Polyalkoxy substituted 4H-chromenes: Synthesis by domino reaction and anticancer activity

Article abstract of DOI:10.1021/co300062e

A series of 4H-chromenes containing various modifications in the ring B and polyalkoxy substituents in the ring E has been synthesized by Knoevenagel-Michael-hetero-Thorpe-Ziegler three-component domino reaction with the overall yield of 45-82%. The targe

Full text of DOI:10.1021/co300062e

Research Article
pubs.acs.org/acscombsci
Polyalkoxy Substituted 4H‑Chromenes: Synthesis by Domino
Reaction and Anticancer Activity
Anatoliy M. Shestopalov,*,† Yuri M. Litvinov,†,§ Lyudmila A. Rodinovskaya,† Oleg R. Malyshev,†
Marina N. Semenova,‡ and Victor V. Semenov†
†N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation
‡N. K. Koltzov Institute of Developmental Biology, RAS, 26 Vavilov Street, 119334, Moscow, Russian Federation
S
* Supporting Information
ABSTRACT: A series of 4H-chromenes containing various
modifications in the ring B and polyalkoxy substituents in the
ring E has been synthesized by Knoevenagel−Michael−hetero-
Thorpe−Ziegler three-component domino reaction with the
overall yield of 45−82%. The targeted molecules were evaluated
in a phenotypic sea urchin embryo assay for antimitotic and
microtubule destabilizing activity. The most active compounds
5{1,5} and 5{5,5} featured sesamol-derived ring B and m-
methoxyphenyl or m-methoxymethylenedioxyphenyl ring E.
Compounds 5{3,1}, 5{1,2}, 5{5,4}, 5{1,5}, and 5{5,5} exhibited strong cytotoxicity in the NCI60 human tumor cell line
anticancer drug screen. Surprisingly, cell growth inhibition caused by these agents was more pronounced in the multidrug
resistant NCI/ADR-RES cells than the parent OVCAR-8 cell line. The results suggest that polyalkoxy substited 4H-chromenes
may prove to be advantageous for further design as anticancer agents.
KEYWORDS: 4H-chromenes, domino reaction, microtubule destabilizing agents, sea urchin embryo, cytotoxicity
INTRODUCTION
■
Microtubules are highly dynamic cytoskeleton organelles that
play an essential role in cell division. At mitosis they rearrange
to form mitotic spindle responsible for proper orientation and
segregation of chromosomes into daughter cells. Hence,
targeting tubulin in rapidly dividing tumor cells is a sound
strategy for anticancer therapy. A number of natural products
and their synthetic derivatives exhibit antiproliferative activity
by interfering with tubulin polymerization and depolymeriza-
tion resulting in mitotic arrest.1 However, the development of
multidrug resistance and high general toxicity limit their use in
oncology. Therefore, there is ongoing need for small molecule
tubulin inhibitors that display better therapeutic window and
overcome multidrug resistance.
4-Aryl-4H-chromenes (Figure 1) were found to exhibit
strong cytotoxicity against human cancer cells involving
microtubule depolymerization, G2/M cell cycle arrest,
caspase-dependent apoptotic cell death, and tumor vasculature
disruption.2−10 Compound EPC2407 (Figure 1) is currently in
Figure 1. Semisynthetic podophyllotoxin derivatives.
phase I/II clinical trials as vascular targeting anticancer agent
and apoptosis inducer for the treatment of patients with
advanced solid tumors.10 4-Aryl-4H-chromenes were identified
due to its strong systemic toxicity, several semisynthetic PT
derivatives, including etoposide (Figure 1), teniposide, and
etopophos, are currently in clinical use for the treatment of
as microtubule destabilizers with binding site at or close to the
colchicine binding site.2 They may be considered as syntheti-
cally feasible structural analogues of natural antimitotic lignan
podophyllotoxin (PT), which is the strong microtubule
destabilizing agent that binds to the colchicine site of tubulin.11
Although the use of PT as anticancer agent was unsuccessful
Received: June 2, 2012
Revised: July 13, 2012
Published: July 23, 2012
© 2012 American Chemical Society
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Research Article
a
Scheme 1. Synthesis of 2-Amino-4H-chromenes 5
a
Reagents and conditions: (i) EtOH, Et3N, reflux, 5−30 min (method A); (ii) EtOH, Et3N, MW 300 W, 4−5 min (method C); (iii) EtOH, Et3N,
reflux, 5−30 min (method B). Insertion: Electronic effects in polyalkoxybenzylidene malononitriles 4.
various malignancies. These compounds exhibit a mechanism
of action entirely different from that of the parent PT, involving
strong DNA-topoisomerase II inhibition, S−G2 cell cycle
arrest, and subsequent apopotosis.12
4H-chromenes via the three-component domino reaction of
polyalkoxybenzaldehydes 1, malononitrile 2, and phenols 3. It
should be noted that in polyalkoxybenzylidene malononitriles 4
the electronic effects of the substituents (Scheme 1, Insertion)
hampered activation of the CC double bond, requiring rather
harsh conditions to undergo Michael reaction. Therefore the
domino reaction was carried out by heating in the microwave
field, affording the yield increase by 8−12%.
Three-component reaction of aldehydes 1, malononitrile 2,
and 3-phenols 3 (Method A, Scheme 1) was carried out by
heating in ethanol in the presence of catalytic amounts of
triethylamine. Under these conditions substituted 4-aryl-4H-
chromenes 5 were obtained in acceptable yields (45−82%).
Most probably, formation of the targeted products 5 proceeded
The stereo structure of PT molecule with four chiral centers
(Figure 1) was reported to be crucial for antitumor activity,13
which makes challenging the structure−activity relationship
(SAR) studies. Numerous attempts have been made to obtain
potent synthetically feasible analogues of PT with a heteroatom
in the ring C to prevent epimerization. Namely, 4-oxa-PT
derivatives featured a single chiral center (I, II; Figure 1),
displayed strong antimitotic and microtubule destabilizing
effects, as well as high cytotoxicity against a panel of human
cancer cell lines.14 Unfortunately, the synthetic approach for 6-
methoxy-4-oxa-PT-derivatives I and II resulted in a low overall
yield (3−6%) due to ambiguous reactivity of tetronic acid in
Mannich reaction.14,15
Considering synthetic feasibility, we aimed at designing a
simple procedure to prepare 4-aryl-4H-chromenes that contain
cyano and amino groups instead of the lactone ring D of PT
(Figure 1), as they could be readily accessible while maintaining
antimitotic potency. The axial orientation of the ring E in PT
molecule was proposed to restrict flexibility at the C1 center
and subsequently enhance antiproliferative effects.13,16 There-
fore, a modification of the ring E by different polyalkoxy
substituents may further hamper its spatial rotation, thus
increasing antimitotic activity of the targeted compounds. In
addition, small hydrophobic moieties at C7 in the ring B of 4H-
chromenes, such as methoxy, amino, dimethylamino, and
ethylamino groups, were found to be beneficial for cancer cell
growth inhibition and caspase-dependent apoptosis.4 Accord-
ingly, in the present study 4-polyalkoxyaryl-4H-chromenes with
the ring B substitued by methoxy, methylenedioxy, and amino
groups have been synthesized.
RESULTS AND DISCUSSION
■
Chemistry. Previously we have designed the three-
component synthesis of 2-amino-4H-pyrans and 2-amino-4H-
chromenes.17,18 Herein, we reported the preparation of 4-aryl-
Figure 2. Diversity of reagents.
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Table 1. 2-Amino-4H-chromenes 5
yield (%)
entry
product
X
R1
R2
OCH3
R3
OCH3
R4
OCH3
method A
method B
method C
53
1
5{3,1}
5{3,2}
5{4,2}
5{2,2}
5{1,2}
5{4,3}
5{7,3}
5{6,3}
5{8,4}
5{5,4}
5{1,5}
5{5,5}
5{4,6}
7-OCH3
H
H
12
53
45
82
74
52
62
70
75
57
82
53
56
2
7-NH2
OCH3
H
OCH3
OCH3
OCH3
H
3
7-NH2
Br
OCH3
52
4
7-NH2
H
H
OCH3
5
7-NH2
H
OCH3
H
H
H
6
7-N(CH3)2
7-N(CH3)2
7-N(CH3)2
7-N(C2H5)2
7-N(C2H5)2
6,7-OCH2O−
6,7-OCH2O−
7,8-C6H4
Br
OCH3
OCH3
OCH3
−OCH2O−
H
7
OCH3
OCH3
H
−OCH2O−
OCH3
OCH3
OCH3
OCH3
OCH3
H
−OCH2O−
−OCH2O−
OCH3
70
77
74
79
8
9
10
11
12
13
H
-OCH2O-
H
-OCH2O-
H
65
H
H
−OCH2O−
OCH3
−OCH2O−
OCH3
Br
as domino reaction including Knoevenagel reaction, Michael
reaction, and hetero-Thorpe−Ziegler reaction. Generally,
domino reactions are widely used in the synthesis of a variety
of organic compounds, including natural products.19 However,
this particular sequence of reactions has not been previously
classified as domino. The introduction of unsaturated nitrile 4,
obtained previously from aldehyde 1 and malononitrile 2, into
the reaction provided the indirect evidence of the domino
mechanism of the compounds interaction (Method B, Scheme
1). Noteworthy, the use of 3-methoxy-4.5-methylenedioxyben-
zonitrile (4, R2 = OCH3, R3−R4 = −OCH2O−) as the chemical
precursor (Method B) increased the yield of the targeted
compound 5{5,4} by ∼8% comparing to the yield of the same
compound in Method A. However, overall yield of the products
in Method B was comparable to that in Method A because the
preliminary synthesis of nitriles 4 by Knoevenagel reaction
proceeded with restricted yield. Application of the microwave
irradiation18 increased the yield by 8−12% (Method C),
simultaneously enhancing the amount of tarry byproduct.
Hence, crystallization of products 5 in the presence of activated
charcoal was required.
Structures of the synthesized compounds were confirmed by
spectral analyses (See Supporting Information). In IR spectra of
compounds 5 characteristic band of conjugated nitrile group
(2184−2197 cm−1), as well as stretching bands of NH2 group
(3113−3480 cm−1), bending bands of NH2 (1605−1664 cm−1)
have been observed. The 1H NMR spectra of the title
compounds featured C(4)H proton singlet in the region
4.45−5.32 ppm and broad singlet of exchangeable NH2 group
(6.36−7.14 ppm). Signals of the corresponding aromatic and
aliphatic protons were present as well. Mass-spectra of all
aminochromenes 5 contained highly intensive peak of
molecular ion.
Table 2. Effects of 2-Amino-4H-chromenes on the Sea
Urchin Embryos and Human Cancer Cells
a
EC (μM)
cleavage
compound alteration
cleavage
arrest
embryo
spinning
cell growth inhibition
(average GI50, μM)
b
PT
0.02
0.005
0.002
0.01
1
0.05
0.05
0.01
0.05
4
0.5
0.2
0.1
0.5
>5
>2
>4
5
c
I
0.631
0.045
c
II
5{3,1}
5{3,2}
5{4,2}
5{2,2}
5{1,2}
5{4,3}
5{7,3}
5{6,3}
5{8,4}
5{5,4}
5{1,5}
5{1,5}-1
5{1,5}-2
5{5,5}
5{4,6}
2
>2
>4
1
2
0.1
0.257
0.5
2
5
1
4
>4
4
0.5
2
0.1
0.5
0.05
0.05
1
4
0.02
0.005
0.1
0.2
0.2
5
0.045
0.065
0.002
0.005
0.2
0.02
0.05
4
0.1
0.2
5
0.389
a
The sea urchin embryo assay was conducted as described in ref 20.
Fertilized eggs and hatched blastulae were exposed to 2-fold
decreasing concentrations of compounds. Duplicate measurements
showed no differences in effective threshold concentration (EC)
b
values. NCI60 anticancer drug screen; GI50: Concentration required
c
for 50% cell growth inhibition. Data from ref 14.
by a molecule (video illustrations are available at http://www.
chemblock.com).
As evidenced from Table 2, 4-aryl-4H-chromenes 5{3,1},
5{1,2}, 5{4,3}, 5{6,3}, 5{8,4}, 5{5,4}, 5{1,5}, 5{5,5}, and
5{4,6} caused noticeable cleavage alteration, cleavage arrest,
and embryo spinning, suggesting their antimitotic microtubule
destabilizing activity. Less potent compounds 5{3,2} and
5{7,3} could be considered as tubulin destabilizers as well,
although they did not cause embryo spinning, since these
compounds induced formation of tuberculate eggs typical of
tubulin destabilizers.20,21
Recent SAR studies of oxa-PT analogues showed that 6-
methoxy moiety in the ring B in combination with the
myristicin-derived or 2,3,4-trimethoxyphenyl ring E resulted in
potent cytotoxic tubulin destabilizing compounds (I, II; Figure
Biological Evaluation: SAR Studies. All synthesized 4H-
chromenes were further evaluated in a phenotypic sea urchin
embryo assay20 for their antimitotic and microtubule
destabilizing activity using PT as a benchmark reference
compound (Table 2). The assay includes (i) fertilized egg
test for antimitotic activity displayed by cleavage alteration/
arrest, and (ii) behavioral monitoring of a free-swimming
blastulae treated immediately after hatching. The lack of
forward movement, settlement to the bottom of the culture
vessel, and rapid spinning of embryos around the animal−
vegetal axis suggests a microtubule destabilizing activity caused
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1, Table 2).14 Similarly, chromene 5{3,1}, with cyano and
amino groups instead of the lactone ring D, displayed high
antimitotic microtubule destabilizing activity in the sea urchin
embryo assay comparable with the effect of the parent PT.
Previously this compound has been identified as strong
proapoptotic agent in the caspase activation assay.4 Replace-
ment of 7-methoxy moiety by 7-amino group resulted in a
marked reduction of activity and inability to induce embryo
spinning (Table 2, 5{3,1} vs 5{3,2}). Chromene 5{1,2}
featuring 3-methoxybenzene ring E was 10 times more active
than 5{3,2}, whereas 5{2,2} containing p-methoxy substituted
ring E exhibited only moderate cleavage alteration effect. It
should be reported previously that the replacement of 3-
methoxy group in the E-ring with bromine enhanced the
potency in caspase activation assay and inhibited cancer cell
growth in submicromolar concentration range.3,4,10a Unfortu-
nately, in our assay we were unable to test compound 5{4,2}
with 2-bromine in the ring E due to its limited solubility in
DMSO and the presence of microscopic crystals in 5−10 mM
stock solutions. Notably, the respective analogue 5{4,3} with 7-
dimethylamino substituent in the ring B showed rather low
antiproliferative antitubulin effect similar to the activity of
5{6,3} with 2,3-dimethoxy-4,5-methylenedioxybenzene ring E.
The corresponding isomer 5{7,3} containing 2,5-dimethoxy-
3,4-methylenedioxybenzene was less potent. Conjugation of
phenyl ring to C7C8 positions of the ring B instead of 7-
dimethylamino group did not influence the activity of Br-
substituted chromenes (Table 2, 5{4,3} and 5{4,6}). For
compounds featuring 7-diethylamino group, the 3-methoxy-4,5-
methylenedioxybenzene analogue 5{5,4} exhibited high activity
in the sea urchin embryo assay, whereas 5{8,4} with 3,4-
dimethoxyphenyl ring E was less potent. Conjugation of
methylenedioxy functionality to C6C7 positions of the ring B
markedly increased antimitotic activity of 4-aryl-4H-chromenes,
yielding the most potent compounds 5{1,5} and 5{5,5} with
EC values of 5 nM (Table 2). In this case both 3-
methoxyphenyl and 3-methoxy-4,5-methylenedioxybenzene
ring E afforded the same antimitotic potency. Chromenes
5{1,5} and 5{5,5} closely resembled PT in the A−B-C ring
assembly. These molecules were found to be more potent than
PT in the sea urchin embryo assay. We suggested that the
replacement of the lactone ring in PT by cyano and amino
groups could be beneficial for antimitotic microtubule
destabilizing activity.
Figure 3. Structures of 5{1,5} enantiomers and compound
EPC2407.10,24 The spatial structures of 5{1,5} are presented by
analogy with EPC2407.
Supporting Information). The isomer 5{1,5}-2 exhibited high
antimitotic effect, inducing cleavage alteration and cleavage
arrest at concentration of 2 and 20 nM, respectively, which was
twice more than the activity of racemic 5{1,5} (Table 2). The
activity of isomer 5{1,5}-1 was 50 times less (EC = 0.1 and 1
μM, respectively). Both enantiomers triggered embryo
spinning, suggesting their microtubule destabilizing properties.
The absolute configuration of these chiral isomers is in
progress.
Chromenes 5{3,1}, 5{1,2}, 5{5,4}, 5{1,5}, and 5{5,5},
determined as strong antimitotics in the sea urchin embryo
assay, were further selected for the NCI60 anticancer drug
screen. These compounds displayed high cytotoxicity against a
panel of human cancer cell lines with GI50 values in the
nanomolar concentration range (Table 2; Table S1, Supporting
Information). Leukemia cells (K-562 and SR), nonsmall cell
lung carcinoma (NCI-H522), CNS carcinoma (SF-295),
melanomas (MDA-MB-435 and UACC-62), and ovarian
carcinoma (NCI/ADR-RES) were the most sensitive cell
lines to 5{5,4} with GI50 < 10 nM. Importantly, the
replacement of the lactone ring by cyano and amino groups
in compounds with 2,3,4-trimethoxyphenyl ring E increased
cytotoxicity (5{3,1} vs I; Table 2; Table S1, Supporting
Information). Generally, the sensitivity of sea urchin embryos
to chromenes was somewhat higher than that of cancer cells,
probably due to the differences between mitotic spindle
microtubules in frequently dividing sea urchin blastomeres vs
predominantly interphase microtubules in cultured cancer cells
altered by these tubulin targeting agents.
The multidrug resistant ovarian cancer cell line NCI/ADR-
RES has been proved to derive from ovarian cancer cell line
OVCAR-8 (http://dtp.nci.nih.gov/docs/misc/common_files/
NCI-ADRres.html). This line overexpresses P-glycoprotein
responsible for the transport of various substrates across cell
membrane, resulting in the type I multidrug resistance
phenotype. Importantly, all tested 4-aryl-4H-chromenes ex-
hibited higher cytotoxicity against NCI/ADR-RES cells than
against the parent OVCAR-8 cells (Table 3), suggesting their
potential to overcome multidrug resistance.
The stereo structure of PT-derived microtubule destabilizers
was reported to contribute significantly to their activity,13 For
4-aza-PTs, only R-enantiomers displayed pronounced cytotox-
icity against cultured insect cells and larvae15 as well as against
HeLa and MCF-7 human cancer cells.22 In our previous studies
R-isomer of 4-aza-PT was found to cause cleavage alteration
and cleavage arrest of the sea urchin embryo at 5 and 50 nM,
respectively, whereas the corresponding S-isomer was less
active exhibiting cleavage abnormalities at 50 nM and cleavage
arrest at 200 nM.23 Similarly, R-isomer of substituted 4H-
chromene (EPC2407, Figure 1, 3) was shown to activate
caspases, induce apoptosis in human cancer cells, and exhibit
vascular disruption effect in nanomolar concentration range,
whereas the caspase activation potency of corresponding S-
isomer was ∼50−100 times less.10,24
CONCLUSION
■
In summary, a novel synthetic approach has been developed to
obtain polyalkoxy substituted 4-aryl-4H-chromenes, namely, a
three-component domino reaction of the respective polyalkox-
ybenzaldehydes, malononitrile, and amino or alkoxyphenols.
The targeted compounds were evaluated using the in vivo
phenotypic sea urchin embryo assay that allowed for rapid and
reproducible identification of antitubulin molecules. A variety of
modifications in the ring B and 4-aryl fragment yielded potent
antimitotic agents with microtubule destabilizing activity. The
Herein, we studied the effect of stereochemistry on the
antimitotic antitubulin activity of compound 5{1,5} in the sea
urchin embryo assay. A racemic 5{1,5} was separated by chiral
HPLC to yield pure enantiomers (Figure 3; Figure S1,
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3 (1 mmol), 5 mL of EtOH and one drop of Et3N were mixed
together in a 10 mL Pyrex glass sample holder (CEM). The
reaction was carried out in a closed vessel using a focused
microwave synthesis system (CEM Discover BenchMate)
under continuous stirring. The incubation time was 4−5 min
with a fixed microwave irradiation power at 300 W and a
maximum temperature at 70 °C (TLC control). The solid
formed after cooling of the reaction mixture to 4 °C for 12 h
was recrystallized from EtOH in the presence of charcoal,
filtered off, washed successively with ethanol (3 mL) and
hexanes (3 mL) and dried.
Table 3. Growth Inhibition of OVCAR-8 and NCI/ADR-
RES Cell Lines
a
cell growth inhibition (GI50, nM)
b
c
compound
OVCAR-8
NCI/ADR-RES
5{3,1}
5{1,2}
5{5,4}
5{1,5}
5{5,5}
34.5
701.0
36.8
19.9
240.0
<10
97.1
30.2
435.0
101.0
a
GI50: Concentration required for 50% cell growth inhibition.
OVCAR-8: ovarian cancer cell line 8. NCI/ADR-RES: P-
b
c
Biology: Materials and Methods. Sea Urchin Embryo
Assay. Adult sea urchins Paracentrotus lividus were collected
from the Mediterranean Sea at the Cyprus coast and kept in an
aerated seawater tank. Gametes were obtained by intracoelomic
injection of 0.5 M KCl. Eggs were washed with filtered seawater
and fertilized by adding drops of a diluted sperm. Embryos
were cultured at room temperature under gentle agitation with
a motor-driven plastic paddle (60 rpm) in filtered seawater.
The embryos were observed with a light microscope Biolam
(LOMO, S.-Petersburg, Russia). For treatment with the test
compounds, 5 mL aliquots of embryo suspension were
transferred to 6-well plates and incubated as a monolayer at a
concentration up to 2000 embryos/mL. Stock solutions of
compounds were prepared in DMSO at 5−10 mM
concentrations, followed by a 10-fold dilution with 95%
EtOH. This procedure enhanced solubility of the test
compounds in the salt-containing medium (seawater), as
evidenced by microscopic examination of the samples. The
maximal tolerated concentrations of DMSO and EtOH in the
in vivo assay were determined to be 0.05% and 1%, respectively.
Higher concentrations of either DMSO (≥0.1%) or EtOH
(>1%) caused nonspecific alteration and retardation of the sea
urchin embryo development independent of the treatment
stage. Podophyllotoxin (Aldrich) served as reference com-
pound.
The antiproliferative activity was assessed by exposing
fertilized eggs (8−20 min after fertilization, 43−55 min before
the first mitotic cycle completion) to 2-fold decreasing
concentrations of the compound. Cleavage alteration and
arrest were clearly detected at 2.5−5.5 h after fertilization. The
effects were quantitatively estimated as a threshold concen-
tration resulting in cleavage alteration and embryo death before
hatching or full mitotic arrest. At these concentrations, all
tested microtubule destabilizers caused 100% cleavage alter-
ation and embryo death before hatching, whereas at 2-fold
lower concentrations, the compounds failed to produce any
effect. For microtubule destabilizing activity, the compounds
were tested on free-swimming blastulae just after hatching (9−
10 h after fertilization), originated from the same embryo
culture. Embryo spinning was observed after 15 min to 20 h of
treatment, depending on the structure and concentration of the
compound. Both spinning and lack of forward movement were
interpreted to be the result of the tubulin destabilizing activity
of a molecule according to previous studies.20 Video
illustrations are available at http://www.chemblock.com. Both
sea urchin embryo assay and DTP NCI60 cell line activity data
are available free of charge via the Internet at http://www.
zelinsky.ru.
glycoprotein-overexpressing multidrug resistant cell line derived from
OVCAR-8.
most active chromenes with EC values of 5 nM featured
sesamol-derived ring B, closely resembling the structure of PT.
Compounds 5{3,1}, 5{1,2}, 5{5,4}, 5{1,5}, and 5{5,5}
markedly inhibited human cancer cell growth in the NCI60
cytotoxicity screen. Interestingly, the multidrug resistant NCI/
ADR-RES cells showed higher sensitivity to these agents than
the parent OVCAR-8 cell line. The results suggest that
synthetically feasible polyalkoxy substited 4H-chromenes may
prove to be advantageous for further design as anticancer
agents.
EXPERIMENTAL PROCEDURES
■
Elemental microanalyses were obtained on an Perkin-Elmer
2400 CHN analyzer. Mass spectra were collected on the Varian
MAT-CH-6 spectrometer with direct sample injection at an
ionization voltage of 70 eV. IR spectra were recorded on IFS-
113v Bruker in KBr pellets (1:200); the frequencies were
1
expressed in cm−1. The H NMR spectra were recorded on
Bruker DRX-500 (500 MHz) and Bruker AM-300 (300 MHz)
using internal standard with DMSO-D6 as the solvent; the
chemical shifts were reported in ppm (δ) and coupling
constants (J) values were given in Hertz (Hz). Melting points
were measured on a Kofler bench. Completion of the reactions
and purity of the obtained products were monitored by thin
layer chromatography on the Silufol UV-254 plates using
hexane-acetone mixture (5:3) as an eluent and iodine vapor as a
stain.
Synthesis of 2-Amino-4H-chromenes 5. General
Procedures. Method A. Aromatic aldehyde 1 (1 mmol),
malononitrile 2 (1 mmol), and the appropriate hydroxyar-
omatic compound 3 (1 mmol) were dissolved in 5 mL of
EtOH, one drop of Et3N was added and the reaction mixture
was refluxed for 5−30 min (TLC control). After cooling of the
reaction mixture to 4 °C for 12 h fine crystalline precipitate was
formed, which was filtered off, washed successively with ethanol
(3 mL) and hexanes (3 mL) and dried.
Method B 5({4,2}, {7,3}, {6,3}, {5,4}). To a mixture of one of
the hydroxyaromatic compounds 3 (1 mmol) and arylidene
malononitrile 4 (1 mmol), which had been preliminary
synthesized from the corresponding aldehyde 1 and malononi-
trile 2, EtOH (5 mL) one drop of Et3N was added and the
reaction mixture was refluxed for 5−30 min (TLC control).
After cooling of the reaction mixture to 4 °C for 12 h fine
crystalline precipitate was formed, which was filtered off,
washed successively with ethanol (3 mL) and hexanes (3 mL)
and dried.
Method C 5({4,2}, {7,3}, {6,3}). Aromatic aldehyde 1 (1
mmol), malononitrile 2 (1 mmol), hydroxyaromatic compound
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caspase-based high throughput screening assay. 4. Structure−activity
relationships of N-alkyl substituted pyrrole fused at the 7,8-positions. J.
Med. Chem. 2008, 51, 417−423.
ASSOCIATED CONTENT
* Supporting Information
Details of experimental procedures and compound character-
ization data for synthesized compounds. This material is
available free of charge via the Internet at http://pubs.acs.org
■
S
(7) Kemnitzer, W.; Jiang, S.; Wang, Y.; Kasibhatla, S.; Crogan-
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AUTHOR INFORMATION
Corresponding Author
*E-mail: amsh@ioc.ac.ru. Phone: +7(499)1358804. Fax:
+7(499)1355328.
■
Present Address
§State Scientific Research Institute of Organic Chemistry and
Technology, 23 Sh. Entuziastov, 111024, Moscow, Russian
Federation
(9) Litvinov, Yu. M.; Shestopalov, A. M. Synthesis, structure,
chemical reactivity, and practical significance of 2-amino-4H-pyrans. In
Advances in Heterocyclic Chemistry; Katritzky, A. R., Ed.; Elsevier:
London, UK, 2011; Vol 103, Chapter 3, pp 175−260.
(10) (a) Cai, S. X.; Drewe, J.; Kemnitzer, W. Discovery of 4-aryl-4H-
chromenes as potent apoptosis inducers using a cell- and caspase-
based anti-cancer screening apoptosis program (ASAP): SAR studies
and the identification of novel vascular disrupting agents. Anti-Cancer
Agents Med. Chem. 2009, 9, 437−456. (b) http://www.epicept.com/
Products_/Product_Pipeline/Cancer/EPC2407/ @2012 EpiCept
Corporation. All Rights Reserved. (c) ClinicalTrials.gov Identifier:
NCT01240590.
Funding
This work was supported by a grant from Chemical Block Ltd.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Cancer Institute (NCI) (Bethesda, MD,
USA) for screening compounds 5{3,1}, 5{1,2}, 5{5,4}, 5{1,5},
and 5{5,5} by the Developmental Therapeutics Program at
NCI (Anticancer Screening Program; http://dtp.cancer.gov).
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2004, 428, 198−202. (b) Gupta, S.; Das, L.; Datta, A. B.; Poddar, A.;
Janik, M. E.; Bhattacharyya, B. Oxalone and lactone moieties of
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ABBREVIATIONS
PT, podophyllotoxin; SAR, structure−activity relationship
■
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