Design, synthesis, and in vitro cancer cell growth inhibition evaluation and antimalarial testing of trioxanes installed in cyclic 2-enoate substructures

Article abstract of DOI:10.1016/j.ejmech.2013.08.008

A novel series of 1,2,4-trioxanes were synthesized from 2H-pyrans via photooxidation, and their antiproliferative and growth factor inhibitory activity has been investigated across a variety of human cancer cell lines. Compounds 5k, 5l, 5s, 7a and 7c exhi

Full text of DOI:10.1016/j.ejmech.2013.08.008

European Journal of Medicinal Chemistry 69 (2013) 294e309
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and in vitro cancer cell growth inhibition evaluation
and antimalarial testing of trioxanes installed in cyclic 2-enoate
substructures
a
b
a
a
a
ꢀ
Md. Imran Hossain , Marta Switalska , Wei Peng , Mariko Takashima , Ning Wang ,
Marcel Kaiser d e, Joanna Wietrzyk b , Shingo Dan
, Takao Yamori ,
,
,
c
,
c
**
***
,
Tsutomu Inokuchi a
*
a Division of Chemistry and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku,
Okayama 700-8530, Japan
b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 R. Weigl Street, 53-114 Wroclaw, Poland
c Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550,
Japan
d Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
e University Basel, Petersplatz 1, CH-4003 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 1,2,4-trioxanes were synthesized from 2H-pyrans via photooxidation, and their anti-
proliferative and growth factor inhibitory activity has been investigated across a variety of human cancer
cell lines. Compounds 5k, 5l, 5s, 7a and 7c exhibited the highest activity and selectivity against a human
Received 11 April 2013
Received in revised form
30 July 2013
Accepted 2 August 2013
Available online 13 August 2013
leukemia (MV4-11) cell line (IC50 ¼ 0.5
m
M). Compound 5o showed the highest growth factor inhibitory
M). A SAR study has confirmed the
activity against a melanoma (LOX-IMVI) cancer cell line (GI50 ¼ 1.0
m
importance of the 1,2,4-trioxane unit as a pharmacophore for anticancer activity. The computer-assisted
database analysis, COMPARE, has suggested that the compounds have unique mechanisms of actions that
were different from those of known anticancer drugs. Some of the selected trioxanes were tested against
the NF54 strain, albeit showing weak antiplasmodial activity. The molecular docking of trioxanes and
Keywords:
Endoperoxides
1,2,4-Trioxanes
Anticancer agents
COMPARE study
Antimalarial
ꢁ
hemin reveals that a short distance (1.30 A) leads to their physical contact. The UVevis spectroscopic
analysis ensured the definite complexation between 1,2,4-trioxanes and hemin. The role of hemin
etrioxane interaction in the hemin-induced oxidative damage has been studied using methylene blue as
a substrate by UVevis spectroscopy.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
For example, as shown in Fig. 1, yingzhosu A, EDBD, arteflene, and
okundoperoxide can kill malaria parasites at nanomolar concen-
Continuous drug resistances in tumors coupled with undesir-
able side effects in patients [1] have exposed the urgency of
searching for new anticancer agents, particularly those with a novel
and selective mechanism of action. The circumvention of these
limitations has been directed to natural as well as synthetic sources
[2,3]. Endoperoxides are found in the terpenes and their derivatives
as secondary metabolites show biologically interesting activities.
trations while they exhibit cytotoxicity toward cancer-affected
mammalian cells in the nanomolar to micromolar range [4e6].
Furthermore, the unique 1,2,4-trioxanes, the peroxygenated acetal-
containing endoperoxide group, are considered today a relevant
pharmacophore over a wide range of biological activities, as
exemplified by well-known antimalarial drugs, arthemisinin and
arterolane [7e9]. The removal of one oxygen or replacement of the
peroxide group with an ether bridge from the 1,2,4-trioxanes
significantly reduces the biological significance, proving the
importance of the peroxide bridge [10e12]. The involvement of the
1,2,4-trioxane moiety in heme has been reported for its antima-
larial activity [13], and the mode of action of 1,2,4-trioxane motifs
for anticancer activity have also been examined [14]. The endo-
peroxide bridge that can be activated and fragmented with
* Corresponding author. Tel.: þ81 86 251 8210; fax: þ81 86 251 8021.
** Corresponding author. Tel.: þ48 71 3709985; fax: þ48 71 3371382.
*** Corresponding author. Tel.: þ81 3 3520 0111; fax: þ81 3 3570 0484.
E-mail addresses: wietrzyk@iitd.pan.wroc.pl (J. Wietrzyk), sdan@jfcr.or.jp
(S. Dan), inokuchi@cc.okayama-u.ac.jp (T. Inokuchi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.008

Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
295
Fig. 1. Structure of some biologically important synthetic and natural endoperoxides.
intracellular iron [Fe(II)] leads to the formation of carbon-centered
electrophilic radical species and reactive oxygen species (ROS) with
involvement of Fenton chemistry [15]. Cancer cells undergoing
rapid proliferation consume large quantities of the essential
nutrient iron through up-regulation of the transferrin receptor,
which might be directed to the similar action of mechanism against
the Plasmodium parasite [16]. Although it is not a conventional
biological target, heme is the master variable in the mechanism of
action of peroxide-containing antimalarial drugs and could be a
potential target for future anticancer drugs [14].
Recently, artemisinin-derived trioxane dimers were prepared
and shown potent as anticancer chemotherapeutics [17e20].
Furthermore, fully synthetic 1,2,4-trioxane products were designed
and shown to be promising for cancer growth inhibition [21].
Stimulated by these features, we have attempted to synthesize a
new series of endoperoxides. Although target-based screening is
now predominant, cell-based screening is still the main technique
for unveiling cytotoxic drugs [22]. Therefore, we applied two-stage
antiproliferative and cell growth inhibition activity tests. All of the
electrocyclization of 2 and
a
,
b
-unsaturated aldehydes, catalyzed by
ethylene-1,2-ammonium diacetate (EDDA) [25]. The trioxanes 5ae
w were synthesized in a high yield by photooxidation of 4aew via
generation of singlet oxygen utilizing a 250 W light bulb and the
photo sensitizer Rose bengal (Scheme 2).
To confirm the structure of the bicyclic trioxane formed by this
method, one of the isomers 5t was subjected to an X-ray crystal-
lographic analysis to determine its relative stereochemistry at the
C2 and C7 positions (Fig. 2). As depicted, one of the isomers of 5t,
isolated by recrystallization, was unambiguously assigned to the
2,7-cis isomer based on the X-ray analysis.
7,7-Disubstituted trioxanes 8 were synthesized by the direct
photooxidation of the corresponding crude 2H-pyrans 7 which
were prepared from the 6,6-diphenyldihydro-2H-pyran-2,4(3H)-
dione (6) and a,b-unsaturated aldehydes 1 according to Scheme 3,
and the results are listed in Table 3.
The synthesis of trioxane in N-protected a,b-unsaturated amides
10aed is shown in Scheme 4, and the corresponding data are listed
in Table 4. The amide analogs of trioxane were prepared by
photooxidation of the respective 2H-pyrans 9 [25,26].
Finally, 1,3-cyclohexanediones were used to produce the
respective 2H-pyrans 12aed according to the reported method
[25], which are subsequently converted into their endoperoxide
structures 13aed by photooxidation as shown in Scheme 5, and the
antiproliferative activities thereof are listed in Table 5.
derivatives are found to be active (IC50 ¼ 0.5e13
mM) during the
primary evaluation against human leukemia (MV4-11), human
lung (A549), colon (HCT116) and normal murine fibroblast (BALB/
3T3) cell lines. Some of the 1,2,4-trioxanes were examined for their
antitumor effects across a panel of 39 human cancer cell lines,
termed JFCR39, which comprised the subpanels of human cancer
cell lines derived from breast, melanoma, lung, colon, central
nervous system (CNS), ovary, stomach, kidney and prostate. An
antitumor spectrum of each compound across JFCR39, or finger-
print was calculated and COMPARE analysis was carried out to
predict the molecular mode of action of a respective antitumor
compound by comparing the fingerprints with those of reference
compounds possessing a known mechanism of action examined
previously [23].
3. Pharmacology
3.1. Antiproliferative activity
The primary antiproliferative activity of the trioxanes was
evaluated against four cell lines, i.e., human leukemia (MV4-11),
lung (A549), colon (HCT116) and normal murine fibroblast (BALB/
3T3) cell lines. The data for the in vitro anticancer activity are re-
ported in Tables 1e5, expressed as the IC50-concentration of the
2. Results and discussion
compound (in mM) that inhibits proliferation of the cells by 50%
2.1. Chemistry
The primary building blocks, polysubstituted 2H-pyrans 2aed,
were prepared according to the reported method [24]. Subse-
quently, the peroxide installation was achieved via production of
singlet oxygen with light and the sensitizer Rose bengal (Scheme 1),
the data on which are listed in Table 1.
The preparation of the trioxanes 5aew is shown in Scheme 2
and results are listed in Table 2. The main scaffold, 2H-pyrans
Scheme 1. Synthesis of 2,3,5-trioxabicyclo[2.2.2]oct-7-ene-8-carboxylate 3. Reagent
and conditions: (i) methyl 3-oxobutanoate, piperidine, AcOH, THP, rt, 24 h; (ii) Rose
4aew, was prepared by the sequence of condensation-6
p-
bengal, O2, hn
, MeOH, 0 ꢀC, 1 h.

296
Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
Table 1
Antiproliferative activity of 3aed against human leukemia MV4-11 cell line.
Entry
Compound
R1
R2
R3
Yield (%)
MV4-11
IC50 M)
(
m
Cisplatin
3a
2.820 ꢁ 0.450
>39
1
2
85
82
3b
7.38 ꢁ 0.37
3
4
3c
R1 ¼ R2
78
85
8.08 ꢁ 2.90
7.79 ꢁ 3.10
3d
compared to the untreated control cells. The IC values were sepa-
rately calculated for each experiment and the mean values ꢁ SD
were calculated from at least 3e5 independent experiments.
The antiproliferative activities of the bicyclic trioxanes 3ae
d against human leukemia (MV4-11) are listed in Table 1. Com-
pounds 3bed demonstrated a moderate activity (IC50 ¼ 7.3e
The diphenyl substituted trioxanes 8aeb show moderate
M) and similar antiproliferative activity (Table 3)
regardless of kind of the aromatic substituted group at C2 of 8.
All of the amide derivatives of the trioxanes, 10, show a good
(IC50 ¼ 3.0e3.79
m
activity with IC50 ¼ 0.5e1
mM (Table 4). The combination of the H-
acceptor N atom and the aromatic group at C-2 and C-7 is an
important feature for the antiproliferative activity in this class of
compounds.
8.0 mM), whereas compound 3a is inactive (IC50 > 39 mM). This
suggests that the aromatic group enhances the activity of entries
3bed.
The ketone derivatives of the trioxanes, compounds 13ced,
show a low activity, whereas compounds 13aeb are nontoxic to the
MV4-11 cell line even though they have an aromatic substituent at
C-2 (Table 5). The H-acceptor oxygen atom is important for the
better antiproliferative activity.
Accordingly, compounds 5a, 5f, 5iel, 5seu, 5w and 10aed were
then chosen for the next study; the antiproliferative activity against
human lung (A549) and colon (HCT116) cancer and normal murine
fibroblast (BALB/3T3) cell lines. These results are summarized in
Table 6. All the tested compounds are cytotoxic against the A549
and HCT116 cell lines, and most of their antiproliferative activities
against the cancer cells are similar to that of cisplatin used as the
control agent (Table 6). Compound 5j shows a selectivity among the
three cell lines, namely against lung cancer cells A549, it has no
The cytotoxic activities of the trioxanes derived from the 1,6-
dioxabicyclo[4.4.0]-3,9-diene-5-ones 5aew are summarized in
Table 2 against the human leukemia MV4-11 cell line, where the
cytotoxic activity of cisplatin is included as a reference compound.
All of the compounds in Table 2 are found to be cytotoxic
(IC50 ¼ 0.549e11.052
lacking the peroxide ring, exhibits a seven times lower activity
(IC50 > 24 M) activity of
mM) against MV4-11, but the 2H-pyran,
m
M) compared to the average (IC50 ¼ 3.6
m
the trioxane counterparts. This suggests that the peroxide moiety is
an important pharmacophore to stop the proliferation of cancer
cells.
Compound 5e shows the lowest cytotoxicity (IC50 ¼ 11.052
Compounds 5bed, 5geh, 5mer and 5v reveal moderate activities
M). On the other hand, a very high anti-
mM).
(IC50 ¼ 3.9e8.5
m
cytotoxic effect (IC50 > 24
normal cell line BALB/3T3, it exhibits a similar activity with the
values of IC50 ¼ 11.7 and 10.0 M respectively.
Compounds 5seu and 10aed reveal similar activities against
the A549 cell line (IC50 ¼ 6.2e8.4 M) compared to the standard
M), but compounds 5a, 5f, 5iel and 5w
mM), whereas against HCT116 and the
proliferative activity was demonstrated with compounds 5a, 5f, 5ie
l, 5seu, and 5w. Among them, the highest potency is exhibited by
m
compound 5k (IC50 ¼ 0.58
m
M), 5l (IC50 ¼ 0.54
mM) and 5s
(IC50 ¼ 0.59
m
M) with the combination of the substituted aromatic
m
group at C-2 and C-7. Compounds 5k, 5l and 5s show 13 times
cisplatin (IC50 ¼ 9.8
m
higher potency compared to its methyl substituted (at C-2) analog,
display lower activities. Against the colon cancer cell line HCT116,
the compound 5b (IC50 ¼ 7.3
m
M). Thus, the aromatic group at C-2
compounds 5a, 5f and 5j show a lower potency than the reference
might improve the antiproliferative activity. This could be more
understandable if we compare the results of entries 5aeh (aliphatic
substituent at C-2) and entries 5iew (aromatic substituent at C-2).
However, the anomalous behavior was observed only for com-
compound (IC50 ¼ 8.5
5se5u, 5w and 10aed reveal a slightly higher potency (IC50 ¼ 5.5e
8.0 M). The toxicity against the normal mice fibroblast BALB/3T3
mM). On the other hand, compounds 5i, 5kel,
m
by compounds 5a, 5f, and 5iel is lower than that of the reference,
cisplatin, however, compounds 5seu, 5w and 10aed are more
toxic.
The second stage anticancer screening of compounds 5h, 5l and
5o was performed in order to determine their cytotoxic and growth
inhibitory activities across the JFCR39 cancer cell line panel. The
compounds were evaluated at five concentration levels (100,10,1.0,
pounds 5a (IC50 ¼ 0.98
m
M) and 5f (IC50 ¼ 0.89
mM), being 5e10
times more potent than the other compounds with a methyl sub-
stituent at C-2.
0.1, and 0.01 mM). The results (the means of GI50, TGI and LC50
values) are summarized in Table 7.
Table 7 shows that compound 5h displayed an activity across
the JFCR39 panel, HBC-5 (breast cancer) and MKN1 (stomach can-
cer) being the most sensitive cell lines of the 39 cell lines
(GI50 ¼ 1.9
pound 5h also showed a strong antitumor effect on HCT116 (colon,
GI50 ¼ 2.0 M, LC50 ¼ 17 M), LOX-IMVI (melanoma, GI50 ¼ 2.1 M,
m
M for both and IC50 ¼ 7.7, 6.4
mM, respectively). Com-
Scheme 2. Synthesis of 7,8-dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one
5. Reagents and conditions: (i) Rose bengal, O2, h
n
, MeOH, 0 ꢀC, 1 h.
m
m
m

Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
297
Table 2
Antiproliferative activity of 5aew against human leukemia MV4-11 cell line.
Entry
Compound
R1
R2
R3
Yield (%)
94
MV4-11
IC50 M)
(
m
Cisplatin
5a
2.820 ꢁ 0.450
0.980 ꢁ 0.155
1
2
5b
92
7.312 ꢁ 0.774
3
4
5
5c
5d
5e
85
90
87
7.759 ꢁ 0.031
6.888 ꢁ 2.100
11.052 ꢁ 3.904
6
7
5f
98
96
0.895 ꢁ 0.465
8.545 ꢁ 1.203
5g
8
5h
97
5.701 ꢁ 1.020
9
5i
5j
90
82
0.779 ꢁ 0.129
0.901 ꢁ 0.275
10
11
12
5k
5l
84
89
0.582 ꢁ 0.100
0.549 ꢁ 0.175
13
14
5m
5n
96
94
4.839 ꢁ 2.071
5.510 ꢁ 1.307
15
5o
88
4.678 ꢁ 1.193
16
17
5p
5q
95
90
4.314 ꢁ 1.976
4.381 ꢁ 1.748
18
5r
92
3.908 ꢁ 1.954
(continued on next page)

298
Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
Table 2 (continued )
Entry
Compound
R1
R2
R3
Yield (%)
MV4-11
IC50 M)
(
m
19
20
5s
5t
93
87
0.593 ꢁ 0.107
0.735 ꢁ 0.253
21
5u
85
0.911 ꢁ 0.331
22
23
cf
5v
81
85
4.205 ꢁ 2.091
0.837 ꢁ 0.126
5w
4m
>24
LC50 ¼ 8
LC50 ¼ 8.1
m
M) and OVCAR-3 (ovarian cancer, GI50 ¼ 2.1
m
M,
is, the higher the r value, the greater the similarity of X to Y.
Generally, an r value 0.5 < r < 0.75 between two agents suggests
they might have a similar mechanism of action [28,29] (Fig. 3).
The COMPARE analysis revealed that compound 5h has a slight
similarity to 6-thioguanine (r ¼ 0.53) and 6-mercaptopurine
(r ¼ 0.50). Similarly, compounds 5l and 5o have a slight similarity
to carmofur (r ¼ 0.49) and 6-thioguanine (r ¼ 0.50), respectively.
However, these compounds did not show higher similarity (r > 0.7)
to reference anticancer drugs, suggesting that they have unique
anticancer mechanisms.
mM). Compounds 5l and 5o were also found to be active
against all of the cancer cell lines in the JFCR39 panel. Compound 5l
has a remarkable effect on HBC-5 (breast, GI50: 2.4
and NCI-H522 (lung cancer, 2.7
M, LC50 ¼ 100
pound 5o demonstrated a mentionable potency against LOX-IMVI
M), OVCAR-4 (ovarian,
M), SK-OV-3 (ovarian, GI50 ¼ 1.9 M,
M, LC50 ¼ 44 M) and
M, LC50 ¼ 22 M).
mM, LC50: 44
mM)
m
mM). Finally, com-
(melanoma, GI50 ¼ 1.0
m
M, LC50 ¼ 7.5
m
GI50 ¼ 1.5
LC50 ¼ 58
m
M, LC50 ¼ 42
m
m
mM), HBC-5 (breast, GI50 ¼ 1.9
m
m
NCI-H522 (lung cancer, IC50 ¼ 2.0
m
m
3.2. COMPARE study
3.3. Antimalarial activity
The COMPARE analysis assesses the correlation coefficient be-
tween the fingerprints of the test compounds and those of various
reference compounds [27]. This system provides an information-
intensive approach to identify the molecular targets of new com-
pounds. The JFCR39 COMPARE analysis-guided assay is a successful
means to find new anticancer drug candidates. The COMPARE
analysis is carried out by calculating the Pearson correlation coef-
ficient (r value) between the fingerprints of compounds X and Y.
The r value is then used to determine the degree of similarity, that
Trioxanes are well-known for their antimalaria activity. The
representative trioxanes from the present compound library were
tested for the antimalaria activity against the Plasmodium falcipa-
rum NF54 strain as well as the cytotoxicity against normal cell lines
and the data are listed in Table 8. Chloroquine is used as a standard
and all of the trioxanes exhibit very weak activity (IC50 ¼ 1.08e
5.6
mono-substituted (C7) ester compounds 5h, 5g, 5l, 5m and 10a
show better antimalarial activity (IC50 ¼ 1.08e1.97 g/mL) than
their disubstituted (8aed) analogs (IC50 ¼ 4.20e9.88 g/mL).
Compounds 13b and 13c that are the keto derivatives of trioxane
g/mL) against the NF54
mg/mL) compared to the standard. Among the tested trioxanes,
m
m
show weak potency (IC50 ¼ 3.41 and 5.62
m
strain and against normal cell lines (4.5 and 5.6 mg/mL).
3.4. Docking of heme and endoperoxides
The hemeetrioxane interaction plays a key role in generating
the reactive oxygen species (ROS), which could cause cellular
damage [15]. In order to study the interactions between heme and
trioxanes (5s), the hemoglobin structure obtained from PDB (1a00)
was selected and the heme extracted. Flexible docking was per-
formed by the software, Molecular Operating Environment, (MOE
2011.10) with all the rotational bonds in both trioxanes and heme as
variables. The distance between the O1 and O4 of the trioxane ring
and Fe2þ is 1.3, 3 A respectively, which indicates a definite inter-
ꢁ
Fig. 2. ORTEP drawing of crystal 5t.
action between heme and the trioxane ring (Fig. 4).

Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
299
Scheme 3. Synthesis of 7,7-diphenyl-7,8-dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one 8. Reagents and conditions: (i) ethylenediammonium diacetate (EDDA, 5 mol%),
MeOH, 2e3 h, 60 ꢀC. (ii) Rose bengal, O2, h , MeOH, 0 ꢀC, 1 h.
n
3.5. Interacting with hemin
drug cisplatin. Finally, the three candidates were employed for
third-step screening against the JFCR39 panel having breast, central
nervous system, colon, lung, melanoma, ovarian, renal, stomach
and prostate cancer subpanels and 39 cell lines were used to
measure the growth factor inhibitory (GI50) and cytotoxicity (IC50).
The computer-assisted database analysis, COMPARE suggested that
the compounds have unique mechanisms of actions which are
different from those of known anticancer drugs. Further modifi-
cation of the structures and targets is currently underway. The
antimalaria activity of the representative trioxanes were examined
and revealed very weak potency against the P. falciparum NF54
strain. The molecular docking of the representative trioxanes 5s, 5r
was performed to study the heminetrioxane interaction. The he-
minetrioxane complexation, as well as the role of this interaction
in the oxidative damage of methylene blue, was examined for the
compounds 5s, 5r and 5u by UVevis spectroscopy.
The binding study of the compounds 5r, 5s and 5u with hemin
was performed using UVevis absorption spectroscopy in 0.1 M tris
buffer (60%, pH 9.0) and DMSO (40%) mixture at ambient temper-
ature. A similar hypochromic effect was observed in other pub-
lished articles [28,29] while the concentrations of the compounds
were increased gradually. Sample concentrations were varied from
0 to 500
mM where the concentration of hemin remains constant
(6 M). Complex formation was monitored after incubation for 12 h
m
and 24 h of the hemin and samples by the decline in absorbance at
402 nm, which essentially remained unchanged. Typical spectral
changes observed for the complexation of hemin-endoperoxides
are shown below: Figs. 5 and 6.
To study the role of heminetrioxane interaction in the hemin-
induced oxidative damage, methylene blue was selected as a sub-
strate to measure the ability of the heme-catalyzed trioxane
oxidizing reaction. The test samples 5r, 5s and 5u were incubated
5. Experimental
for 2 h at 37 ꢀC with hemin (6
mM) and methylene blue (10 mM) in
0.1 M tris buffer (60%, pH 9.0) and DMSO (40%) mixture. Subse-
quently, the absorption of the resulting mixture was determined
spectrophotometrically [30,31] (Fig. 7).
5.1. General methods
The commercially obtained reagents were used without further
purification. The IR spectrawere recorded on a Shimadzu FTIR-8400S
spectrometer. The 1H NMR, 13C NMR and 19F NMR spectra were
measured on the Varian INOVA-600 or Varian INOVA-400 spec-
trometer with CDCl3 as a solvent unless otherwise indicated. High
resolution mass spectra were obtained on a Bruker micrOTOF II-SKA
spectrometer. The UV spectra were recorded with Hitachi U-2910
spectrophotometer in a 1 cm path length quartz cuvette at indicated
wavelength, and the sample was dissolved in phosphate buffer with
2.5% DMSO. Melting points were determined on a J-Science RFS-10
hot stage microscope. The scaffolds 3aed, 4aew, 6 and 9aed were
prepared by the method we previously reported [24].
4. Conclusion
This study described the synthesis of endoperoxides from 1,6-
dioxa- or 1-oxa-6-azabicyclo[4.4.0]-3,9-dien-5-ones and 1-oxabi
cyclo[4.4.0]-3,9-dien-5-ones and the three-step antiproliferative
activities were evaluated. The primary screening was done with 36
candidates against the human leukemia (MV4-11) cancer cell line
and 14 of them exhibited a higher potency (IC50 ¼ 0.5e1.0
mM) than
that of the control cisplatin (IC50 ¼ 2.8 M). The compound without
m
the peroxide ring was also tested and demonstrated more than 50
times less activity than its trioxane congeners, which suggests the
important structure activity relationship of the endoperoxides. The
most active compounds from the primary screening were chosen
for the second step screening against the human lung (A549) and
colon cancer (HCT116) and normal murine fibroblast (BALB/3T3)
cell lines. Most of them exhibit a better activity than the reference
5.2. Chemistry
5.2.1. General procedure for the synthesis of 3aed, 5aew, 6aed
and 9aed
To a solution of 2H-pyrans (1.0 mmol) in MeOH (10 ml), Rose
bengal (15 mg) was added and cooled to 0 ꢀC. The reaction flask was
fitted with oxygen gas balloon and excited externally with 250 W
Table 3
Antiproliferative activity of 8aed against human leukemia MV4-11 cell line.
Entry Compound
R1
R2
Yield (%) MV4-11
IC50 M)
(m
Cisplatin
2.820 ꢁ 0.450
3.79 ꢁ 0.019
1
2
8a
8b
76
74
3.78 ꢁ 1.092
3
4
8c
92
81
3.60 ꢁ 0.910
2.99 ꢁ 0.281
Scheme 4. Synthesis of N-protected 5-oxo-2,3,7,8-tetrahydro-3,8a-epidioxypyrano
8d
[3,2-c]pyridine-6(5H)-carboxylate 7. Reagents and conditions: (i) Rose bengal, O2, hn,
MeOH, 0 ꢀC, 1 h.

300
Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
Table 4
5.2.6. 7-(4-Bromophenyl)-2,3-dimethyl-7,8-dihydro-2H-3,8a-
epidioxypyrano[4,3-b]pyran-5(3H)-one (5a)
Antiproliferative activity of 10aed against human leukemia MV4-11 cell line.
White solids in 94% yield, mp: 169e170 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3072, 2982, 2937, 2899, 1732, 1649, 1595, 1491,
Entry Compound
R1
R2
Yield (%) MV4-11
IC50 M)
(m
1381, 1276, 1188 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.58e7.50 (m,
Cisplatin
2.820 ꢁ 0.450
0.501 ꢁ 0.062
1
2
10a
10b
86
85
2H), 7.40e7.35 (m, 1H), 7.33e7.21 (m, 2H), 5.42 (dd, J ¼ 9.0, 5.0 Hz)
and 5.32 (dd, J ¼ 12.3, 1.9 Hz) (total 1H), 4.37 (dq, J ¼ 16.3, 6.6 Hz,
1H), 2.50e2.33 (m) and 2.32e2.22 (m) (total 1H), 1.57 (s, 1H) and
1.50 (s, 2H), 1.48e1.41 (m, 1H), 1.03 (dt, J ¼ 6.6, 3.3 Hz, 2H); 13C NMR
0.661 ꢁ 0.332
(101 MHz, CDCl3)
d 140.3 (duplicated), 136.0, 132.0, 131.7 (dupli-
cated), 127.7 (duplicated), 123.0, 95.2, 94.8, 78.5, 78.3, 76.2, 75.4,
36.8, 36.4, 17.3 (duplicated), 16.98. Anal. Calcd for C16H15BrO5: C,
52.34; H, 4.12. Found: C, 52.40; H, 3.92.
3
4
10c
10d
87
84
0.517 ꢁ 0.009
1.045 ꢁ 0.438
5.2.7. 7-(3-Chlorophenyl)-2,3-dimethyl-7,8-dihydropyrano[4,3-b]
pyran-5(2H)-one (5b)
White solids in 92% yield, mp: 152e154 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3063, 2978, 2931, 2890, 1728, 1647, 1593, 1438,
halogen lamps for 1e2 h at 0 ꢀC. The solution was brought to room
temperature and solvent was removed in vacuum to afford the
crude residue, which was purified by flash column chromatography.
1383, 1276, 1190 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.40 (d,
J ¼ 9.1 Hz, 2H), 7.37e7.33 (m, 2H), 7.28e7.22 (m, 1H), 5.33 (dd,
J ¼ 12.3, 1.8 Hz, 1H), 4.36 (q, J ¼ 6.6 Hz, 1H), 2.45 (dd, J ¼ 15.4,
12.3 Hz, 1H), 2.29 (dd, J ¼ 15.3, 2.0 Hz, 1H), 1.58 (s, 1H), 1.51 (s, 2H),
1.49e1.42 (m, 1H), 1.03 (d, J ¼ 6.6 Hz, 2H); 13C NMR (101 MHz,
5.2.2. Methyl 1-ethyl-4-methyl-6-propyl-2,3,5-trioxabicyclo[2.2.2]
oct-7-ene-8-carboxylate (3a) [24]
CDCl3)
d 160.8, 141.9, 140.4, 138.9, 134.8, 131.7, 130.1, 129.1, 126.3,
White solids in 85% yield, mp: 137e138 ꢀC (ethyl acetateehex-
124.1, 95.1, 78.5, 76.2 (duplicated), 36.8 (duplicated), 17.2 (dupli-
cated), 16.9, 15.0. Anal. Calcd for C16H15ClO5: C, 59.54; H, 4.68.
Found: C, 59.12; H, 4.20.
ane); 1H NMR (600 MHz, CDCl3)
d
0.89 (t, J ¼ 7.2 Hz, 3H), 1.03 (m,
1H), 1.04 (t, J ¼ 7.2 Hz, 3H), 1.30 (m, 2H), 1.45 (m, 1H), 1.76 (s, 3H),
1.79 (m, 1H), 1.92 (m, 1H), 3.81 (s, 3H), 4.20 (dd, J ¼ 10.2, 2.4 Hz, 1H),
7.17 (s, 1H); 13C NMR (150.8 MHz, CDCl3)
d 6.8, 13.9, 18.0, 19.6, 24.8,
5.2.8. 7-(3-Methoxyphenyl)-2,3-dimethyl-7,8-dihydro-2H-3,8a-
epidioxypyrano[4,3-b]pyran-5(3H)-one (5c)
33.3, 51.9, 76.8, 78.7, 97.0, 136.4, 138.9, 162.5.
White solids in 85% yield, mp: 128e129 ꢀC (ethyl acetatee
hexane); IR (KBr) nmax ¼ 3074, 2980, 2937, 2835, 1732, 1645, 1600,
5.2.3. Methyl 1-ethyl-4-phenyl-6-propyl-2,3,5-trioxabicyclo[2.2.2]
1494, 1373, 1274, 1188 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.39 (s,
oct-7-ene-8-carboxylate (3b) [24]
Yield 82%; 1H NMR (600 MHz, CDCl3)
1.11 (t, J ¼ 7.2 Hz, 3H), 1.19 (m, 1H), 1.44 (m, 2H), 1.62 (m, 1H), 1.88
(m, 1H), 2.00 (m, 1H), 3.54 (s, 3H), 4.41 (d, d, J ¼ 10.8, 2.4 Hz, 1H),
7.23 (s, 1H), 7.39 (m, 3H), 7.54 (m, 2H); 13C NMR (150.8 MHz, CDCl3)
d
0.97 (t, J ¼ 7.2 Hz, 3H),
1H), 7.32 (t, J ¼ 7.9 Hz, 1H), 6.93 (ddd, J ¼ 10.7, 6.6, 4.8 Hz, 3H), 5.34
(dd, J ¼ 12.3, 2.0 Hz, 1H), 4.37 (q, J ¼ 6.7 Hz, 1H), 3.83 (d, J ¼ 1.5 Hz,
3H), 2.49 (dd, J ¼ 15.4, 12.3 Hz) and 2.36e2.26 (m) (total 1H), 1.59
(s) and 1.52 (s) (total 3H),1.49e1.42 (m) and 1.05 (t, J ¼ 7.3 Hz) (total
3H); 13C NMR (101 MHz, CDCl3)
d 161.2, 159.9, 140.1, 138.4, 131.9,
d
6.9,13.9,18.1, 24.9, 33.3, 51.9, 77.3, 79.4, 97.4,126.3 (2C),127.7 (2C),
129.2, 134.5, 138.36, 138.43, 162.4.
129.9, 118.2 (duplicated), 114.7, 111.4, 95.3, 78.5, 76.2, 73.8, 55.3,
36.9, 17.3 (duplicated), 16.9. Anal. Calcd for C17H18O6: C, 64.14; H,
5.70. Found: C, 64.24; H, 5.24.
5.2.4. Methyl 2-methyl-2,5,6,7,8,8a-hexahydro-2,4a-
epidioxychromene-3-carboxylate (3c) [24]
Yield 78%; 1H NMR (600 MHz, CDCl3)
d 0.87e1.69 (m, 4H), 1.73
5.2.9. 7-(4-Fluorophenyl)-2,3-dimethyl-7,8-dihydro-2H-3,8a-
epidioxypyrano[4,3-b]pyran-5(3H)-one (5d)
(s) and 1.75 (s) (total 3H), 1.80e2.11 (m, 4H), 3.77 (s) and 3.79 (s)
(total 3H), 3.40 and 4.09 (dd, J ¼ 12.0, 5.4 Hz) (total 1H), 7.13 (s) and
White solids in 90% yield, mp: 145e147 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3074, 2985, 2937, 2832, 1736, 1649, 1606, 1516,
7.22 (s) (total 1H); 13C NMR (150.8 MHz, CDCl3)
d 19.5, 19.7, 20.0,
1383, 1286, 1188 cmꢂ1; 1H NMR (400 MHz, CDCl3)
d 7.4e7.3 (m, 3H),
22.5, 22.8, 23.1, 28.4, 29.2, 30.2, 30.3, 51.9, 73.3, 73.7, 75.8, 76.7, 97.1,
98.2, 134.9, 136.1, 139.2, 140.3, 162.4, 162.7.
7.10 (td, J ¼ 8.6, 1.5 Hz, 2H), 5.44 (dd, J ¼ 8.2, 5.9 Hz) and 5.34 (dd,
J ¼ 12.3, 1.8 Hz) (total 1H), 4.37 (dt, J ¼ 19.9, 6.6 Hz, 1H), 2.5e2.3 (m)
and 2.27 (ddd, J ¼ 9.4, 2.1, 1.1 Hz) (total 2H), 1.58 (s) and 1.52 (s)
(total 3H), 1.40 (m) and 1.04 (t, J ¼ 6.5 Hz) (total 3H); 13C NMR
5.2.5. Methyl 1,4-dimethyl-6-phenyl-2,3,5-trioxabicyclo[2.2.2]oct-
7-ene-8-carboxylate (3d) [24]
Yield 85%; 1H NMR (600 MHz, CDCl3)
d
1.32 (s, 3H), 1.91 (s, 3H),
3.86 (s, 3H), 5.18 (s, 1H), 7.03 (s, 1H), 7.11 (m, 2H), 7.32 (m, 3H); 13
NMR (150.8 MHz, CDCl3) 17.8, 19.5, 52.1, 77.1, 80.1, 98.2, 127.4 (2C),
128.3 (2C), 128.7, 136.1, 136.7, 139.7, 162.5.
(151 MHz, CDCl3)
d 163.7, 162.1, 161.0, 141.8, 140.3, 140.1, 132.8
C
(duplicated), 131.82, 128.1e127.9 (m), 115.9, 115.7, 95.2, 94.94
(duplicated), 78.5, 76.30 (duplicated), 75.4, 73.8, 36.8 (duplicated),
36.4, 17.3 (duplicated), 16.9, 15.0. HRMS (FAB) calcd for [M þ H]þ
d
Scheme 5. Synthesis of 2,3,7,8-tetrahydro-3,8a-epidioxychromen-5(6H)-one 13. Reagents and conditions: (i) ethylenediammonium diacetate (EDDA, 5 mol%), MeOH, 2e3 h, 60 ꢀC.
(ii) Rose bengal, O2, h
, MeOH, 0 ꢀC, 1 h.
n

Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
301
Table 5
Antiproliferative activity of 13aed against human leukemia MV4-11 cell line.
Entry
Compound
R1
R2
R3
Yield (%)
MV4-11
IC50 M)
(
m
Cisplatin
13a
2.820 ꢁ 0.450
>31
1
2
H
H
96
97
13b
>33
3
4
13c
13d
94
96
6.532 ꢁ 0.660
6.500 ꢁ 0.951
307.0904. Found 307.0990. Anal. Calcd for C16H15FO5: C, 62.74; H,
4.94. Found: C, 62.46; H, 4.99.
17.35 (duplicated). Anal. Calcd for C17H15F3O5: C, 57.31; H, 4.24.
Found: C, 57.14; H, 3.98.
5.2.10. 7-(3,4-Dimethoxyphenyl)-2,3-dimethyl-7,8-dihydro-2H-
3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5e)
5.2.12. 3-(4-Fluorophenyl)-3,4,6,7,8,9-hexahydro-4a,9a-
epidioxypyrano[4,3-b]chromen-1(5aH)-one (5g)
White solids in 85% yield, mp: 136e137 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3068, 2980, 2937, 2837, 1732, 1649, 1595, 1519,
White solids in 96% yield, mp: 161e162 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3076, 2941, 2864, 1732, 1645, 1606, 1514, 1381,
1465, 1271, 1188 cmꢂ1
;
1H NMR (300 MHz, CDCl3)
d
7.37 (d,
1273, 1226, 1190, 1035 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.49 (d,
J ¼ 6.2 Hz, 1H), 6.99e6.81 (m, 3H), 5.40 (dd, J ¼ 10.4, 3.5 Hz) and
5.30 (dd, J ¼ 12.3, 1.9 Hz) (total 1H), 4.38 (dd, J ¼ 11.7, 6.6 Hz, 1H),
3.99e3.81 (m, 6H), 2.51 (dd, J ¼ 15.4, 12.3 Hz) and 2.41e2.21 (m)
(total 2H), 1.58 (s) and 1.50 (s) (total 3H), 1.04 (dd, J ¼ 6.6, 4.8 Hz,
J ¼ 5.7 Hz) and 7.36 (ddd, J ¼ 7.3, 5.4, 2.6 Hz) (total 3H), 7.14e7.03
(m, 2H), 5.48e5.39 (m) and 5.39e5.31 (m) (total 1H), 4.22 (td,
J ¼ 12.6, 4.9 Hz) and 3.57 (ddd, J ¼ 20.6, 11.2, 5.0 Hz) (total 1H), 2.45
(td, J ¼ 15.6, 12.3 Hz) and 2.32e2.24 (m) (total 2H), 2.24e2.15 (m,
1H), 2.15e2.03 (m, 1H), 2.03e1.70 (m, 3H), 1.70e1.52 (m) and 1.47
(ddd, J ¼ 13.4, 8.3, 3.5 Hz) (total 2H), 1.40e1.22 (m, 1H) and 1.00
(ddd, J ¼ 25.6, 13.0, 3.9 Hz) (total 1H); 13C NMR (101 MHz, CDCl3)
3H); 13C NMR (101 MHz, CDCl3)
d 161.3, 149.5, 149.2, 140.0 (dupli-
cated), 131.9, 129.3, 118.7 (duplicated), 110.9, 109.1, 95.3, 78.3
(duplicated), 76.2, 75.4, 55.9, 36.7, 36.3, 17.3 (duplicated). Anal.
Calcd for C18H20O7: C, 62.06; H, 5.79. Found: C, 62.02; H, 5.51.
d
164.1, 161.7, 161.4, 161.1, 141.1, 140.3, 140.0, 132.8, 131.6, 130.5, 128.0
(duplicated), 115.9, 115.7, 96.0, 95.1, 78.4, 78.1, 77.5, 76.3 (dupli-
cated), 75.4, 75.1, 74.2, 36.8 (triply), 30.9, 30.1 (quadruplicated),
29.2 (duplicated), 28.5, 28.2, 23.0e22.5 (m), 22.4, 19.9. Anal. Calcd
for C18H17FO5: C, 65.05; H, 5.16. Found: C, 64.91; H, 4.96.
5.2.11. 3-Methyl-2-phenyl-7-(4-(trifluoromethyl)phenyl)-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5f)
White solids in 86% yield, mp: 173e175 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3056, 2985, 2941, 2891, 1728, 1653, 1624, 1329,
1276, 1190 cmꢂ1
;
1H NMR (300 MHz, CDCl3)
d
7.68 (d, J ¼ 8.3 Hz,
5.2.13. 3-(4-(Trifluoromethyl)phenyl)-3,4,6,7,8,9-hexahydro-4a,9a-
epidioxypyrano[4,3-b]chromen-1(5aH)-one (5h)
2H), 7.57e7.48 (m, 2H), 7.41 (s, 1H), 5.50e5.36 (m, 1H), 4.36 (dd,
J ¼ 6.6, 0.7 Hz) and 3.77 (d, J ¼ 6.4 Hz) (total 1H), 2.55e2.24 (m, 2H),
1.58 (s) and 1.51 (s) (total 3H), 1.4 (d, J ¼ 6.6 Hz) and 1.03 (d,
White solids in 97% yield, mp: 156e157 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3068, 2951, 2864, 1735, 1649, 1624, 1431, 1327,
J ¼ 6.6 Hz) (total 3H); 13C NMR (151 MHz, CDCl3)
d
160.4, 140.9,
1288, 1174, 1068 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.65 (d,
140.4, 131.6, 126.31, 125.89 (duplicated), 94.8, 78.3, 76.1, 75.4, 36.5,
J ¼ 8.3 Hz, 2H), 7.53 (d, J ¼ 8.5 Hz, 2H), 6.02e5.92 (m, 1H), 5.45 (dd,
J ¼ 12.0, 4.1 Hz, 1H), 5.05 (ddd, J ¼ 16.8, 11.4, 5.2 Hz, 1H), 2.83e2.66
(m, 1H), 2.63e2.47 (m, 1H), 2.40 (dd, J ¼ 14.1, 1.9 Hz, 1H), 2.21e2.03
(m, 1H), 2.01e1.87 (m, 2H), 1.85e1.62 (m, 2H), 1.55e1.25 (m, 2H);
Table 6
13C NMR (101 MHz, CDCl3)
d 164.8 (duplicated), 163.4 (duplicated),
Antiproliferative activity of compounds 5a, 5f, 5iel, 5tew and 10aed against normal
mice fibroblast BALB/3T3 and cancer cell lines A549 and HCT116.
142.3,131.5 (duplicated), 126.1 (duplicated),125.6 (quadruplicated),
125.2, 122.5, 109.2, 108.9, 99.6, 99.3, 80.5, 80.3, 75.5 (duplicated),
35.0 (duplicated), 34.0, 33.0, 32.8, 26.8 (duplicated), 24.5 (dupli-
cated). Anal. Calcd for C19H17F3O5: C, 59.69; H, 4.48. Found: C, 59.76;
H, 3.96.
Compound
A549
IC50
HCT116
IC50 M)
Balb/3T3
IC50 M)
(
m
M)
(
m
(m
Cisplatin
5a
5f
5i
5j
5k
5l
5s
5t
9.870 ꢁ 2.400
13.017 ꢁ 5.664
13.92 ꢁ 5.052
10.92 ꢁ 4.74
>24
11.773 ꢁ 4.310
10.243 ꢁ 3.184
8.282 ꢁ 0.186
8.439 ꢁ 0.799
7.729 ꢁ 1.361
11.575 ꢁ 0.105
6.220 ꢁ 0.056
6.323 ꢁ 0.194
6.664 ꢁ 0.227
7.200 ꢁ 0.545
8.500 ꢁ 0.540
9.201 ꢁ 0.811
9.489 ꢁ 0.785
7.433 ꢁ 0.341
11.744 ꢁ 4.142
8.082 ꢁ 0.101
7.665 ꢁ 0.313
7.37 ꢁ 0.232
8.700 ꢁ 0.970
12.010 ꢁ 2.995
12.461 ꢁ 1.263
9.011 ꢁ 0.932
10.087 ꢁ 0.779
9.061 ꢁ 1.681
8.967 ꢁ 0.819
8.212 ꢁ 0.162
7.357 ꢁ 0.658
7.202 ꢁ 0.812
7.527 ꢁ 0.210
6.673 ꢁ 0.529
6.868 ꢁ 0.544
5.464 ꢁ 1.262
6.210 ꢁ 1.896
5.2.14. 3-Methyl-2-phenyl-7-(4-(trifluoromethyl)phenyl)-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5i)
White solids in 90% yield, mp: 173e175 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3066, 2991, 2939, 2893,1732, 1646,1454,1329,
1276, 1186 cmꢂ1
;
1H NMR (600 MHz, CDCl3)
d
7.70 (d, J ¼ 8.1 Hz,
7.496 ꢁ 0.869
6.785 ꢁ 1.163
7.084 ꢁ 0.316
5.539 ꢁ 0.831
5.562 ꢁ 0.466
6.023 ꢁ 1.014
7.300 ꢁ 0.521
5u
2H), 7.58 (t, J ¼ 6.4 Hz, 2H), 7.42e7.29 (m, 4H), 7.15e7.04 (m, 2H),
5.72 (d, J ¼ 11.3 Hz) and 5.52 (d, J ¼ 11.8 Hz) (total 1H), 5.28 (d,
J ¼ 34.5 Hz, 1H), 2.63 (dd, J ¼ 15.5, 12.2 Hz, 1H), 2.54e2.32 (m, 1H),
5w
10a
10b
10c
10d
1.40 (d, J ¼ 11.1 Hz, 3H); 13C NMR (151 MHz, CDCl3)
d 160.5, 141.6,
141.3, 140.8, 134.8, 132.1, 129.2 (duplicated), 128.6 (duplicated),

302
Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
Table 7
Growth inhibitory (GI50) and cytotoxic (IC50) activities of compounds 5h, 5l and 5o across the JFCR39 panel.
Tissue of origin
Breast
Cell line
5h
5l
5o
GI50
(
mM)
TGI (
m
M)
LC50
(
mM)
GI50
(
m
M)
TGI (
m
M)
LC50
(
mM)
GI50
(
m
M)
TGI (
m
M)
LC50 (mM)
HBC-4
BSY-1
HBC-5
MCF-7
MDA-MB-231
U251
SF-268
SF-295
SF-539
SNB-75
SNB-78
HCC2998
KM-12
3.8
2.9
1.9
6.4
2.9
17
6.5
17
2.3
11
12
3.8
12
3.5
2.6
2.0
12
14.0
8.5
3.9
23
7.3
32
25
33
5.2
25
31
17
6.0
15
9.0
4.9
31
50
5.4
38
38
22
6.5
4.1
4.1
6.2
7.6
21
33
32
28
44
3.5
7.2
34
29
33
31
27
38.0
32.0
7.7
66
27
64
86
62
28
56
77
48
58
80
50
17
84
11
12
2.4
11
12
18
18
18
8.4
12
14
15
25
15
4.4
4.1
18
20
2.7
19
18
14
14
3.1
3.4
9.2
5.3
12
16
16
16
19
4.5
10
14
14
15
15
15
24
26
8.0
27
26
36
41
33
23
28
34
30
51
48
24
20
40
48
8.6
38
38
31
36
12
10
24
17
41
29
30
29
47
17
31
40
31
44
29
28
54
57
44
69
58
73
94
62
55
3.7
5.0
1.9
10
5.4
16
14
18
2.8
12
13
6.3
18
5.2
3.7
3.4
17
18
2.0
20
18
10
4.4
1
2.4
1.5
2.8
9.4
1.9
17
15
18
2.1
3.4
11
16
14
16
13
13
17
4.1
29
18
33
36
33
5.4
28
34
22
39
27
17
13
36
40
5.0
41
37
27
20
9
5.1
11
7.4
38
33
31
29
45
4.4
14
36
30
37
31
27
38
46
8.8
87
51
68
92
60
14
64
90
62
85
100
100
70
79
92
22
82
72
74
95
7.5
13
42
27
100
58
59
56
100
9.1
CNS
67
84
62
Colon
Lung
100
100
100
100
93
100
100
77
78
69
92
60
42
59
42
100
54
56
55
100
49
97
HT-29
HCT-15
HCT116
NCI-H23
NCI-H226
NCI-H522
NCI-H460
A549
DMS273
DMS114
LOX-IMVI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
RXF-631L
ACHN
St-4
MKN1
MKN7
MKN28
MKN45
MKN74
DU145
20
2.1
18
100
100
78
83
63
27
8.0
8.1
31
27
86
58
61
58
18
6.4
2.3
2.1
2.1
2.3
2.8
4.8
18
17
14
18
1.9
2.6
9.3
15
9.7
16
Melanoma
Ovarian
Renal
Stomach
100
6.4
5.5
100
56
100
58
90
100
57
96
57
100
68
100
54
Prostate
PC-3
13
56
55
56
CNS: central nervous system; GI50: 50% growth inhibition concentration (mM); TGI: total growth inhibition concentration (mM); LC50: lethal concentration (mM).
127.8 (duplicated), 126.3 (duplicated), 125.93 (duplicated), 95.9,
81.8, 80.5, 78.8, 78.6, 76.2 (duplicated), 36.75 (duplicated), 17.68
(duplicated). Anal. Calcd for C22H17F3O5: C, 63.16; H, 4.10. Found: C,
63.24; H, 3.84.
(m, 2H), 5.61 (dd, J ¼ 9.3, 4.7 Hz) and 5.43 (dd, J ¼ 12.2, 1.9 Hz)
(total 1H), 5.21 (d, J ¼ 15.7 Hz,1H), 3.80 (d, J ¼ 3.4 Hz, 3H), 2.63 (dd,
J ¼ 15.5, 12.2 Hz) and 2.48e2.32 (m) (total 2H), 1.57 (d, J ¼ 3.2 Hz)
and 1.37 (d, J ¼ 5.2 Hz) (total 3H); 13C NMR (101 MHz, CDCl3)
d
164.2, 160.9, 160.2, 141.5, 141.3, 132.7, 132.2, 128.6 (duplicated),
5.2.15. 7-(3,4-Dimethoxyphenyl)-3-methyl-2-phenyl-7,8-dihydro-
2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5j)
128.0 (duplicated), 127.0 (duplicated), 116.0, 115.8, 114.0 (dupli-
cated), 96.1, 95.6, 81.5, 80.3, 78.8, 76.8e76.2 (m), 55.3 (duplicated),
36.6 (duplicated). Anal. Calcd for C22H19FO6: C, 66.33; H, 4.81.
Found: C, 66.25; H, 4.61.
White solids in 82% yield, mp: 155e156 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3063, 2941, 2833, 1735, 1647, 1519, 1454, 1267,
1238,1190,1026 cmꢂ1,1H NMR (400 MHz, CDCl3)
d
7.34 (ddd, J ¼ 21.0,
12.7, 8.4 Hz, 4H), 7.17e7.08 (m, 2H), 7.01e6.93 (m, 2H), 6.88 (d,
J ¼ 8.5 Hz,1H), 5.59 (d, J ¼ 1.6 Hz) and 5.40 (dd, J ¼ 12.2,1.8 Hz) (total
1H), 5.30 (s) and 5.24 (s) (total 1H), 3.95e3.86 (m, 6H), 2.68 (dd,
J ¼ 15.5, 12.3 Hz) and 2.48e2.37 (m) (total 2H), 1.58 (s) and 1.39 (d,
5.2.17. 7-(3-Chlorophenyl)-2-(4-methoxyphenyl)-3-methyl-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5l)
White solids in 89% yield, mp: 153e154 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3061, 2968, 2841, 1735, 1637, 1612, 1516, 1383,
J ¼ 8.0 Hz) (total 3H); 13C NMR (101 MHz, CDCl3)
d
160.5, 141.6,141.3,
1283, 1274, 1190, 1074 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.46 (s,
134.8, 132.1, 129.2, 128.6 (duplicated), 127.3 (duplicated), 126.3
(duplicated), 125.9 (duplicated), 95.9, 81.8, 80.5, 78.8, 76.2, 36.7, 17.6.
Anal. Calcd for C23H22O7: C, 67.31; H, 5.40. Found: C, 67.40; H, 4.95.
1H), 7.41e7.32 (m, 4H), 7.10e6.99 (m, 2H), 6.86 (t, J ¼ 9.2 Hz, 2H),
5.60 (dd, J ¼ 11.3, 2.5 Hz) and 5.42 (dd, J ¼ 12.2, 1.9 Hz) (total 1H),
5.21 (d, J ¼ 21.6 Hz, 1H), 3.80 (d, J ¼ 4.4 Hz, 3H), 2.61 (dd, J ¼ 15.5,
12.2 Hz) and 2.51e2.31 (m) (total 2H), 1.58 (s) and 1.37 (d,
5.2.16. 7-(4-Fluorophenyl)-2-(4-methoxyphenyl)-3-methyl-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5k)
White solids in 84% yield, mp: 151e152 ꢀC (ethyl acetatee
hexane); IR (KBr) nmax ¼ 3076, 2937, 2843, 1722, 1612, 1514, 1383,
J ¼ 7.0 Hz) (total 3H); 13C NMR (101 MHz, CDCl3)
d 160.7, 160.2
(duplicated), 141.6, 141.4, 138.8, 134.9, 132.2, 130.2, 129.2, 128.6
(duplicated), 126.9, 126.3 (duplicated), 124.1 (duplicated), 114.0
(duplicated), 96.0, 95.6, 81.5, 80.2, 78.9, 78.7, 76.2 (duplicated), 55.3
(duplicated), 36.7 (duplicated), 17.7 (duplicated). Anal. Calcd for
1282, 1226, 1195, 1033 cmꢂ1 1H NMR (300 MHz, CDCl3)
; d 7.47e
7.40 (m, 2H), 7.33 (d, J ¼ 10.1 Hz, 1H), 7.16e6.98 (m, 4H), 6.94e6.78
C22H19ClO6: C, 63.70; H, 4.62. Found: C, 63.62; H, 4.40.

Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
303
Fig. 3. Growth inhibitory- and cytotoxic activities of compounds 5l and 5h across a panel of JFCR39 cell lines. The mean graph was produced by computer processing of the 50%
growth inhibition (GI50) and the 50% lethal concentration (LC50) values. Logarithm of the GI50 and the LC50 values for each cell line are indicated. The X-axis shows the difference in
logarithmic scale between the mean of Log GI50/Log LC50 values for all 39 cell lines (MG-MID, expressed as 0 in the fingerprint) and the Log GI50/Log LC50 for each cell line in the
JFCR39 panel. Columns to the right of 0 indicate the sensitivity of the cell lines to a given compound and columns to the left indicate their resistance. MG-MID ¼ mean of Log GI50
Log LC50 values for all 39 cell lines; delta ¼ difference between the MG-MID and the Log GI50/Log LC50 value for the most sensitive cell line; range ¼ difference between the Log GI50
Log LC50 values for the most resistant cell line and the most sensitive cell line.
/
/
5.2.18. 2-(4-Methoxyphenyl)-3-methyl-7-(4-(trifluoromethyl)
phenyl)-7,8-dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-
one (5m)
(duplicated), 126.9, 126.36 (duplicated), 125.9 (duplicated),
114.0 (duplicated), 95.9, 95.5, 81.5, 80.3, 78.9, 78.7, 76.22
(duplicated), 55.3 (duplicated), 36.7 (duplicated), 17.6 (dupli-
cated). Anal. Calcd for C23H19F3O6: C, 61.61; H, 4.27. Found: C,
61.55; H, 3.96.
White solids in 96% yield, mp: 158e159 ꢀC (ethyl acetatee
hexane); IR (KBr) nmax ¼ 3066, 2939, 2843, 1726, 1647, 1614,
1514, 1327, 1280, 1172, 1124, 1066 cmꢂ1 1H NMR (400 MHz,
;
CDCl3)
d
7.70 (d, J ¼ 8.2 Hz, 2H), 7.57 (d, J ¼ 8.2 Hz, 2H), 7.35 (d,
5.2.19. 2-(4-Bromophenyl)-3-methyl-7-(4-(trifluoromethyl)
phenyl)-7,8-dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-
one (5n)
J ¼ 11.6 Hz, 1H), 7.03 (tt, J ¼ 9.6, 2.4 Hz, 2H), 6.90e6.80 (m, 2H),
5.69 (dd, J ¼ 11.5, 2.4 Hz) and 5.53e5.48 (m) (total 1H), 5.23 (s)
and 5.19 (s) (total 1H), 3.80 (d, J ¼ 5.4 Hz, 3H), 2.61 (dd, J ¼ 15.5,
12.1 Hz) and 2.43 (dddd, J ¼ 18.8, 17.4, 9.5, 7.6 Hz) (total 2H),
1.57 (s) and 1.38 (d, J ¼ 6.6 Hz) (total 3H); 13C NMR (151 MHz,
White solids in 94% yield, mp: 169e171 ꢀC (ethyl acetatee
hexane); IR (KBr) nmax ¼ 3070, 2987, 2828, 1720, 1647, 1521, 1383,
1225, 1168, 1066 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.70 (d,
CDCl3)
d
160.6, 160.2, 141.8, 141.6, 140.8, 132.1, 131.3, 128.6
J ¼ 8.2 Hz, 2H), 7.58 (dd, J ¼ 8.2, 2.0 Hz, 2H), 7.53e7.43 (m, 2H),
7.30 (d, J ¼ 10.6 Hz, 1H), 7.04e6.92 (m, 2H), 5.69 (dd, J ¼ 11.5,
2.4 Hz) and 5.51 (dd, J ¼ 12.1, 1.9 Hz) (total 1H), 5.23 (d,
J ¼ 21.6 Hz, 1H), 2.62 (dd, J ¼ 15.5, 12.1 Hz) and 2.53e2.36 (m)
(total 2H), 1.40 (d, J ¼ 5.4 Hz, 3H); 13C NMR (101 MHz, CDCl3)
Table 8
Antimalaria activity of trioxanes against P. falciparum NF54 and cytotoxicity against
L6.
d
160.4, 160.1, 141.2, 140.9, 140.7, 134.0 (duplicated), 132.3, 131.8
(duplicated), 128.9 (duplicated), 126.4, 126.1 (duplicated), 125.1,
123.5 (duplicated), 95.9, 95.5, 81.1, 79.9, 78.4 (duplicated), 76.2
(duplicated), 36.6 (duplicated), 29.6, 17.62 (duplicated). Anal.
Calcd for C22H16BrF3O5: C, 53.14; H, 3.24. Found: C, 53.09; H,
3.07.
Compound
P. falc. NF54 IC50 mg/mL
Cytotox. L6 IC50 mg/mL
Chloroquine
0.002
1.17
1.08
3.28
1.68
1.38
5.25
4.20
9.88
5.62
1.97
3.41
3.10
0.007
0.44
0.50
0.52
0.50
0.46
3.49
1.90
2.03
1.67
1.16
4.5
5g
5h
5j
5k
5l
8a
8b
8c
8d
10a
13b
13c
5.2.20. 7-(3-Methoxyphenyl)-2-(4-methoxyphenyl)-3-methyl-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5o)
White solids in 88% yield, mp: 157e158 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3063, 2997, 2935, 2839, 1724, 1643, 1612, 1516,
1383, 1278, 1247, 1174, 1028 cmꢂ1 1H NMR (400 MHz, CDCl3)
;
d
7.29e7.36 (m, 3H), 7.06e7.02 (m, 2H), 6.96 (d, J ¼ 13.2, 2H), 6.82e
5.6
6.92 (m, 2H), 5.57 (q, J ¼ 4.4 Hz) and 5.40 (dd, J ¼ 10.0, 4 Hz) (total

304
Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
5.23 (s) and 5.18 (s) (total 1H), 3.80 (d, J ¼ 4.4 Hz, 3H), 2.61 (dd,
J ¼ 15.5, 12.2 Hz) and 2.40 (ddd, J ¼ 15.5, 7.1, 2.1 Hz) (total 2H),
1.37 (d, J ¼ 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 160.8, 160.2,
141.6, 141.3, 135.4, 135.0, 132.2, 129.1, 128.6 (duplicated), 127.4
(duplicated), 126.9, 114.0 (duplicated), 96.0, 81.5, 80.3, 78.7
(duplicated), 76.37 (duplicated), 55.3, 36.6 (duplicated), 17.7
(duplicated) Anal. Calcd for C22H19ClO6: C, 63.70; H, 4.62. Found:
C, 63.57; H, 4.58.
5.2.22. 7-(3,4-Dimethoxyphenyl)-2-(4-methoxyphenyl)-3-methyl-
7,8-dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5q)
White solids in 90% yield, mp: 144e145 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3068, 2993, 2937, 2837, 1727, 1641, 1612, 1516,
1448, 1381, 1261, 1238, 1184, 1026 cmꢂ1; 1H NMR (400 MHz, CDCl3)
d
7.34 (s) and 7.31 (s) (total 1H), 7.05 (dd, J ¼ 8.6, 6.3 Hz, 2H), 7.00e
6.92 (m, 2H), 6.91e6.81 (m, 3H), 5.57 (s) and 5.39 (dd, J ¼ 12.2,
1.8 Hz) (total 1H), 5.24 (s) and 5.18 (s) (total 1H), 3.96e3.86 (m, 6H),
3.84e3.76 (m, 3H), 2.67 (dd, J ¼ 15.5, 12.3 Hz, 1H), 2.48e2.35 (m,
1H),1.37 (d, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 161.2, 160.1,
149.6, 149.2, 141.3, 141.0, 132.4, 129.2, 128.6 (duplicated), 127.1
(duplicated), 118.7 (duplicated), 113.9 (duplicated), 111.0, 109.1
(duplicated), 96.2, 81.5, 80.2, 78.7 (duplicated), 55.9 (duplicated),
55.3 (duplicated), 36.6 (duplicated), 17.7 (duplicated). Anal. Calcd
for C24H24O8: C, 65.45; H, 5.49. Found: C, 65.30; H, 5.35.
5.2.23. 7-(3-Chlorophenyl)-3-methyl-2-(4-nitrophenyl)-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5r)
White solids in 90% yield, mp: 167e168 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3080, 2985, 2860, 1732, 1608, 1521, 1350, 1276,
1192, 1035 cmꢂ1
;
1H NMR (400 MHz, CDCl3)
d
8.22 (d, J ¼ 8.7 Hz,
1H), 8.16 (d, J ¼ 8.7 Hz, 1H), 7.47 (d, J ¼ 1.2 Hz, 1H), 7.42e7.21 (m,
6H), 5.63 (dd, J ¼ 11.1, 2.8 Hz), 5.48e5.39 (m) and 5.36 (s) (total 2H),
2.68 (dd, J ¼ 15.5, 12.1 Hz) and 2.54e2.38 (m) (total 2H), 1.43 (d,
J ¼ 3.4 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 160.2, 159.9, 148.3,
141.9 (duplicated), 140.2 (duplicated), 138.5 (duplicated), 134.9,
132.5, 130.2 (duplicated), 129.3 (duplicated), 128.3 (duplicated),
126.2 (duplicated), 124.2, 124.0, 123.7 (duplicated), 95.9, 95.5, 80.6,
79.5, 78.2 (duplicated), 76.2, 36.3 (duplicated), 17.6 (duplicated).
Anal. Calcd for C21H16ClNO7: C, 58.68; H, 3.75; N, 3.26. Found: C,
58.55; H, 3.63; N, 3.16.
5.2.24. 7-(4-Chlorophenyl)-3-methyl-2-(4-nitrophenyl)-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5s)
White solids in 93% yield, mp: 180e181 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3072, 2987, 2939, 1732, 1606, 1523, 1350, 1276,
Fig. 4. Molecular docking of trioxane derivatives and heme. (a) and (b) refer to the
binding modes of 5s with heme, the green lines represents the distance of Fe2þ (black)
4
to the O1 (1.31 A) and O (3 A) of the trioxane. (c) and (d) represents the binding modes
ꢁ
ꢁ
of 5r with heme, the distances from the green lines represents the distance of Fe2þ
1192, 1014 cmꢂ1 1H NMR (300 MHz, CDCl3)
; d 8.27e8.15 (m, 2H),
4
(black) to the O1 (1.30 A) and O (3.03 A) of the trioxane. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article.)
ꢁ
ꢁ
7.46e7.22 (m, 7H), 5.63 (dd, J ¼ 10.4, 3.6 Hz) and 5.48e5.39 (m) and
5.36 (s) (total 2H), 2.65 (dd, J ¼ 15.5, 12.0 Hz) and 2.56e2.34 (m)
(total 2H), 1.43 (d, J ¼ 4.0 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d
160.0, 148.4, 141.9 (duplicated), 140.2 (duplicated), 135.1 (dupli-
1H), 5.23 (s) and 5.18 (s) (total 1H), 3.81 (t, J ¼ 4 Hz, 6H). 2.63e2.70
(m) and 2.35e2.48 (m) (total 2H), 1.38 (d, J ¼ 8.4 Hz, 3H); 13C NMR
cated), 132.5, 129.2 (duplicated), 128.3 (duplicated), 127.4 (dupli-
cated), 123.7 (duplicated), 95.61, 80.7, 79.5, 78.2 (duplicated), 76.4,
36.4 (duplicated), 17.6 (duplicated). Anal. Calcd for C21H16ClNO7: C,
58.68; H, 3.75; N, 3.26. Found: C, 58.68; H, 3.38; N, 3.13.
(101 MHz, CDCl3)
d 161.2, 160.1, 141.2, 140.9, 132.4, 128.7 (triply),
127.7 (duplicated), 127.1, 114.2, 113.9 (duplicated), 96.2, 95.8, 81.5,
80.2, 78.7 (duplicated), 55.32 (duplicated), 36.4 (duplicated), 17.72
(duplicated). Anal. Calcd for C23H22O7: C, 67.31; H, 5.40. Found: C,
67.15; H, 5.10.
5.2.25. 7-(4-Methoxyphenyl)-3-methyl-2-(4-nitrophenyl)-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5t)
White solids in 87% yield, mp: 177e178 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3080, 2937, 2839, 1728, 1612, 1519, 1350, 1251,
5.2.21. 7-(4-Chlorophenyl)-2-(4-methoxyphenyl)-3-methyl-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5p)
White solids in 95% yield, mp: 154e155 ꢀC (ethyl acetatee
hexane); IR (KBr) nmax ¼ 3068, 2982, 2935, 2839, 1724, 1647, 1612,
1180, 1033 cmꢂ1; 1H NMR (300 MHz, CDCl3)
d
8.21 (ddt, J ¼ 11.1, 9.2,
2.1 Hz, 2H), 7.40e7.19 (m, 5H), 6.99e6.90 (m, 2H), 5.60 (dd, J ¼ 9.2,
4.9 Hz) and 5.42 (dd, J ¼ 11.6, 2.4 Hz) and 5.36 (s) (total 2H), 3.83 (s,
3H), 2.71 (dd, J ¼ 15.5, 12.0 Hz) and 2.53e2.36 (m) (total 2H), 1.43
1514, 1381, 1278, 1240, 1182, 1033 cmꢂ1 1H NMR (400 MHz,
;
CDCl3)
d
7.44e7.30 (m, 5H), 7.08e6.98 (m, 2H), 6.89e6.80 (m, 2H),
(d, J ¼ 4.9 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 160.7, 160.2, 148.3,
5.61 (dd, J ¼ 11.0, 3.1 Hz), 5.43 (dd, J ¼ 12.2, 2.0 Hz) (total 1H),
141.9, 139.8, 132.8, 128.5 (duplicated), 127.7, 123.7 (duplicated),

Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
305
Fig. 5. UVevis absorption spectra of the compounds 5r, 5s and 5u after the incubation for 12 h at 37 ꢀC in 0.1 M tris buffer (60%, pH 9.0) and DMSO (40%) mixture. The curves 1e7
for the Chemin ¼ 6 M and C5r, ¼ 0, 25, 50, 150, 300, 400 and 500 M to the final solution. A gradual hypochromic shift of the absorptions is observed as the compounds
concentrations are increases.
m
m
5s, 5u
114.2, 96.1, 80.6, 78.2, 55.39, 36.2, 17.6. Anal. Calcd for C22H19NO8: C,
62.12; H, 4.50; N, 3.29. Found: C, 62.33; H, 4.42; N, 3.19.
(duplicated), 132.3, 131.82 (duplicated), 129.2e128.5 (m), 127.4
(duplicated), 123.4, 95.9, 95.5, 81.1, 79.9, 78.4 (duplicated), 76.3
(duplicated), 36.5 (duplicated), 17.6 (duplicated). Anal. Calcd for
5.2.26. 7-(3,4-Dimethoxyphenyl)-3-methyl-2-(4-nitrophenyl)-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5u)
White solids in 85% yield, mp: 174e175 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3076, 2939, 2835, 1730, 1608, 1523, 1352, 1274,
C21H16BrClO5: C, 54.39; H, 3.48. Found: C, 54.47; H, 3.34.
5.2.28. 7-(3-Bromophenyl)-3-methyl-2-(4-nitrophenyl)-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5w)
White solids in 84% yield, mp: 164e165 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3080, 2985, 2937, 1732, 1651, 1606, 1521, 1350,
1190, 1024 cmꢂ1 1H NMR (300 MHz, CDCl3)
; d 8.29e8.12 (m, 2H),
7.30 (dt, J ¼ 17.6, 8.5 Hz, 3H), 7.02e6.84 (m, 3H), 5.59 (dd, J ¼ 8.7,
5.4 Hz) and 5.45e5.32 (m) (total 2H), 3.91 (d, J ¼ 4.8 Hz, 6H), 2.70
(dd, J ¼ 15.5, 12.1 Hz) and 2.54e2.37 (m) (total 2H), 1.43 (d,
1276, 1192, 1035 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 8.30e8.19 (m,
1H), 8.19e8.08 (m, 1H), 7.63 (dd, J ¼ 3.8, 1.8 Hz, 1H), 7.57e7.47 (m,
1H), 7.44e7.17 (m, 5H), 5.62 (dd, J ¼ 11.2, 2.7 Hz) and 5.50e5.30 (m)
(total 2H), 2.67 (dd, J ¼ 15.5, 12.1 Hz) and 2.57e2.36 (m) (total 2H),
J ¼ 4.5 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 160.7, 149.7, 149.3,
148.3, 142.2 (duplicated), 139.9, 132.7, 128.9, 128.4, 123.7 (dupli-
cated), 118.7 (duplicated), 111.0, 109.1, 96.1, 95.7, 80.6, 78.1 (dupli-
cated), 55.9, 36.4, 17.6. Anal. Calcd for C23H21NO9: C, 60.66; H, 4.65;
N, 3.08. Found: C, 60.75; H, 4.53; N, 3.05.
1.44 (d, J ¼ 4.0 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 160.2, 148.3,
141.9 (duplicated), 140.2 (duplicated), 138.8, 132.5 (triply), 130.5,
129.1 (duplicated), 128.3 (duplicated), 124.7, 124.5, 123.7 (dupli-
cated), 123.0, 95.9, 95.5, 80.6, 79.5, 78.2 (duplicated), 76.1, 36.4
(duplicated), 17.62 (duplicated). Anal. Calcd for C21H16BrNO7: C,
53.18; H, 3.40; N, 2.95. Found: C, 53.03; H, 3.19; N, 2.76.
5.2.27. 2-(4-Bromophenyl)-7-(4-chlorophenyl)-3-methyl-7,8-
dihydro-2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (5v)
White solids in 81% yield, mp: 178e179 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3070, 2987, 2901, 1720, 1647, 1494, 1383, 1282,
5.2.29. 3-Methyl-2-(4-nitrophenyl)-7,7-diphenyl-7,8-dihydro-2H-
3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (8a)
1197, 1070 cmꢂ1 1H NMR (300 MHz, CDCl3)
; d 7.49e7.29 (m, 6H),
7.28e7.20 (m, 1H), 7.00e6.90 (m, 2H), 5.56 (s) and 5.39 (dd, J ¼ 12.1,
2.1 Hz) (total 1H), 5.19 (d, J ¼ 15.5 Hz,1H), 2.58 (dd, J ¼ 15.5,12.1 Hz)
and 2.43e2.28 (m) (total 2H), 1.35 (d, J ¼ 4.4 Hz, 3H); 13C NMR
White solids in 76% yield, mp: 202e203 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3070, 2993, 2931, 1724, 1643, 1521, 1450, 1346,
1267 cmꢂ1; 1H NMR (400 MHz, CDCl3)
(dd, J ¼ 7.5, 2.2 Hz, 2H), 7.45e7.28 (m, 8H), 7.07 (s, 1H), 6.35 (d,
d
7.86 (d, J ¼ 8.8 Hz, 2H), 7.60
(101 MHz, CDCl3)
d
160.6, 140.9, 140.7, 135.2, 135.0, 134.0

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Fig. 6. UVevis absorption spectra of the mixture of hemin and compounds 5r, 5s and 5u after the incubation for 24 h at 37 ꢀC in 0.1 M tris buffer (60%, pH 9.0) and DMSO (40%)
M to the final solution. A gradual hypochromic shift of the absorptions at 402 nm
mixture. The curves 1e7 are for the Chemin ¼ 6
m
M and C5r, 5s, 5u ¼ 0, 25, 50, 150, 300, 400 and 500
m
is observed as the compounds concentrations are increases.
J ¼ 8.5 Hz, 2H), 5.24 (s, 1H), 3.40 (dd, J ¼ 15.4, 0.5 Hz, 1H), 2.93 (d,
1182 cmꢂ1; 1H NMR (600 MHz, CDCl3)
d 7.59e7.55 (m, 2H), 7.41 (dd,
J ¼ 15.5 Hz,1H),1.35 (s, 3H); 13C NMR (101 MHz, CDCl3)
d
160.3,142.4,
J ¼ 8.4, 1.0 Hz, 2H), 7.39e7.31 (m, 5H), 7.29 (d, J ¼ 7.3 Hz, 1H), 7.09 (s,
1H), 7.01e6.97 (m, 2H), 6.16 (d, J ¼ 8.4 Hz, 2H), 5.10 (s, 1H), 3.37 (d,
J ¼ 15.5 Hz, 1H), 2.91 (d, J ¼ 15.5 Hz, 1H), 1.31 (s, 3H); 13C NMR
141.9, 141.2, 139.7, 131.7, 128.5 (duplicated), 127.9, 127.1, 125.4, 123.2,
95.43, 85.94, 80.17, 78.21, 38.2, 17.6; Anal. Calcd for C27H21NO7: C,
68.78; H, 4.49; N, 2.97. Found: C, 68.81; H, 4.55; N, 2.88.
(151 MHz, CDCl3)
d 160.6, 142.6, 141.2, 140.4, 134.48, 133.4, 131.4,
128.6, 128.3, 127.0, 125.4, 95.4, 85.9, 80.4, 78.4, 38.41 (s, 5H), 31.5,
22.6, 17.6, 14.1. Anal. Calcd for C27H21ClO5: C, 70.36; H, 4.59. Found:
C, 70.78; H, 5.22.
5.2.30. 2-(4-Methoxyphenyl)-3-methyl-7,7-diphenyl-7,8-dihydro-
2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (8b)
White solids in 82% yield, mp: 175e176 ꢀC (ethyl acetatee
hexane); IR (KBr) nmax ¼ 3055, 2999, 2837, 1739, 1645, 1514, 1388,
1271, 1174 cmꢂ1
;
1H NMR (600 MHz, CDCl3)
d
7.36 (dd, J ¼ 8.3,
5.2.32. Methyl 4-(3-methyl-5-oxo-7,7-diphenyl-3,5,7,8-tetrahydro-
2H-3,8a-epidioxypyrano[4,3-b]pyran-2-yl)benzoate (8d)
1.0 Hz, 2H), 7.30 (t, J ¼ 7.7 Hz, 2H), 7.27e7.11 (m, 8H), 6.87e6.78
(m, 2H), 6.23 (s, 1H), 5.64 (s, 1H), 3.84 (s, 3H), 3.23 (d, J ¼ 17.6 Hz,
1H), 3.12 (d, J ¼ 17.6 Hz, 1H), 1.56 (d, J ¼ 7.8 Hz, 3H); 13C NMR
White solids in 76% yield, mp: 200e201 ꢀC (ethyl acetatee
hexane); IR (KBr) nmax ¼ 3063, 2955, 1722, 1651, 1492, 1450, 1348,
1197 cmꢂ1
;
1H NMR (600 MHz, CDCl3)
d
7.68 (d, J ¼ 8.4 Hz, 2H),
(151 MHz, CDCl3)
d 164.5, 161.9, 160.2, 143.4, 142.5, 130.1, 129.3,
7.60e7.55 (m, 2H), 7.43e7.40 (m, 2H), 7.40e7.31 (m, 5H), 7.29 (d,
J ¼ 7.3 Hz, 1H), 7.08 (s, 1H), 6.30 (d, J ¼ 8.2 Hz, 2H), 5.19 (s, 1H),
3.90 (s, 3H), 3.38 (d, J ¼ 15.4 Hz, 1H), 2.93 (d, J ¼ 15.4 Hz, 1H), 1.31
128.43 (duplicated), 127.66 (duplicated), 125.8, 125.4, 125.1, 114.1,
113.2, 102.0, 83.9, 82.93, 55.3, 37.3, 19.2; Anal. Calcd for C28H24O6:
C, 73.67; H, N, 5.30. Found: C, 73.79; H, 5.31.
(s, 3H); 13C NMR (151 MHz, CDCl3)
d 166.4, 160.6, 142.6, 141.2,
140.3, 139.8, 131.5, 130.3, 129.2, 128.64 (duplicated), 128.4, 128.2,
127.03 (duplicated), 125.4, 95.4, 85.9, 80.7, 78.4, 52.2, 38.4, 17.6;
Anal. Calcd for C29H24O7: C, 71.89; H, 4.99. Found: C, 71.20; H,
4.56.
5.2.31. 2-(4-Chlorophenyl)-3-methyl-7,7-diphenyl-7,8-dihydro-
2H-3,8a-epidioxypyrano[4,3-b]pyran-5(3H)-one (8c)
White solids in 88% yield, mp: 180e181 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3063, 3005, 2955,1724,1645,1448,1348,1280,

Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
307
Fig. 7. UVevis absorption spectra of the mixture of hemin, methylene blue and compounds 5r, 5s and 5u after the incubation for 2 h at 37 ꢀC in 0.1 M tris buffer (60%, pH 9.0) and
DMSO (40%) mixture. The curves 1e10 are for the Chemin ¼ 6 M, CM. ¼ 10 M, C5r, ¼ 0, 5, 10, 25, 50, 100, 200, 300, 500 and 700 M to the final solution. A regular
hypochromic shift of the absorptions at 400 nm is observed for hemin and at 290 and 668 nm for methylene blue as the compounds concentrations are increases.
m
m
m
blue
5s, 5u
5.2.33. tert-Butyl 7-(4-chlorophenyl)-3-methyl-2-(4-nitrophenyl)-
5-oxo-2,3,7,8-tetrahydro-3,8a-epidioxypyrano[3,2-c]pyridine-
6(5H)-carboxylate (10a)
128.6 (m), 128.2, 127.7, 127.4, 127.1, 126.8, 113.8, 113.3, 95.9 (dupli-
cated), 84.5 (duplicated), 81.0, 80.5, 78.9, 55.2 (duplicated), 54.7e
54.5 (m), 34.6, 27.8, 17.7. Anal. Calcd for C27H28ClNO7: C, 63.10; H,
5.49; N, 2.73. Found: C, 63.05; H, 5.28; N, 2.83.
White solids in 86% yield, mp: 163e164 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3084, 2983, 2927, 1776, 1720, 1645, 1521, 1384,
1288, 1149 cmꢂ1
;
1H NMR (600 MHz, CDCl3)
d
7.95 (d, J ¼ 8.6 Hz,
5.2.35. tert-Butyl 7-(4-chlorophenyl)-3-methyl-5-oxo-2-phenyl-
2,3,7,8-tetrahydro-3,8a-epidioxypyrano[3,2-c]pyridine-6(5H)-
carboxylate (10c)
2H), 7.37 (d, J ¼ 8.5 Hz, 2H), 7.27 (d, J ¼ 8.0 Hz, 2H), 7.06 (s, 1H), 6.49
(d, J ¼ 8.6 Hz, 2H), 5.73 (s, 1H), 5.16 (s, 1H), 2.89 (dd, J ¼ 15.0, 4.2 Hz,
1H), 2.62 (dd, J ¼ 15.0, 4.9 Hz, 1H), 1.51 (d, J ¼ 12.6 Hz, 9H), 1.35 (s,
White solids in 87% yield, mp: 109e110 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3071, 2982, 2935, 2839, 1770, 1722, 1639, 1514,
3H); 13C NMR (151 MHz, CDCl3)
d 159.2, 151.8, 147.9, 142.1, 138.3,
137.9, 137.5, 135.7, 133.6, 128.9, 128.8, 128.4, 127.7 (duplicated),
126.7, 123.6,123.1, 95.7 (duplicated), 84.8, 80.1, 79.8, 78.2, 60.3, 55.1,
54.4, 35.6, 34.2, 27.8, 17.6, 17.49, 14.1. Anal. Calcd for C26H25ClN2O8:
C, 59.04; H, 4.76; N, 5.30. Found: C, 58.96; H, 4.59; N, 5.23.
1359, 1288, 1151, 1001 cmꢂ1; 1H NMR (400 MHz, CDCl3)
d 7.38e7.16
(m, 6H), 7.09 (ddd, J ¼ 11.0, 6.5, 5.2 Hz, 3H), 6.42e6.36 (m, 1H), 5.66
(t, J ¼ 4.7 Hz, 1H), 5.07 (d, J ¼ 4.5 Hz, 1H), 2.94e2.78 (m, 1H), 2.59
(dd, J ¼ 15.0, 4.9 Hz, 1H), 1.59 (s) and 1.49 (d, J ¼ 2.9 Hz) (total 9H),
1.32 (s) and 1.26 (s) (total 3H); 13C NMR (101 MHz, CDCl3)
d 159.7,
5.2.34. tert-Butyl 7-(4-chlorophenyl)-2-(4-methoxyphenyl)-3-
methyl-5-oxo-2,3,7,8-tetrahydro-3,8a-epidioxypyrano[3,2-c]
pyridine-6(5H)-carboxylate (10b)
151.9, 139.5, 138.9, 137.6, 135.9, 135.0, 133.1, 129.1e128.4 (m), 127.9,
127.6 (duplicated), 126.91 (duplicated), 95.9, 84.5 (duplicated), 81.3,
80.8, 78.8, 78.3, 55.3, 54.7, 35.7, 34.6, 27.8 (duplicated), 17.6
(duplicated). Anal. Calcd for C26H26ClNO6: C, 64.53; H, 5.42; N, 2.89.
Found: C, 64.49; H, 5.38; N, 2.61.
White solids in 87% yield, mp: 157e158 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3060, 2983, 2883, 1770, 1716, 1639, 1492, 1369,
1294, 1153, 1012 cmꢂ1 1H NMR (600 MHz, CDCl3)
; d 7.35e7.31 (m,
2H), 7.31e7.27 (m, 1H), 7.25 (d, J ¼ 9.0 Hz, 2H), 7.13 (s, 1H), 7.06 (d,
J ¼ 8.5 Hz) and 7.01 (d, J ¼ 8.7 Hz) (total 1H), 6.61 (d, J ¼ 8.7 Hz, 1H),
6.28 (d, J ¼ 8.7 Hz, 1H), 5.66 (t, J ¼ 4.6 Hz) and 5.61 (dd, J ¼ 9.5,
5.6 Hz) (total 1H), 5.01 (d, J ¼ 9.3 Hz, 1H), 3.75 (s, 3H), 2.84 (dd,
J ¼ 15.0, 4.6 Hz, 1H), 2.57 (dd, J ¼ 15.0, 4.9 Hz, 1H), 1.54e1.43 (m,
5.2.36. tert-Butyl 7-(4-chlorophenyl)-2,3-dimethyl-5-oxo-2,3,7,8-
tetrahydro-3,8a-epidioxypyrano[3,2-c]pyridine-6(5H)-carboxylate
(10d)
White solids in 84% yield, mp: 153e155 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3075, 2982, 2935, 1770, 1722, 1643, 1492, 1359,
9H), 1.30 (s, 3H); 13C NMR (151 MHz, CDCl3)
d
160.0, 159.7 (dupli-
1288, 1151, 1001 cmꢂ1 1H NMR (600 MHz, CDCl3)
; d 7.16 (ddd,
cated), 151.9, 151.6, 139.6, 139.1, 138.1, 137.6, 135.3, 133.7, 128.9e
J ¼ 7.5, 5.1, 2.8 Hz, 3H), 7.05e6.95 (m, 2H), 5.35 (t, J ¼ 5.1 Hz) and

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Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
5.25 (s) (total 1H), 3.99 (dd, J ¼ 6.6, 0.5 Hz) and 3.30 (d, J ¼ 6.3 Hz)
(total 1H), 2.37e2.25 (m, 2H), 1.29 (d, J ¼ 3.9 Hz) and 1.24 (d,
J ¼ 12.5 Hz) (total 12H), 0.52 (d, J ¼ 6.6 Hz, 3H); 13C NMR (151 MHz,
6. Biological testing assay
6.1. Cell line
CDCl3)
d 159.9, 159.6, 151.4 (duplicated), 139.5, 138.1, 138.0, 137.7,
135.0, 133.9, 133.4 (duplicated), 128.6 (duplicated), 127.3 (dupli-
cated), 94.6 (duplicated), 84.4 (duplicated), 78.3, 76.1, 75.6, 73.0,
55.5 (duplicated), 35.5, 35.2, 31.5, 27.6 (duplicated), 17.3, 16.9
(duplicated), 14.6, 14.1. Anal. Calcd for C21H24ClNO6: C, 59.79; H,
5.73; N, 3.32. Found: C, 60.10; H, 5.70; N, 3.28.
Established in vitro, human leukemia cell line: MV4-11 (biphe-
notypic B myelomonocytic leukemia) was used. This line was ob-
tained from American Type Culture Collection (Rockville, Maryland,
USA) and is being maintained at the Institute of Immunology and
Experimental Therapy, Wroclaw, Poland.
MV4-11 cells were cultured in the RPMI 1640 medium (Gibco,
5.2.37. 3-Methyl-2-(4-nitrophenyl)-2,3,7,8-tetrahydro-3,8a-
epidioxychromen-5(6H)-one (13a)
Scotland, UK) supplemented with 2 mM L-glutamine and 1.0 mM
sodium pyruvate, 10% fetal bovine serum (all from SigmaeAldrich
Chemie GmbH, Steinheim, Germany), 100 units/ml penicillin, and
White solids in 96% yield, mp: 153e154 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3074, 2943, 2881, 1701, 1626, 1606, 1519, 1348,
100
mg/mL streptomycin (both from Polfa, Tarchomin S.A.,
1274, 1109, 1049 cmꢂ1
;
1H NMR (400 MHz, CDCl3)
d
8.19e8.13 (m,
Poland). The cell line was grown at 37 ꢀC with 5% CO2 humidified
atmosphere.
2H), 7.25 (dd, J ¼ 6.7, 1.8 Hz, 2H), 6.99 (s, 1H), 5.30 (s, 1H), 2.59 (dd,
J ¼ 8.6, 5.4 Hz, 2H), 2.19 (dd, J ¼ 5.1, 3.4 Hz, 2H), 2.04 (d, J ¼ 5.1 Hz,
HCT116andA549cellswereculturedintheRPMI1640þOptiMEM
2H), 1.37 (s, 3H); 13C NMR (101 MHz, CDCl3)
d
193.4, 148.2, 142.8,
(50:50) medium (Gibco, Scotland, UK) supplemented with 2 mM L-
140.1, 135.3, 129.9, 128.3, 123.5, 123.3, 98.6, 79.9, 77.6, 38.5, 29.9,
18.2, 17.7. Anal. Calcd for C16H15NO6: C, 60.57; H, 4.77; N, 4.41.
Found: C, 60.77; H, 4.51; N, 4.35.
glutamine and 5% fetal bovine serum (all from SigmaeAldrich Ger-
many), BALB/3T3 cells were cultured in Dulbecco medium (IIET)
supplemented with 2 mM
10% fetal bovine serum (all from SigmaeAldrich Germany). All culture
medium was supplemented with 100 units/ml penicillin and 100 g/
L-glutamine and 1.0 mM sodium pyruvate,
5.2.38. 2-(4-Methoxyphenyl)-3-methyl-2,3,7,8-tetrahydro-3,8a-
epidioxychromen-5(6H)-one (13b)
m
mL streptomycin (both from Polfa, Tarchomin S.A., Poland). All cell
White solids in 97% yield, mp: 130e131 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 2995, 2962, 2881, 1697, 1608, 1514, 1383, 1246,
lines were grown at 37 ꢀC with 5% CO2 humidified atmosphere.
1178, 1028 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 7.08 (s, 1H), 7.00e
6.2. Antiproliferative assay in vitro
6.94 (m, 2H), 6.85e6.75 (m, 2H), 5.13 (s, 1H), 3.77 (s, 3H), 2.64e2.47
(m, 2H), 2.24e2.12 (m, 2H), 2.08e1.91 (m, 2H),1.31 (s, 3H); 13C NMR
Test solutions of the compounds tested (1 mg/ml) were pre-
(101 MHz, CDCl3) d 193.9,159.9,139.9,136.6,128.6,127.8,113.7, 98.7,
80.6, 78.1, 55.2, 38.6, 30.1, 18.2, 17.81. Anal. Calcd for C17H18O5: C,
67.54; H, 6.00. Found: C, 67.22; H, 5.92.
pared by dissolving the substances in 100
with 900 l of tissue culture medium. Afterward, the tested com-
pounds were diluted in culture medium to reach the final con-
centrations of 10, 1, 0.1, 0.01 g/mL.
ml of DMSO completed
m
m
5.2.39. 3-Methyl-2-(4-nitrophenyl)-7-phenyl-2,3,7,8-tetrahydro-
3,8a-epidioxychromen-5(6H)-one (13c)
Twenty four hours prior to the addition of the tested com-
pounds, the cells were plated in 96-well plates (Sarstedt, Germany)
at a density of 1 ꢃ104 cells per well. The assay was performed after
72 h of exposure to varying concentrations of the tested agents. The
in vitro cytotoxic effect of all agents was examined using the MTT or
SRB assay [32].
The results were calculated as an IC50 (inhibitory concentration
50) e the dose of tested agent which inhibits proliferation of 50%
of the cancer cell population. IC values were calculated for each
experiment separately and mean values SD are presented in Tables 1
and 2. Each compound in each concentration was tested in triplicate
in a single experiment, which was repeated 3e5 times.
White solids in 94% yield, mp: 175e176 ꢀC (ethyl acetateehex-
ane); IR (KBr) nmax ¼ 3070, 2937, 2895, 1685, 1616, 1521, 1348, 1263,
1182, 1028 cmꢂ1; 1H NMR (400 MHz, CDCl3)
d
8.18 (ddt, J ¼ 11.1, 9.2,
2.1 Hz, 2H), 7.38 (dd, J ¼ 10.1, 4.6 Hz, 2H), 7.34e7.21 (m, 5H), 7.07 (d,
J ¼ 11.7 Hz, 1H), 5.34 (d, J ¼ 15.3 Hz, 1H), 3.39e3.27 (m, 1H), 2.90
(ddd, J ¼ 18.1, 3.8, 1.9 Hz, 1H), 2.81e2.68 (m, 1H), 2.57e2.41 (m) and
2.32e2.21 (m) (total 2H),1.41 (d, J ¼ 9.5 Hz, 3H); 13C NMR (101 MHz,
CDCl3)
d 193.0, 148.2, 142.6, 141.5, 139.4, 135.8, 135.5, 129.03
(duplicated), 128.5 (duplicated), 127.43 (duplicated), 126.58
(duplicated), 123.6 (duplicated), 98.4, 98.7, 80.2, 79.4, 77.8, 46.1
(duplicated), 37.1 (duplicated), 36.5, 35.8, 17.7 (duplicated). Anal.
Calcd for C22H19NO6: C, 67.17; H, 4.87; N, 3.56. Found: C, 66.92; H,
4.56; N, 3.48.
6.3. MTT assay
5.2.40. 2-(4-Methoxyphenyl)-3-methyl-7-phenyl-2,3,7,8-
tetrahydro-3,8a-epidioxychromen-5(6H)-one (13d)
This technique was applied for the cytotoxicity screening against
MV4-11 leukemia cells growing in suspension culture. An assay was
performed after 72-h exposure to varying concentrations (from
0.001 to 10 g/ml) of the tested agents. For the last 3e4 h of incu-
White solids in 96% yield, mp: 175e176 ꢀC (ethyl acetatee
hexane); IR (KBr) nmax ¼ 3064, 2935, 2837, 1787, 1614, 1514, 1377,
1247, 1176, 1033 cmꢂ1
;
1H NMR (400 MHz, CDCl3)
d
7.40e7.32 (m,
bation 20 ml of MTT solution were added to each well (MTT: 3-(4,5-
2H), 7.32e7.21 (m, 3H), 7.16 (d, J ¼ 17.8 Hz, 1H), 7.05e6.98 (m,
2H), 6.89e6.79 (m, 2H), 5.17 (d, J ¼ 13.9 Hz, 1H), 3.79 (s, 3H),
3.51e3.41 (m) and 3.36e3.24 (m) (total 1H), 2.92e2.82 (m, 1H),
2.75e2.63 (m, 1H), 2.51e2.38 (m) and 2.23 (dd, J ¼ 14.0, 13.2 Hz)
(total 2H), 1.35 (d, J ¼ 8.6 Hz, 3H); 13C NMR (101 MHz, CDCl3)
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; stock
solution: 5 mg/ml, Sigma, Germany). The mitochondria of viable
cells reduce the pale yellow MTT to a navy blue formazan: the more
viable cells are present in well, the more MTT will be reduced to
formazan. When incubation time was completed, 80 ml of the lysing
d
193.4, 193.0, 160.0, 141.8 (duplicated), 139.2 (duplicated), 137.1,
mixture were added to each well (lysing mixture: 225 ml dime-
thylformamide, POCh, Gliwice, Poland, 67.5 g sodium dodecyl sul-
fate, Sigma, Germany, and 275 ml of distilled water). After 24 h,
when formazan crystals had been dissolved, the optical densities of
the samples were read on an Multiskan RC photometer at 570 nm
wavelength.
136.8, 128.9, 128.6 (duplicated), 127.78 (duplicated), 127.2
(duplicated), 126.5, 113.8 (duplicated), 98.5, 98.3, 81.0, 80.1, 78.3,
78.1, 55.2, 46.3 (duplicated), 37.3 (duplicated), 36.4, 36.0, 17.8
(duplicated). Anal. Calcd for C23H22O5: C, 73.00; H, 5.86. Found: C,
72.93; H, 5.56.

Md.I. Hossain et al. / European Journal of Medicinal Chemistry 69 (2013) 294e309
309
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6.6. In vitro cytotoxicity against L6 cells
Assays were performed on the basis of the reported method
[34].
Acknowledgments
We are grateful to Okayama University for its support and to the
Advanced Science Research Center for the NMR experiments, EA by
Mr. M. Kobayashi and Ms. M. Kosaka, and X-ray analyses by Dr. H.
Ota. We are thankful to Prof. S. Nakashima and Prof. J. Nokami
(Okayama University of Science) for HRMS analyses, and Prof. J.
Futami for UV analyses. This study was partially supported by the
Adaptable and Seamless Technology Transfer Program of JST.
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