Catecholic flavonoids acting as telomerase inhibitors

Article abstract of DOI:10.1021/jm040810b

In recent years telomerase has been identified as a new promising target in oncology and consequently new telomerase inhibitors have been intensely explored as anticancer agents. Focused screening of several polyhydroxylated flavonoids has allowed us to i

Full text of DOI:10.1021/jm040810b

6466
J. Med. Chem. 2004, 47, 6466-6475
Catecholic Flavonoids Acting as Telomerase Inhibitors
Maria Menichincheri,*,† Dario Ballinari,‡ Alberto Bargiotti,† Luisella Bonomini,§ Walter Ceccarelli,†
Roberto D’Alessio,† Antonella Fretta,§ Juergen Moll,‡ Paolo Polucci,† Chiara Soncini,‡ Marcellino Tibolla,†
Jean-Yves Trosset,† and Ermes Vanotti†
Departments of Chemistry, Pharmacology, and Biology, Oncology BU-Nerviano Medical Sciences, Viale Pasteur 10,
20014 Nerviano (MI), Italy
Received March 18, 2004
In recent years telomerase has been identified as a new promising target in oncology and
consequently new telomerase inhibitors have been intensely explored as anticancer agents.
Focused screening of several polyhydroxylated flavonoids has allowed us to identify 7,8,3′,4′-
tetrahydroxyflavone 1 as a new telomerase inhibitor with an interesting in vitro activity in a
Flash-Plate assay (IC50 ) 0.2 µM) that has been confirmed in the classical TRAP assay. Starting
from this compound, we developed a medicinal chemistry program to optimize our lead, and in
particular to replace one of the two catechols with potential bioisosteres. From this study, new
structural analogues characterized by submicromolar potencies have been obtained. Their
synthesis and biological activity are described.
Chart 1. Structures of Flavonoid Leads
Introduction
The ends of chromosomes in higher eukaryotes are
built up of telomeres, which consist of tandem arrays
of hexameric TTAGGG sequences ranging from ap-
proximately 15 to 20 kb in normal human cells.1
Telomeres protect the ends of chromosomes, and their
stability and length are regulated by telomerase, which
is a ribonucleoprotein responsible for adding telomere
repeats to the 3′-ends. The enzyme consists of two
components, an RNA, which serves as a template for
the telomere repeats, and the catalytic subunit, which
has reverse transcriptase activity. Telomeres shorten
after each round of replication and serve as a “mitotic
clock”, which in normal cells limits the number of
replications. This block can be bypassed in cancer cells
by upregulation of telomerase, leading to unlimited
proliferative capacity.2,3 Telomerase is an attractive
target for a potentially selective anticancer therapy due
to its specific expression in tumors (>85%) compared
to a low expression in normal somatic cells.4
within the flavonoid family have been described in the
literature (epigallocatechin gallate and apigenin, Chart
1).
Therefore, inhibition of telomerase has been ap-
proached through different strategies, ranging from
antisense-based inhibitors to random screening of syn-
thetic and natural products.5,6 Among the latter, several
compounds have been reported in the literature, includ-
ing rubromycins,7 alterperylenol,8 diazaphilonic acid,9
epigallocatechin gallate,10 and apigenin.11
Flavonoids have been known for a long time to exert
multiple biological effects (bioflavonoids), in particular
to act as anticancer agents12-14 and as inhibitors of other
DNA polymerases like reverse transcriptases.15,16 As
already mentioned, examples of telomerase inhibitors
In particular the interesting activity of epigallocat-
echin gallate has been reconfirmed in our in vitro assay
(IC50 ) 1 µM; for details on the assay, see below). For
this reason we performed a focused screening of several
polyhydroxylated compounds to identify new hits for
telomerase inhibition. This effort, which took advantage
of commercially available flavonoids, has resulted in the
identification of the tetrahydroxy flavone 1 (Chart 1)
with submicromolar potency in the in vitro assay
(IC50 ) 0.2 µM). This compound is characterized by a
biscatecholic substitution pattern that we argued to be
probably essential for biological activity. On the other
hand, these highly oxygenated functionalities were also
thought to represent an intrinsic limitation for drug
development. In fact. many flavonoids are often char-
acterized by a labile nature due to their polyhydroxyl-
ated templates. Thus we started a medicinal chemistry
program with the final goal to highlight the essential
* To whom correspondence should be addressed: phone +390331-
581928; fax +390331581757; e-mail maria.menichincheri@
nervianoms.com.
† Department of Chemistry.
‡ Department of Pharmacology.
§ Department of Biology.
10.1021/jm040810b CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/12/2004

Catecholic Flavonoids Acting as Telomerase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6467
Scheme 1a
Figure 1. Structures of strictly related analogues of tetrahy-
droxyflavone 1.
a Reagents: (i) tBu(Me)2SiCl, DIEA, DMF, rt; (ii) LiHMDS,
THF, -78 °C to rt; (iii) CH3COOH, H2SO4, 100 °C.
appropriate hydroxyacetophenone is first converted into
a benzoyl ester that, after treatment with base, yields
a 1,3-diketone. Treatment of the diketone with acid
leads to the desired flavone. As reported in Scheme 3,
the chloro derivative 14 was obtained by reaction of the
commercially available 2,3-dimethoxy-2-hydroxyace-
tophenone (12b) with N-chlorosuccinimide in hot acetic
acid.23 Base-catalyzed condensation of 14 with the
commercial 3,4-dimethoxybenzoyl chloride (15) fur-
nished the intermediate benzoyl ester 16, which was
converted to the 1,3-diketone 17. Contemporary cyclo-
dehydration and demethylation with a mixture of hy-
driodic and acetic acids at reflux gave the final flavone
3a.24 The 3-substituted flavones 3b-d were prepared
according to the routes reported in Scheme 4. The
common intermediate 1,3-diketone 19 was obtained
likewise from the commercially available 2,3-dimethoxy-
2-hydroxyacetophenone (12b) and 2,4-dimethoxybenzoyl
chloride (15). Reaction of 19 with N-fluorodibenzosul-
fonimide gave intermediate 20,25 which was trans-
formed by cyclodehydration under acidic conditions into
3-fluorotetramethoxy flavone 21, which was converted
to the corresponding tetrahydroxy derivative 3b with
boron tribromide.26 Treatment of 19 with thionyl chlo-
ride in dioxane at reflux afforded the 3-chlorotetra-
methoxy flavone 22,27 which, after reaction with boron
tribromide yielded derivative 3c. Compound 22 was also
used to prepare the 3-cyanotetramethoxy flavone 23, in
acceptable yield, by treatment with cuprous cyanide at
220 °C.28 Subsequent demethylation with boron tribro-
mide provided the final flavone 3d. In Scheme 5 the
synthesis of flavone 4b is reported. Condensation of the
acid chloride 24 with the commercially available 3,4-
dimethoxy-2-hydroxyacetophenone (12b), in the pres-
ence of pyridine, furnished the benzoyl ester 25. This
was converted to the 1,3-diketone 26 by use of pyridine/
KOH. Ring closure and amide hydrolysis of 26 with
concentrated hydrochloric acid furnished flavone 28.29
Reduction of the nitro group by reaction with zinc and
nickel(II) chloride hexahydrate in hot N,N-dimethyl-
formamide (DMF) afforded the final compound 4b.
Flavone 4b was also used as key intermediate for the
synthesis of a series of desired products shown in
Scheme 6. Demethylation of 4b with hydrobromic acid
at reflux gave the dihydroxy flavone 4c,30 while com-
Figure 2. Structures of bioisosteric catechol analogues of
tetrahydroxyflavone 1.
Figure 3. Structures of alternative scaffolds of tetrahydroxy-
flavone 1.
structural features for the biological activity of flavone
1 and to identify alternative moieties to circumvent
potentially unstable catechols.
Biological results on compound 1 as well as the
synthesis and the biological activity of the analogues
prepared will be reported.
Chemistry
In Figures 1-3, chemical structures of analogues
submitted to the in vitro telomerase inhibition assay are
reported. Compounds 1a-1d, 1f, 2a, and 5a have been
obtained from commercial sources. Compounds 1e, 2b,
and 5b have been prepared according to methods
reported in the literature.17-19
Schemes 1 and 2 show the synthetic pathways leading
to the target derivatives 2d and 2e,c. According to the
literature,18 reaction of the appropriate methyl O-
silyloxylated benzoates 8 and 11 with the commercially
available hydroxyacetophenones 9 and 12a,b afforded
the intermediate diketones 10 and 13a,b that were
subjected to cyclodehydration with 0.5% sulfuric acid
in acetic acid at 100 °C. Under these reaction conditions,
the tert-butyldimethylsilyl protecting groups were also
cleaved to generate the desired partially methylated
flavones 2d and 2e,c. In Scheme 3 the synthesis of
flavone 3a is reported. We have used the most common
method of synthesizing flavones, known as the Baker-
Venkataraman transformation.20-22 In this process, an

6468 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Menichincheri et al.
Scheme 2a
a Reagents: (i) LiHMDS, THF, -78 °C to rt; (ii) CH3COOH, H2SO4, 100 °C.
Scheme 3a
a Reagents: (i) NCS, CH3COOH, 80 °C; (ii) pyridine, rt; (iii) KOH, pyridine, 50 °C; (iv) Hl, CH3COOH, reflux.
pound 4a was obtained, from 4b, by use of acetyl
chloride. The series of 2-[2-substituted-1H-benzimidazol-
5(6)-yl]-4H-1-benzopyran-4-ones (4d,h,j) were achieved
by a Phillips-type reaction from the corresponding
aliphatic carboxylic acids (see Scheme 6). Demethylation
of 4d,h with hydrobromic acid at reflux afforded com-
pounds 4e,i. Treatment of 4b with 1,1′-carbonyldiim-
idazole supplies the benzimidazole derivative 4f,32 while
the use of 1,1′-oxalyldiimidazole instead of 1,1′-carbon-
yldiimidazole produced flavone 4k.33 The corresponding
dihydroxy derivatives 4g,l were achieved by reaction
with hydrobromic acid at reflux.
The tetrahydroxyflavone 1 has been recognized as a
potent telomerase inhibitor in this Flash-Plate (FP)
assay (IC50 ) 0.2 µM). In addition, compound 1 showed
50% inhibition at 1 µM also in the conventional polym-
erase chain reaction- (PCR-) based cell-free TRAP assay,
with A431 cell lysates as a source of telomerase,35,36 and
in an intact cell assay, which determines the inhibition
of intracellular telomerase activity (manuscript in prepa-
ration). Tetrahydroxyflavone 1 has also been tested in
a selectivity assay, based on T3 RNA polymerase, to
examine its specificcty of inhibition. In this assay it did
not show inhibitory activity up to 2 µM.
These preliminary results on compound 1 triggered
a more extensive investigation on this chemical class,
and in particular in assessing the role of the hydroxyls
in the enzymatic interaction and their replacement with
more attractive substituents.
First of all, we considered a set of analogues with a
reduced number of hydroxyls. As can be noticed from
Table 1, the minimal structural requirement for activity
is the simultaneous presence of hydroxyls at positions
7 and 8 (1b) (see numbering system in Chart 1). Im-
provement of activity can be achieved with additional
hydroxyls on ring C, and in particular, low micromolar
potency is ensured only if 4′-OH is present (1a), while
the maximum of activity is reached with the biscat-
echolic pattern (1). On the contrary, when one of the
two hydroxyls at position 7 or 8 is removed, a complete
drop of activity is observed (1d and 1e).
Results and Discussion
For the identification of telomerase inhibitors, an
assay that quantifies the incorporation of nucleotides
by telomerase in a microtiter format has been used,
allowing high-throughput screening.34 In particular, the
elongation of a 5′-biotinylated primer in the presence
of the corresponding dNTPs necessary for the telomeric
sequence (dGTP, dATP, and TTP), was evaluated by
hybridization in solution with a 3′-radiolabeled comple-
mentary oligonucleotide. The extent of hybridization
was then quantitated by immobilizing the hybridized
oligonucleotides in a streptavidin-coated Flash-Plate.
Telomerase activity is proportional to the radioactivity
measured, and the inhibitory activity of the compounds
was evaluated as IC50 (see Experimental Section for
details).

Catecholic Flavonoids Acting as Telomerase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6469
Scheme 4a
a Reagents: (i) pyridine, rt; (ii) KOH, pyridine, rt; (iii) (PhSO2)2NF, DCM, rt; (iv) CH3COOH, H2SO4, 90 °C; (v) BBr3, DCM, rt;
(vi) SOCl2, dioxane, reflux; (vii) CuCN, NMP, 220 °C.
Table 1. Flavone Substituents and Telomerase Inhibition Data
donor/acceptor properties. As can be seen in Table 1,
the tetramethoxy derivative is completely inactive (2a),
showing the importance of free OH in the interaction
with the enzyme. Compound 2b, in which the catecholic
moiety on ring A is restored, has shown moderate
activity, while 2c, where the catechol on ring C is
present, is inactive, further confirming the importance
for the activity of hydroxyls at position 7 and 8.
Interestingly, when hydroxyl group at position 7 is
methylated (compound 2e), micromolar potency is ob-
served. This result seems to suggest that the free OH
at that position is required for optimal activity but not
critical. The H-bond acceptor nature of the methoxy
group can be also tolerated. Analogously, when only the
4′-OH is methylated (compound 2d), activity in the
micromolar range is observed.
Once the importance of the biscatechol for optimal
activity was established, modulation of the electron
density of this electron-rich template was a further
object of our study. Various electron-withdrawing sub-
stituents (F, Cl, and CN) were introduced first onto
position 3 (ring B, compounds 3b-d, Table 1), thus
interfering with the enone moiety, and then onto posi-
tion 6 (Cl) (ring A, compound 3a, Table 1), thus
modifying the acidity of the proximal OH. Surprisingly,
both sets of substitution did not affect sensibly the
activity. This evidence suggests that the flavonoid
skeleton might be not sensitive to modifications and
might also be replaced by other scaffolds.
(FP assay)
R7
OH
R8
OH
OH
OH
OH
H
R3′
R4′
OH
OH
H
H
OH
OH
OH
R3
R6 IC50a (µM)
1
OH
H
H
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
0.2
3
1a OH
1b OH
1c OH
1d OH
H
H
H
H
H
H
H
H
H
H
H
H
F
36
50
.40
.40
.40
.40
11.7
.40
11
1e
1f
H
H
OH
H
2a OCH3 OCH3 OCH3 OCH3
2b OH
OH
OCH3 OCH3
2c OCH3 OCH3 OH
OH
2d OH
OH
OH
OH
OH
OH
OH
OH
OCH3
OH
2e OCH3 OH
7.8
0.82
0.6
0.8
0.13
7.4
3a OH
3b OH
3c OH
3d OH
OH
OH
OH
OH
OH
OH
OH
Cl
CN
H
H
H
OH
4a OCH3 OCH3 NH2
NH2
4b OCH3 OCH3 NHAc NHAc
.40
3.6
4c OH
OH
NH2
NH2
a Concentration required to inhibit telomerase activity by 50%.
Then we investigated the effect of partial or total
methylation of the hydroxyl groups as a way to possibly
prevent any oxidative degradation and, at the same
time, to understand the importance of their H-bond
The next step involved modifications of both reactive
catechol moieties. In particular, it was decided to replace

6470 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Menichincheri et al.
Scheme 5a
a Reagents: (i) pyridine, 0 °C to rt; (ii) KOH, pyridine, 60 °C; (iii) CH3COOH, CH3COONa, reflux; (iv) HCl, reflux; (v) Zn dust, NiCl2‚6H2O,
DMF, MeOH, 70 °C.
Scheme 6a
a Reagents: (i) 48% HBr, reflux; (ii) CH3COCl, TEA, THF, 0 °C to rt; (iii) HCOOH/4 N HCl, reflux; (iv) 1,1′-carbonyldimidazole, THF,
0 °C to rt; (v) 1,1′-oxalyldiimidazole, DMF, rt; (vi) CH3COOH/4 N HCl, reflux; (vii) (CH3)2N(CH2)3COOH‚HCl, 4 N HCl, reflux.
catechol on ring C first, as it proved to be less critical
for the activity and thus possibly more flexible toward
modifications. Our plan was to replace it with a conve-
nient heterocyclic ring.

Catecholic Flavonoids Acting as Telomerase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6471
Table 2. Flavone Substituents and Telomerase Inhibition Data
(FP Assay)
Table 3. Chalcone Substituents and Telomerase Inhibition
Data (FP Assay)
R1
R2
R3
R4
R5
IC50a (µM)
5a
5b
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
6
R7
R8
Y
IC50a (µM)
1.7
4d
4e
4f
4g
4h
4i
4j
4k
4l
OCH3
OH
OCH3
OH
OCH3
OH
OCH3
OCH3
OH
OCH3
OH
OCH3
OH
OCH3
OH
OCH3
OCH3
OH
CH
2.2
4
19
1.2
22.1
.40
0.47
.40
.40
CH
a Concentration required to inhibit telomerase activity by 50%.
COH
COH
CMe
CMe
The effect of more flexible scaffolds on the activity was
also studied by opening the benzopyranone, thus giving
rise to chalcones (Table 3, 5a,b). Both compounds are
weaker inhibitors with respect to their cyclic parent
compound 1, the best one (5b) being almost 10-fold less
active. This result suggests a preference for cyclic
templates.
C(CH2)3N(CH3)2
CH(OH)CO
CH(OH)CO
a Concentration required to inhibit telomerase activity by 50%.
Bioisosteres of catechols have already been reported
in the literature and they deal mainly with nitrogen-
containing heterocyclic derivatives. For example, novel
ligands of dopamine receptors have been identified by
replacing the catechol moiety of dopamine by benzim-
idazole-2-thione and 2,3-dihydroquinoxaline.37 Analo-
gously, benzimidazole derivatives of catecholamines
succeeded in showing agonistic activity at the adrenergic
receptors, thus confirming the bioisosteric role of benz-
imidazole with respect to catechol.38
Therefore, we focused our attention on the imidazole
ring, both unsubstituted and substituted at position 2,
and on the piperazinedione ring, as reported in Table 2
(compounds 4d-l). The corresponding precursors are
compounds 4a-c, reported in Table 1. Compound 4a,
the 3′,4′ diamino analogue of 1 protected as dimethoxy
at positions 7-8, and the fully deprotected 4c were
surprisingly almost equally active in the micromolar
range, while the diacetyl derivative 4b turned out to
be inactive, as expected.
Analogously, compounds 4d and 4e, bearing an un-
substituted imidazole at ring C, are almost equally
active. Instead, when oxygen was introduced at position
2 of the imidazole ring, dimethoxy derivative 4f turned
out to be 1 order of magnitude less active than the
corresponding dihydroxy 4g, consistent with the hy-
pothesis that the catechol on ring A is more important
for a good enzymatic interaction. However the insertion
of alkylic substituents on the imidazole completely
changes the activity trend. In fact, in the case of methyl,
compound 4h (7,8-dimethoxy derivative) shows a weak
potency, while the dihydroxy 4i is completely inactive.
Despite the lack of information on the mode of action
of these compounds, we were interested in checking the
possibility of an interaction with the DNA polyanionic
backbone through a group charged at physiological pH.
Thus we introduced at position 2 of imidazole the short
dimethylaminopropyl chain (compound 4j), present in
some G-quadruplex interacting compounds reported in
the literature.39-41 Interestingly, derivative 4j showed
a very good potency compared to its parent compound
4d, suggesting some additional interactions with the
enzyme. This result supports our hypothesis, and fur-
ther studies are currently in progress.
Conclusion
In conclusion, although the complete lack of structural
information on the enzymatic target has hampered a
full understanding of drug interaction at the active site
and thus a rational approach to analogue design, new
telomerase inhibitors have been discovered by the use
of a Flash-Plate assay suitable for high-throughput
screening. In fact we have identified a new telomerase
inhibitor with a catecholic flavonoid structure (com-
pound 1) endowed with an interesting activity profile.
Medicinal chemistry studies on this lead compound
allowed the understanding of bioactivity determinants.
In addition, preparation of new catecholic derivatives
still endowed with submicromolar potencies has been
achieved (compounds 3a-d).
Finally, replacement of catechol on ring C with
bioisosteric substitutents yielded the analogue 4j char-
acterized by submicromolar potency and improved
chemical profile. It represents a new lead structure to
be considered for further investigation.
Experimental Section
1H NMR spectra were recorded with a Varian 300 or 400
spectrometer. Chemical shifts are reported as δ values (parts
per million, ppm) downfield from internal Me4Si in the solvent
shown. The following NMR abbreviations are used: br (broad),
s (singlet), d (doublet), t (triplet), m (multiplet), ex (exchange-
able with D2O). Other abbreviations: ESI, electrospray ioniza-
tion; MS, mass spectrometry; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; THF, tetrahydrofuran. All products
reported showed 1H NMR spectra in agreement with the
assigned structures. ESI MS spectra were recorded on a ion-
trap mass LCQ-Deca Finningan spectrometer. Elemental
analyses were performed on a EA 1110 CHNS-O C. E.
Instruments elemental analyzer and agreed with theoretical
values to within (0.4%. Compounds 1, 1a,b, 1d, and 2a were
purchased from Apin Chemicals Ltd. Compound 1c was
purchased from Indofine Chemical Co, Ltd., and compound 1f
was from Lancaster Synthesis Ltd.
T3 RNA polymerase inhibition has been evaluated by using
the T3 RNA polymerase kit (Stratagene) and adapting the
instructions of kit.
The conventional PCR-based cell-free TRAP assay has been
run by using TRAP-eze kit (ONCOR) according to the instruc-
tions of the manufacturer.
Methyl 3-{[tert-Butyl(dimethyl)silyl]oxy}-4-methoxy-
benzoate (8). To a solution of methyl 3-hydroxy-4-methoxy-
benzoate (1.5 g, 8.2 mmol) in dry DMF (20 mL), under argon
Finally, piperazinedione insertion on ring C resulted
in a complete lack of activity, for both dimethoxy and
dihydroxy derivatives (compounds 4k and 4l).

6472 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Menichincheri et al.
at 0 °C, N-ethyldiisopropylamine (2.8 mL, 16.4 mmol) is added
dropwise, followed by a solution of tert-butylchlorodimethyl-
silane (1.55 g, 10.3 mmol) in dry DMF (10 mL). The reaction
mixture is stirred at room temperature overnight, iced water
is added, and the precipitate is filtered, washed with water,
and dried to yield 8 as a white powder (98%). 1H NMR (DMSO-
d6) δ 0.12 (6H, s), 0.95 (9H, s), 3.80 (3H, s), 3.84 (3H, s),
7.08 (1H, d), 7.33 (1H, s), 7.60 (1H, d). MS (ESI) m/z 297
[(M + H)+].
1-(3-{[tert-Butyl(dimethyl)silyl]oxy}-4-methoxyphenyl)-
3-(2,3,4-trihydroxyphenyl)-1,3-propanedione (10). To a
solution of 2,3,4-trihydroxyacetophenone (0.5 g, 2.97 mmol) in
dry THF (20 mL), cooled to -78 °C under argon, lithium bis-
(trimethylsilyl)amide (1M solution in THF, 14.8 mL) is added
dropwise in 15 min, and the solution is stirred at -78 °C for
1 h and at -10 °C for 2 h. To the mixture, cooled to -78 °C, a
solution of 8 (0.88 g, 2.97 mmol) in dry THF (5 mL) is added
dropwise, and the reaction mixture is stirred at -78 °C for 1
h and at room temperature overnight. The mixture is poured
into ice, 20% aqueous HCl (5 mL) is added (pH 2.5), and the
precipitate is extracted with ethyl acetate. The organic layer
is separated and washed with brine, dried, and concentrated
to yield dark oil that is stirred in isopropyl ether/hexane (1:1)
and filtered. The solid is washed with hexane and dried to
obtain 10 (45%). 1H NMR (DMSO-d6) δ 0.17 (6H, s), 1.01 (9H,
s), 3.88 (3H, s), 4.53 (2H, s), 6.56 (1H, d), 7.05 (1H, d), 7.18
(1H, d), 7.62 (1H, d), 7.84 (1H, d), 10.41 (3H, br s, ex). MS
(ESI) m/z 433 [(M + H)+].
By analogous procedures, compounds 13a,b were prepared:
1-(3,4-Di-{[tert-butyl(dimethyl)silyl]oxy}phenyl)-3-(2,3-
dihydroxy-4-methoxyphenyl)-1,3-propanedione (13a).
Yield 75%. 1H NMR (DMSO-d6) δ 0.25 (12H, s), 1.02 (18H, s),
3.76 (3H, s), 4.50 (2H, s), 6.77 (1H, d), 7.10-7.16 (2H, m), 7.41
(2H, br s, ex), 7.60 (1H, d), 7.72 (1H, d). MS (ESI) m/z 547
[(M + H)+].
1-(3,4-Di-{[tert-butyl(dimethyl)silyl]oxy}phenyl)-3-(2-
hydroxy-3,4-dimethoxyphenyl)-1,3-propanedione (13b).
Yield 80%. 1H NMR (DMSO-d6) δ 0.25 (12H, s), 1.04 (18H, s),
3.86 (3H, s), 3.96 (3H, s), 4.53 (2H, s), 6.63 (1H, d), 7.12-7.18
(2H, m), 7.63 (1H, d), 7.75 (1H, d), 10.90 (1H, br s, ex). MS
(ESI) m/z 561 [(M + H)+].
solid (61%). 1H NMR (CDCl3) δ 2.59 (3H, s), 3.91 (3H, s), 4.08
(3H, s), 7.52 (1H, s). MS (ESI) m/z 231 [(M + H)+].
6-Acetyl-4-chloro-2,3-dimethoxyphenyl 3,4-dimethoxy-
benzoate (16). To a solution of 14 (0.4 g, 1.73 mmol) in
anhydrous pyridine (5 mL), under argon, 3,4-dimethoxybenzoyl
chloride (0.52 g, 2.59 mmol) is added in a period of 15 min.
The mixture is stirred for 2 h at room temperature and then
acidified with 2 N HCl, extracted with ethyl acetate, washed
with water, dried, and evaporated under reduced pressure. The
crude reaction product is purified by flash chromatography
(eluant, hexane/ethyl acetate 7:3) to yield 16 as white solid
1
(88%). H NMR (DMSO-d6) δ 2.45 (3H, s), 3.78 (3H, s), 3.82
(3H, s), 3.88 (3H, s), 3.92 (3H, s), 7.18 (1H, d), 7.55 (1H, d),
7.78-7.82 (2H, m). MS (ESI) m/z 395 [(M + H)+].
By analogous procedures and starting from the properly
substituted ethanones and aroyl chlorides, the following
compounds were prepared:
6-Acetyl-2,3-dimethoxyphenyl-3,4-dimethoxybenzo-
1
ate (18). Yield 80%. H NMR (DMSO-d6) δ 2.42 (3H, s), 3.68
(3H, s), 3.83 (3H, s), 3.87 (3H, s), 3.92 (3H, s), 7.11 (1H, d),
7.13 (1H, d), 7.55 (1H, d), 7.71 (1H, d), 7.78 (1H, d). MS (ESI)
m/z 361 [(M + H)+].
6-Acetyl-2,3-dimethoxyphenyl-4-(acetylamino)-3-ni-
trobenzoate (25). Yield 78%. 1H NMR (CDCl3) δ 2.35 (3H,
s), 2.45 (3H, s), 3.80 (3H, s), 3.95 (3H, s), 6.90 (1H, d), 7.65
(1H, d), 8.43 (1H, d), 9.00 (1H, d), 9.10 (1H, s), 10.60 (1H, br
s, ex). MS (ESI) m/z 403 [(M + H)+].
1-(5-Chloro-2-hydroxy-3,4-dimethoxyphenyl)-3-(3,4-di-
methoxyphenyl)-1,3-propanedione (17). To a solution of
6-acetyl-4-chloro-2,3-dimethoxyphenyl-3,4-dimethoxybenzo-
ate (16) (0.58 g, 1.47 mmol) in anhydrous pyridine (5 mL),
stirred at 50 °C, powdered potassium hydroxide (0.12 g, 2.25
mmol) is added. After 1 h, the reaction mixture is cooled,
acidified with 2 N HCl, extracted with ethyl acetate, washed
with water, dried, and evaporated under reduced pressure. The
crude reaction product is purified by flash chromatography
(eluant hexane/ethyl acetate 7:3) to yield 17 as yellow solid
(62%). 1H NMR (DMSO-d6) δ 3.80 (3H, s), 3.82-3.90 (6H, m),
3.88 (3H, s), 7.10 (1H, d), 7.25 (1H, s), 7.55 (1H, d), 7.75 (1H,
d), 7.95 (1H, s), 11.40-11.60 (2H, m, ex). MS (ESI) m/z 395
[(M + H)+].
7,8-Dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-chro-
men-4-one (2d). Compound 10 is dissolved in glacial acetic
acid (10 mL), 96% sulfuric acid (0.05 mL) is added, and the
solution is stirred at 100 °C for 1 h. The mixture is poured
into ice and the precipitate is extracted with ethyl acetate. The
organic layer is separated and washed with brine, dried over
sodium sulfate, and concentrated to yield 2d as a dark solid
that is crystallized from dichloromethane/methanol (42%). 1H
NMR (DMSO-d6) δ 3.29 (3H, s), 6.63 (1H, s), 6.91(1H, d), 7.07
(1H, d), 7.36 (1H, d), 7.50 (1H, d), 9.35 (1H, s), 9.38 (1H, s),
10.26 (1H, s). MS (ESI) m/z 301 [(M + H)+]. Anal. (C16H12O6)
C, H.
By analogous procedures and starting from the appropriate
aryl benzoates, the following compounds were prepared:
1-(3,4-Dimethoxyphenyl)-3-(2-hydroxy-3,4-dimethoxy-
phenyl)-1,3-propanedione (19). Yield 78%. 1H NMR (DMSO-
d6) δ 3.60-4.00 (12H, m), 4.75 (2H, s), 6.70 (1H, m), 7.00 (1H,
m), 7.20 (1H, s), 7.70 (1H, m), 11.80 (1H, s, ex). MS (ESI) m/z
361 [(M + H)+].
N-{4-[3-(2-Hydroxy-3,4-dimethoxyphenyl)-3-oxopro-
panoyl]-2-nitrophenyl}acetamide (26). Yield 65%. 1H NMR
(CDCl3) δ 2.38 (3H, s), 3.90 (3H, s), 3.98 (3H, s), 6.60 (1H, d),
6.78 (1H, s), 7.60 (1H, d), 8.18 (1H, d), 8.80 (1H, d), 9.00 (1H,
d), 10.55 (1H, br s, ex), 11.50 (1H, br s, ex). MS (ESI) m/z 403
[(M + H)+].
By analogous procedures, compounds 2e and 2c were
prepared:
6-Chloro-2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-4H-
chromen-4-one (3a) A suspension of 6-chloro-2-(3,4-dimeth-
oxyphenyl)-7,8-dimethoxy-4H-chromen-4-one (17) (0.11 g, 0.29
mmol) in a mixture of aqueous hydriodic acid (57%, 4 mL) and
glacial acetic acid (4 mL) is refluxed for 15 h. After cooling,
the yellow precipitate is filtered and washed with acetic acid
and water. The solid is suspended into an aqueous NaHSO3
solution and extracted with 1-butanol. The organic phase is
washed with water, dried, and evaporated under reduced
pressure. The crude product is washed with hot absolute
ethanol and ether and dried in a vacuum to yield 3a as a yellow
8-Hydroxy-7-methoxy-2-(3,4-dihydroxyphenyl)-4H-chro-
1
men-4-one (2e). Yield 63%. H NMR (DMSO-d6) δ 3.93 (3H,
s), 6.60 (1H, s), 6.87 (1H, d), 7.15 (1H, d), 7.40-7.50 (3H, m),
9.38 (1H, br s, ex), 9.55 (1H, br s, ex), 9.75 (1H, br s, ex). MS
(ESI) m/z 301 [(M + H)+]. Anal. (C16H12O6) C, H.
7,8-Dimethoxy-2-(3,4-dihydroxyphenyl)-4H-chromen-
4-one (2c). Yield 86%. 1H NMR (DMSO-d6) δ 3.94 (6H, s), 6.65
(1H, s), 6.89 (1H, d), 7.23 (1H, d), 7.39 (1H, d), 7.43 (1H, d),
7.73 (1H, d), 9.40 (1H, br s, ex), 9.80 (1H, br s, ex). MS (ESI)
m/z 315 [(M + H)+]. Anal. (C17H14O6) C, H.
1-(5-Chloro-2-hydroxy-3,4-dimethoxyphenyl)etha-
none (14). A solution of 3,4-dimethoxy-2-hydroxyacetophenone
(0.2 g, 1.02 mmol) and N-chlorosuccinimide (0.16 g, 1.22 mmol)
in glacial acetic acid (5 mL) is stirred at 80 °C for 6 h. After
cooling, the solution is diluted with water and extracted with
ethyl acetate. The organic phase is washed with water and
brine, dried, and evaporated under reduced pressure. The
crude reaction product is purified by flash chromatography
(eluant, hexane/ethyl acetate 9:1) to yield 14 as a yellowish
1
solid (38%). H NMR (DMSO-d6) δ 6.61 (1H, s), 6.87 (1H, d,),
7.43 (1H, s), 7.50-7.55 (2H, m). MS (ESI) m/z 321 [(M + H)+].
Anal. (C15H9ClO6) C, H.
1-(3,4-Dimethoxyphenyl)-2-fluoro-3-(2-hydroxy-3,4-di-
methoxyphenyl)-1,3-propanedione (20). To a solution of
1-(3,4-dimethoxyphenyl)-3-(2-hydroxy-3,4-dimethoxyphenyl)-
1,3-propanedione (19) (0.36 g, 1 mmol) in anhydrous dichlo-
romethane (20 mL), under argon, a solution of N-fluorodiben-
zosulfonimide (0.41 g, 1.3 mmol) in anhydrous dichloromethane

Catecholic Flavonoids Acting as Telomerase Inhibitors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6473
(20 mL) is added, and the solution is stirred at room temper-
ature for 7 days. After solvent removal, the crude product is
purified by flash chromatography (eluant, dichloromethane/
methanol 100:1), to obtain 20 (28%). 1H NMR (DMSO-d6) δ
3.73 (3H, s), 3.79 (3H, s), 3.80 (3H, s), 3.86 (3H, s), 5.70 (1H,
d), 6.80 (1H, d), 7.04 (1H, d), 7.23 (1H, d), 7.27 (1H, d), 7.52
(1H, d), 8.07 (1H, s). MS (ESI) m/z 379 [(M + H)+].
acetate, and dried to yield 27 as a yellow solid (67%). 1H NMR
(DMSO-d6) δ 2.10 (3H, s), 3.95-3.97 (6H, m), 7.08 (1H, s), 7.30
(1H, d), 7.80 (1H, d), 7.90 (1H, d), 8.35 (1H, d), 8.55 (1H, d),
10.50 (1H, br s, ex). MS (ESI) m/z 385 [(M + H)+].
2-(4-Amino-3-nitrophenyl)-7,8-dimethoxy-4H-chromen-
4-one (28). A suspension of 27 (0.2 g, 0.52 mmol) in 9 N HCl
(5 mL) is refluxed for 2 h. After cooling, the precipitate is
filtered, washed with water, methanol, and ether, and dried
in a vacuum to give 28 as a yellow solid (92%). 1H NMR
(DMSO-d6) δ 3.95-3.97 (6H, m), 6.80 (1H, s), 7.15 (1H, d), 7.25
(1H, d), 7.75 (1H, d), 7.95 (1H, br s, ex), 8.05 (1H, d), 8.65
(1H, d). MS (ESI) m/z 343 [(M + H)+].
2-(3,4-Dimethoxyphenyl)-3-fluoro-7,8-dimethoxy-4H-
chromen-4-one (21). Product 20 (0.02 g, 0.05 mmol) is
dissolved in glacial acetic acid (1 mL), 96% H2SO4 (0.01 mL)
is added, and the solution is refluxed for 1 h. Water (10 mL)
is added, and the precipitate is filtered, washed to neutrality,
1
and dried to yield 21 (82%). H NMR (DMSO-d6) δ 3.85 (3H,
2-(3,4-Diaminophenyl)-7,8-dimethoxy-4H-chromen-4-
one (4b). Product 28 (0.1 g, 0.29 mmol) and NiCl2‚6H2O (0.14
g, 0.58 mmol) are suspended in a mixture of methanol/DMF
(4:1, 5 mL), and Zn powder (0.15 g, 2.32 mmol) is added in
portions with stirring. The solution is then heated at 70 °C
for 2 h. The precipitate is separated by filtration while hot
and washed with methanol. The filtrate and the washing are
combined and the solvent is evaporated under reduced pres-
sure. The crude product is then suspended in a mixture of
methanol/water (8:2, 10 mL), stirred for 30 min, filtered,
washed with water, and dried in a vacuum to yield 4b as a
s), 3.86 (3H, s), 3.94 (3H, s), 3.96 (3H, s), 7.23 (1H, d), 7.32
(1H, d), 7.51 (1H, d), 7.60 (1H, d), 7.83 (1H, d). MS (ESI) m/z
361 [(M + H)+]. Anal. (C19H17FO6) C, H.
3-Chloro-2-(3,4-dimethoxyphenyl)-7,8-dimethoxy-4H-
chromen-4-one (22). A portion of 1-(3,4-dimethoxyphenyl)-
3-(2-hydroxy-3,4-dimethoxyphenyl)-1,3-propanedione (19) (1 g,
2.77 mmol) is dissolved in dry dioxane (20 mL), sulfuryl
chloride (0.25 mL, 3.1 mmol) is added, and the mixture is
refluxed for 1 h. The mixture is cooled to room temperature.
(white precipitate), iced water is added (70 mL), and the
precipitate is filtered, washed thoroughly with water, and
dried. The solid is stirred in isopropyl ether (2 × 40 mL) and
filtered. After crystallization from dichloromethane/isopropyl
ether, 22, as a white solid, is obtained (77%). 1H NMR (DMSO-
d6) δ 3.81 (3H, s), 3.86 (3H, s), 3.89 (3H, s), 3.95 (3H, s), 7.18
(1H, d), 7.32 (1H, d), 7.50 (1H, d), 7.55 (1H, d), 7.82 (1H, d).
MS (ESI) m/z 377 [(M + H)+]. Anal. (C19H17ClO6) C, H.
3-Cyano-2-(3,4-dimethoxyphenyl)-7,8-dimethoxy-4H-
chromen-4-one (23). Compound 22 (0.38 g, 1 mmol) is
suspended in 1-methyl-2-pyrrolidinone (5 mL), cuprous cyanide
(0.16 g, 1.8 mmol) is added, and the mixture is refluxed
overnight. The solvent is evaporated under vacuum and the
crude material is purified by flash chromatography (eluant,
dichloromethane/ethyl acetate 95:5) to yield 23 (54%). 1H NMR
(DMSO-d6) δ 3.90-4.00 (12H, m), 7.30 (1H, d), 7.45 (1H, d),
7.65 (1H, d), 7.75 (1H, d), 7.80 (1H, d). MS (ESI) m/z 368
[(M + H)+]. Anal. (C20H17NO6) C, H, N.
1
yellow solid (79%). H NMR (DMSO-d6) δ 3.95-3.97 (6H, m),
4.80 (2H, br s), 5.35 (2H, br s, ex), 6.45 (1H, s), 6.60 (1H, d),
7.15-7.25 (3H, m), 7.70 (1H, d). MS (ESI) m/z 313 [(M + H)+].
Anal. (C17H16N2O4) C, H, N.
N-[2-(Acetylamino)-4-(7,8-dimethoxy-4-oxo-4H-chromen-
2-yl)phenyl]acetamide (4a). A suspension of 4b (0.15 g, 0.48
mmol) and trietylamine (0.53 mL, 3.84 mmol) in dry tetrahy-
drofuran (3 mL) is cooled (0 °C), and acetyl chloride (0.14 mL,
1.92 mmol) is added with stirring. The cooling bath is removed
and stirring is continued overnight. The reaction mixture is
then filtered and the solid is washed with tetrahydrofuran and
dried. The crude product is suspended in water (15 mL), stirred
at room temperature for 30 min, filtered, washed with water,
and dried in a vacuum at 50 °C to yield 4a as a yellow solid
(33%). 1H NMR (DMSO-d6) δ 2.05 (3H, s), 2.10 (3H, s), 3.95-
3.98 (6H, m), 6.80 (1H, s), 7.25 (1H, d), 7.75 (1H, d), 7.80-
7.90 (2H, m), 8.30 (1H, s), 9.50-9.52 (2H, m, ex). MS (ESI)
m/z 397 [(M + H)+]. Anal. (C21H20N2O6) C, H, N.
2-(1H-Benzimidazol-5-yl)-7,8-dimethoxy-4H-chromen-
4-one (4d). A suspension of 2-(3,4-diaminophenyl)-7,8-dimeth-
oxy-4H-chromen-4-one (4b) (0.2 g, 0.64 mmol) in a mixture of
4 N HCl (5 mL) and formic acid (1 mL) is heated at 100 °C for
2 h. After cooling, the reaction mixture is carefully neutralized
with sodium bicarbonate powder and the solid precipitated is
filtered, washed with water, methanol, and ether, and dried
in a vacuum to obtain 4d as a white solid (77%). 1H NMR
(DMSO-d6) δ 3.96 (3H, s), 3.98 (3H, s), 6.95 (1H, s), 7.25 (1H,
d), 7.70-8.00 (4H, m), 8.40 (1H, s), 11.90 (1H, br s, ex). MS
(ESI) m/z 323 [(M + H)+]. Anal. (C18H14N2O4) C, H, N.
2-(2-Hydroxy-1H-benzimidazol-5-yl)-7,8-dimethoxy-4H-
chromen-4-one (4f). A suspension of 2-(3,4-diaminophenyl)-
7,8-dimethoxy-4H-chromen-4-one (4b) (0.1 g, 0.32 mmol) in dry
tetrahydrofuran (5 mL) is cooled (0 °C) and N,N′-carbonyldi-
imidazole (0.062 g, 0.38 mmol) is added rapidly with stirring.
The cooling bath is removed and stirring is continued over-
night. The reaction mixture is then filtered and the solid is
washed with tetrahydrofuran, methanol, and ether and dried
in a vacuum to yield 4f as a yellow solid (80%). 1H NMR
(DMSO-d6) δ 3.95-3.98 (6H, m), 6.80 (1H, s), 7.10 (1H, d), 7.25
(1H, d), 7.55 (1H, d), 7.70 (1H, d), 7.80 (1H, d), 10.90 (1H, br
s, ex), 11.0 (1H, br s, ex). MS (ESI) m/z 339 [(M + H)+]. Anal.
(C18H14N2O5) C, H, N.
7,8-Dimethoxy-2-(2-methyl-1H-benzimidazol-5-yl)-4H-
chromen-4-one (4h). A suspension of 2-(3,4-diaminophenyl)-
7,8-dimethoxy-4H-chromen-4-one (4b) (0.5 g, 1.60 mmol) in a
mixture of 4 N HCl (5 mL) and glacial acetic acid (2.5 mL) is
heated at 100 °C for 2 h. After cooling, the reaction mixture is
carefully neutralized with sodium bicarbonate powder and the
solid precipitated is extracted with chloroform (3 × 50 mL).
The organic phase is dried, filtered, and evaporated. The crude
product is purified by use of boiling methanol to obtain pure
3-Chloro-2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-4H-
chromen-4-one (3c). To a portion (0.15 g, 0.398 mmol) of
3-chloro-2-(3,4-dimethoxyphenyl)-7,8-dimethoxy-4H-chromen-
4-one (22), dissolved in anhydrous dichloromethane (15 mL)
and cooled to 0 °C, a 1 M solution of BBr3 in dichloromethane
(4.8 mL) is dropped slowly; the solution is stirred at room
temperature for 2 h and then is diluted with iced water. The
pH is arranged to 6 with 5% Na2HPO4, the mixture is extracted
with ethyl acetate, and the organic layer is separated and
washed with brine, dried, and concentrated to yield, after
crystallization from dichloromethane/methanol/ethyl acetate,
1
3c as yellow solid (42%). H NMR (DMSO-d6) δ 6.90 (1H, d),
6.97 (1H, d), 7.31 (1H, d), 7.38 (1H, d), 7.42 (1H, d), 9.22 (1H,
s), 9.35 (1H, s), 9.80 (1H, s), 10.60 (1H, s). MS (ESI) m/z 321
[(M + H)+]. Anal. (C15H9ClO6) C, H.
By analogous procedures and starting from the appropriate
precursors 21, 23, 31, and 34, the corresponding following
compounds were prepared:
3-Fluoro-2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-4H-
chromen-4-one (3b). Yield 71%. 1H NMR (DMSO-d6) δ 6.90-
7.01 (2H, m), 7.41-7.52 (3H, m), 9.45-9.46 (2H, m. ex), 9.79
(1H, s, ex), 10.44 (1H, s, ex). MS (ESI) m/z 305 [(M + H)+].
Anal. (C15H9FO6) C, H.
3-Cyano-2-(3,4-dihydroxyphenyl)-7,8-dihydroxy-4H-
chromen-4-one (3d). Yield 56%. 1H NMR (DMSO-d6) δ 6.94-
7.04 (2H, m), 7.41 (1H, d), 7.50-7.60 (2H, m), 9.43 (1H, s, ex),
9.47 (1H, s, ex), 9.85 (1H, s, ex), 10.80 (1H, s, ex). MS (ESI)
m/z 312 [(M + H)+]. Anal. (C16H9NO6) C, H, N.
N-[4-(7,8-Dimethoxy-4-oxo-4H-chromen-2-yl)-2-nitro-
phenyl]acetamide (27). A suspension of N-{4-[3-(2-hydroxy-
3,4-dimethoxyphenyl)-3-oxopropanoyl]-2-nitrophenyl}aceta-
mide (26) (0.25 g, 0.62 mmol) and sodium acetate (0.5 g) in
glacial acetic acid (5 mL) is refluxed for 2 h. After cooling, the
precipitate is filtered, washed with acetic acid, water, and ethyl

6474 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Menichincheri et al.
1
4h (43%). H NMR (DMSO-d6) δ 2.55 (3H, s), 3.95-4.00 (6H,
CGTCGAGCAGAGTT). The reaction was started by adding to
the above mixture telomerase enzyme obtained from cellular
extracts of HEK293 cells overexpressing telomerase and
enriched by affinity purification (kindly provided by Geron).
The specific activity of the enzyme extract was adjusted to 5
fmol of dNTP incorporation min-1 µL-1 diluted in HTB
containing 0.5 µM BSA.
The combined HTB, deoxynucleotides 5′-triphosphate, and
the primers were aliquoted and distributed to individual wells
of a 96-well microtiter plate. The test compounds, diluted in
DMSO, were added individually to the wells at a dilution of
0.01-10 µM before the telomerase enzyme extract was added
to start the reaction.
After 2 h of incubation at 37 ° C, the polymerization of
telomeric repeats to the end of the primers was determined
by hybridization in solution with 5 µg/mL 33P-labeled (3′-
dCCCTAACCCTAACCC), with a specific activity of 2500 Ci/
mmol. This probe has been synthethized using a commercial
kit (Amersham Biosciences), diluted in a buffer containing 1
M Tris-HCl, pH 7.0, 20× SSC (0.3 M sodium citrate, pH 7.0,
containing 3 M NaCl), and 0.5 M ethylenediaminetetraacetic
acid (EDTA). The hybridized oligonucleotides were then im-
mobilized for quantification by transferring the reaction
mixture to a streptavidin-coated Flash-Plate (purchased from
NEN/Perkin-Elmer/Wallac Inc). After 2 h of incubation at
37 °C, the solution was removed and the wells were washed
extensively with 2× SSC and 0.1% SDS. Finally, the amount
of radiolabeled oligonucleotide was quantified by scintillation
counting on a Packard TopCount.
m), 6.80 (1H, s), 6.95 (1H, s), 7.25 (1H, d), 7.60 (1H, br s), 7.80
(1H, d), 7.85 (1H, d), 8.20 (1H, br s, ex). MS (ESI) m/z 337
[(M + H)+]. Anal. (C19H16N2O4) C, H, N.
2-{2-[3-(Dimethylamino)propyl]-1H-benzimidazol-5-
yl}-7,8-dimethoxy-4H-chromen-4-one (4j). A suspension of
2-(3,4-diaminophenyl)-7,8-dimethoxy-4H-chromen-4-one (4b)
(0.2 g, 0.64 mmol) and 4-(dimethylamino)butyric acid hydro-
chloride (3.2 g, 19.2 mmol) in 4 N HCl (10 mL) is heated at
reflux for 48 h. After cooling, to the mixture sodium bicarbon-
ate powder is carefully added until a basic condition is reached,
and the solid precipitated is extracted with dichloromethane
(3 × 20 mL). The organic phase is dried, filtered, and evap-
orated. The crude product is purified by flash chromatography
(eluant dichloromethane/methanol in different ratios, 9:1, 8:2,
1
7:3, and 1:1) to yield 4j as a yellowish solid (35%). H NMR
(CDCl3) δ 2.00-2.10 (2H, m), 2.45 (6H, s), 2.60 (2H, t), 3.20
(2H, t), 4.00 (3H, s), 4.08 (3H, s), 6.80 (1H, s), 7.05 (1H, d),
7.60 (1H, d), 7.80 (1H, d), 7.95 (1H, d), 8.20 (1H, s). MS (ESI)
m/z 408 [(M + H)+]. Anal. (C23H25N3O4) C, H, N.
6-(7,8-Dimethoxy-4-oxo-4H-chromen-2-yl)-3-hydroxy-
quinoxalin-2(1H)-one (4k). To a solution of 2-(3,4-diami-
nophenyl)-7,8-dimethoxy-4H-chromen-4-one (4b) (0.1 g, 0.32
mmol) in anhydrous DMF (1 mL), at room temperature, 1,1′-
oxalyldiimidazole (0.091 g, 0.48 mmol) is added and the
mixture is stirred for 24 h. Methanol (5 mL) is added and the
suspension is stirred at 50 °C for 30 min. After cooling, the
yellow solid precipitated is filtered, washed with methanol and
ether, and dried in a vacuum to obtain 4k as a yellowish solid
(51%). 1H NMR (DMSO-d6) δ 3.90-4.00 (6H, m), 6.80 (1H, s),
7.20-7.30 (2H, m), 7.70-7.85 (3H, m). MS (ESI) m/z 367
[(M + H)+]. Anal. (C19H14N2O6) C, H, N.
2-(3,4-Diaminophenyl)-7,8-dihydroxy-4H-chromen-4-
one (4c). A suspension of 2-(3,4-diaminophenyl)-7,8-dimethox
y-4H-chromen-4-one (4b) (0.1 g, 0.32 mmol) in aqueous
hydrobromic acid (48%, 1 mL) is refluxed for 10 h. After
cooling, the reaction mixture is diluted with water, neutralized
with 20% NaHCO3, and extracted with 1-butanol. The organic
phase is washed with water, dried, evaporated under reduced
pressure, and dried in a vacuum to obtain 4c as a yellow solid
(40%). 1H NMR (DMSO-d6) δ 5.60-6.00 (4H, m), 6.40 (1H, s),
6.65 (1H, d), 6.90 (1H, d), 7.30-7.40 (3H, m), 9.20 (1H, br s,
ex), 10.20 (1H, br s, ex). MS (ESI) m/z 285 [(M + H)+]. Anal.
(C15H12N2O4) C, H, N.
By analogous procedures and starting from the appropriate
chromen-4-ones, the following compounds were prepared:
2-(1H-Benzimidazol-5-yl)-7,8-dihydroxy-4H-chromen-
4-one (4e). Yield 27%. 1H NMR (DMSO-d6) δ 6.88-6.92 (2H,
m), 7.40 (1H, d), 7.80 (1H, d), 8.10 (1H, d), 8.50 (1H, s), 8.80
(2H, br s, ex). MS (ESI) m/z 295 [(M + H)+]. Anal. (C16H10N2O4)
C, H, N.
7,8-Dihydroxy-2-(2-hydroxy-1H-benzimidazol-5-yl)-4H-
chromen-4-one (4g). Yield 60%. 1H NMR (DMSO-d6) δ 6.70
(1H, s), 6.95 (1H, d), 7.05 (1H, d), 7.38 (1H, d), 7.65 (1H, s),
7.75 (1H, d), 9.40 (1H, s), 10.25 (1H, s), 10.90 (1H, br s, ex),
10.95 (1H, br s, ex). MS (ESI) m/z 311 [(M + H)+]. Anal.
(C16H10N2O5) C, H, N.
Acknowledgment. We thank Geron for providing
telomerase purified extracts for the FP assay and for
1
collaboration. We also thank Vittorio Pinciroli for H
NMR spectra and Maristella Colombo for MS.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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