Synthesis and biological evaluation of new bischromone derivatives with antiproliferative activity

Article abstract of DOI:10.1002/ardp.201200220

The synthesis of new bischromone derivatives (4a-c and 5a-c) as potential anticancer drugs is described. The difference in the reactivity between 4-oxo-4H-chromene-3-carboxylic acid 2 (or its methyl ester 3) and 4-oxo-4H-chromene-3-carbonyl chloride 1 with three different polyamines: 3,3′-diamino-N-methyldipropylamine (a), 1,4-bis(3-aminopropyl)piperazine (b), 4,9-dioxa-1,12-dodecanediamine (c) resulted in the formation of two different groups of products, compounds 4a-c and 5a-c, designed in agreement with the bisintercalators' structural requirements. The transformation of 4-oxo-4H-chromene-3-carboxylic acid into 2H-chromene-2,4(3H)-diones (5) was confirmed by the NMR and XRD experiments. Compounds 4a and 5a were evaluated in vitro in the highly aggressive melanoma cell line A375. An enhanced induction of apoptosis and cell cycle arrest clearly revealed that compound 5a was more potent than 4a. Compound 5a was also more active in diminishing the adhesive potential of melanoma cells. Current studies support the notion that small changes in the three-dimensional structure of molecules might have a substantial impact on their biological activity. New bischromone derivatives resulting in two different product groups (compounds 4a-c and 5a-c) are described as potential anticancer drugs. N,N′-[(Methylimino)dipropane-3,1-diyl]bis(4- oxo-4H-chromene-3-carboxamide) 4a and (3,3′)-3,3′-{(methylimino) bis[propane-3,1-diyliminomethylylidene]}bis(2H-chromene-2,4(3H)-dione) 5a were evaluated in vitro in the highly aggressive melanoma cell line A375, with 5a showing higher potency in the induction of apoptosis and cell cycle arrest. Compound 5a was also more active in diminishing the adhesive potential of melanoma cells. Copyright

Full text of DOI:10.1002/ardp.201200220

34
Arch. Pharm. Chem. Life Sci. 2013, 346, 34–43
Full Paper
Synthesis and Biological Evaluation of New Bischromone
Derivatives with Antiproliferative Activity
Agata Szulawska-Mroczek1, Marta Szumilak2, Malgorzata Szczesio3, Andrzej Olczak3,
Ryszard B. Nazarski4, Wieslawa Lewgowd2, Malgorzata Czyz1, and Andrzej Stanczak2
1 Faculty of Medicine, Department of Molecular Biology of Cancer, Medical University of Lodz, Lodz, Poland
2 Faculty of Pharmacy, Department of Hospital Pharmacy, Medical University of Lodz, Lodz, Poland
3 Faculty of Chemistry, Institute of General and Ecological Chemistry, Lodz University of Technology, Lodz,
Poland
4 Laboratory of Molecular Spectroscopy, Faculty of Chemistry, Department of Organic Chemistry,
University of Lodz, Lodz, Poland
The synthesis of new bischromone derivatives (4a–c and 5a–c) as potential anticancer drugs
is described. The difference in the reactivity between 4-oxo-4H-chromene-3-carboxylic acid 2
(or its methyl ester 3) and 4-oxo-4H-chromene-3-carbonyl chloride 1 with three different
polyamines: 3,30-diamino-N-methyldipropylamine (a), 1,4-bis(3-aminopropyl)piperazine (b), 4,9-
dioxa-1,12-dodecanediamine (c) resulted in the formation of two different groups of products,
compounds 4a–c and 5a–c, designed in agreement with the bisintercalators’ structural
requirements. The transformation of 4-oxo-4H-chromene-3-carboxylic acid into 2H-chromene-
2,4(3H)-diones (5) was confirmed by the NMR and XRD experiments. Compounds 4a and 5a were
evaluated in vitro in the highly aggressive melanoma cell line A375. An enhanced induction of
apoptosis and cell cycle arrest clearly revealed that compound 5a was more potent than 4a.
Compound 5a was also more active in diminishing the adhesive potential of melanoma cells.
Current studies support the notion that small changes in the three-dimensional structure of
molecules might have a substantial impact on their biological activity.
Keywords: Antiproliferative activity / A375 melanoma cells / Bischromone derivatives
Received: May 22, 2012; Revised: September 10, 2012; Accepted: September 19, 2012
DOI 10.1002/ardp.201200220
Supporting Information available online
:
Introduction
possibly the coplanarity of the two chromone (1,4-benzopyrone)
rings [5].
The structure of the chromone ring meets the require-
4-Oxo-4H-1-benzopyran derivatives were found to have a wide
range of biological properties including the anti-inflammatory
[1], antimicrobial [2], anticancer [3], and anti-HIV [4] activity.
Bischromones constitute a group of compounds which are
also pharmaceutically important. Their activity depends on
the position of attachment, the nature of the linker and
ments for the terminal moiety of bisintercalators. The con-
nection of such two almost planar unsaturated systems with
the polyamine linker may lead to the formation of molecules
which exhibit specific biological properties, e.g., antitumor
activity and are capable of binding to DNA via a bisinterca-
lative binding mode (simultaneous insertion of two planar
systems between the adjacent base-pairs according to a neigh-
bor exclusion principle) [6, 7]. Bisintercalators constitute a
versatile and very promising group of compounds extensively
studied by many research groups worldwide. Lots of them
turned out to be potent anticancer drugs, e.g., elinafide
Correspondence: Wieslawa Lewgowd, Faculty of Pharmacy,
Department of Hospital Pharmacy, Medical University of Lodz,
1 Muszynskiego Street, 90-151 Lodz, Poland.
E-mail: wieslawa.lewgowd@umed.lodz.pl
Fax: þ48 42 677 92 52
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Arch. Pharm. Chem. Life Sci. 2013, 346, 34–43
Bischromone Derivatives with Antiproliferative Activity
35
(LU79553), bisnaphthalimide which progressed to clinical
trials against solid tumors [8].
carbonyl chloride 1 was synthesized from 4-oxo-4H-chro-
mene-3-carbaldehyde obtained by Wilsmeier–Haack reaction,
according to published procedures [10–12]. 4-Oxo-4H-1-ben-
zopyran-3-carboxylic acid 2 was prepared by hydrolysis of 4-
oxo-4H-chromene-3-carbonyl chloride 1, whereas conden-
sation of 1 with methanol at room temperature gave methyl
4-oxo-4H-1-benzopyran-3-carboxylate 3.
Taking into consideration our earlier studies on potential
anticancer drugs [9], we decided to design the bischromone
derivatives structurally related to known bisintercalators,
containing two chromone rings tethered at the 3,30 position
by three different polyamines: 3,30-diamino-N-methyldipro-
pylamine (a), 1,4-bis(3-aminopropyl)piperazine (b), and 4,9-
dioxa-1,12-dodecanediamine (c). To get information on the
biological activity of the synthesized compounds we planned
to evaluate their action on melanoma cell line A375 in vitro.
As a consequence of the diverse reactivity of 4-oxo-4H-chro-
mene-3-carbonyl chloride 1 and 4-oxo-4H-chromene-3-carbox-
ylic acid 2 (as well as its methyl ester 3) with primary
polyamines (a–c), two different groups of compounds 4a–c
and 5a–c were synthesized. The reaction of 1 with polyamines
(a–c) led to bis(4-oxo-4H-chromene-3-carboxamides) 4a–c [13].
Instead, the products obtained from the reaction of 2 with
the polyamines (a–c) in the presence of 1,10-carbonyldiimi-
dazole (CDI) were identified as the stereoisomeric mixture
(E/Z ꢀ 2:1) of 2H-chromene-2,4(3H)-dione derivatives 5a–c.
Results and discussion
Chemistry
The compounds were prepared by the procedure depicted in
Scheme 1. The starting material, 4-oxo-4H-chromene-3-
Scheme 1. The synthetic pathway to the
compounds 4a–c and 5a–c under study.
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The two geometrical isomers of compounds 5a–c were also
formed in the transformation of methyl 4-oxo-4H-1-benzo-
pyran-3-carboxylate 3 under the influence of polyamines
(a–c), as described in Ref. [13].
E isomer of 5, respectively. These two signals were exchanged
to the singlets by shaking with D2O. An isomeric E/Z ratio,
determined in this way from the 300 MHz 1H NMR spectra of
both isomers of 5 in the mixture, was established as ꢀ2:1.
The 13C NMR spectra revealed that each of the isomers (E)-5
and (Z)-5 is represented in solution by their two confor-
mations, which also appear in the ratio of ꢀ2:1.
The purity and structure of all synthesized compounds
were fully confirmed by physical data, elemental analyses,
1
IR, and H/13C NMR spectroscopy in solution. Especially the
2D correlation 1H–1H COSY and 1H–13C HETCOR exper-
iments, supported with additional DFT-level GIAO [14] based
calculations of NMR chemical shifts [15], allowed the com-
plete identification and assignment of all carbon and hydro-
gen atoms in the E/Z-isomers of 5. Indeed, the 1H NMR spectra
of isomeric products 5 in CDCl3 solution, instead of a charac-
teristic NH amide triplet at ꢀ9.4 ppm, exhibited two very
broad low-field signals at ꢀ11.9 and ꢀ10.5 ppm (disappear-
ing on shaking with D2O), which were univocally assigned
[15] to the exchangeable NH protons involved in an intra-
molecular H-bond with the keto and lactone oxygen
atoms, respectively (Scheme 1). Simultaneously, two doublets
were observed at ꢀ8.5 ppm (J ¼ 15.2 Hz) and ꢀ8.4 ppm
Both isomers of 5 were obtained as a consequence of the
two-way transformation of the g-pyrone ring occurring under
the influence of the nucleophilic attack of primary amines
at C2 of the CDI-activated carboxylic group of 4-oxo-4H-chro-
mene-3-carboxylic acid (2). This rearrangement was also
described by Alderete et al. [16], where the reactivity of
the acid was enhanced with dimethylsulfamoyl chloride.
The proposed mechanism for the transformation of 5 in
the presence of CDI is shown in Scheme 2. The conversion
into 2H-chromene-2,4(3H)-diones was also observed for the
methyl 4-oxo-4H-1-benzopyran-3-carboxylate derivative as
described by Budzisz et al. [13].
The aforementioned change into the two geometric iso-
mers was confirmed by the XRD experiments for 5a
–
(J ¼ 14.3 Hz) due to the exocyclic CH proton in the Z and
–
Scheme 2. A proposed mechanism for the reaction of compounds 5a–c according to Ref. [16].
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Arch. Pharm. Chem. Life Sci. 2013, 346, 34–43
Bischromone Derivatives with Antiproliferative Activity
37
Only 4a (as hydrochloride) and 5a both stable under exper-
imental conditions were subjected to biological in vitro
evaluation. Biological activity was investigated on the highly
aggressive melanoma cell line A375. The antiproliferative
activity was determined by the MTT-based assay and the acid
phosphatase activity (APA) assay. Cell death was visualized by
fluorescence microscopy and apoptosis was quantified by
flow cytometric analysis.
To determine the drug concentration required to inhibit
the growth of melanoma cells by 50% (IC50), the tetrazolium
derivative reduction (MTT) assay was used. Metabolic activity
of adherent melanoma cells in relation to control cells was
assessed. We found a significant difference in the activity
of the tested compounds. Compound 5a was found to have
higher antiproliferative activity, with an IC50 value of
16 ꢂ 2.2 mM (Fig. 2). A lower effect on cellular proliferation
was observed after treatment with compound 4a with an IC50
value of 46.8 ꢂ 3.2 mM. To confirm the ability of these com-
pounds to inhibit melanoma cell proliferation, the APA assay
was used. In this method, both adherent and floating cells
Figure 1. Disorder identified in the crystal structure of compound
5a.
both at room and low temperatures. Its molecular structure
is shown in Supporting Information Fig. S1, while the
details of the structure determination are presented in
Supporting Information Table S1. The structure reveals
disorder in the crystal state caused by the presence of
two different isomers (E and Z; Fig. 1). The populations
of these stereoisomers, as determined for the low-tempera-
ture structure, are presented in Table 1 and are very
similar to that found for the room-temperature structures.
The total ratio of E to Z is 2:1. The intramolecular H-bond
N5A-H ꢁ ꢁ ꢁ O8A and N5C-H ꢁ ꢁ ꢁ O16C {S11(6) in the nomencla-
ture of the graph set theory [17]} stabilizes the coplanarity
of the cyclic systems with C6–N5–H fragments. The
end groups of the molecule are almost parallel to each
other (ꢀ58), which allows for stacking interaction in the
crystal (Supporting Information Fig. S2). Apart from
an internal H-bond, the N5 atom is a donor for intermole-
cular H-bonds N5-H ꢁ ꢁ ꢁ O8 and N5-H ꢁ ꢁ ꢁ O16 (Supporting
Information Table S2), which forms two chains C11(14) on
the first level and the ring R22(12) on the second level of a
graph set.
Biological in vitro evaluation
Table 1. Populations of E and Z isomers of (3,30)-3,30-
{(methylimino)bis[propane-3,1-diyliminomethylylidene]}bis(2H-
chromene-2,4(3H)-dione) (5a) in the crystal state.
I cyclic system
II cyclic system
A
B
C
D
Isomers
Occupancy (%)
Z
52
E
48
E
86
Z
14
Figure 2. The influence of newly synthesized compounds 4a and
5a on A375 cell proliferation.
A, B, C, and D denote disordered cyclic groups (Fig. 1).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 34–43
were analyzed. The same culture conditions, time of cultur-
ing and drug concentration range were retained. A similar
effect on inhibition of A375 cell proliferation was observed
after treatment with compound 4a. For comparison, 100 mM
4a inhibited the growth of A375 cells to 31 and 41% of control
using the MTT and APA methods, respectively. Much bigger
differences were observed after treatment with 5a. For
example, this compound at 100 mM inhibited the growth
of melanoma cells to 4.6% of control when the MTT assay
was used, and to 47% of control when the APA assay was
applied. These results suggested that the newly synthesized
compound 5a was active at inhibition of both cell prolifer-
ation and adhesive potential of A375 cells.
100 mM was used.
The cell cycle analysis was performed in adherent A375
cells exposed to the newly synthesized compounds 4a and 5a
(Fig. 4). Flow cytometric analysis of the DNA content was
performed to analyze the accumulation of A375 cells in
the cell cycle phases.
We found that the new compound 5a induced a reduction
in the percentages of cells in the G0/G1 and G2/M phases, and
an accumulation of cells in the S phase of the cell cycle,
irrespective of the concentration. When the concentration of
50 mM was used, an accumulation of cells in hypodiploid
subG1 was additionally observed. Compound 4a used at 20
and 50 mM did not introduce any changes in the cell cycle
distribution when compared with control cells. Our results
suggested that the newly synthesized compound 5a was
rather a cytostatic than a cytotoxic agent, whereas compound
4a was inactive at the same concentrations. To confirm these
To evaluate the ability of the new compounds to detach
A375 cells from the surface, a more detailed analysis was
performed. In the MTT assay, only adherent cells were ana-
lyzed since cells which lost contact with the surface (floating
cells) were removed during the procedure. In the APA assay,
both adherent and floating cells were analyzed. The percen-
tages of floating viable cells were calculated by subtracting
the percentages of adherent cells (MTT assay) from the per-
centages of all cells (APA assay). As shown in Fig. 3, compound
5a reduced the adhesive potential of A375 melanoma cells in
a concentration dependent manner. After incubation with 5a
at 50 and 100 mM, about 30% and more than 40% of A375
cells, respectively, lost contact with the surface, but remained
viable (Fig. 3). In contrast to 5a, compound 4a did not influ-
ence the adhesion process and only about 10% of the A375
cells were detached from the surface when compound 4a at
Figure 3. The influence of the new compounds 4a and 5a on the
adhesive potential of A375 melanoma cells. The percentages of
A375 cells which were detached from the surface (floating viable
cells) after 44 h of incubation with the new compounds were calcu-
lated by subtracting the percentages of the adherent cells from the
percentages of all cells (adherent and floating). The percentage of
adherent viable cells was determined by MTT assay while the per-
centage of adherent and floating viable cells was assessed by APA
assay, as described in the Experimental section.
Figure 4. Effects of the new compounds 4a and 5a on cell cycle
distribution. DNA content measured by flow cytometry at two differ-
ent concentrations after 24 h of treatment with the new compounds.
Percentages of the cells in each fraction were calculated using the
ModFit 3.0 software. Representative histograms of one out of three
independent experiments are shown.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 34–43
Bischromone Derivatives with Antiproliferative Activity
39
observations, cell death analysis was performed.
7 and 9% of necrotic cells were noted after treatment with
To examine whether the new compounds induced apop-
tosis in melanoma A375 cells, we employed a flow cytometric
analysis of annexin V/PI-stained cells (Fig. 5A), and fluor-
75 mM compounds 4a and 5a, respectively.
escence microscopy for acridine orange (AO)/ethidium bro- Conclusion
mide (EB)-stained cells (Fig. 5B). The percentages of apoptotic
(annexin-positive) and necrotic (exclusively PI-positive) cells
were determined after 22 h of treatment with compounds 4a
and 5a at two concentrations. The exposure of A375 cells to
4a at a concentration close to the IC50 value and higher, did
not induce significantly the percentage of apoptotic cells.
About 11% of annexin V-positive cells were noted after treat-
ment with 50 mM concentration. A higher concentration of
4a slightly increased the apoptotic cells to 16% at 75 mM
concentration. On the contrary, compound 5a induced a
higher increase in the amount of apoptotic cells. About 20
and 30% of A375 cells treated with 5a at the concentration 50
and 75 mM, respectively, were annexin V positive. Those cells
showed morphological features of apoptosis in AO/EB stain-
ing. Our result demonstrated that both compounds only
slightly increased the percentages of necrotic cells. About
The present work describes the synthesis of six bischromone
derivatives 4a–c and 5a–c, structurally related to bisinterca-
lators. The condensation of 4-oxo-4H-chromene-3-carbonyl
chloride 1 with corresponding polyamines (a–c) gave com-
pounds 4a–c, respectively, which was in agreement with
previous studies. On the other hand, the reaction of 4-oxo-
4H-chromene-3-carboxylic acid 2 with corresponding poly-
amines (a–c) in the presence of CDI resulted in the formation
of stereoisomeric (E/Z ꢀ 2:1) mixtures of compounds 5a–c,
respectively. Geometric isomers of 5a–c were also formed
in the transformation of methyl 4-oxo-4H-1-benzopyran-3-
carboxylate 3 under the influence of the polyamines (a–c) used.
The structure of all synthesized compounds was fully con-
1
firmed by physical data, elemental analyses, IR, H/13C NMR
spectroscopy in solution, and XRD experiments for the
Figure 5. Cell death analysis of A375 melanoma cell line after treatment with new compounds 4a and 5a. (A) Externalization of phospha-
tidylserine was determined by annexin V/PI staining and analyzed by flow cytometry. Representative histograms from one typical experiment
are shown. (B) Bars showing average percentages of annexin V- and PI-positive cells are presented. The data are means ꢂ SD of three
independent experiments (ꢃp < 0.05, ꢃꢃp < 0.01). (C) Cells were stained with nucleic acid selective fluorochromes: membrane-permeable
AO and impermeable EB. Representative microscopic fields presenting non-apoptotic, apoptotic and necrotic cells are shown.
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A. Szulawska-Mroczek et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 34–43
carbonate filter cake in water and filtering off the product.
Recrystallization from DMF/H2O gave pure amides.
crystal of 5a.
The outcome of our chemical investigations clearly con-
firmed a significant influence of the electron-withdrawing
substituents at the 3-position of the chromone system on the
reactivity of the g-pyrone ring with respect to nitrogen nucle-
ophiles. It also showed that in the case of 4-oxo-4H-1-benzo-
pyran-3-carboxylic acid (2), the presence of CDI did not favor
the amide formation but gave the stereoisomeric (E/Z ꢀ 2:1)
mixture of 2H-chromene-2,4(3H)-dione derivatives (5a–c).
Methyl 4-oxo-4H-1-benzopyran-3-carboxylate (3) underwent
similar transformations.
N,N0-[(Methylimino)dipropane-3,1-diyl]bis(4-oxo-4H-
chromene-3-carboxamide) (4a)
Yield: 55% as yellow crystals, mp 108.5–110.58C; IR (KBr) n: 1667,
1614 (C O) cmꢄ1
.
1H NMR (300 MHz, CDCl3) dH: 9.39
–
–
–
–
(t, J ꢀ 5.5 Hz, 2H, 2 CONH), 8.96 (s, 2H, OCH ), 8.27 (dd,
J ¼ 8.1, 1.7 Hz, 2H, CHar), 7.69–7.76 (m, 2H, CHar), 7.43–7.55
(m, 4H, CHar), 3.53 (pq, J ꢀ 6.5 Hz, 4H, NCH2), 2.49
[t, J ¼ 6.9 Hz, 4H, CH2N(CH3)], 2.26 (s, 3H, NCH3), 1.77–1.88
(m, 4H, CH2CH2CH2) ppm. 13C NMR (75 MHz, CDCl3) dC: 177.11
–
–
–
(C O
), 162.74 (C O
), 162.21 (OCH ), 156.08
–
–
–
unsaturated keto
amide
When biological activity is concerned, the observed differ-
ences in antiproliferative activity of the examined com-
pounds 4a and 5a were significant, with 5a being more
active, especially when the induction of apoptosis and cell
cycle arrest are considered. Compound 5a was also more
potent in diminishing the adhesive potential of melanoma
cells. The current studies support the notion that small
changes in the three-dimensional structure of the molecules
might have a substantial impact on their biological activity.
Our results suggest that selected bischromone derivatives
may be worthy of future research for designing new entities
with an increased cytotoxic/cytostatic activity.
(Cq), 134.52, 126.30 (3 ꢅ CHar), 124.39 (Cq), 118.46 (CHar),
116.02 (Cq), 55.62 (CH2), 42.22 (CH3), 37.85, 27.39 (2 ꢅ CH2)
1
ppm. Anal. calcd. for (C27H27N O ꢅ = H O) C, 65.05, H, 5.66,
2
3
6
2
N, 8.42. Found: C, 64.90, H, 5.46, N, 8.59.
N,N0-(Piperazine-1,4-diyldipropane-3,1-diyl)bis(4-oxo-4H-
chromene-3-carboxamide) (4b)
Yield: 60% as pale yellow crystals, mp 201.3–202.38C; IR (KBr)
ꢄ1
–
–
n: 1664, 1613 (C O) cm
.
1H NMR (300 MHz, CDCl3) dH: 9.38
–
(t, J ꢀ 5.4 Hz, 2H, 2CONH), 8.99 (s, 2H, OCH ), 8.30 (dd, J ¼ 7.9,
–
1.4 Hz, 2H, CHar), 7.75–7.79 (m, 2H, CHar), 7.50–7.53 (m, 4H, CHar),
3.53 (pq, J ꢀ 6.8 Hz, 4H, NCH2), 2.47–2.51 (cluster, 8H CH2
piperazine þ 4H CH2N), 1.82–1.88 (m, 4H, CH2CH2CH2) ppm.
–
13C NMR (75 MHz, CDCl3) dC: 177.17 (C O
keto),
–
unsaturated
–
–
162.77 (C O
), 162.24 (OCH ), 156.14 (C ), 134.53, 126.22
–
–
amide
q
Experimental
(3 ꢅ CHar), 124.37 (Cq), 118.42 (CHar), 115.99 (Cq), 56.09,
53.20, 37.74, 26.74 (5 ꢅ CH2) ppm. Anal. calcd. for
(C30H32N O ꢅ 21= H O) C, 61.10, H, 6.32, N, 9.50. Found:
2
4
6
2
General
C, 60.95, H, 6.33, N, 9.67.
Reagents and solvents were purchased from common commer-
cial suppliers. The purity of the products was routinely con-
firmed by TLC on Merck plates (Kieselgel 60 F254); the
appropriate solvents were used, and spots were visualized
under UV light. The melting points were measured on an
Electrothermal apparatus in open capillaries and are uncor-
rected. Elemental analyses were carried out with a Perkin
Elmer series II CHNS/O Analyzer 2400 and were within ꢂ0.4%
of the theoretical values. IR spectra were recorded in KBr using
a Mattson Infinity Series FT-IR spectrophotometer. 1H and 13C
NMR spectra were acquired on a Varian Mercury-300 spec-
trometer (at 300.06/75.45 MHz, for the 1H/13C nuclei) in CDCl3
solution with TMS as internal standard. Chemical shifts are
expressed in d (ppm) and the coupling constants J in Hertz
(Hz). The following abbreviations were used to describe the signal
patterns when appropriate: s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), vbr (very broad), and p (pseudo).
N,N-[Butane-1,4-diylbis(oxypropane-3,1-diyl)]bis(4-oxo-
4H-chromene-3-carboxamide) (4c)
Yield: 73% as yellow crystals, mp 110.5–112.18C; IR (KBr) n: 1667,
1615 (C O) cmꢄ1
.
1H NMR (300 MHz, CDCl3) dH: 9.37
–
–
–
–
(t, J ꢀ 5.5 Hz, 2H, 2 CONH), 8.95 (s, 2H, OCH ), 8.27 (dd,
J ¼ 7.9, 1.4 Hz, 2H, CHar), 7.70–7.78 (m, 2H, CHar), 7.51–7.57
(m, 2H, CHar), 7.48 (t, J ¼ 7.5 Hz, 2H, CHar), 3.48–3.58 (cluster,
6H, NCH2 þ 6H CH2O), 1.86–1.93 (m, 4H, CH2CH2CH2), 1.67–1.72
(m, 4H, CH2CH2CH2CH2) ppm. 13C NMR (75 MHz, CDCl3) dC:
–
–
–
–
177.08 (C O
), 162.73 (C O
), 162.16 (OCH ),
amide
–
–
unsaturated keto
156.09 (Cq), 134.49, 126.18 (3 ꢅ CHar), 124.33 (Cq), 118.38
(CHar), 115.96 (Cq), 70.95, 68.58, 36.90, 29.61, 26.46 (5 ꢅ CH2)
ppm. Anal. calcd. for (C30H32N2O8) C, 65.68, H, 5.87, N, 5.11.
Found: C, 65.40, H, 5.68, N, 5.24.
General procedure for the synthesis of 5a–c
Chemistry
General procedure for the synthesis of 4a–c
From 4-oxo-4H-chromene-3-carboxylic acid (2)
A
solution of 4-oxo-4H-chromene-3-carbonyl chloride 1 in
10 mmol of 2 and 10 mmol of 1,1-carbonyl-diimidazole (CDI) in
DMF (100 mL) were stirred for 1 h at room temperature. Then the
appropriate polyamines (a–c) (6 mmol) were added and stirring
was continued for an additional 2 h. The mixture was filtered,
the solvent was removed under reduced pressure, water was
added and the whole was left for 12 h at þ58C. The solids
(precipitate) were filtered off, washed with H2O, and crystallized
from DMF/H2O.
200 mL of methylene chloride was added for a period of
5 min to a stirred mixture of appropriate polyamines (a–c),
powdered potassium carbonate and 100 mL of methylene
chloride, keeping the temperature at 258C with ice bath cooling.
After 1 h, the solids were filtered off and the filtrate was con-
centrated. Additional material was recovered by dissolving the
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 346, 34–43
Bischromone Derivatives with Antiproliferative Activity
41
–
–
–
–
d : 178.60 (C O ), 164.85 (C O
C
), 160.79 ( CHN), 154.79 (C ),
q
–
–
keto
ester
From methyl 4-oxo-4H-1-benzopyran-3-carboxylate (3)
The appropriate polyamines (a–c) (6 mmol) in MeOH (0.5 mL)
were added at room temperature to a solution of 3 (10 mmol) in
MeOH (5 mL). The solid crude products, which were precipitated
after 24 h, were filtered off, dried, and crystallized from MeOH.
134.20, 126.39, 124.04 (3 ꢅ CHar), 120.91 (Cq), 117.23 (CHar),
96.68 (Cq), 55.23, 54.82 (2 ꢅ CH2), 53.10 (2 ꢅ CH2), 49.88, 26.38
(2 ꢅ CH2) ppm. Anal. calcd. for C30H32N4O6: C, 66.16, H, 5.87,
N, 10.28. Found: C, 65.92, H, 6.11, N, 10.67.
(3,30)-3,30-{(Methylimino)bis[propane-3,1-
diyliminomethylylidene]}bis(2H-chromene-2,4(3H)-dione)
(5a)
From methyl 4-oxo-4H-1-benzopyran-3-carboxylate (3)
Yield: 55% as white crystals, mp 233.6–234.18C (from MeOH); its
analytical, IR, and 1H/13C NMR spectroscopic data were in full
accordance with the results described above.
From 4-oxo-4H-chromene-3-carboxylic acid (2)
Yield: 96.5% as dark red crystals of an E/Z (ꢀ2:1) isomeric mixture
(3,30)-3,30-[(1,16)-6,11-Dioxa-2,15-diazahexadecane-
1,16-diylidene]bis(2H-chromene-2,4(3H)-dione) (5c)
of 5a, mp 167.8–168.58C (from DMF/H2O); IR (KBr) n: 1700, 1638
(C O) cmꢄ1. 1H NMR (300 MHz, CDCl3) dH: ꢀ11.95 (vbr ps, 1.33H,
–
–
–
–
–
From 4-oxo-4H-chromene-3-carboxylic acid (2)
Yield: 60% as white crystals of an E/Z (ꢀ2:1) isomeric mixture of
CHNH, E forms), ꢀ10.55 (vbr asymmetric massif, 0.67H, CHNH,
–
–
–
Z forms), 8.56 (d, J ¼ 15.2 Hz, 0.67H, CHNH, Z forms), 8.42
–
–
5c, mp 168.3–170.38C (from DMF/H2O); IR (KBr) n: 1697, 1647,
(d, J ¼ 14.3 Hz, 1.33H, CHNH, E forms), 8.11–8.09 (m, 0.67H,
ꢄ1
1608 (C O) cm
–
.
1H NMR (300 MHz, CDCl3) dH: ꢀ11.89 (vbr s,
–
–
–
CHar, Z forms), 8.08–8.06 (m, 1.33H, CHar, E forms), 7.58–7.50
(m, 2H, CHar), 7.26–7.18 (m, 4H, CHar), 3.79 (pq, J ꢀ 6.3 Hz, 1.33H,
NCH2, Z forms), 3.74 (pq, J ꢀ 6.3 Hz, 2.67H, NCH2, E forms), 2.57–
2.49 [m, 4H, CH2N(CH3)], 2.21 (s, 3H, NCH3), 1.99–1.89 (m, 4H,
CH2CH2CH2) ppm. 13C NMR (75 MHz, CDCl3) (a) the E isomer (only
–
–
–
1.33H, CHNH, E forms), ꢀ10.38 (vbr s, 0.67H, CHNH, Z forms),
–
–
8.54 (dd, J ¼ 15.1, 4.3 Hz, 0.67H, CHNH,
Z forms), 8.40
–
–
(dd, J ¼ 14.2, 4.6 Hz, 1.33H, CHNH, E forms), 8.11 (dd, J ¼ 7.8,
1.4 Hz, 0.67H, CHar, Z forms), 8.03 (dd, J ¼ 7.8, 1.5 Hz, 1.33H,
CHar, E forms), 7.60–7.54 (m, 2H, CHar), 7.28–7.22 (m, 4H, CHar),
3.76–3.67 (m, 4H, CH2O), 3.60–3.54 (m, 4H, NCH2), 3.54–3.50 (m,
4H, OCH2), 2.02–1.95 (m, 4H, CH2CH2CH2), 1.79–1.70 (m, 4H,
CH2CH2CH2CH2) ppm. 13C NMR (75 MHz, CDCl3) (a) the E isomer
(only lines of the major conformation are given) dC: 181.15
lines of the major conformation are given) d : 180.98 (C Oketo),
–
C
–
–
–
–
164.05 (C O
), 162.24 ( CHN), 154.76 (C ), 134.16, 125.78,
q
ester
123.93 (3 ꢅ CHar), 120.69 (Cq), 117.25 (CHar), 96.87 (Cq), 55.85,
49.78 (2 ꢅ CH2), 40.91 (CH3), 27.55 (CH2) ppm; (b) the Z isomer
(only lines of the major conformation are given) dC: 178.46
–
–
–
–
–
–
(C O ), 163.96 (C O
keto
), 162.43 ( CHN), 154.91 (C ), 134.14,
q
(C O ), 164.94 (C O
keto
), 160.58 ( CHN), 154.64 (C ), 134.21,
q
–
–
–
–
–
–
ester
ester
125.66, 123.84 (3 ꢅ CHar), 120.71 (Cq), 117.27 (CHar), 96.86 (Cq),
126.37, 124.09 (3 ꢅ CHar), 120.86 (Cq), 117.25 (CHar), 96.69 (Cq),
56.53, 50.38 (2 ꢅ CH2), 40.77 (CH3), 27.17 (CH2) ppm. Anal. calcd.
for C27H27N3O6: C, 66.24, H, 5.51, N, 8.58. Found: C, 65.90, H, 5.46,
N, 8.59.
71.21, 67.76, 48.75, 29.96, 26.43 (5 ꢅ CH2) ppm; (b) the Z isomer
–
–
(only lines of the major conformation are given) d : 178.50 (C
C
–
–
Oketo), 164.98 (C O
), 160.68 ( CHN), 154.77 (C ), 134.17,
–
–
q
126.38, 124.02 (3 ꢅ eCstHerar), 120.90 (Cq), 117.19 (CHar), 96.73 (Cq),
71.31, 67.23, 49.16, 29.87, 26.36 (5 ꢅ CH2) ppm. Anal. calcd. for
C30H32N2O8: C, 65.68, H, 5.83, N, 5.10. Found: C, 65.30, H, 5.74, N,
5.22.
From methyl 4-oxo-4H-1-benzopyran-3-carboxylate (3)
Yield: 42% as yellow crystals mp 167.9–168.58C (from MeOH); its
analytical, IR, and 1H/13C NMR spectroscopic data were in full
accordance with the results described above.
From methyl 4-oxo-4H-1-benzopyran-3-carboxylate (3)
Yield: 50% as white crystals, mp 168.5–170.38C (from MeOH); its
analytical, IR, and 1H/13C NMR spectroscopic data were in full
accordance with the results described in above.
(3)-3-[({3-[4-(3-{[(2,4-Dioxo-2H-chromen-3(4H)-ylidene)-
methyl]amino}propyl)piperazin-1-yl]propyl}amino)-
methylene]-2H-chromene-2,4(3H)-dione (5b)
X-ray crystallography
From 4-oxo-4H-chromene-3-carboxylic acid (2)
Yield: 73.5% as creamy crystals of an E/Z (ꢀ2:1) isomeric mixture
The crystals suitable for an X-ray experiment were obtained by
the slow evaporation method from DMF solution. The data were
collected with a Bruker CCD APEX II diffractometer using the Mo
radiation. The experiment was carried out at ꢄ1738C (100 K).
Data processing was performed by means of Bruker packages of
programs [18]. The structure was solved by direct methods using
SHELXTL [19] and refined by full-matrix least squares [20]. Non-
hydrogen atoms were refined anisotropically except that belong-
ing to the least populated cyclic system. All H-atoms were refined
isotropically at calculated positions, by using the riding model.
Supporting Information Tables S1 and S2 as well as Supporting
Information Fig. S1 and Fig. 1 were prepared with Platon [21],
while Supporting Information Fig. S2 was prepared with the
Mercury software [22]. Final coordinates and details of the
measurement, solution, and refinement were deposited with
the Cambridge Crystallographic Data Centre No. CCDC
829311. All details of crystal data may be obtained free of
of 5b, mp 233.6ꢄ–1234.28C (from DMF/H2O); IR (KBr) n: 1693, 1640,
1605 (C O) cm
–
.
1H NMR (300 MHz, CDCl3) dH: ꢀ11.90 (vbr s,
–
–
–
–
1.33H, CHNH, E forms), ꢀ10.45 (vbr s, 0.67H, CHNH, Z forms),
–
–
–
8.58 (d, J ¼ 15.1 Hz, 0.67H, CHNH, Z forms), 8.44 (d, J ¼ 14.2 Hz,
–
1.33H, CHNH, E forms), 8.12 (dd, J ¼ 7.8, 1.6 Hz, 0.67H CH , Z
–
ar
forms), 8.04 (dd, J ¼ 7.8, 1.3 Hz, 1.33 H CHar, E forms), 7.60–7.55
(m, 2H, CHar), 7.29–7.24 (m, 4H, CHar), 3.70 (q, J ¼ 6.1 Hz, 1.33H,
NCH2, Z forms), 3.65 (q, J ¼ 6.3 Hz, 2.67H NCH2, E forms), 2.65–
2.43 (m, 12H, CH2N þ CH2 piperazine), 1.93–1.85 (m, 4H,
CH2CH2CH2) ppm.13C NMR (75 MHz, CDCl3) (a) the E isomer (only
–
lines of the major conformation are given) d : 181.02 (C Oketo),
–
C
–
–
164.07 (C O
), 162.50 ( CHN), 154.90 (C ), 134.15, 125.67,
q
–
–
ester
123.90 (3 ꢅ CHar), 120.75 (Cq), 117.28 (CHar), 96.81 (Cq), 55.84,
54.81 (2 ꢅ CH2), 52.90 (2 ꢅ CH2), 49.52, 26.48 (2 ꢅ CH2) ppm;
(b) the Z isomer (only lines of the major conformation are given)
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

42
A. Szulawska-Mroczek et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 34–43
charge via www.ccdc.cam.ac.uk/conts/retrieving.html or from
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: þ44 1223 336033; or
deposit@ccdc.cam.ac.uk.
Acridine orange/ethidium bromide staining
Cell death was monitored using fluorescent dyes: AO and EB.
Briefly, A375 cells were cultured for 22 h with or without the
tested compounds at indicated concentrations, then collected by
centrifugation and resuspended in 30 mL of staining solution, a
mixture (1:1) of EB (100 mg/mL) and AO (100 mg/mL). They were
examined by ultraviolet fluorescence microscopy (Olympus
BX 41).
Biological in vitro evaluation
Cell line and cell culture conditions
A375, a human melanoma cell line with a high metastatic
potential, was grown in a humidified atmosphere of 5% CO2
at 378C in RPMI 1640 (Lonza, Switzerland), supplemented with
10% fetal bovine serum (FBS), penicillin (10 U/mL), and strepto-
mycin (50 mg/mL). For experiments, the culture medium was
substituted with fresh medium containing 0.5% FBS. In exper-
iments, cells in the logarithmic phase of growth were used.
Statistical analysis
Data represent means ꢂ SD from at least three separate exper-
iments. The significance of any apparent difference in mean
values for any tested parameter was validated by a Student’s
paired t-test. The difference was considered significant if the
p-value was <0.05.
Analysis of cell proliferation
This study was supported by the Medical University in Lodz, Poland,
Research Programs no. 503/3-011-03/503-01, no. 503/1-156-01/503-01, and
sponsored by a Polpharma Science Foundation. The authors wish to thank
Dr. Marta Stasiak for her help with a flow cytometric analysis, Dr. Michal
Wozniak for stimulating discussions, and Karolina Niewinna for excellent
technical assistance.
Cell proliferation was measured by two methods: a colorimetric
tetrazolium derivative reduction (MTT) and the APA assay. Cells
were seeded in 24-well plates, and 6 h later adherent cells were
exposed to tested compounds at indicated concentrations for
44 h. To determine IC50 values, the concentration of tested
compounds causing 50% inhibition of cell growth, the MTT assay
was used as described previously [9]. To confirm the influence of
newly synthesized compounds on the proliferation potential of
A375 melanoma cells, the APA assay was used. Briefly, cells on
plates were centrifuged (2500 rpm, 10 min) and washed with
PBS. To each well 0.1 M sodium acetate (pH 5.0), 0.1% Triton X-
100 and 5 mM p-nitrophenyl phosphate were added. After 2 h of
incubation at 378C 1 M NaOH was added. The absorbance at
405 nm was recorded using a microplate reader (Infinite
M200 PRO, TECAN, Austria). In the MTT assay, only adherent
cells were assessed, whereas in the APA assay both adherent and
floating cells were analyzed.
The authors have declared no conflict of interest.
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www.archpharm.com