Bis(4-hydroxy-2H-chromen-2-one): Synthesis and effects on leukemic cell lines proliferation and NF-κB regulation

Article abstract of DOI:10.1016/j.bmc.2014.03.046

Synthesis of the bis-4-hydroxycoumarin-type compound, 3,3′-[3-(2- hydroxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-2H-chromen-2-one), was performed by two alternative pathways, either involving a basic organocatalyzed 1,4-conjugate addition tandem reaction of 4-hydroxycoumarin on chromone-3-carboxylic acid, or a double condensation of 4-hydroxycoumarin on ω-formyl-2′-hydroxyacetophenone. The anti-proliferative effects of the bis-4-hydroxycoumarin-type compound on human K-562 (chronic myeloid leukaemia) and JURKAT (acute T-cell leukaemia) cell lines using trypan blue staining, as well as its involvement in nuclear factor-kappa B (NF-κB) regulation analyzed by luciferase reporter gene assay, gene expression analysis and western blots were analysed. This compound inhibited TNFα-induced NF-κB activation in K-562 (IC₅₀ 17.5 μM) and JURKAT (IC ₅₀ 19.0 μM) cell lines, after 8 h of incubation. Interestingly, it exerted mainly cytostatic effects at low doses on both cell lines tested, whereas it decreased JURKAT cell viability starting at 50 μM from 24 h of treatment. Importantly, it did not affect the viability of peripheral blood mononuclear cells (PBMCs) from healthy donors, even at concentrations above 100 μM.

Full text of DOI:10.1016/j.bmc.2014.03.046

Bioorganic & Medicinal Chemistry 22 (2014) 3008–3015
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bis(4-hydroxy-2H-chromen-2-one): Synthesis and effects
on leukemic cell lines proliferation and NF-jB regulation
Oualid Talhi a,1, Michael Schnekenburger b,1, Jana Panning b,c, Diana G. C. Pinto a, José A. Fernandes d,
Filipe A. Almeida Paz d, Claus Jacob c, Marc Diederich e, , Artur M. S. Silva a,
⇑
⇑
a QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
b Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, L-2540 Luxembourg, Luxembourg
c School of Pharmacy, Building B 2.1., Room 1.13, University of Saarland, Campus, 66123 Saarbrucken, Germany
d CICECO, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
e College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Synthesis of the bis-4-hydroxycoumarin-type compound, 3,30-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-
diyl]bis(4-hydroxy-2H-chromen-2-one), was performed by two alternative pathways, either involving a
basic organocatalyzed 1,4-conjugate addition tandem reaction of 4-hydroxycoumarin on chromone-3-
carboxylic acid, or a double condensation of 4-hydroxycoumarin on
The anti-proliferative effects of the bis-4-hydroxycoumarin-type compound on human K-562 (chronic
myeloid leukaemia) and JURKAT (acute T-cell leukaemia) cell lines using trypan blue staining, as well
Article history:
Received 11 February 2014
Revised 27 March 2014
Accepted 30 March 2014
Available online 6 April 2014
x
-formyl-20-hydroxyacetophenone.
Keywords:
Biscoumarins
2D-NMR
X-ray crystallography
Leukaemia cell lines
as its involvement in nuclear factor-kappa B (NF-
gene expression analysis and western blots were analysed. This compound inhibited TNF
NF- B activation in K-562 (IC50 17.5 M) and JURKAT (IC50 19.0 M) cell lines, after 8 h of incubation.
Interestingly, it exerted mainly cytostatic effects at low doses on both cell lines tested, whereas it
decreased JURKAT cell viability starting at 50 M from 24 h of treatment. Importantly, it did not affect
the viability of peripheral blood mononuclear cells (PBMCs) from healthy donors, even at concentrations
above 100 M.
j
B) regulation analyzed by luciferase reporter gene assay,
a
-induced
j
l
l
l
NF-jB inhibition
l
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
inflammatory cytokines, I
ylates I
degraded by the proteasome. The released NF-
tor then translocates to the nucleus and transactivates target genes
involved in cancer initiation, promotion and progression.4–6
A large majority (79.8%) of anticancer drugs developed between
1981 and 2010 derive from natural origins.7 The flavonoid family is
a prominent group of natural compounds showing anti-prolifera-
tive effects; the flavone quercetin, for example, is able to inhibit
the growth of several tumour cell lines,8 while chalcones were
j
B kinase (IKK) is activated and phosphor-
j
B. As a consequence I
j
B is ubiquitinated and subsequently
B transcription fac-
Cancer is characterized by uncontrolled growth and spreading of
abnormal cells. Smoking, alcohol intake, obesity and physical inac-
tivity are seen as main cancer risk factors. Also genetic predisposi-
tion, inflammatory environment, activation of oncogenes and
deactivation of tumour suppressor genes contribute to development
of this disease.1–3 A central molecular mechanism involved in
inflammatory and innate immune response is the transcription fac-
j
tor nuclear factor kappa-B cell (NF-
including, relA (p65), relB, cRel, NF-
Canonical NF- B pathway is activated by Toll-like-, tumour necrosis
factor (TNF- ) or T-cell receptors. RelA-p50 dimers are associated
with inhibitor of B (I B) retaining them in an inactive form in the
j
j
B) comprising five members
B1 (p50) and NF- B2 (p52).
j
described as NF-jB pathway inhibitors, inducing cell death via
j
apoptosis.9,10 Other interesting bioorganic compounds including
polysulfides from garlic,11 curcumin from the rhizome of Curcuma
longa12–15 and the sesquiterpene heteronemin from the marine
organism Hyrtios erecta,16 possess anti-tumour, anti-inflammatory
and anti-tumorigenic abilities based on the anti-proliferative, pro-
apoptotic character and/or cytotoxicity leading to cell death by
apoptosis or autophagy depending on the cellular context.
a
j
j
cytoplasm. After stimulation of cell surface receptors with pro-
⇑
Corresponding authors. Tel.: +82 2 880 8919 (M.D.); tel.: +351 234 370714; fax:
+351 234 370084 (A.M.S.S.).
Coumarins constitute an important category of natural products
as more than thousand coumarin derivatives have been described
E-mail addresses: marcdiederich@snu.ac.kr (M. Diederich), artur.silva@ua.pt
(A.M.S. Silva).
1
Equal contribution.
http://dx.doi.org/10.1016/j.bmc.2014.03.046
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

O. Talhi et al. / Bioorg. Med. Chem. 22 (2014) 3008–3015
3009
B
A
O
O
HO
O
O
O
HO
O
O
4-PPy
O
O
O
H
O
O
HOOC
OH
OH
O
OH
HOOC
O-
2
3
1
O
HO
- H2O
- CO2
4PPy
OH
O
O
OH
O
O
O
O
O
O
O
O
O
O
OH
O
O
O
O
O
O
O-
O
4
O
O-
OH
HO
O
OH
Intermediate
Scheme 1. Synthesis of biscoumarin 4.
and isolated from more than 800 plant species.17 Certain deriva-
tives exhibit useful pharmaceutical activities, as they were credited
with potent antioxidant, anti-inflammatory, anticancer and mol-
luscicidal activities.18 They induce apoptosis by cytochrome c
release and caspase activation.19 Recent reports described the
selective cytotoxicity to cancer cells of some structurally simple
coumarins, such as 7,8-dihydroxy-4-methylcoumarin, 7,8-diacet-
oxy-4-methylcoumarin20 and 7-hydroxycoumarin,21 which inhibit
proliferation of lung cancer cells without affecting the normal
growth of healthy peripheral blood mononuclear cells (PBMCs).
In the quest for novel natural-mimicking compounds, we describe
here the anticancer and anti-inflammatory activities of a new syn-
thetic bis-4-hydroxycoumarin-type compound, which has been
elaborated by updated synthetic routes.
HMBC
NOESY
O
O
H
O
4'
2'
3'
O
HO
2'
3'
4'
HO
H
H
1
OH
H
1
6'''
OH
CH2
O
2
2
CH2
O
O
3
O
3
O
O
H
O
OH
Figure 1. Main HMBC correlations and NOE effects observed in the 2D NMR spectra
of the biscoumarin 4.
2. Results and discussion
2.1. Chemistry
The well-known biological activities of coumarins oriented us
towards a biscoumarin scaffold as a potential biologically active
dyad presenting a quite symmetrical geometry and containing
the relevant bioactive benzopyran-2-one ring. The target
biscoumarin 4 was first synthesized by reacting two equivalents
of 4-hydroxycoumarin 1 on chromone-3-carboxylic acid 2 using
a catalytic amount of 4-pyrrolidinopyridine (4PPy) in refluxing
chloroform. Mechanistically, an initial 1,4-conjugate addition/
decarboxylation tandem process has led to the conjugated
intermediate which suffers a subsequent classical Michael addition
on the existing
a,b-unsaturated ketone system giving rise to the
biscoumarin 4 (72%) (Scheme 1, pathway A).
The action of 4-hydroxycoumarin 1 on aliphatic and aromatic
aldehydes has been reported in the literature giving similar bis-
coumarin structures.11 In consequence, we decided to study the
Figure 2. Schematic representation of the molecular unit present in the crystal
structure of the biscoumarin compound 4. Non-hydrogen atoms are represented as
thermal ellipsoids drawn at the 50% probability level and hydrogen atoms as small
spheres with arbitrary radii. The atomic labelling is provided for all non-hydrogen
atoms. Dashed green and gold lines represent, respectively, strong O–Hꢀ ꢀ ꢀO and
weak C–Hꢀ ꢀ ꢀO hydrogen bonds. Hydrogen bonding geometry: O2–H2ꢀ ꢀ ꢀO1,
dOꢀ ꢀ ꢀO = 2.575(5) Å, <(OHO) = 152°; O5–H5ꢀ ꢀ ꢀO6, dOꢀ ꢀ ꢀO = 2.614(5) Å, <(OHO) = 158°;
O8–H8ꢀ ꢀ ꢀO3, dOꢀ ꢀ ꢀO = 2.665(5) Å, <(OHO) = 158°; C2–H2Bꢀ ꢀ ꢀO5, dOꢀ ꢀ ꢀO = 3.145(5) Å,
<(CHO) = 125°. The inset (top view of the molecule) depicts the tilted nature of the
3-(2-hydroxyphenyl)propion-1-yl unit with respect to the sp3 average plane of the
central C1–C2 moiety.
reaction of 4-hydroxycoumarin
1 on the cheap x-formyl-2-
hydroxyacetophenone 3, utilizing our optimized conditions
(catalytic amount of 4PPy in refluxing chloroform). Following this
protocol, we prepared biscoumarin 4 with a higher yield (83%),
which is readily isolated in a pure state after solvent removal under
vacuum and direct re-crystallisation from ethanol (Scheme 1,
pathway B). This alternative and economic reaction follows a
succession of organo-base promoted double condensations of two
4-hydroxycoumarin units 1 through their activated 3-C methylene

3010
O. Talhi et al. / Bioorg. Med. Chem. 22 (2014) 3008–3015
group on the aldehyde function in
x-formyl-2-hydroxyacetophe-
of both coumarin moieties (Fig. S1 of ESI). When the 1H NMR spec-
none 3.
trum was acquired at 50 °C (CDCl3), all signals were tentatively
resolved and H-2 clearly appears as the expected doublet, while
the signals of both hydroxyl protons (40-OH/400-OH) appear very
close 11.61 and 11.95 ppm (Fig. S2 of ESI). This result indicates that
an increase in the temperature greatly affects the mobility of the
molecule moieties, making the structure more symmetric.
Alongside with HSQC, the HMBC experiment of biscoumarin 4
allows the assignments of several non-protonated and quaternary
carbons resonances. H-1 showed correlations with C-30/300 and the
carbonyl carbons in the structure 4, including C-3 (202.6 ppm) of
the 3-(20-hydroxyphenyl)propion-1-yl fragment, C-20/200 and C-40/
4 (164.1 and 165.3) of the 4-hydroxycoumarin units (Fig. 1, HMBC).
In addition, H-2 shows weak connectivity with C-3 and C-30/300
enabling the assignment of this latter to the signals at 105.3 and
Structural elucidation of 4 was first made on the basis of exten-
sive 1D and 2D NMR techniques. From 1H NMR spectrum (CDCl3)
we concluded that a pair of 4-hydroxycoumarin nuclei and a 3-(20-
hydroxyphenyl)-3-oxopropyl moiety is involved in the structure of
the obtained compound. Resonances of the aromatic protons of both
4-hydroxycoumarin nuclei appear as broad and unresolved signals,
while both exchangeable protons of hydroxyl groups (40-OH/400-OH)
occur at different chemical shift values (11.45 and 12.10 ppm).
These data seem to indicate that both 4-hydroxycoumarin rings
may not be absolutely positioned in a symmetrical bilateral manner
relative to the sp3 medium plane of the tetrahedral carbon C-1
(26.7 ppm in the 13C NMR spectrum). Moreover, the H-2 signal
appears as a pseudo triplet, which also confirms some asymmetry
24h 48h 72h
G2/M
S
G1
A
C
1.6E+06
1.2E+06
8.0E+05
4.0E+05
K-562
JURKAT
100%
75%
50%
25%
100%
75%
50%
25%
*
*
8h
0.0E+00
1.5E+06
0%
0%
100%
100%
1.0E+06
5.0E+05
0.0E+00
75%
50%
25%
75%
50%
25%
**
**
**
**
**
**
**
**
**
*
*
*
24h
0
10
25
50
100
0%
0%
µ
100%
100%
B
24 h
48 h
72 h
100
75
75%
50%
25%
75%
50%
25%
**
48h
72h
*
50
25
0%
100%
0%
100%
0
100
**
75%
50%
25%
0%
75%
50%
25%
0%
**
**
**
75
50
25
0
**
**
0
10
25
50
100
0
10
25
50
100
10
25
50
100
Compound 4 ( µM)
Compound 4 ( µM)
Compound 4 ( µM)
24h 48h
D
100
75
50
25
0
10
50
100
Compound 4 ( µM)
Figure 3. Compound 4 decreases cancer cell proliferation by blocking cell cycle progression in G0/G1 phase. K-562 and JURKAT cells were treated with the indicated
concentrations of compound 4. (A) Cell viability and (B) proliferation were assessed after 24, 48 and 72 h. (C) Cell cycle distribution was analyzed between 8 and 72 h. (D)
PBMCs from healthy donors were incubated with compound 4 and then cell viability was evaluated after 24 and 48 h of treatment. Data are the mean SD of three
⁄
⁄⁄
independent experiments. and indicate p <0.05 and p <0.01 versus untreated cells, respectively.

O. Talhi et al. / Bioorg. Med. Chem. 22 (2014) 3008–3015
3011
106.0 ppm, once again confirming the absence of symmetry at
room temperature (Figs. S3–S6 of ESI).
Instead, this moiety is engaged in a weak intramolecular C–Hꢀ ꢀ ꢀO
hydrogen bond interaction with a neighbouring hydroxyl group of
a 4-hydroxycoumarin (dashed gold line in Fig. 2). Noteworthy,
despite the weak nature and poor directionality of this supramolec-
ular interaction, it seems to minimize overall steric hindrance of the
molecule, being most likely also present in solution: as shown from
the NOESY NMR studies, this weak interaction helps to promote
tilting of the whole 3-(20-hydroxyphenyl)propion-1-yl moiety with
respect to the sp3 average plane of the central tertiary carbon atom
(tilt angle of ca. 34°). The combined effect of the intramolecular O–
Hꢀ ꢀ ꢀO hydrogen bonds involving the 4-hydroxycoumarin moieties
and this weak supramolecular C–Hꢀ ꢀ ꢀO interaction explains why,
both in the solid state and in solution, compound 4 does not exhibit
any local symmetry element (such as a possible mirror plane bisect-
ing the 3-(20-hydroxyphenyl)propion-1-yl moiety and containing
the tertiary carbon atom).
In the NOESY experiment of biscoumarin 4 (Fig. 1), the main NOE
effects are observed between H-6000 and H-2 and also between both
40/400-OH protons and H-1 and H-2. However, 2000-OH proton pre-
sents only NOE cross effect with one of the two 40/400-OH protons
which is a clear indication that the coumarin units are not disposed
in a symmetric manner around the central tertiary carbon atom at
room temperature (Fig. S7 of ESI). This assumption was unequivo-
cally confirmed using single-crystal X-ray diffraction studies, which
showed that at ꢁ123 °C compound 4 crystallizes in the centrosym-
metric P21/n space group with the asymmetric unit being com-
posed of a whole molecular unit as depicted in Figure 2. The two
4-hydroxycoumarin moieties attached to the tertiary carbon atom
are mutually rotated. In this way the hydroxyl groups are engaged
in strong and highly directional intra-molecular O–Hꢀ ꢀ ꢀO hydrogen
bonds with the carbonyl moieties of the neighbouring 4-hydroxy-
coumarin unit, ultimately boosting the structural robustness of this
molecule. The same type of interaction is also observed within the
3-(20-hydroxyphenyl)propion-1-yl moiety, but the presence of the
methylene C-2 moiety prevents the existence of strong supramo-
lecular interactions with the substituent 4-hydroxycoumarins.
2.2. Biological assays
2.2.1. Compound 4 affects cancer cell proliferation
Compound 4 was evaluated for its effect on cancer cell prolifer-
ation and viability in human chronic myeloid leukaemia K-562 and
Figure 4. Compound 4 inhibits TNF-
plasmid and a Renilla luciferase control reporter plasmid. 24h post-transfection cells were treated with various concentrations of compound 4 for 2 h and then cultured in a
medium with or without 20 ng/ml TNF- for an additional 6 h. Data are collected as the ratio of firefly:Renilla luciferase signal activities with cells treated TNF- set to 100%.
Results represent the mean SD of three independent measurements performed in quadruplicate. (B) K-562 cells were treated with various concentrations of compound 4 for
2 h and then cultured with or without 20 ng/ml TNF- for an additional 6 h. Total RNA was isolated and analyzed by qRT-PCR using primers specific for ICAM1 and CFLAR.
Results represent the ratio targeted gene/b-actin mRNA. Data are the mean SD of 3 independent experiments. (C) JURKAT cells were treated with 40 M compound 4 for 2 h
and then cultured in a medium with or without 20 ng/ml TNF- for the indicated time. Cytoplasmic and nuclear fractions were then analyzed by western blot. Protein loading,
and purity of nuclear and cytosolic fractions were checked by immunodetection of lamin B and -tubulin. Blots are representative of three independent experiments. In all
experiments: and indicate p <0.05 and p <0.01 versus untreated cells, respectively; and indicate p <0.05 and p <0.01 versus TNF- -treated cells.
a-induced NF-jB transactivation. (A) K-562 and JURKAT cell lines were co-transfected with a NF-jB-responsive luciferase reporter
a
a
a
l
a
a
]]
⁄
⁄⁄
]
a

3012
O. Talhi et al. / Bioorg. Med. Chem. 22 (2014) 3008–3015
acute T-cell leukaemia JURKAT cell lines (Fig. 3A and B). Results
showed that treatments with compound 4 affected cell prolifera-
tion in both cell lines with JURKAT cells being more sensitive than
OH
OH
O
OH
O
O
K-652 cells. Indeed, we observed a moderate effect at 10
48 h of treatment only in JURKAT cells and then a more pro-
nounced effect in both cell lines starting from 25 M and 24 h of
treatment. The drastic decrease of proliferation observed from
50 M was accompanied by a relatively restrained reduction of cell
lM after
+
O
O
O
l
OH
5
O
OH
O
4
1
l
viability but solely in JURKAT cells. Decreased tumour cell prolifer-
Scheme 2. Chemical substructures of the biscoumarin 4.
ation was associated to an inhibition of cell cycle progression in
G0/G1 phase in both cell lines starting at 8 h from 50
lM and at
reported to block the UVB-induced activation of NF-
j
j
B and suppress
B by inhibiting
24 h from 25 in JURAKT and K-562 cells, respectively
lM
the transcriptional activity and translocation of NF-
(Fig. 3C). Nevertheless, compound 4 did not alter the viability of
PBMCs isolated from healthy donors (Fig. 3D).
IjBa
degradation and IKK phosphorylation, respectively.23,24 Fur-
thermore, it has been shown that increased IjB phosphorylation
and NF- B nuclear translocation induced by ischemia/reperfusion
j
2.2.2. Compound 4 inhibits TNFa-induced NF-jB activation
injuryweresignificantlydecreasedfollowing pre-treatment withost-
hole, a natural derivative of coumarin found in several plants.25 Based
on these findings, we believe that our novel dicoumarinic compound
could further provide increased inhibition potential. However, clearly
It iswellestablishedthatNF- B signaling pathway isimplicatedin
the regulation of inflammation, proliferation and apoptosis. In addi-
tion, its activation is linked to cancer development and progression.
j
Therefore, we evaluated interference of compound 4 with TNF-
induced NF- B transactivation. We observed that compound 4 abro-
gated TNF- -induced NF- B activation in a dose-dependent manner
with IC50 values of 17.5 and 19.0 M in K-562 and JURKAT cells,
a-
the initial(s) target(s) of coumarin-mediated NF-jB inhibition
j
remains to be elucidated.
a
j
We selected here two leukaemia cell lines where activation of
l
NF-j
B is under the control of differential molecular mechanisms.
respectively (Fig. 4A). These values are within the range of IC50 values
previously reported by our laboratory for various compounds against
TNF-
imentalconditions(Table1). Inordertofurther confirmtheinhibitory
effect of compound 4 on TNF- -induced NF- B signaling, we per-
formed gene expression analyses of NF- B target genes in K-562 cells
(Fig. 4B). Results demonstrated that compound 4 dose-dependently
impaired expression of ICAM (intercellular adhesion molecule) 1
and caspase 8 and FADD-like apoptosis regulator (CFLAR; c-Flip)
a-induced NF-jB activation in K-562 cells using the same exper-
A
a
j
0 µM
2.0E+06
j
10 µM
100 µM
1.5E+06
Compound 5
Compound 1
1.0E+06
5.0E+05
genes induced by TNF-
translocation form cytoplasm to nucleus requires degradation of its
cytoplasmic inhibitor I B, we determined the effect of compound 4
on I integrity as well as on translocation of p50 and p65 NF-
subunits (Fig. 4C).Western blot analyses revealed that compound 4
prevented TNF- -induced I degradation and the subsequent
translocation of NF- B subunits into the nucleus. In the past few
years, several natural and synthetic coumarin derived compounds
were reported to act as NF- B inhibitors. For instance, decursinol
a. Since NF-jB activation and its subsequent
0.0E+00
2.0E+06
j
jBa
jB
1.5E+06
1.0E+06
5.0E+05
0.0E+00
a
jBa
j
j
0
24
48
72
angelate, a coumarin compound isolated from Angelica gigas, blocks
the transmigration and inflammatory activation of cancer cells
through inhibition of phosphoinositide 3-kinase (PI3K), extracellular
Hours
B
140
120
100
80
signal-regulated kinases (ERK) and NF-j
B activation.22 Decursin and
nodakenin, two other coumarins isolated from Angelicae giga, were
Table 1
60
Inhibitors of TNF-
a-induced NF-jB activation in K-562 cells
40
Compound
IC50
(lM)
Refs.
20
4-Hydroxychalcone
4-Methoxychalcone
Altersolanol A
Butein
Calomelanone
Curcumin
Embellicine A
Embellicine B
Flavokawain C
Goniothalamin
Heteronamin
Homobutein
Isoliquiritigenin
Phloretin
Sanguinarine
Tetraacetylaltersolanol
UNBS1450
24
29
0.8
38
11
8
3
0.4
8
(Orlikova, 2012)16
(Orlikova, 2012)16
(Teiten, 2013)27
0
Compound (µM)
0
0
10
100
10
100
(Orlikova, 2012)16
(Orlikova, 2012)16
(Duvoix, 2003)13
(Ebrahim, 2013)28
(Ebrahim, 2013)28
(Orlikova, 2012)16
(Orlikova, 2013)29
(Schumacher, 2010)16
(Orlikova, 2012)16
(Orlikova, 2012)16
(Orlikova, 2012)16
(Duvoix, 2004)28
(Teiten, 2013)27
Compound 1
TNF-α (20ng/ml)
Compound 5
Figure 5. Effect of compound 1 and 5 on cell proliferation and NF-jB transcrip-
tional activity. (A) K-562 cells were treated with the indicated concentrations of
compound and cell proliferation was evaluated after 24, 48 and 72 h. Data are the
mean SD of three independent experiments. (B) K-562 cells were co-transfected
with a NF-jB-responsive luciferase reporter plasmid and the Renilla luciferase
control reporter plasmid. 24 h post-transfection cells were treated with various
concentration of compound 1 and 5 for 2 h and then cultured in a medium with or
without 20 ng/ml TNF-
firefly:Renilla luciferase signal activities with cell treated TNF-
7
5
38
32
41
2
4.6
0.03
4
a
for an additional 6 h. Data are collected as the ratio of
set to 100%. Results
(Juncker, 2011)30
(Duvoix, 2004)28
a
represent the mean SD of three independent measurements performed in
b-Lapachone
⁄⁄
quadruplicate. indicates p <0.01 versus untreated cells.

O. Talhi et al. / Bioorg. Med. Chem. 22 (2014) 3008–3015
3013
Whereas JURKAT cells activate NF-
j
B via a canonical pathway
on a Hampton Research CryoLoop31 with the help of a Stemi 2000
stereomicroscope equipped with Carl Zeiss lenses. Data were col-
lected at 150(2) K on a Bruker X8 Kappa APEX II charge-coupled
involving degradation of the I B inhibitor of the NF-
j
j
B transcrip-
tion factor allowing translocation into the nucleus and transactiva-
tion of target genes, K-562 cells rely on oncokinase Bcr-Abl.
According to Baldwin and co-workers expression of Bcr-Abl leads
device (CCD) area-detector diffractometer (MoK graphite-mono-
a
chromated radiation, k = 0.71073 Å) controlled by the APEX2 soft-
ware package,32 and equipped with an Oxford Cryosystems Series
700 cryostream monitored remotely using the software interface
Cryopad.33 Images were processed using the software package
SAINT+,34 and data were corrected for absorption by the multi-scan
semi-empirical method implemented in SADABS.35 The crystal
structure was solved using the direct methods algorithm imple-
mented in SHELXS-97,36,37 allowing the immediate location of
most of non-hydrogen atoms composing the molecular unit of
compound 4. All remaining non-hydrogen atoms were located
from difference Fourier maps calculated from successive full-
matrix least squares refinement cycles on F2 using SHELXL-
97.37,38 Non-hydrogen atoms were successfully refined using
anisotropic displacement parameters.
Hydrogen atoms bound to carbon were placed at their idealized
positions using appropriate HFIX instructions in SHELXL: 43 for the
aromatic, 23 for the –CH2– groups and 13 for the tertiary carbon
atom. The hydrogen atoms associated with the –OH groups were
directly located from difference Fourier maps and were included
in the structural model with the O–H distances restrained to
0.95(1) Å so to ensure a chemically reasonable geometry for these
moieties. All these hydrogen atoms were included in subsequent
refinement cycles in riding-motion approximation with isotropic
thermal displacements parameters (Uiso) fixed at 1.2 (those bound
to carbon) or 1.5 (for the –OH groups) times Ueq of the atom to
which they are attached.
to activation of NF-
translocation of NF-
function of the RelA/p65 subunit of NF-
tion is dependent on the tyrosine kinase activity of Bcr-Abl’.26 In
our hands and in other labs, degradation of I B inhibitor is not
j
j
B-dependent transcription by causing nuclear
B as well as by increasing the transactivation
B. Importantly, this activa-
j
j
observable in K-562 cell line and accordingly was not investigated
here. We essentially investigate effects of anticancer compounds
on leukaemia and lymphoma cell models and as chronic myeloge-
nous leukaemia patients show increased resistance against clini-
cally used Gleevec (Imatinib) Bcr-Abl inhibitor, additional novel
inhibitors are urgently required.
2.2.3. Substructures 1 and 5 present in compound 4 fail to
produce biological effects
In order to clearly demonstrate that the newly synthetized
hybrid compound 4 possesses an inhibitory potential superior to
the substructures present in its structure 4, namely 4-hydroxy-
coumarin 1 and 20-hydroxypropiophenone 5 (Scheme 2), we then
investigated the effect of such compounds on cell viability and
TNF-a-activation of NF-jB transcriptional activity in K-562 cells
(Fig. 5A and B). Results revealed that both substructures failed to
produce the effects induced by compound 4.
3. Conclusions
The last difference Fourier map synthesis showed the highest
peak (1.274 eÅꢁ3) and deepest hole (ꢁ0.363 eÅꢁ3) located at
1.10 Å and 0.58 Å from O5 and O7, respectively. Structural draw-
ings have been created using the software package Crystal Impact
Diamond.39
Our results underline two alternative synthetic methods of the bis-
4-hydroxycoumarin-type compound 3,30-[3-(2-hydroxyphenyl)-3-
oxopropane-1,1-diyl]bis(4-hydroxy-2H-chromen-2-one. We demon-
strated that the new compound inhibits proliferation of K-562 and
JUKAT leukaemia cell lines associated to an accumulation of cells in
G0/G1 phase of cell cycle without affecting the viability of healthy
PBMCs. In addition, this compound blocks the activation of the pro-
Crystal data for 3,30-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-
diyl]bis(4-hydroxy-2H-chromen-2-one 4: C27H18O8, MW = 470.41
g molꢁ1, colourless block (0.16 ꢂ 0.08 ꢂ 0.05 mm3), monoclinic,
space group P21/n, a = 11.1193(13) Å, b = 14.1801(16) Å, c =
14.4024(14) Å, b = 112.559(7), V = 2097.1(4) Å3, Z = 4, Dc = 1.490
inflammatory NF-jB pathway. We have also rationalized the concept
of the active molecule built from inactive constructive fragments, as
substructures of the biscoumarin-type compound, 20-hydroxyphenyl-
propione and 4-hydroxycoumarin, do not show any activity.
g cmꢁ3 ) = 0.111 mmꢁ1, 19163 reflections were collected
, l(Mo-Ka
in the range 2.10 6 h 6 25.35, index ranges ꢁ12 6 h 6 13,
ꢁ11 6 k 6 17, ꢁ17 6 l 6 17, of which 3847 were independent
(Rint = 0.0753), completeness to h = 25.35° = 100.0%, final R1 ([I >
2r(I)]) = 0.0879, wR2 ([I>2r(I)]) = 0.2354, R1 (all data) = 0.1214,
wR2 (all data) = 0.2613. CCDC 920034.
4. Experimental section
4.1. General
Melting points are measured on Büchi B-540 equipment and are
uncorrected. NMR spectra were recorded on Bruker Avance 300
spectrometer (300.13 MHz for 1H and 75.47 MHz for 13C), with
CDCl3 as solvent and TMS as the internal standard. Chemical shifts
(d) are reported in ppm and coupling constants (J) in Hz which all
are calculated using MESTRENOVA 8 (Free Trail License) analytical
4.3. Synthesis of 3,30-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-
diyl]bis(4-hydroxy-2H-chromen-2-one) 4
4-Hydroxycoumarin 1 (2.00 g, 12.34 mmol) is added to chro-
mone-3-carboxylic acid 2 (1.174 g, 6.17 mmol, 0.5 equiv, pathway
x
-formyl-20-hydroxyacetophenone 3 (1.013 g, 6.17 mmol,
chemistry software suite for NMR, LC, GC and MS. Unequivocal 13
C
A) or
0.5 equiv, pathway B) in chloroform (20 mL). A catalytic amount
of 4PPy (0.09 g, 0.62 mmol, 0.05 equiv) is dropped into the solu-
tion, which is allowed to reflux under stirring overnight. After
TLC monitoring, the solvent is evaporated and the resulting resin-
ous solid is directly recrystallized from ethanol to afford the bis-
coumarin 4: C27H18O8 (yellowish white solid, MW = 470.43 g/
mol, 2.410 g, yield 83%, mp = 223–225 °C).
assignments were made with the aid of 2D HSQC and HMBC exper-
iments (delays for one bond and long-range JC/H couplings were
optimized for 145 and 7 Hz, respectively). Compounds 1, 2, 3, 5
and 4-pyrrolidinopyridine (4PPy) have been purchased form
Sigma–Aldrich.
4.2. Single-crystal X-ray diffraction studies
1H NMR (300.13 MHz, CDCl3): d = 4.21 (pseudo t, J = 6.9 Hz, 2H,
H-2), 5.44 (t, J = 6.7 Hz, 1H, H-1), 6.86–6.97 (m, 2H, H-3000, H-5000),
7.368 (br s, 2H, H-60/600), 7.369 (dd, J = 7.8 and 0.9 Hz, 2H, H-80/
800), 7.46 (ddd, J = 7.8, 7.2 and 1.4 Hz, 1H, H-4000), 7.60 (t, J = 7.8 Hz,
2H, H-70/700), 7.87 (dd, J = 8.1 and 1.4 Hz, 1H, H-6000), 8.01 (br s,
Crystals of compound 4 were harvested from the crystallization
vial and were immersed in highly viscous FOMBLIN
Y
perfluoropolyether vacuum oil (LVAC 140/13) purchased from
Sigma–Aldrich. A suitable single-crystal was selected and mounted

3014
O. Talhi et al. / Bioorg. Med. Chem. 22 (2014) 3008–3015
2H, H-50/500), 11.45 and 12.10 (2 br s, 2H, 40/400-OH), 11.92 (s, 1H,
2000-OH) ppm.
4.9. Statistical analysis
1H NMR (300.13 MHz, 50 °C, CDCl3): d = 4.18 (d, J = 6.7 Hz, 2H,
H-2), 5.44 (t, J = 6.7 Hz, 1H, H-1), 6.87–6.94 (m, 2H, H-3000, H-5000),
7.32–7.37 (m, 4H, H-60/600, H-80/800), 7.44 (ddd, J = 7.7, 7.2 and
1.6 Hz, 1H, H-4000), 7.60 (dt, J = 7.6 and 1.6 Hz, 2H, H-70/700), 7.85
(dd, J = 8.1 and 1.6 Hz, 1H, H-6000), 8.01 (br d, 2H, J = 7.9 Hz, H-50/
500), 11.61 and 11.95 (2 br s, 2H, 40/400-OH), 11.85 (s, 1H, 2000-OH)
ppm.
Differences between mean values were tested for statistical
significance by the two-tailed Student’s t-test. p-Values below
0.05 were considered as statistically significant.
Acknowledgments
We would like to thank Fundação para a Ciência e a Tecnologia
(FCT, Portugal), the European Union, QREN, FEDER, COMPETE, for
funding the Organic Chemistry Research Unit (QOPNA) (Project
PEst-C/QUI/UI0062/2013), Laboratório Associado Centro de Inves-
tigação em Materiais Cerâmicos e Compósitos—CICECO (Project
PEst-C/CTM/LA0011/2013) and the Portuguese National NMR
Network (RNRMN). European Community’s Seventh Framework
Programme (FP7/2007-20139 under Grant agreement n° 215009)
is also acknowledged for financial support. We further wish to
thank FCT for the post-doctoral Grant SFRH/BD/63736/2009 (to
J.A.F.) and for specific funding toward the purchase of the single-
crystal X-ray diffractometer. MiS is supported by a ‘Waxweiler
Grant for cancer prevention research’ from the Action Lions
‘Vaincre le Cancer’. The work performed at Laboratoire de Biologie
Moléculaire et Cellulaire du Cancer was supported by Télévie
Luxembourg, the «Recherche Cancer et Sang» foundation, and the
«Recherches Scientifiques Luxembourg» and «Een Häerz fir
Kriibskrank Kanner» associations. M.D. is supported by the NRF
by the MEST of Korea for Tumor Microenvironment GCRC
2012-0001184 Grant, by the Seoul National University (SNU)
Research Grant, by the Research Settlement Fund for the new fac-
ulty of SNU and by the Research Institute of Pharmaceutical
Sciences.
13C NMR (75.47 MHz, CDCl3): d = 26.7 (C-1), 37.8 (C-2), 105.3
and 106.0 (C-30,300), 116.6 (C-80,800), 118.5 (C-3000), 119.0 (C-1000),
119.1 (C-5000), 124.0 (C-100,1000), 124.4 (C-50,500), 125.0 (C-60,600),
129.6 (C-6000), 132.8 (C-70,700), 136.7 (C-4000), 152.2 (C-90/900), 162.2
(C-2000), 164.1 and 165.3 (C-20,200, C-40,400), 202.6 (C-3) ppm.
HRMS (ESI+): m/z calcd for [C27H18O8+Na]+: 493.0899; found:
493.0904. Anal. Calcd: C, 68.94; H, 3.86. Found: C, 68.96; H, 3.89.
4.4. Cell culture, treatments and viability assay
The human K-562 (chronic myeloid leukaemia) and JURKAT
(acute T-cell leukaemia) cell lines were obtained from the Deut-
sche Sammlung von. Mikroorganismen und Zellkulturen. PBMCs
were obtained as previously reported.40 All cell types were
cultured in RPMI 1640 (Lonza) supplemented with 10% heat-inac-
tivated foetal calf serum (Lonza) and 1% antibiotic-antimycotic
(Lonza) at 37 °C in humid atmosphere and 5% CO2.
Compounds 1, 4 and 5 were dissolved at 50 mM in DMSO
(Sigma–Aldrich), aliquoted and stored at ꢁ20 °C upon treatment.
TNF
a
(Sigma–Aldrich) was dissolved in 5% (w/v) BSA in 1ꢂ PBS
to a concentration of 10 lg/mL. Cells in exponential phase were
treated at a concentration of 200,000 cells/mL. Control cells were
treated with the same volume of DMSO as the one required for
compounds. Cell proliferation and viability were measured as pre-
viously reported.40
Supplementary data
Supplementary data (1H, 13C, HSQC, HMBC and NOESY NMR
spectra) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmc.2014.03.046.
4.5. Analysis of cell cycle distribution
Cell cycle analyses were performed as previously described.41
4.6. Transient transfection and luciferase reporter gene assay
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