1H, J1,2a = 3.7 Hz, H-1), 4.60 (ABq, 1H, J =
≡
≡
16.2 Hz, OCH2C C), 4.58 (ABq, 1H, J = 16.2 Hz, OCH2C C),
¯
¯
3.98 (br q, 1H, J5,6 = 6.6 Hz, H-5), 3.62–3.68 (m, 2H, H-3, H-4),
2.05 (ddd, 1H, J2a,2e = J2a,3 = 12.7 Hz, J1,2a = 3.7 Hz, H-2a), 1.76
(br dd, 1H, J2a,2e = 12.7 Hz, J2e,3 = 4.6 Hz, H-2e), 1.18 (d, 3H, J5,6
=
6.6 Hz, H-6); 13C NMR (100 MHz, CD3OD, dC) 147.9 (Ar), 142.3
(Ar), 138.8 (Ar), 134.9 (Ar), 130.1 (Ar), 129.8 (2, Ar), 129.4 (2,
Ar), 126.6 (Ar), 126.2 (Ar), 124.0 (Ar), 123.2 (Ar), 114.1 (Ar), 97.5
≡
≡
(C-1), 92.0 ( C), 81.0 ( C), 72.1 (C-5), 68.6 (C-4), 55.7 (OCH2),
47.7 (C-3), 33.6 (C-2), 17.3 (C-6). HRMS (ESI) calcd for (M + H)
C23H24NO3S: 394.1471. Found: 394.1470. Purity: > 99%.
Microwave procedure for the preparation of compound 11
3-(1-Tosyl-2-phenylindol-3-yl)-prop-2-ynyl 3-amino- 2,3,6-
trideoxy-a-L-lyxo-hexopyranoside (3). Pure 3 was obtained by
column chromatography (1% Et3N in 10:1 CH2Cl2–CH3OH)
To a solution of piperidine (0.75 mL), DMF (0.25 mL),
PdCl2(PPh3)2 (11 mg, 20 mol%), 15 (38 mg, 0.079 mmol), and
3718 | Org. Biomol. Chem., 2009, 7, 3709–3722
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The Royal Society of Chemistry 2009
©

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in 61% yield: solid off-white paste, Rf 0.45 (1% Et3N in 7:1
3-(2-Phenyl-benzo[b]thiophene-3-yl)-prop-2-ynyl 3-amino-2,3,6-
trideoxy-a-L-arabino-hexopyranoside (6). Pure 6 was obtained by
column chromatography on Iatrobeads (7:1 CH2Cl2–CH3OH) in
77% yield: yellow oil, Rf 0.27 (7:1 CH2Cl2–CH3OH); IR: n 3311
-1
≡
CH2Cl2–CH3OH); IR: n 3358 (br, N–H, O–H), 2221 (C C) cm ;
1
[a]23D)–73.9 (c 0.2, CH3OH); H NMR (400 MHz, CD3OD, dH)
8.22–8.26 (m, 1H, Ar), 7.38–7.52 (m, 7H, Ar), 7.29–7.33 (m, 1H,
Ar), 7.20–7.24 (m, 2H, Ar), 7.08–7.12 (m, 2H, Ar), 4.97 (br d, 1H,
-1
23
≡
(N–H), 3265 (N–H), 3056 (O–H), 2218 (C C) cm ; [a] D)–88.3
(c 0.2, CH3OH); 1H NMR (400 MHz, CD3OD, dH) 7.88–7.93 (m,
2H, Ar), 7.82–7.85 (m, 1H, Ar), 7.76–7.80 (m, 1H, Ar), 7.32–7.45
(m, 5H, Ar), 5.09 (br d, 1H, J1,2a = 3.6 Hz, H-1), 4.52 (s, 2H,
≡
J1,2a = 3.7 Hz, H-1), 4.35 (ABq, 1H, J = 16.2 Hz, OCH2C C),
¯
≡
4.32 (ABq, 1H, J = 16.2 Hz, OCH2C C), 3.82 (br q, 1H, J5,6
=
¯
6.6 Hz, H-5), 3.61 (br s, 1H, H-4), 3.53 (ddd, 1H, J2a,3 = 12.8 Hz,
J2e,3 = 4.7 Hz, J3,4 = 2.9 Hz, H-3), 2.23 (s, 3H, PhCH3), 1.96
(ddd, 1H, J2a,2e = J2a,3 = 12.8 Hz, J1,2a = 3.7 Hz, H-2a), 1.76 (br
≡
OCH2C C), 3.67 (dq, 1H, J4,5 = 9.2 Hz, J5,6 = 6.2 Hz, H-5), 3.03–
¯
3.11 (m, 1H, H-3), 2.90 (dd, 1H, J3,4 = J4,5 = 9.2 Hz, H-4), 2.04
dd, 1H, J2a,2e = 12.8 Hz, J2e,3 = 4.7 Hz, H-2e), 1.18 (d, 3H, J5,6
=
(br dd, 1H, J2a,2e = 13.1 Hz, J2e,3 = 3.8 Hz, H-2e), 1.62 (ddd, 1H,
6.6 Hz, H-6); 13C NMR (100 MHz, CD3OD, dC) 147.0 (Ar), 145.3
(Ar), 138.3 (Ar), 135.6 (Ar), 132.3 (2, Ar), 132.0 (Ar), 131.9 (Ar),
130.6 (2, Ar), 130.3 (Ar), 128.6 (2, Ar), 127.9 (2, Ar), 127.0 (Ar),
126.1 (Ar), 120.9 (Ar), 117.6 (Ar), 108.6 (Ar), 96.2 (C-1), 91.7
J2a,2e = J2a,3 = 13.1 Hz, J1,2a = 3.6 Hz, H-2a), 1.23 (d, 3H, J5,6 =
6.2 Hz, H-6); 13C NMR (100 MHz, CD3OD, dC) 148.0 (Ar), 142.3
(Ar), 138.8 (Ar), 134.8 (Ar), 130.1 (Ar), 129.8 (2, Ar), 129.3 (2,
Ar), 126.6 (Ar), 126.2 (Ar), 124.0 (Ar), 123.2 (Ar), 114.0 (Ar), 96.6
≡
≡
≡
≡
( C), 78.7 ( C), 67.8 (C-4/C-5), 67.6 (C-5/C-4), 55.8 (OCH2),
48.6 (C-3), 29.3 (C-2), 21.6 (PhCH3), 16.9 (C-6). HRMS (ESI)
calcd for (M + H) C30H31N2O5S: 531.1948. Found: 531.1945.
Purity: > 99%.
(C-1), 91.8 ( C), 81.2 ( C), 78.4 (C-4), 70.1 (C-5), 55.6 (OCH2),
50.6 (C-3), 37.6 (C-2), 18.2 (C-6). HRMS (ESI) calcd for (M + H)
C23H24NO3S: 394.1471. Found: 394.1469. Purity: > 99%.
3-(1-Tosyl-2-phenylindol-3-yl)-prop-2-ynyl 3-amino- 2,3,6-
trideoxy-a-L-arabino-hexopyranoside (7). Pure 7 was obtained
by column chromatography on Iatrobeads (7:1 CH2Cl2–CH3OH)
in 77% yield: yellow oil, Rf 0.27 (7:1 CH2Cl2–CH3OH); IR: n
3-(2-Phenylindol-3-yl)-prop-2-ynyl 3-amino-2,3,6- trideoxy-a-L-
lyxo-hexopyranoside (4). Pure 4 was obtained by column chro-
matography on Iatrobeads (1% Et3N in 7:1 CH2Cl2–CH3OH) in
89% yield: white semi-solid, Rf 0.29 (1% Et3N in 6:1 CH2Cl2–
-1
23
≡
3128 (br, N–H, O–H), 2228 (C C) cm ; [a] D)–64.0 (c 0.1,
-1
23
1
≡
CH3OH); IR: n 3283 (br, N–H, O–H), 2217 (C C) cm ; [a] D)–
CH3OH); H NMR (400 MHz, CD3OD, dH) 8.24–8.26 (m, 1H,
1
104.1 (c 0.4, CH3OH); H NMR (400 MHz, CD3OD, dH) 8.06–
Ar), 7.39–7.52 (m, 7H, Ar), 7.31–7.34 (m, 1H, Ar), 7.23–7.25 (m,
8.09 (m, 2H, Ar), 7.58–7.61 (m, 1H, Ar), 7.44–7.49 (m, 2H,
Ar), 7.33–7.41 (m, 2H, Ar), 7.15–7.19 (m, 1H, Ar), 7.07–7.11
(m, 1H, Ar), 5.20 (br d, 1H, J1,2a = 2.9 Hz, H-1), 4.56 (ABq,
2H, Ar), 7.11–7.14 (m, 2H, Ar), 4.91 (br d, 1H, J1,2a = 3.6 Hz,
≡
H-1), 4.35 (s, 2H, OCH2C C), 3.60 (dq, 1H, J4,5 = 9.6 Hz,
¯
J5,6 = 6.3 Hz, H-5), 3.24–3.31 (m, 1H, H-3), 3.10 (dd, 1H, J3,4
J4,5 = 9.6 Hz, H-4), 2.27 (s, 3H, PhCH3), 2.07 (br dd, 1H, J2a,2e
12.8 Hz, J2e,3 = 3.7 Hz, H-2e), 1.76 (ddd, 1H, J2a,2e = J2a,3
=
=
=
≡
1H, J = 15.9 Hz, OCH2C C), 4.54 (ABq, 1H, J = 15.9 Hz,
¯
≡
OCH2C C), 3.98 (br q, 1H, J5,6 = 6.6 Hz, H-5), 3.45 (br s, 1H,
¯
H-4), 3.10–3.18 (m, 1H, H-3), 1.72–1.83 (m, 2H, H-2a, H-2e),
12.8 Hz, J1,2a = 3.6 Hz, H-2a), 1.18 (d, 3H, J5,6 = 6.3 Hz, H-6);
13C NMR (100 MHz, CD3OD, dC) 147.0 (Ar), 145.4 (Ar), 138.3
(Ar), 135.7 (Ar), 132.3 (2, Ar), 132.0 (Ar), 131.9 (Ar), 130.6 (2,
Ar), 130.3 (Ar), 128.5 (2, Ar), 127.9 (2, Ar), 127.0 (Ar), 126.0
1.19 (d, 3H, J5,6 = 6.6 Hz, H-6); 13C NMR (100 MHz, CD3OD,
dC) 141.4 (Ar), 137.3 (Ar), 133.3 (Ar), 131.7 (Ar), 129.7 (2, Ar),
129.2 (Ar), 127.7 (2, Ar), 124.0 (Ar), 121.4 (Ar), 120.3 (Ar), 112.4
≡
≡
≡
(Ar), 97.1 (C-1), 95.1 (Ar), 89.8 ( C), 82.2 ( C), 71.9 (C-4), 68.5
(C-5), 56.1 (OCH2), 47.8 (C-3), 33.4 (C-2), 17.3 (C-6). HRMS
(ESI) calcd for (M + H) C23H25N2O3: 377.1860. Found: 377.1859.
Purity: >99%.
(Ar), 120.9 (Ar), 117.6 (Ar), 108.6 (Ar), 95.6 (C-1), 91.4 ( C),
≡
78.8 ( C), 74.7 (C-4), 70.1 (C-5), 55.6 (OCH2), 49.6 (C-3), 35.2
(C-2), 21.5 (PhCH3), 17.9 (C-6). HRMS (ESI) calcd for (M + H)
C30H31N2O5S: 531.1948. Found: 531.1946. Purity: > 99%.
3-(2-Phenyl-benzo[b]furan-3-yl)-prop-2-ynyl-3-amino-2,3,6-
trideoxy-a-L-arabino-hexopyranoside (5). Pure 5 was obtained by
column chromatography on Iatrobeads (7:1 CH2Cl2–CH3OH) in
77% yield: colorless syrup, Rf 0.27 (7:1 CH2Cl2–CH3OH); IR n
3328 (N–H), 3276 (N–H), 3058(O–H), 2221.7(C C) cm ; [a] D)–
89.5 (c 2.4, CH2Cl2); 1H NMR (400 MHz, CD3OD, dH) 8.20–8.24
(m, 2H, Ar), 7.58–7.61 (m, 1H, Ar), 7.45–7.52 (m, 3H, Ar), 7.38–
7.42 (m, 1H, Ar), 7.32–7.36 (m, 1H, Ar), 7.27–7.31 (m, 1H, Ar),
3-(2-Phenylindol-3-yl)-prop-2-ynyl 3-amino-2,3,6-trideoxy-a-L-
arabino-hexopyranoside (8). Pure 8 was obtained by column
chromatography on Iatrobeads (7:1 CH2Cl2–CH3OH) in 94%
yield: yellow syrup, Rf 0.17 (5:1 CH2Cl2–CH3OH); n 3286 (br,
N–H, O–H), 2218 (C C) cm ; [a] D)–81.9 (c 0.3, CH3OH); H
NMR (400 MHz, CD3OD, dH) 8.08–8.10 (m, 2H, Ar), 7.58–7.61
(m, 1H, Ar), 7.45–7.48 (m, 2H, Ar), 7.38–7.41 (m, 1H, Ar), 7.34–
7.37 (m, 1H, Ar), 7.15–7.19 (m, 1H, Ar), 7.08–7.11 (m, 1H, Ar),
-1
23
-1
23
1
≡
≡
≡
≡
5.15 (br d, 1H, J1,2a = 3.6 Hz, H-1), 4.60 (s, 2H, OCH2C C), 3.71
5.17 (br d, 1H, J1,2a = 3.7 Hz, H-1), 4.56 (s, 2H, OCH2C C), 3.70
¯
¯
(dq, 1H, J4,5 = 9.3 Hz, J5,6 = 6.3 Hz, H-5), 3.02–3.12 (m, 1H,
(dq, 1H, J4,5 = 9.3 Hz, J5,6 = 6.3 Hz, H-5), 3.00–3.10 (m, 1H,
H-3), 2.89 (dd, 1H, J3,4 = J4,5 = 9.3 Hz, H-4), 2.08 (br dd, 1H,
H-3), 2.87 (dd, 1H, J3,4 = J4,5 = 9.3 Hz, H-4), 2.05 (br dd, 1H,
J2a,2e = 13.1 Hz, J2e,3 = 3.7 Hz, H-2e), 1.56 (ddd, 1H, J2a,2e = J2a,3
=
J2a,2e = 13.1 Hz, J2e,3 = 3.9 Hz, H-2e), 1.62 (ddd, 1H, J2a,2e = J2a,3 =
13.1 Hz, J1,2a = 3.6 Hz, H-2a), 1.25 (d, 3H, J5,6 = 6.3 Hz, H-6);
13C NMR (100 MHz, CD3OD, dC) 157.9 (Ar), 154.8 (Ar), 131.1
(Ar), 131.0 (Ar), 130.6 (Ar), 129.8 (2, Ar), 126.9 (2, Ar), 126.8
(Ar), 124.7 (Ar), 121.1 (Ar), 112.2 (Ar), 99.5 (Ar), 97.0 (C-1), 94.3
13.1 Hz, J1,2a = 3.7 Hz, H-2a), 1.25 (d, 3H, J5,6 = 6.3 Hz, H-6);
13C NMR (100 MHz, CD3OD, dC) 141.5 (Ar), 137.3 (Ar), 133.2
(Ar), 131.7 (Ar), 129.7 (2, Ar), 129.2 (Ar), 127.7 (2, Ar), 124.0 (Ar),
≡
121.4 (Ar), 120.3 (Ar), 112.4 (Ar), 96.5 (C-1), 95.0 (Ar), 89.7 ( C),
82.3 ( C), 79.0 (C-4), 70.0 (C-5), 56.0 (OCH2), 50.5 (C-3), 38.0
(C-2), 18.2 (C-6). HRMS (ESI) calcd for (M + H) C23H25N2O3:
377.1860. Found: 377.1858. Purity: 97.7%.
≡
≡
≡
( C), 79.1 (C-4), 78.3 ( C), 70.1 (C-5), 55.8 (OCH2), 50.5 (C-3),
38.2 (C-2), 18.2 (C-6). HRMS (ESI) calcd for (M + H) C23H24NO4:
378.1700. Found: 378.1703. Purity: > 99%.
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3-(2-Phenyl-benzo[b]furan-3-yl)-prop-2-ynyl 4-amino-2,3,4,6-
tetradeoxy-a-L-threo-hexopyranoside (9). Pure 9 was obtained
by column chromatography (1:1 hexanes–EtOAc→10:1 CH2Cl2–
CH3OH) in 85% yield: yellowish syrup, Rf 0.28 (10:1 CH2Cl2–
Ar), 130.2 (Ar), 128.5 (2, Ar), 127.9 (2, Ar), 127.0 (Ar), 126.0
≡
(Ar), 120.9 (Ar), 117.6 (Ar), 108.8 (Ar), 97.1 (C-1), 92.2 ( C),
≡
78.3 ( C), 67.6 (C-5), 55.4 (OCH2), 49.4 (C-4), 26.5 (C-3), 24.1
(C-2), 21.5 (PhCH3), 17.7 (C-6). HRMS (ESI) calcd for (M + H)
-1
23
≡
CH3OH); IR: n 3383 (N–H), 2222 (C C) cm ; [a] D)–93.7
(c 1.9, CH3OH); 1H NMR (500 MHz, CD3OD, dH) 8.17–8.22 (m,
2H, Ar), 7.56–7.60 (m, 1H, Ar), 7.43–7.50 (m, 3H, Ar), 7.36–7.40
(m, 1H, Ar), 7.25–7.34 (m, 2H, Ar), 5.09 (br d, 1H, J1,2a = 4.0 Hz,
≡
C30H31N2O4S: 515.1999. Found: 515.2000. Purity: > 99%.
3-(2-Phenylindol-3-yl)-prop-2-ynyl 4-amino-2,3,4,6-tetradeoxy-
a-L-threo-hexopyranoside (12). Pure 12 was obtained by column
chromatography (10:1, CH2Cl2–CH3OH) in 75% yield: yellowish
oil, Rf 0.18 (10:1 CH2Cl2–CH3OH); IR: n 3356 (N–H), 2218
H-1), 4.58 (s, 2H, OCH2C C), 4.07 (dq, 1H, J5,6 = 6.6 Hz, J4,5
=
¯
1.5 Hz, H-5), 2.68 (br s, 1H, H-4), 2.09 (dddd, 1H, J3a,3e = 13.8 Hz,
J2a,3a = 13.8 Hz, J3a,4 = 4.0 Hz, J2e,3a = 4.0 Hz, H-3a), 1.97 (dddd,
1H, J2a,2e = 13.8 Hz, J2a,3a = 13.8 Hz, J1,2a = 4.0 Hz, J2a,3e = 4.0 Hz,
H-2a), 1.58–1.64 (m, 1H, H-3e), 1.51–1.57 (m, 1H, H-2e), 1.08
(d, 3H, J5,6 = 6.6 Hz, H-6); 13C NMR (125 MHz, CD3OD, dC)
157.8 (Ar), 154.8 (Ar), 131.1 (Ar), 131.0 (Ar), 130.5 (Ar), 129.8
(2, Ar), 126.9 (2, Ar), 126.7 (Ar), 124.7 (Ar), 121.1 (Ar), 112.2
-1
23
1
≡
(C C) cm ; [a] D)–92.9 (c 0.9, CH2Cl2); H NMR (500 MHz,
CD3OD, dH) 8.06–8.10 (m, 2H, Ar), 7.58–7.62 (m, 1H, Ar), 7.43–
7.48 (m, 2H, Ar), 7.38–7.41 (m, 1H, Ar), 7.32–7.37 (m, 1H, Ar),
7.14–7.18 (m, 1H, Ar), 7.07–7.12 (m, 1H, Ar), 5.12 (br d, 1H,
≡
J1,2a = 3.9 Hz, H-1), 4.57 (s, 2H, OCH2C C), 4.06 (br q, 1H,
¯
J5,6 = 6.6 Hz, H-5), 2.68 (br s, 1H, H-4), 2.09 (dddd, 1H, J3a,3e
=
≡
≡
13.8 Hz, J2a,3a = 13.8 Hz, J3a,4 = 3.9 Hz, J2e,3a = 3.9 Hz, H-3a),
1.93 (dddd, 1H, J2a,2e = 13.8 Hz, J2a,3a = 13.8 Hz, J1,2a = 3.9 Hz,
J2a,3e = 3.9 Hz, H-2a), 1.58–1.64 (m, 1H, H-3e), 1.50–1.56 (m, 1H,
H-2e), 1.09 (d, 3H, J5,6 = 6.6 Hz, H-6); 13C NMR (125 MHz,
CD3OD, dC) 141.4 (Ar), 137.3 (Ar), 133.3 (Ar), 129.7 (Ar), 129.7
(2, Ar), 129.2 (Ar), 127.7 (2, Ar), 124.0 (Ar), 121.4 (Ar), 120.3
(Ar), 99.6 (Ar), 97.3 (C-1), 94.6 ( C), 78.2 ( C), 67.8 (C-5), 55.7
(OCH2), 49.4 (C-4), 26.8 (C-2), 24.2 (C-3), 17.8 (C-6). HRMS
(ESI) calcd for (M + H) C23H24NO3: 362.1751. Found: 362.1753.
Purity: > 99%.
3-(2-Phenyl-benzo[b]thiophen-3-yl)-prop-2-ynyl
4-amino-2,3,
4,6-tetradeoxy-a-L-threo-hexopyranoside (10). Pure 10 was
obtained by column chromatography (1:1 hexanes–EtOAc →
10:1 CH2Cl2–CH3OH) in 76% yield: yellow oil, Rf 0.25 (10:1
≡
≡
(Ar), 112.4 (Ar), 96.9 (C-1), 95.1 (Ar), 89.9 ( C), 82.1 ( C), 67.7
(C-5), 56.0 (OCH2), 49.4 (C-4), 26.8 (C-3), 24.3 (C-2), 17.8 (C-6).
HRMS (ESI) calcd for (M + H) C23H25N2O2: 361.1911. Found:
369.1910. Purity: > 99%.
-1
23
≡
CH2Cl2–CH3OH); IR: n 3379 (N–H), 2216 (C C) cm ; [a] D)–
97.2 (c 2.4, CH2Cl2); 1H NMR (500 MHz, CD3OD, dH) 7.91–7.95
(m, 2H, Ar), 7.83–7.87 (m, 1H, Ar), 7.77–7.81 (m, 1H, Ar),
7.33–7.45 (m, 5H, Ar), 5.03 (br d, 1H, J1,2a = 4.0 Hz, H-1), 4.57
DNA binding fluorescence assay
≡
(s, 2H, OCH2C C), 4.06 (dq, 1H, J5,6 = 6.6 Hz, J4,5 = 1.7 Hz,
The fluorescence was measured according to the literature re-
ported procedure.36a,37,40 Sheared herring sperm DNA (10 mg/mL
in 10 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, pH 7.5) was
purchased from Promega and diluted with BPE (bis-phosphate
EDTA) buffer to 2 mg/mL as the stock solution. BPE buffer
consists of 6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM Na2EDTA,
pH 7.0 in Milli-Q water. Molecular-biological grade DMSO and
ethidium bromide (EtBr) were purchased from Sigma-Aldrich. A
stock solution of EtBr (1 mM) was prepared in BPE buffer. The
test analogues were dissolved in DMSO to make 10 mM stock
solutions and further diluted with DMSO, as needed, to the desired
concentration prior to the assay. Fluorescence was measured on a
PTI (Photon Technology International) MP1 fluorescence system.
¯
H-5), 2.64 (br s, 1H, H-4), 2.04 (dddd, 1H, J3a,3e = 13.9 Hz, J2a,3a
=
13.9 Hz, J3a,4 = 4.0 Hz, J2e,3a = 4.0 Hz, H-3a), 1.93 (dddd, 1H,
J2a,2e = 13.9 Hz, J2a,3a = 13.9 Hz, J1,2a = 4.0 Hz, J2a,3e = 4.0 Hz,
H-2a), 1.56–1.62 (m, 1H, H-3e), 1.47–1.53 (m, 1H, H-2e), 1.14
(d, 3H, J5,6 = 6.6 Hz, H-6); 13C NMR (125 MHz, CD3OD, dC)
147.8 (Ar), 142.3 (Ar), 138.8 (Ar), 134.9 (Ar), 130.1 (Ar), 129.8
(2, Ar), 129.4 (2, Ar), 126.5 (Ar), 126.2 (Ar), 124.1 (Ar), 123.2
≡
≡
(Ar), 114.1 (Ar), 97.2 (C-1), 92.2 ( C), 81.0 ( C), 67.9 (C-5), 55.6
(OCH2), 49.6 (C-4), 26.8 (C-3), 24.2 (C-2), 17.8 (C-6). HRMS
(ESI) calcd for (M + H) C23H24NO2S: 378.1522. Found: 378.1520.
Purity: > 99%.
3-(1-Tosyl-2-phenylindol-3-yl)-prop-2-ynyl
4-amino-2,3,4,6-
tetradeoxy-a-L-threo-hexopyranoside (11). Pure 11 was obtained
by column chromatography (1:1, hexanes–EtOAc → 10:1,
CH2Cl2–CH3OH) in 19% yield: yellowish oil, Rf 0.20 (10:1
Direct fluorescence measurement
-1
23
≡
CH2Cl2–CH3OH); IR: n 3374 (N–H), 2227 (C C) cm ; [a] D)–
72.7 (c 0.2, CH2Cl2); 1H NMR (500 MHz, CD3OD, dH) 8.23–8.26
(m, 1H, Ar), 7.48–7.53 (m, 3H, Ar), 7.38–7.47 (m, 4H, Ar),
7.30–7.36 (m, 1H, Ar), 7.22–7.25 (m, 2H, Ar), 7.10–7.14 (d, 2H,
J = 8.0 Hz, Ar), 4.83 (1H, H-1, overlap with CD3OH signal),
To a fluorescence microcell was added 99 mL of a solution of a test
compound (50 mM) in BPE buffer containing less than 1% DMSO,
and its emission curve was measured from 335 nm to 450 nm at an
excitation wavelength of 320 nm at ambient temperature. To the
above ligand solution was added 1 mL of a 2 mg/mL stock solution
of herring sperm DNA (hsDNA) in BPE buffer. After gentle
vortexing and a short-time equilibration (~ 1 min), the emission
scan was performed under the same set of parameters until the
error between the maxima of two continuous emission curves
was < 10%. The fluorescence intensity at the emission maximum
before the addition of DNA was set as 100% to normalize the
intensity obtained after the addition of the nucleic acid. Under
the above assay conditions, no fluorescence was observed for any
of the reagents, except for the test compounds. Each compound
≡
4.35 (ABq, 1H, J = 16.3 Hz, OCH2C C), 4.32 (ABq, 1H, J =
¯
≡
16.3 Hz, OCH2C C), 3.90 (dq, 1H, J5,6 = 6.6 Hz, J4,5 = 1.6 Hz,
¯
H-5), 2.66 (br s, 1H, H-4), 2.26 (s, 3H, PhCH3), 1.98 (dddd,
1H, J3a,3e = 14.0 Hz, J2a,3a = 14.0 Hz, J3a,4 = 4.2 Hz, J2e,3a
=
4.2 Hz, H-3a), 1.93 (dddd, 1H, J2a,2e = 14.0 Hz, J2a,3a = 14.0 Hz,
J1,2a = 4.2 Hz, J2a,3e = 4.2 Hz, H-2a), 1.54–1.60 (m, 1H, H-3e),
1.38–1.44 (m, 1H, H-2e), 0.99 (d, 3H, J5,6 = 6.6 Hz, H-6); 13C
NMR (125 MHz, CD3OD, dC) 146.9 (Ar), 145.2 (Ar), 138.4 (Ar),
135.6 (Ar), 132.3 (2, Ar), 132.0(3) (Ar), 131.9(9) (Ar), 130.6 (2,
3720 | Org. Biomol. Chem., 2009, 7, 3709–3722
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was assayed in duplicate, and the values shown in the table reflect
an average of this data.
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1994, 269, 31051–31058.
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D. G. I. Kingston and D. J. Newman, CRC Press: Boca Raton, FL,
2005, Ch. 16, pp. 299–320.
Single-point fluorescent intercalator displacement (FID) assay
Three individual fluorescence measurements were required for
this assay. First, to a fluorescence microcell was added 98.5 mL
of an ethidium bromide (EtBr) solution in BPE buffer (5 mM),
and its emission curve was measured from 540 nm to 700 nm
at an excitation wavelength of 520 nm. Second, to the above
EtBr solution was added 1 mL of a 2 mg/mL stock solution of
hsDNA in BPE buffer. After mixing and a short-time equilibration
(~ 10 sec), the emission scan was performed under the same
set of parameters until the error between the maxima of two
continuous emission curves was < 10%. The intensity difference at
the emission maximum from the two previous measurements was
set as 100% for normalization. Finally, 0.5 mL of a 10 mM stock
solution of a test compound in DMSO was added to the mixture
in the cell. After mixing and equilibration (~ 3 min), the emission
scan was performed until steady measurments were obtained. The
normalized intensity difference at the emission maximum between
the first measurement and this measurement was used to represent
the percentage of remaining EtBr. Alternatively, the normalized
intensity difference between the second and third measurements
was used to represent the percentage of displaced EtBr. Under the
above assay conditions, no fluorescence was observed for all the
reagents and their combinations except for EtBr. Each compound
was assayed in triplicate, and the values shown in the table reflect
an average of this data.
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Multiple-point fluorescent intercalator displacement (FID) assay
The assay is nearly identical to the above single-point assay. The
major difference is that in step 3, 0.2 mL aliquots of a 10 mM
stock solution of a test compound in DMSO was added to
the fluorescing solution in the cell, and the fluorescence was
measured after each addition until a 50% reduction of fluorescence
occurred, corresponding to a 50% displacement of EtBr by the test
compound. The apparent binding constant of the test ligand was
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EtBr
[
]
]
K Ligand = K EtBr
×
Ligand
[
50%
where [Ligand]50% is the concentration of ligand that gives a 50%
reduction of fluorescence, [EtBr] is the concentration of EtBr, and
KEtBr is the binding constant for ethidium bromide.
Acknowledgements
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This work was supported by the University of Alberta, the Natural
Sciences and Engineering Research Council of Canada (NSERC),
the Alberta Ingenuity Centre for Carbohydrate Science (TLL),
and by grant R01 CA65875 from the National Cancer Institute
(to RSC). WS was the recipient of a Studentship from the Alberta
Ingenuity Fund.
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