Total synthesis of taspine and a symmetrical analogue: Study of binding to G-quadruplex DNA by ESI-MS

Article abstract of DOI:10.1002/ejoc.201201034

Taspine is an alkaloid found in the latex of certain trees belonging to the family of Euphorbiaceae, commonly called "Sangre de Drago" (Dragon's blood). Its structure and antineoplastic properties are well known. Here we report the total synthesis of tasp

Full text of DOI:10.1002/ejoc.201201034

FULL PAPER
DOI: 10.1002/ejoc.201201034
Total Synthesis of Taspine and a Symmetrical Analogue: Study of Binding to
G-Quadruplex DNA by ESI-MS
Alessandro Altieri,[a] Marco Franceschin,[a] Daniele Nocioni,[a] Antonello Alvino,[a]
Valentina Casagrande,[a] Maria Luisa Scarpati,[a] and Armandodoriano Bianco*[a]
Keywords: Natural products / Total synthesis / Alkaloids / DNA recognition / DNA structures
Taspine is an alkaloid found in the latex of certain trees be-
longing to the family of Euphorbiaceae, commonly called
“Sangre de Drago” (Dragon’s blood). Its structure and anti-
illin. The successful synthetic strategy was then applied to
the synthesis of a symmetrical analogue TAS2C. Both the
synthesised compounds were tested by ESI-MS (electrospray
neoplastic properties are well known. Here we report the to- ionisation mass spectrometry) for their ability to bind dif-
tal synthesis of taspine, starting from ferulic acid and isovan- ferent quadruplex and duplex DNA structures.
Introduction
In Peru, the therapeutic use of the sap coming out after
the incision of some trees is still very popular among the
local population. It is collected principally in the jungle re-
gion of Pucallpa-Tingo Maria from a tree belonging to the
Euphorbiaceae family, Croton draconoides.[1] This latex is
commonly called “Sangre de Drago” (Dragon’s blood) be-
cause of its characteristic red colour, similar to blood. It
has been shown to contain several alkaloids, including mag-
noflorine, isoboldine, norisoboldine, and taspine, which are
believed to be responsible for its main curative proprieties.
In recent years, many studies have shown that taspine (Fig-
ure 1) accounts for about 9% by weight of the dry latex.
Moreover, taspine is the component responsible for the ma-
jority of the pharmaceutical properties of the latex.[2] These
properties include bacteriostasis, wound healing,[3] cytotoxi-
city, immunosuppression,[4] and acetylcholinesterase inhibi-
tion. Moreover, it has been shown that isolated taspine in-
hibits the proliferation and migration of endothelian cells
(HUVECs) in vitro, and the neurovascularisation of
chicken chorioallantoic membranesin vivo.[5–6] It is also an
important inhibitor of VEGF and bFGF cells.[7] These fin-
dings suggest that taspine is a promising candidate for use
as an angiogenesis inhibitor and, more widely, as an anti-
cancer agent.
Figure 1. Taspine (9) and TAS2C (14).
Starting from these considerations, our study was fo-
cussed on a new strategy for the synthesis of this alkaloid
and related compounds. To date, in the literature, two syn-
theses have been reported that allow access to the alkaloid
itself.[8,9]
We have used an ESI-MS assay to evaluate the ability of
compounds to bind to G-quadruplex DNA, in order to ex-
plore the possible basis of their biological action.[10–11] G-
quadruplexes are unusual DNA secondary structures based
on Hoogsteen G–G-paired hydrophobic planar ring-sys-
tems comprising four guanine units.[12] Many studies have
shown that G-quadruplex structures appear to be involved
in several important biological processes, such as DNA rep-
lication, gene expression and recombination, and cell trans-
formation. Stabilising these structures can inhibit the ex-
pression of certain oncogenes, whose promoters are G-rich
sequences. Or it can inhibit enzymes that act directly on
duplex DNA sequences rich in guanine, such as the
telomerase, whose activation has been observed in ca. 90%
of all human tumors. Molecules capable of stabilising G-
quadruplexes are characterised by an aromatic core that fa-
[a] Dipartimento di Chimica, Università di Roma “La Sapienza”,
Piazzale A. Moro 5, 00185 Roma, Italy
Fax: +39-06-49913841
E-mail: armandodoriano.bianco@uniroma1.it
Homepage: http://www.chem.uniroma1.it/persone/armando
doriano-bianco
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201034.
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A. Bianco et al.
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vours stacking interactions with the G-tetrads and, in most ately positioned on the aromatic rings.[14,17] A large excess
cases, by basic side-chains that interact with DNA of monomer 2 over compound 1 was used to encourage the
grooves.[13] Taspine has these structural features, and this desired asymmetric coupling. Since under these conditions,
could therefore be its mechanism of action as an antitumor homo-coupling of the main component of the mixture is
agent.
likely to occur, the choice of monomer 2 as the compound
in excess, instead of monomer 1, was due to the presence
of a bulky protecting group (a benzyl group) that would
minimise the homo-coupling.
Results and Discussion
The next steps were the deprotection of the hydroxy and
carboxyl functions, and then dehydration leading to the
typical dilactonic structure of taspine, which is responsible
for its low solubility. Degradation of the side-chain involves
the rearrangement of the acyl azide into the corresponding
isocyanate, which is then hydrolysed to amine. To convert
compound 6 into the corresponding chloride (i.e., 7), we
suspended it in a refluxing solution of dioxane and SOCl2
until complete dissolution of the reagent was observed. The
crude product was subjected to side-chain degradation fol-
lowing a previously reported procedure for a modified Cur-
tius rearrangement,[18] and the corresponding amine 8 was
obtained. Finally our target was achieved by reductive am-
ination, the Eschweiler–Clarke methylation, using excess
formic acid and formaldehyde, thereby obtaining the de-
sired tertiary amine (i.e., 9, taspine).
Synthesis of Taspine and its Analogue TAS2C
Taspine has a particular molecular structure character-
ised by a biphenyl-bislactone skeleton, plus a 2-(dimeth-
ylamino)ethyl side-chain attached to only one of the two
rings. Therefore, we decided to follow a synthetic pathway
starting from the preparation of two appropriately func-
tionalised monomers that can be joined to give an asym-
metric biphenyl structure. The synthetic strategy is summa-
rised in Scheme 1.
We used commercially available ferulic acid and isovanil-
lin as starting materials. Monomers 1 and 2 were synthe-
sised by optimising previously reported protocols.[14–16] The
coupling of these two units was carried out by Ullmann
coupling in Cu/DMF using the bromine atoms appropri-
Scheme 1. Synthesis of taspine.
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Taspine and a Symmetrical Analogue
Scheme 2. Synthesis of TAS2C.
After completing the synthesis of the desired alkaloid, we modes and energies of interaction. We used three different
used a similar strategy to synthesise a symmetrical analogue oligonucleotides that can form G-quadruplex structures,
(TAS2C, Scheme 2). In this case, a symmetrical Ullmann and a self-complementary single-stranded oligonucleotide
coupling of monomer 1 was performed to form the basis able to fold in a double-stranded structure, as a model of
for a symmetrical core. In contrast to the synthetic route duplex DNA. In particular, we used an oligonucleotide rep-
used for taspine, a single cleavage step was sufficient to ob- resenting four human telomeric repeats (HTelo21, 5Ј-
tain the free functional groups ready for the subsequent de- GGGTTAGGGTTAGGGTTAGGG-3Ј), two oligonucleo-
hydration. That reaction led to the dilactonic core similarly tides taken from G-rich promoter sequences of two human
to the case of taspine. Finally, two symmetrical basic side- oncogens (c-myc, 5Ј-TGGGGAGGGTGGGGAGGGT
chains were installed, similarly to our previously synthe- GGGGAAGG-3Ј, and c-kit, 5Ј-AGGGAGGGCGCT
sised G-quadruplex ligands;[19–21] in this case 1-(2-amino- GGGAGGAGGGGC-3Ј), and a model oligonucleotide for
ethyl)piperidine was used.
duplex DNA (DK66, 5Ј-CGTAAATTTACG-3Ј). The de-
tailed experimental conditions are reported in the Exp. Sec-
tion. The association constants were calculated directly
from the corresponding peaks found in the mass spectra,
since the relative intensities in the spectrum are assumed to
Study of the Binding Affinities of the Synthesised
Compounds by ESI-MS
Here we report a study of the non-covalent interactions be proportional to the relative concentrations in the injected
of different G-quadruplex structures and of duplex DNA solution, as previously reported.[22] The association con-
with the taspine alkaloid and its synthetic analogue by ESI- stants K1, K2 and K3, related to the equilibria for the forma-
MS, a suitable and powerful technique for the determi- tion of drug–DNA complexes with 1:1, 2:1, and 3:1, stoi-
nation of stoichiometries and relative binding affinities. The chiometries respectively, as well as the corresponding total
most striking advantage of ESI-MS is that, under certain percentage of bound DNA, are reported in Table 1, with
conditions, it allows the transfer of non-covalently bound the relative standard deviations.
complexes into the gas phase without disruption of the
The data show that taspine has a moderate binding affin-
complex itself, and therefore allows the determination of ity towards the human telomeric repeats HTelo21 and the
the stoichiometry and, in particularly favourable cases, the model duplex oligonucleotide DK66. The observed stoi-
Table 1. K1, K2 and K3 values (reported on a logarithmic scale) and percentage of bound DNA calculated at 4:1 drug/DNA ratio,
as described in the Exp. Section for the indicated oligonucleotides, with standard deviations reported over at least three independent
experiments.
Taspine
logK1
TAS2C
logK1
logK2
logK3
% DNA bound
logK2
% DNA bound
HTelo21
c-myc
c-kit
4.5Ϯ0.1
4.9Ϯ0.1
5.1Ϯ0.2
4.5Ϯ0.1
4.3Ϯ0.2
4.5Ϯ0.1
4.7Ϯ0.1
4.2Ϯ0.2
–
43Ϯ2
73Ϯ6
76Ϯ7
42Ϯ7
4.2Ϯ0.1
4.7Ϯ0.1
4.0Ϯ0.1
4.3Ϯ0.1
4.2Ϯ0.1
4.5Ϯ0.1
–
26Ϯ4
56Ϯ3
17Ϯ3
33Ϯ2
4.2Ϯ0.2
4.3Ϯ0.1
–
Dk66
3.8Ϯ0.1
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A. Bianco et al.
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(see Supporting Information).[15,16] All compounds were obtained
as colourless amorphous powders.
chiometries for these complexes were 1:1 and 2:1. While we
were writing this manuscript, a paper by Neidle and co-
workers[23] appeared in which the telomeric binding ability
of taspine within a series of anticancer products was de-
scribed. This property is confirmed by our own results, but
furthermore, we observed a stronger affinity towards the
two human oncogene promoters c-kit and c-myc. In fact,
the complexes between taspine and these oligonucleotides
have higher binding constants and higher percentages of
DNA bound. This feature reflects the ability of taspine to
bind these sequences with a stoichiometry of up to 3:1,
leading to percentages of bound DNA higher than 70. This
behaviour suggests a potential selectivity of taspine for G-
rich oncogene promoters over both telomeric and duplex
DNA. The synthetic derivative TAS2C shows a generally
lower affinity towards oligonucleotides, since lower binding
constants and lower percentages of bound DNA have been
found. Its complexes with DK66, HTelo21, and c-myc
showed 1:1 and 2:1 stoichiometries, whereas only 1:1 drug–
DNA complexes have been found in presence of the c-kit
oncogene sequence.
Copper Bronze Activation: Copper bronze (500 mg) was added to a
stirred solution of iodine in acetone (2%; i.e., 160 mg in 10 mL) at
room temp. After 20 min, the suspension was filtered and washed
with the minimum amount of acetone. Then, the product was sus-
pended in a 1:1 mixture (5 mL) of HCl (37%) and acetone. After
10 min, the mixture was filtered again and washed with copious
acetone. Finally, it was dried in a vacuum desiccator for about 30–
40 min. Once activated, copper bronze should be used immedi-
ately.[24]
Synthesis of Taspine
Dimethyl 6Ј-(Benzyloxy)-5,5Ј-dimethoxy-6-[(methoxycarbonyl)oxy]-
3-(3-methoxy-3-oxopropyl)biphenyl-2,2Ј-dicarboxylate (3): A mix-
ture of 1 (300 mg, 0.87 mmol) and 2 (1.0 g, 3.86 mmol) was dis-
solved in anhydrous dimethylformamide (DMF; 1.5 mL). The solu-
tion was warmed at 140 °C under a nitrogen atmosphere, and a
suspension of freshly activated copper bronze (600 mg) in DMF
(1.0 mL) was added over 30 min. The reaction mixture was heated
at reflux for 30 min until ester 1 had completely reacted. After cool-
ing to 25 °C, the solution was filtered through a Gooch sintered
funnel, washed repeatedly with acetone, and dried under vacuum.
After chromatography on silica gel, 3 (283 mg 64%) was obtained.
1H NMR (300 MHz, CDCl3): δ = 7.81 (d, J = 8.7 Hz, 1 H, aro-
matic), 7.01 (m, 5 H, CH2-Ph), 6.96 (d, J = 8.7 Hz, 1 H, aromatic),
6.84 (s, 1 H, aromatic), 4.81 (s, 1 H, CH2-Ph), 4.80 (s, 1 H, CH2-
Ph), 3.90, 3.85, 3.65, 3.63, 3.57, 3.44 (s, 18 H, -OCH3), 3.02 (t, J =
6.1 Hz, 2 H, -CH2CH2CO-), 2.68 (m, 2 H, -CH2CH2CO-) ppm.
13C NMR (75 MHz, CDCl3): δ = 173.4, 168.0, 166.6, (-CO-), 156.3
(aromatic), 152.7 (carbonate), 152.5, 146.1, 138.4, 138.1, 136.5,
131.9, 131.5, 128.0, 127.5, 127.3, 127.3, 125.7, 124.1, 123.5, 112.9,
113.8, 111.5 (aromatic), 74.3 (CH2-Ph), 56.3, 56.0, 55.4, 51.8, 51.7,
51.7, (-OCH3), 36.2 (-CH2CH2CO-), 30.2 (-CH2CH2CO-) ppm. MS
(ESI): m/z = 619.59 [M + Na]+.
Conclusions
We have reported the total synthesis of the naturally oc-
curring alkaloid taspine, starting from ferulic acid and iso-
vanillin. The proposed approach is general, and it could be
successfully used in the synthesis of compounds with sim-
ilar dilactonic moieties. This versatile strategy has in fact
also been applied here to the synthesis of a symmetrical
analogue, TAS2C. The ability of both taspine and TAS2C
to bind different G-quadruplex and duplex DNA structures
has been tested by ESI-MS. Taspine shows a higher affinity
in binding DNA than TAS2C. The reported data provide
evidence for a moderate interaction of this alkaloid with G-
quadruplex human telomeric sequences, as also shown by
Neidle,[23] as well as with duplex oligonucleotides. Further-
more, taspine emerges as an even stronger ligand for G-
rich sequences of human oncogene promoters, since higher
values of percentage bound DNA have been found for such
oligonucleotides. These results suggest, then, a moderate
selectivity towards the topology of G-quadruplex-forming
sequences over both duplex and human telomeric DNA,
which encourages us to investigate the detailed mechanism
of action of this promising anticancer compound, and to
design more potent analogues, accessible by modifying the
versatile synthetic pathway.
Dimethyl 6Ј-Hydroxy-5,5Ј-dimethoxy-6-[(methoxycarbonyl)oxy]-3-
(3-methoxy-3-oxopropyl)biphenyl-2,2Ј-dicarboxylate (4): A solution
of 3 (450 mg, 0.76 mmol) in methanol/dioxane (4:1; 8 mL) was
treated with H2 and Pd/C (5%, 20 mg) at 25 °C at atmospheric
pressure for 12 h. Then the suspension was filtered through a
Gooch G4 sintered funnel and the solvents were evaporated under
vacuum to obtain 4 (382 mg, quantitative yield). 1H NMR
(300 MHz, CDCl3): δ = 7.61 (d, J = 8.7 Hz, 1 H, aromatic), 6.88
(d, J = 8.7 Hz, 1 H, aromatic), 3.93, 3.88, 3.69, 3.67, 3.57, 3.43 (s,
18 H, -OCH3), 3.02 (t, J = 6.1 Hz, 2 H, -CH2CH2CO-), 2.70 (t, J
= 6.1 Hz, 2 H, -CH2CH2CO) ppm. 13C NMR (75 MHz, CDCl3): δ
= 173.4, 168.1, 166.6 (-CO-), 155.8 (carbonate), 153.1, 152.4, 149.9,
143.6, 138.3, 136.5, 130.6, 125.9, 123.6, 123.4, 113.1, 109.7 (aro-
matic), 56.3, 56.3, 55.6, 51.9 (OCH3), 36.1 (-CH2CH2CO-), 30.0 (-
CH2CH2CO-) ppm. MS (ESI): m/z = 506.47 [M + H]+.
3-(3,8-Dimethoxy-5,10-dioxo-5,10-dihydrochromeno[5,4,3-cde]-
chromen-1-yl)propanoic Acid (6): Compound 4 (30.2 mg,
0.06 mmol) was dissolved in THF (3 mL). Then, aqueous NaOH
(0.5 mL, 2 n) was added, and the mixture was stirred at reflux tem-
perature for 2 h. After cooling, the solution was neutralised with
HCl (0.5 n) and the solvent was removed under vacuum. The re-
sulting white solid, consisting of 5, was redissolved in anhydrous
THF and concentrated. H2SO4 (0.3 mL) was added dropwise. After
stirring for 12 h at room temp., a new white precipitate was ob-
tained and isolated by filtration. The solid was thoroughly washed
Experimental Section
Synthesis, General: All commercially available reagents and sol-
vents were purchased from Fluka and Sigma–Aldrich, and were
used without further purification. TLC glass plates (silica gel 60
F254) and silica gel 60 (0.040–0.063 mm) were purchased from
Merck. 1H and 13C NMR spectra were performed with Varian
Gemini 200 and Varian Mercury 300 instruments. ESI-MS spectra
were recorded with a Micromass Q-TOF MICRO spectrometer.
Starting compounds 1 and 2 were prepared as described previously
1
with water and dried under vacuum to give 6 (19.0 mg, 86%). H
NMR (300 MHz, CF3COOD): δ = 8.52 (d, J = 8.7 Hz, 1 H, aro-
matic), 7.65 (d, J = 8.7 Hz, 1 H, aromatic), 7.59 (s, 1 H, aromatic),
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Taspine and a Symmetrical Analogue
4.31 (s, 3 H, -OCH3), 4.30 (s, 3 H, -OCH3), 3.87 (t, J = 7.4 Hz, 2
H, -CH2CH2CO), 3.13 (t, J = 7.4 Hz, 2 H, -CH2CH2CO-) ppm.
13C NMR (75 MHz, CF3COOD): δ = 158.3, 155.3, 148.4, 131.5,
3,3Ј-(3,8-Dimethoxy-5,10-dioxo-5,10-dihydrochromeno[5,4,3-cde]-
chromene-1,6-diyl)dipropanoic Acid (12): Compound 10 (45 mg,
0.07 mmol) was dissolved in THF (3 mL), then aqueous NaOH
125.8, 122.1, 121.8, 120.1, 118.4, 117.3, 114.6, 110.9 (aromatic), (2 n, 0.5 mL) was added, and the mixture was heated at reflux 2 h.
58.8, 58.5 (OCH3), 37.0 (-CH2CH2CO-), 32.7 (-CH2CH2CO-) ppm. After cooling, the solution was neutralised with HCl (0.5 n), and
MS (ESI): m/z = 368.31 [M – H]–.
the solvents were removed under vacuum. The resulting white solid
(i.e., 11), was redissolved in anhydrous THF (4 mL), and concen-
trated H2SO4 (0.3 mL) was added dropwise. After stirring for 12 h
at room temp., the newly formed white precipitate was obtained by
filtration and was washed thoroughly with water. After drying un-
der vacuum, 12 (27.5 mg, 90%) was obtained. 1H NMR (300 MHz,
CF3COOD): δ = 7.29 (s, 2 H, aromatic), 3.97 (s, 6 H, OCH3), 3.54
(t, J = 7.2 Hz, 4 H, -CH2CH2CO-), 3.00 (t, J = 7.2 Hz, 4 H,
-CH2CH2CO-) ppm. 13C NMR (75 MHz, CF3COOD): δ = 180.9,
(-CO-), 152.4, 145.5, 136.5, 126.0, 117.4, 112.2 (aromatic), 56.1
(-OCH3), 34.4 (-CH2CH2CO-), 30.2 (-CH2CH2CO-) ppm. MS
(ESI): m/z = 441.38 [M – H]–.
1-(2-Aminoethyl)-3,8-dimethoxychromeno[5,4,3-cde]chromene-5,10-
dione (8): Compound 6 (22 mg, 0.06 mmol) was suspended in anhy-
drous 1,4-dioxane (3 mL). Then SOCl2 (0.3 mL) was added and
heated to reflux temperature under argon until complete dissol-
ution was observed. After evaporation of the solvent, crude chlor-
ide 7 was dissolved in toluene (3 mL) in presence of benzyltrieth-
ylammonium chloride (5 mg). The solution was heated for 5 min
at 50 °C, then NaN3 (8.5 mg, 0.12 mmol) was added. The reaction
mixture was heated at 80 °C for 90 min, then a slightly alkaline
solution (0.02 n, 1.0 mL) was added, and the mixture was stirred
12 h. at room temp. The solution volume was reduced, water was
added, and the crude product was extracted with ethyl acetate. The
organic phase was washed with water until the aqueous phase was
neutral, and it was then finally washed with brine. After drying
with Na2SO4, the solvents were evaporated under vacuum to give
crude 8, which was used in the next synthetic step without further
purification. MS (ESI): m/z = 342.27 [M + H]+.
3,3Ј-(3,8-Dimethoxy-5,10-dioxo-5,10-dihydrochromeno[5,4,3-cde]-
chromene-1,6-diyl)bis[N-(2-piperidin-1-ylethyl)propanamide]
(14):
Compound 12 (20 mg, 0.05 mmol) was suspended in anhydrous
1,4-dioxane (3 mL). Then SOCl2 (0.5 mL) was added and the mix-
ture was heated at reflux under argon until complete dissolution
was observed. After cooling to room temp., the solvents were evap-
orated under vacuum. The resulting crude chloride (i.e., 13), was
dissolved in 1,4-dioxane (3 mL), and 1-(2-aminoethyl)piperidine
(14 μL, 2.2 equiv.) was added. The mixture was stirred for 1 h at
60 °C, then HCl (0.5 n) was added. The aqueous phase was first
extracted with ethyl acetate, then basified with NaOH (0.5 n) and
extracted a second time with ethyl acetate. The organic phase was
finally washed with water and dried under vacuum to give 14
(27.89 mg, 93%) as a white solid that was soluble in slightly acidic
1-[2-(Dimethylamino)ethyl]-3,8-dimethoxychromeno[5,4,3-cde]-
chromene-5,10-dione (taspine, 9): Formic acid (1 mL) was added to
the mixture obtained in the previous step, and the resulting mixture
was stirred for 10 min. Then, formaldehyde solution (37%; 1 mL)
was added, and the mixture was stirred for 4 h at 100 °C. After
cooling to room temp., HCl (2 n, 1 mL) was added, and the sol-
vents were evaporated under vacuum. Finally, NaOH (2 n, 0.3 mL)
was added to the crude product, and the mixture was extracted
with ethyl acetate. The organic phase was washed with water and
finally with brine. After drying with Na2SO4, the solvents were
evaporated under reduced pressure to give 9 (16.2 mg, 74%), which
was identical to an authentic sample of taspine. 1H NMR
(300 MHz, CDCl3): δ = 8.17 (d, J = 6.6 Hz, 1 H, aromatic), 7.44
(d, J = 6.6 Hz, 1 H, aromatic), 7.27 (s, 1 H, aromatic), 4.08 (s, 3
H, -OCH3), 4.05 (s, 3 H, -OCH3), 3.83 (t, J = 6.1 Hz, 2 H,
-CH2CH2CO-), 3.24 (t, J = 6.1 Hz, 2 H, -CH2CH2CO-), 2.87 [s, 3
H, N(CH3)2], 2.86 [s, 3 H, N(CH3)2] ppm. 13C NMR (75 MHz,
CDCl3): δ = 158.6, 157.5 (-CO-), 152.9, 152.0, 144.2, 137.9, 136.6,
126.8, 119.1, 118.5, 116.3, 113.4, 111.6, 109.1 (aromatic), 60.1
(-NCH3 ), 56.6, 56.4 (OCH3 ), 45.0 (-CH2 CH2 N-), 32.9
(-CH2CH2N-) ppm. HRMS: calcd. for C20H20NO6 [M + H]+
370.1291; found 370.1282. C20H19NO6 (369.37): calcd. C 65.03, H
5.18, N 3.79; found C 65.20, H 5.06, N 3.64.
1
solution. H NMR (300 MHz, CF3COOD): δ = 7.49 (s, 2 H, aro-
matic), 4.24 (s, 6 H, OCH3), 3.98 (m, 4 H, CH2), 3.90 (m, 4 H,
CH2), 3.78 (t, J = 7 Hz, 4 H, CH2), 3.52 (m, 4 H, CH2), 3.10 (m,
4 H, CH2), 2.94 (t, J = 7 Hz, 4 H, CH2), 2.07 (m, 8 H, CH2), 1.67
(m, 4 H, CH2) ppm. 13C NMR (75 MHz, CF3COOD): δ = 170.9
(-CO-), 157.7 (-CO-), 157.0, 145.8, 144.2, 140.3, 137.2, 125.6 (aro-
matic), 59.0, 56.9 (-OCH3), 55.8 (-CONHCH2-), 37.8
(-CH2CH2CO-), 36.3 (-CH2CH2CO-), 23.7, 21.6 (-CH2CH2CH2)
ppm. HRMS: calcd. for C36H46N4O8 [M + H]+ 662.7945; found
662.7955. C36H46N4O8 (662.78): calcd. C 65.24, H 7.00, N 8.45;
found C 65.15, H 7.05, N 8.41.
Determination of the Complexes Between Ligands and Quadruplex/
Duplex DNA by Electrospray Ionisation Mass Spectrometry (ESI-
MS): Single-stranded oligonucleotides were purchased from Euro-
fins MWG Operon (Ebersberg, Germany). Their sequences were:
HTelo21 (5Ј-GGGTTAGGGTTAGGGTTAGGG-3Ј)
c-myc (5Ј-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3Ј)
c-kit (5Ј-AGGGAGGGCGCTGGGAGGAGGGGC-3Ј)
DK66 (5Ј-CGTAAATTTACG-3Ј)
ESI-MS experiments were performed as described previously.[21]
ESI-MS spectra were recorded with a Micromass (now Waters) Q-
TOF MICRO spectrometer in the negative ionisation mode. The
rate of sample infusion into the mass spectrometer was 5 μL/min,
Synthesis of TAS2C
Dimethyl 5,5Ј-Dimethoxy-6,6Ј-bis[(methoxycarbonyl)oxy]-3,3Ј-bis(3-
methoxy-3-oxopropyl)biphenyl-2,2Ј-dicarboxylate (10): Activated
copper bronze (80 mg) was added to a solution of 1 (102 mg,
0.25 mmol), dissolved in anhydrous DMF (1 mL). The reaction was
carried out at reflux under nitrogen. After 2 h, the solution was
filtered through a Gooch G4 sintered funnel and washed repeatedly
with acetone. The filter residue was purified by column chromatog-
1
raphy on silica gel to give 10 (62 mg, 76%). H NMR (300 MHz,
CDCl3): δ = 6.87 (s, 2 H, aromatic), 3.87 (s, 6 H, -OCH3), 3.76 (s, and the capillary voltage was set to –2.6 kV. The source tempera-
6 H, OCH3), 3.66 (s, 6 H, -OCH3), 3.43 (s, 6 H, -OCH3), 3.02 (t, J ture was adjusted to 70 °C, the cone voltage to 40 V and the colli-
= 7.5 Hz, 4 H, -CH2 CH2 CO-), 2.66 (t, J = 7.5 Hz, 4 H, sion energy to 3 V. Data were analysed using the MassLynx soft-
-CH2CH2CO-) ppm. 13C NMR (75 MHz, CDCl3): δ = 173.3, 167.1
ware developed by Waters. Samples were prepared by mixing ap-
(-CO-), 152.5, 139.1, 137.0, 129.4, 125.3, 114.0, (aromatic), 56.3,
propriate volumes of 150 mm ammonium acetate buffer, 50 μm an-
55.5, 51.8, 51.7 (-OCH3), 36.2 (-CH2CH2CO-), 30.2 (-CH2CH2- nealed oligonucleotide stock solution, and 100 μm stock solutions
CO-) ppm. MS (ESI): m/z = 673.60 [M + Na]+.
of the specified ligand and methanol. The final concentration of
Eur. J. Org. Chem. 2013, 191–196
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FULL PAPER
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and the final volume of the sample was 50 μL. After the binding
equilibrium in ammonium acetate was established, methanol was
added to the mixture just before injection, in order to obtain a
more stable electrospray signal. As a reference, samples containing
only 5 μm DNA with no drug were prepared. To minimise random
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For drug–DNA complexes with 1:1, 2:1, and 3:1 stoichiometries,
which were shown to be the main species present in solution in all
the experiments, complex formation can be represented by three
distinct equilibria, which are described by the following equations:
K1 = [1:1]/([DNA] [drug]); K2 = [2:1]/([1:1] [drug]); K3 = [3:1]/([2:1]
[drug]); where [DNA], [drug], [1:1] [2:1], and [3:1] represent the re-
spective concentrations of the different species in solution: DNA
(duplex or quadruplex, depending on the oligonucleotide used), the
ligand, the 1:1, 2:1, and 3:1 drug–DNA complexes at equilibrium.
The association constants K1, K2, and K3 can be calculated directly
from the relative intensities of the corresponding peaks found in
the mass spectra, with the assumption that the response factors of
the oligonucleotides alone and of the drug–DNA complexes are the
same, so that the relative intensities in the spectrum are assumed
to be proportional to the relative concentrations in the injected
solution.[9] The percentage of bound DNA was calculated accord-
ing to an equation developed by Brodbelt and co-workers,[11] which
represents the percentage of DNA bound ligand: % bound DNA
= 100ϫ([1:1] + [2:1] + [3:1])/([DNA] + [1:1] + [2:1] + [3:1]).
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Supporting Information (see footnote on the first page of this arti-
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Acknowledgments
This research was financially supported by Ministero dell’Univer-
sità e della Ricerca (MIUR) (PRIN 2009).
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Received: August 1, 2012
Published Online: November 21, 2012
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Eur. J. Org. Chem. 2013, 191–196