Synthesis and cellular characterization of novel isoxazolo- and thiazolohydrazinylidene-chroman-2,4-diones on cancer and non-cancer cell growth and death

Article abstract of DOI:10.1016/j.bmc.2014.03.026

Coumarins are extensively studied anticoagulants that exert additional effects such as anticancerogenic and even anti-inflammatory. In order to find new drugs with anticancer activities, we report here the synthesis and the structural analysis of new coumarin derivatives which combine the coumarin core and five member heterocycles in hydrazinylidene-chroman-2,4-diones. The derivatives were prepared by derivatization of the appropriate heterocyclic amines which were used as electrophiles to attack the coumarin ring. The structures were characterized by spectroscopic techniques including IR, NMR, 2D-NMR and MS. These derivatives were further characterized especially in terms of a potential cytotoxic and apoptogenic effect in several cancer cell lines including the breast and prostate cancer cell lines MCF-7, MDA-MB-231, PC-3, LNCaP, and the monocytic leukemia cell line U937. Cell viability was determined after 48 h and 72 h of treatment with the novel compounds by MTT assay and the 50% inhibitory concentrations (EC₅₀ values) were determined. Out of the 8 novel compounds screened for reduced cell viability, 4c, 4d and 4e were found to be the most promising and effective ones having EC₅₀ values that were several fold reduced when compared to the reference substance 4-hydroxycoumarin. However, the effects were cancer cell line dependent. The breast cancer MDA-MB-231 cells, the prostate cancer LNCaP cells, and U937 cells were most sensitive, MCF-7 cells were less sensitive, and PC-3 cells were more resistant. Reduced cell viability was accompanied by increased apoptosis as shown by PARP-1 cleavage and reduced activity of the survival protein kinase Akt. In summary, this study has identified three novel coumarin derivatives that in comparison to 4-hydroxycoumarin have a higher efficiency to reduce cancer cell viability and trigger apoptosis and therefore may represent interesting novel drug candidates.

Full text of DOI:10.1016/j.bmc.2014.03.026

Bioorganic & Medicinal Chemistry 22 (2014) 2655–2661
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cellular characterization of novel isoxazolo- and
thiazolohydrazinylidene-chroman-2,4-diones on cancer and
non-cancer cell growth and death
Ahmed Jashari a, , Faik Imeri b, , Lulzime Ballazhi a,b, Agim Shabani a, Bozhana Mikhova c, Gerald Dräger d,
Emil Popovski e, Andrea Huwiler b,
⇑
a Group of Chemistry, Faculty of Natural Sciences & Mathematics, State University of Tetova, 1200 Tetova, Macedonia
b Institute of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland
c Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Build. 9, 1113 Sofia, Bulgaria
d Institute of Organic Chemistry, Leibniz Universität Hannover, D-30167 Hannover, Germany
e Institute of Chemistry, Faculty of Natural Sciences & Mathematics, Ss. Cyril & Methodius University, PO Box 162, 1000 Skopje, Macedonia
a r t i c l e i n f o
a b s t r a c t
Article history:
Coumarins are extensively studied anticoagulants that exert additional effects such as anticancerogenic
and even anti-inflammatory. In order to find new drugs with anticancer activities, we report here the syn-
thesis and the structural analysis of new coumarin derivatives which combine the coumarin core and five
member heterocycles in hydrazinylidene-chroman-2,4-diones. The derivatives were prepared by deriva-
tization of the appropriate heterocyclic amines which were used as electrophiles to attack the coumarin
ring. The structures were characterized by spectroscopic techniques including IR, NMR, 2D-NMR and MS.
These derivatives were further characterized especially in terms of a potential cytotoxic and apoptogenic
effect in several cancer cell lines including the breast and prostate cancer cell lines MCF-7, MDA-MB-231,
PC-3, LNCaP, and the monocytic leukemia cell line U937. Cell viability was determined after 48 h and 72 h
of treatment with the novel compounds by MTT assay and the 50% inhibitory concentrations (EC50 val-
ues) were determined. Out of the 8 novel compounds screened for reduced cell viability, 4c, 4d and 4e
were found to be the most promising and effective ones having EC50 values that were several fold reduced
when compared to the reference substance 4-hydroxycoumarin. However, the effects were cancer cell
line dependent. The breast cancer MDA-MB-231 cells, the prostate cancer LNCaP cells, and U937 cells
were most sensitive, MCF-7 cells were less sensitive, and PC-3 cells were more resistant. Reduced cell via-
bility was accompanied by increased apoptosis as shown by PARP-1 cleavage and reduced activity of the
survival protein kinase Akt.
Received 17 December 2013
Revised 12 March 2014
Accepted 16 March 2014
Available online 24 March 2014
Keywords:
Chromandiones
Cancer cells
Apoptosis
PARP-1
Akt
In summary, this study has identified three novel coumarin derivatives that in comparison to 4-
hydroxycoumarin have a higher efficiency to reduce cancer cell viability and trigger apoptosis and there-
fore may represent interesting novel drug candidates.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
coumarin, has been extensively studied both in biochemical and
pharmaceutical fields.3–5
Coumarins are a group of heterocyclic compounds synthesized
by numerous plant species as well as by some bacteria and fungi.1,2
According to their chemical structure, they belong to the family of
benzopyrones. So far, more than 1300 different coumarins have
been identified. The most representative molecule, that is
Over the past decades, many studies have reported that couma-
rins and derivatives exert a plethora of biological activities includ-
ing anti-microbial, anti-viral, anti-coagulant, anti-inflammatory,
and anti-cancer effects.6–13 Best known is the anti-coagulant effect
of the 4-hydroxycoumarin derivative warfarin ((RS)-4-hydroxy-3-
(3-oxo-1-phenyl-butyl)-coumarin) that reached market approval
early on.14–16 A beneficial effect of warfarin in cancer patients lead-
ing to prolonged survival was shown by Zacharski and col-
leagues.17 Meanwhile, many studies have reported a beneficial
effect of coumarins on other cancer types including malignant
⇑
Corresponding author. Tel.: +41 31 632 32 14.
E-mail address: Huwiler@pki.unibe.ch (A. Huwiler).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmc.2014.03.026
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2656
A. Jashari et al. / Bioorg. Med. Chem. 22 (2014) 2655–2661
melanoma, leukemia, renal cell carcinoma, prostate and breast
cancer cells progression.18–20 Also, certain platinum(II) complexes
azole). 13C NMR (DMSO-d6, d/ppm): 157.6 (C2-coum.), 125.7 (C3-
coum.), 178.4 (C4-coum.), 126.8 (C5-coum.), 124.8 (C6-coum.),
136.9 (C7-coum.), 117.4 (C8-coum.), 120.4 (C4a-coum.), 154.1
(C8a-coum.), 162.7 (C3-isoxazole), 94.1(C4-isoxazole), 172.0 (C5-
isoxazole), 12.3 (CH3-isoxazole). TOF-MS-ES+ (m/z): 272.0677
[M+H]+, 294.0440 [M+Na]+, C13H10N3O4.
of aminocoumarins showed very good in vitro cytotoxicity.21
A
variety of mechanisms have been proposed such as interfering
with estrogen synthesis, interfering with cell cycle progression or
even acting as inhibitors of cytochrome P450 1.22
Also, a number of coumarins with substituents in position 7
(usually some electron-releasing group) and position 3, especially
imines,23,24 were reported and, in general their photosensitivity
was tested. Finally, a number of nitrogen-rich compounds were
found to be good chemotherapeutic agents25 especially if a thiazole
ring was introduced as shown by Gouda et al.26 Recent studies
have suggested that several coumarin derivatives showed antipro-
liferative activity in various tumor cells.13,20,27 Considering those
inputs, coumarin as a very versatile biological agent, and nitro-
gen-rich heterocyclic compounds as good chemotherapeutic
agents, it was tempting to combine these moieties and evaluate
their activity. Therefore, in this study, we have synthesized several
novel 3-substituted thiazolo and isoxazolo hydrazinylidene-chro-
man-2,4-dione compounds. These compounds were tested for cell
viability in different cancer and non-cancer cell lines. We found
that three of the novel compounds effectively reduced cell viability
in a concentration-dependent manner. A decrease in the level of
phospho-Akt and an increase in the level of PARP-1 cleavage
strongly argues for the induction of the intrinsic pathway of
apoptosis.
2.1.3. 3-[2-(Thiazol-2-yl)hydrazinylidene]chroman-2,4-dione
(4c)
Orange-red crystals (82%), mp 209–211 °C. FTIR (KBr, m
/cmÀ1):
3625-3250 (NH, stretching, broad), 3125, 3080 (CH, aromatic
stretching), 1765 (C@O, stretching), 1623, 1606, 1521 (aromatic
deformations). 1H NMR (DMSO-d6, d/ppm, J/Hz): 8.00 dd (8.2, 1.6,
H5-coum.), 7.30–7.45 m (H6&H8-coum.), 7.81 ddd (8.0, 8.0, 1.8,
H7-coum.), 7.70 d (2.5, H4-thiazole), 7.57 d (2.5, H5-thiazole).
13C NMR (DMSO-d6, d/ppm): 157.3 (C2-coum.), 125.0 (C3-coum.),
177.9 (C4-coum.), 126.7 (C5-coum.), 124.8 (C6-coum.), 136.8 (C7-
coum.), 117.4 (C8-coum.), 120.4 (C4a-coum.), 154.0 (C8a-coum.),
166.3 (C2-thiazole), 140.4 (C4-thiazole), 117.6 (C5-thiazole). TOF-
MS-ES+ (m/z): 274. 0345 [M+H]+, 296.0160 [M+Na]+, C12H7N3O3S.
2.1.4. 3-[2-(5-Methylthiazol-2-yl)hydrazinylidene]chroman-2,4-
dione (4d)
Red crystals (63%), mp 218–220 °C. FTIR (KBr, m
/cmÀ1): 3616-
3175 (NH, stretching, broad), 3120 (CH, aromatic stretching),
1736 (C@O, stretching), 1617, 1544 (aromatic deformations). 1H
NMR (DMSO-d6, d/ppm, J/Hz): 8.00 dd (8.0, 1.7, H5-coum.),
7.30–7.45 m (H6&H8-coum. &H4-thiazole), 7.78 ddd (8.0, 8.0,
1.5, H7-coum.), 2.44 d (1.2, CH3-thiazole). 13C NMR (DMSO-d6,
d/ppm): 157.3 (C2-coum.), 124.6 (C3-coum.), 177.6 (C4-coum.),
126.6 (C5-coum.), 124.7 (C6-coum.), 136.7 (C7-coum.), 117.3
(C8-coum.), 120.4 (C4a-coum.), 153.9 (C8a-coum.), 164.5 (C2-thi-
azole), 137.5 (C4-thiazole), 131.7 (C5-thiazole), 11.9 (CH3-thia-
zole). TOF-MS-ES+ (m/z): 288.0403 [M+H]+, 310.0294 [M+Na]+,
2. Materials and methods
2.1. General synthetic procedure
The heterocyclic amines (1a–h, 10 mmol) were dissolved in
10 mL water followed by addition of 40 mL of 6 M HCl and the sys-
tems were cooled in the ice-salt bath down to À10 °C. Afterwards,
an aqueous solution of NaNO2 (10 mmol, 0.7 g/5 mL H2O) was
added slowly drop by drop and stirred vigorously on a magnetic
stirrer. After 15 min, fresh solution of 4-hydroxycoumarin (3,
10 mmol, 1.62 g) in 10 mL NaOH (10 wt.) was added. Intensively
colored and voluminous precipitates (4a–h) were obtained imme-
diately which were stirred 15 min. in the bath and 30 min. on room
temperature. Finally, they were filtrated by vacuum, washed 3
times with distilled water and dried on air. The purification was
carried out by the technique of recrystallization using ethanol as
solvent.
C13H9N3O3S.
2.1.5. 3-[2-(4,5-Dimethylthiazol-2-yl)hydrazinylidene]chroman-
2,4-dione (4e)
Carmine-red crystals (65%), mp 206–208 °C. FTIR (KBr, m
/cmÀ1):
3629-3075 (NH, stretching, broad), 1752 (C@O, stretching), 1616,
1558 (aromatic deformations). 1H NMR (DMSO-d6, d/ppm, J/Hz):
8.01 dd (7.8, 1.4, H5-coum.), 7.25-7.45 m (H6&H8-coum.), 7.77
ddd (8.0, 8.0, 1.8, H7-coum.), 2.34 s (5-CH3-thiazole), 2.23 s (4-
CH3-thiazole). 13C NMR (DMSO-d6, d/ppm): 157.3 (C2-coum.),
124.0 (C3-coum.), 177.2 (C4-coum.), 126.7 (C5-coum.), 124.7 (C6-
coum.), 136.5 (C7-coum.), 117.3 (C8-coum.), 120.6 (C4a-coum.),
153.9 (C8a-coum.), 163.9 (C2-thiazole), 145.0 (C5-thiazole), 144.5
(C4-thiazole), 11.2 (5-CH3-thiazole), 14.0 (4-CH3-thiazole). TOF-
MS-ES+ (m/z): 302.0834 [M+H]+, 324.0802 [M+Na]+, C14H11N3O3S.
2.1.1. 3-[2-(4H-1,2,4-Triazol-3-yl)hydrazinylidene]chroman-2,4-
dione (4a)
Yellow powder (93%), mp 225–227 °C. FTIR (KBr,
m
/cmÀ1):
3616-3175 (NH, stretching, broad), 3120 (CH, aromatic stretching),
1736 (C@O, stretching), 1617, 1544 (aromatic deformations). 1H
NMR (DMSO-d6, d/ppm, J/Hz): 8.01 dd (7.5, 1.5, H5-coum.), 7.30–
7.45 m (H6&H8-coum.), 7.80 dd (7.5, 1.5, H7-coum.), 8.63 br s
(H5-triazole), 10.77 s (NH-triazole). 13C NMR (DMSO-d6, d/ppm):
157.9 (C2-coum.), 124.4 (C3-coum.), 178.6 (C4-coum.), 126.7 (C5-
coum.), 124.8 (C6-coum.), 136.2 (C7-coum.), 117.3 (C8-coum.),
120.3 (C4a-coum.), 154.0 (C8a-coum.), 164.0 (C2-triazole), 145.3
(C5-triazole). TOF-MS-ES+ (m/z): 280.0582 [M+Na]+, C11H7N5O3.
2.1.6. 3-[2-(5-tert-Butylisoxazol-3-yl)hydrazinylidene]chroman-
2,4-dione (4f)
Yellow crystals (86%), mp 225–227 °C. FTIR (KBr,
m
/cmÀ1):
3616–3175 (NH, stretching, broad), 3120 (CH, aromatic stretching),
1736 (C@O, stretching), 1617, 1544 (aromatic deformations). 1H
NMR (DMSO-d6, d/ppm, J/Hz): 8.00 dd (8.0, 1.5, H5-coum.), 7.30–
7.45 m (H6&H8-coum.), 7.80 ddd (8.0, 8.0, 1.5, H7-coum.), 6.58 s
(H4-isoxazole), 1.35 s (tert-CH3-isoxazole). 13C NMR (DMSO-d6, d/
ppm): 157.6 (C2-coum.), 125.7 (C3-coum.), 178.3 (C4-coum.),
126.8 (C5-coum.), 124.9 (C6-coum.), 137.0 (C7-coum.), 117.4
(C8-coum.), 120.4 (C4a-coum.), 154.1 (C8a-coum.), 162.4 (C3-
isoxazole), 91.2 (C4-isoxazole), 182.8 (C5-isoxazole), 32.1
(tert-C(CH3)3-isoxazole), 28.2 (tert-C(CH3)3-isoxazole). TOF-MS-ES+
(m/z): 314.1189 [M+H]+, 336.0926 [M+Na]+, 649.2048 [2M+Na]+
C16H15N3O4.
2.1.2. 3-[2-(5-Methylisoxazol-3-yl)hydrazinylidene]chroman-
2,4-dione (4b)
Yellow small needles (92%), mp 203–205 °C. FTIR (KBr, m
/cmÀ1):
3620–3053 (NH, stretching, broad), 1740 (C@O, stretching), 1602,
1525 (aromatic deformations). 1H NMR (DMSO-d6, d/ppm, J/Hz):
8.00 dd (7.8, 1.7, H5-coum.), 7.30–7.45 m (H6&H8-coum.), 7.80
ddd (8.0, 8.0, 1.5, H7-coum.), 6.63 (H4-isoxazole), 2.46 s (CH3-isox-

A. Jashari et al. / Bioorg. Med. Chem. 22 (2014) 2655–2661
2657
2.1.7. 3-[2-(5-Bromothiazol-2-yl)hydrazinylidene]chroman-2,4-
dione (4g)
mycin (100
l
g/mL), 2 mM N-acetyl-
L
-alanyl-
L
-glutamine and
10 mM HEPES. Prostate cancer cells PC-3 were maintained in DMEM
medium with 10% FCS, HEPES 1M (5 mL), PS (5 mL). LNCaP cells
were maintained in RPMI medium with 10% FCS, HEPES 1M
(5 mL), PS (5 mL). All cell lines were incubated at 37 °C in an atmo-
sphere containing 5% CO2. For experiments, cells from exponentially
growing culture were used.
Orange crystals (69%), mp 217–219 °C. FTIR (KBr,
m
/cmÀ1):
3624–3164 (NH, stretching, broad), 1738 (C@O, stretching), 1617,
1560 (aromatic deformations). 1H NMR (DMSO-d6, d/ppm, J/Hz):
7.99 dd (8.0, 1.7, H5-coum.), 7.30–7.45 m (H6&H8-coum.), 7.77
dd (7.5, 1.7, H7-coum.), 7.77 s (H4-thiazole). 13C NMR (DMSO-d6,
d/ppm): 157.2 (C2-coum.), 125.3 (C3-coum.), 177.3 (C4-coum.),
126.8 (C5-coum.), 124.7 (C6-coum.), 136.6 (C7-coum.), 117.3 (C8-
coum.), 120.7 (C4a-coum.), 154.0 (C8a-coum.), 163.1 (C2-thiazole),
142.3 (C4-thiazole), 105 (C5-thiazole). TOF-MS-ES+ (m/z):
353.9615 [M+H]+, 375.9648 [M+Na]+, C12H6BrN3O3S.
2.5. Cell viability assay
The effect of 4-hydroxycoumarin and novel derivatives on the
viability of the different cell lines was determined by an MTT
(3[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
assay. In brief, 5000 cells were plated into each well of a 96-well
plate in a volume of 100
to attach overnight and then treated with the different compounds
in increasing concentrations (1–500 M). After 48 h and 72 h of
incubation, 20 L of a 5 mg/mL MTT solution was added to each
well and incubated for further 4 h at 37 °C. Thereafter, 100 L of
4 mM HCl, 0.1% Nonidet P-40 (in isopropanol) was added to each
well to solubilize MTT crystals. The plates were covered with a foil
and vortexed on an orbital shaker for 15 min. Then, absorbance at
590 nm was measured in a SpectraMax microplate reader. All
experiments were performed at least 3 times, with 4 wells for each
concentration of the tested agents. Cell survival (% of control) was
calculated relative to untreated controls. The 50% inhibitory con-
centration (EC50) was determined as the anticancer drug concen-
tration causing 50% reduction in cell viability and calculated from
the viability curves by using the Bliss‘s software (Bliss Co, CA). Cell
survival was calculated using the following formula: survival
rate = (mean experimental absorbance/mean control absor-
bance) Â 100%. The viability of U937 cells was measured by Alamar
blue.
2.1.8. 3-[2-(Isoxazol-3-yl)hydrazinylidene]chroman-2,4-dione
(4h)
lL growth medium. Cells were allowed
Yellow crystals (74%), mp 218–221 °C. FTIR (KBr,
m
/cmÀ1):
3611-3198 (NH, stretching, broad), 3107 (CH, aromatic stretching),
1741 (C@O, stretching), 1626, 1572 (aromatic deformations). 1H
NMR (DMSO-d6, d/ppm, J/Hz): 8.00 dd (7.8, 1.7, H5-coum.), 7.32–
7.42 m (H6&H8-coum.), 7.80 ddd (8.0, 8.0, 1.8, H7-coum.), 6.92 d
(1.6, H4-isoxazole), 8.99 d (1.6, H5-isoxazole). 13C NMR (DMSO-
d6, d/ppm): 157.6 (C2-coum.), 126.0 (C3-coum.), 178.6 (C4-coum.),
126.8 (C5-coum.), 124.9 (C6-coum.), 137.0 (C7-coum.), 117.4 (C8-
coum.), 120.4 (C4a-coum.), 154.1 (C8a-coum.), 162.2 (C3-isoxaz-
ole), 97.3 (C4-isoxazole), 162.4 (C5-isoxazole). TOF-MS-ES+ (m/z):
258.0629 [M+H]+, 280.0565 [M+Na]+, C12H7N3O4
l
l
l
2.2. Characterization of synthesized compounds
Melting points were determined on a Reichert heating plate and
were uncorrected. Infrared spectra (KBr pellets) were measured on
a Perkin–Elmer System 2000 FT IR. The NMR spectra were run on a
Bruker-250 DRX Spectrometer using standard Bruker Topspin soft-
ware. DMSO-d6 was used as a solvent and the chemical shifts were
referenced to the residual solvent signal (2.5 ppm for 1H and
39.5 ppm for 13C spectra). The signals were assigned with the
aim of 1H, 13C, DEPT, COSY, HMQC and HMBC spectra. The digital
resolution of the 1D-spectra was 0.12 Hz/Pt for 1H and 1.4 Hz/Pt
for 13C. Mass spectra were measured with Q-TOF premier (MICRO-
MASS) spectrometer (ESI mode) in combination with a WATERS
Acquity UPLC system equipped with a WATERS Acquity UPLC
2.6. Western blot analysis
Stimulated cells were homogenized in lysis buffer28 and centri-
fuged for 10 min at 14,000g. The supernatant was taken for protein
determination. 30 lg of protein were separated by SDS–PAGE,
transferred to a nitrocellulose membrane and Western blot analy-
sis was performed as previously described29 using antibodies as
indicated in the figure legends.
BEH C18 1.7 lm column (solvent A: water + 0.1% {v/v} formic acid,
solvent B: MeOH + 0.1% {v/v} formic acid; flow rate = 0.4 mL/min;
gradient {t [min]/solvent B [%]}: {0/5} {2.5/95} {6.5/95} {6.6/5}
{8/5}). All the reagents and solvents were obtained from commer-
cial sources and were used without further purification.
2.7. Statistical analysis
Statistical analysis was performed using one-way analysis of
variance (ANOVA) followed by a Bonferroni’s post hoc test for mul-
tiple comparisons (GraphPad InStat version 3.00 for Windows NT,
GraphPad Software, San Diego, CA, USA).
2.3. Chemicals for cell culture studies
All synthesized compounds were dissolved in DMSO as 10 mM
stock solutions and stored at -20 °C. Further dilutions were made
in complete Dulbecco’s modified eagle’s medium (DMEM) contain-
ing 10% fetal bovine serum (FBS). MTT 3-(4, 5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazoliumbromide were purchased from Sigma
(St. Louis, MO, US). All cell media and supplements were from
Invitrogen, Basel, Switzerland.
3. Results
In order to synthesize novel derivatives of coumarines, we used
the fact based on HSAB30,31 that electrophiles are attacking the
coumarin core at the position 3. We expected that diazonium ions
as electrophilic particles will attack right at this position.
For this aim, 8 salts (2a–h) from their corresponding five mem-
ber heterocyclic amines (1a–h, Scheme 1) were prepared. A basic
aqueous solution (NaOHaq, 10%) of 4-hydroxycoumarin (3, schema
2) was prepared and added dropwise to the freshly prepared salts
(2a–h). Precipitates of hydrazinylidene-chroman-2,4-diones (4a–
h) with characteristic intensive colors were obtained (Scheme 2).
The reactions were monitored by the TLC technique using different
eluents. Total spectral assignation, by IR, 1H NMR, 13C NMR,
2D-NMR and MS was performed.
2.4. Cell culturing
The human breast cancer cell line MCF-7 was maintained in
DMEM containing 10% FBS, supplemented with glutamax (0.03%)
and 100 lg/mL benzyl penicillin, 100 U/mL streptomycin. The hu-
man endothelial cell line (EA.hy 926), the breast cancer cell line
MDA-MB-231 and U937 (human leukemic monocyte lym-
phoma cell line) were cultured in RPMI medium containing 4.5 g/L
glucose, 10% fetal bovine serum, penicillin (100 units/mL), strepto-

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A. Jashari et al. / Bioorg. Med. Chem. 22 (2014) 2655–2661
R3
Y
and multiplet at about 8.07, 7.80 and 7.35 ppm on 1:1:2 ratio
R3
Y
R3
Y
R2
R1
R2
R1
R2
R1
can be seen, which is similar to that of 3 for the four aromatic pro-
tons of the benzene ring. On the other side, the signal at 5.37 ppm
from the hydrogen at the position 3 of compound 3 is absent in all
derivatives which confirms that the substitution had taken please
right in this position, as expected. Additionally, the HMBC, HMQC
and MS were successfully performed to confirm that the structures
of those coupled derivatives matches the proposed structures
(4a–h, Scheme 2). Additionally, mono-crystals from one of the
derivatives (4f) were successfully obtained that allowed
single-crystal X-ray diffractogram.32 By the means of diffractome-
try the hydrazinylidene structure of the synthesized derivatives
was revealed.
NaNO2 / HCl
-10 °C
N
N
NH2
N
N
X
X
X
N
N
N
1a - h
2a - h
1a: X=N; Y=NH; R2=H
1b: X=O; Y=C; R2=CH3; R3=H
1c: X=C; Y=S; R1=R2=H
1e: X=C; Y=S; R1=R2=CH3
1f: X=O; Y=C; R2=terc-Bu; R3=H
1g: X=C; Y=S; R1=H; R2=Br
1h: X=O; Y=C; R2=R3=H
1d: X=C; Y=S; R1=H; R2=CH3
Scheme 1. Derivatization of heterocyclic amines.
To see whether these novel isoxazolo- and thiazolo derivatives
of coumarin had a superior cellular effect compared to the
4-hydroxycoumarin, we tested their effects on cell viability of a
panel of different human cell lines including cancer cell lines of
different histotypes and non-cancer cell lines such as the human
endothelial cell line EA.hy 926. All compounds were treated with
R3
Y
R3
Y
R2
R1
R2
R1
N
N
N
NH2
X
X
N
N
OH
O
2a - h
H
O
increasing concentrations from 1 to 500 lM and the 50% inhibitory
concentration was thereafter calculated from the viability curves.
EC50 values at 48 h and at 72 h are summarized in Table 1A and
B, respectively.
Results show that the lead compound 4-hydroxycoumarin is
not very cytotoxic in any of the cell types tested. EC50 values are
3
R2
R3
Y
X
R1
N
O
HN
N
in a very high concentration range of 100–200 lM in all cell types
tested. The derivatives 4a and 4b which had a nitrogen or oxygen
in the position 5 of the five-membered heterocycle did not reveal
any higher cytotoxic effect than 4-HC (3). Interestingly, the deriv-
atives 4c, 4d and 4e having sulfur in position 3 of the five mem-
bered heterocycle (Scheme 3) showed a more potent effect on
cell viability than 4-HC (3) although this was strikingly dependent
on the cell type tested. Whereas the estrogen-dependent breast
cancer cell line MCF-7, the androgen-independent prostate cancer
cell line PC-3 and the human endothelial cell line EA.hy926 were
rather resistant to treatment with 4c, 4d, and 4e and EC50 values
O
O
4a - h
4a: X=N; Y=NH; R2=H
4e: X=C; Y=S; R1=R2=CH3
4f: X=O; Y=C; R2=terc-Bu; R3=H
4g: X=C; Y=S; R1=H; R2=Br
4h: X=O; Y=C; R2=R3=H
4b: X=O; Y=C; R2=CH3; R3=H
4c: X=C; Y=S; R1=R2=H
4d: X=C; Y=S; R1=H; R2=CH3
Scheme 2. Synthesis of hydrazinylidene-chroman-2,4-diones.
remained between 100 and 200 lM, the breast cancer cell line
MDA-MB231 cell line, the prostate cancer cell line LNCaP, and
the human monocytic leukemia cell line U937 were several time
In the IR spectra’s of those derivatives it can be noticed charac-
teristic C@O band in the narrow region from 1766–1736 cmÀ1. In
the 1H NMR in general doublet of doublets, triplet of doublets
more sensitive to treatment. EC50 values dropped to 20–60
lM
after 48 h of treatment, and to 10–50 M after 72 h of treatment.
l
Table 1
Inhibitory effects of 4-hydroxycoumarin and of the synthesized derivatives on the viability of various human cancer and non-cancer cell lines
MCF-7
MDA-MB-231
EA.hy926
LNCaP
PC-3
U937
A
48 h
4-HC
4a
>200
>200
>200
>200
>200
>200
100.51 10.26
>200
150.46 12.92
>200
>200
>200
4b
4c
4d
4e
4f
4g
>200
>200
>200
>200
>200
>200
99.94 0.53
100.40 0.41
99.94 0.51
>200
64.84 6.65
59.14 6.18
50.43 5.74
>200
128.27 6.54
153.93 1.74
120.35 1.04
163.45 8.32
150.43 3.12
29.96 8.41
20.46 8.92
20.07 11.47
>200
98.08 6.73
118.10 3.14
82.93 4.55
>200
49.72 3.51
49.97 0.42
50.05 0.54
>200
99.78 0.25
99.58 1.33
>200
>200
117.57 3.53
B
72 h
4-HC
4a
4b
4c
4d
4e
4f
199.82 2.88
49.69 0.88
100.29 2.96
19.95 0.90
19.60 5.96
20.15 0.80
100.03 3.18
50.21 2.18
100.26 18.40
49.68 5.18
50.26 0.92
49.96 6.50
9.91 0.38
9.82 0.41
50.26 0.92
49.60 2.01
>200
120.27 9.06
>200
>200
19.69 5.27
20.12 4.07
20.09 3.67
>200
173.31 11.27
>200
>200
95.32 6.92
118.10 9.68
109.32 9.66
>200
164.12 2.78
154.41 7.68
128.90 7.01
49.89 0.25
28.38 2.17
29.68 1.72
119.71 6.75
107.99 5.41
95.12 1.21
150.34 6.71
96.54 5.03
77.27 2.15
72.77 3.28
149.96 1.51
156.06 1.02
4g
>200
>200
The different cell lines were treated for either 48 h (1A) or 72 h (1B) with the indicated compounds at concentrations between 1
l
M and 500
lM and cell viability was
determined by the MTT method as described in the Section 2. EC50 values were calculated for each compound. Values are means S.D. of at least three independent
experiments.

A. Jashari et al. / Bioorg. Med. Chem. 22 (2014) 2655–2661
2659
in position 4, was also less effective than the series of 4c, 4d and
4e on all cell lines tested although after 72 h of treatment MCF-7
and MDA-MB231 cells responded more to 4g (EC50 value at 50 lM).
We further tested whether the reduced viability of cells was due
to increased apoptosis. To this end, the breast cancer cell lines
MDA-MB231 and MCF-7, and the prostate cancer cell lines LNCaP
and PC-3, and the monocytic leukemia cell line U937 were treated
with different concentrations of 4c, 4d, 4e, and in comparison with
4-HC (3). PARP-1 cleavage, which is considered a marker of apop-
tosis, was determined by Western blot analysis. Figure 1A shows
that in MDA-MB231 cells, all concentrations of 4c, 4d and 4e led
to a reduced protein expression of 116 kDa full-length PARP with
a concentration-dependent appearance of a cleavage product at
86 kDa. In parallel, we measured the protein expression and phos-
phorylation level of the protein kinase Akt. Akt is a well known
protein kinase involved in cell growth and survival and reduced
activity often correlates with reduced proliferation and increased
apoptosis. The level of phospho-Thr308 Akt is well accepted to
Scheme 3. Structures of the most potent compounds.
After 72 h of treatment the MCF-7 cells also turned sensitive to 4c,
4d, and 4e treatment underlining the concentration- and time-
dependent cytotoxic effect of the compounds. Derivative 4f resem-
bling structure 4b, that is oxygen in position 5 of the pentacycle,
but replacing methyl for tert-butyl at position 4, was hardly
effective. Finally, derivative 4g which resembled the active
sulfur-containing 4c, 4d, and 4e compounds but had a bromide
Figure 1. Effect of novel 4-hydroxycoumarin derivatives on PARP-1 cleavage and activity of the survival kinase Akt in various cancer cell lines. The breast cancer cell lines
MDA-MB-231 (A) and MCF-7 (B), the prostate cancer cell lines LNCaP (C) and PC-3 (D) and the monocytic leukemia cell line U937, (E) cells were treated for 48 h with either
vehicle (0.1% DMSO, co) or the indicated concentrations of 4c, 4d, 4e, and 4-HC (in lM). Protein extracts were prepared and equal amounts of protein (30 lg) were separated
by 8% SDS–PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed by using antibodies against PARP-1 (upper panels), phospho-Thr308-Akt
(middle panels) and the house-keeping protein GAPDH (lower panels). Data show one representative blot.

2660
A. Jashari et al. / Bioorg. Med. Chem. 22 (2014) 2655–2661
reflect Akt activity. We found that phospho-Thr308 Akt levels were
downregulated by 4c, 4d and 4e when compared to control and to
4-HC (3) treated cells. In the more resistant cell line MCF-7, 4c, 4d
and 4e were less effective in downregulating phospho-Akt. Only 4c
and 4d at higher concentrations downregulated phospho-Akt,
whereas 4e and 4-HC (3) had no major effect (Fig. 1B). Still,
PARP-1 cleavage occurred in MCF-7 by all three compounds 4c,
4d and 4e, but not by 4-HC (3).
The androgen-dependent prostate cancer cell line LNCaP
showed PARP-1 cleavage upon treatment with all compounds with
the exception of 4-HC (Fig. 1C). In parallel, phospho-Thr308 Akt was
decreased in a concentration-dependent manner by the three com-
pounds 4c, 4d and 4e, but not by 4-HC (3). In contrast to the LNCaP
cells, the androgen-independent prostate cancer cell line PC-3,
which was found to be resistant to 4c, 4e and 4d treatment in
the viability assay (Table 1), was also more resistant to apoptosis
induction as shown in Figure 1D. Hardly any cleaved PARP-1 frag-
ment was seen upon 4c, 4d and 4e treatment (Fig. 1 D). However,
phospho-Thr308 Akt was downregulated by 4c, 4d and 4e when
compared to control and 4-HC treated cells. Finally, the monocy-
toic leukemia cell line U937, which was sensitive to 4c, 4d and
4e treatment in the viability assay (Table 1), consistently showed
enhanced PARP-1 cleavage and reduced phospho-Thr308 Akt upon
treatment (Fig. 1E).
ER-negative MDA-MB-231 cell line than in the p53- and ER-po-
sitive MCF-7 cell line. Although the MDA-MB-231 cell line is
considered highly tumorigenic, neither p53 nor ER seem to be
predictive for drug susceptibility. Notably, MDA-MB-231 cells
were also reported to be more sensitive to taxane treatment
than MCF-7.41 Recently, a study reported on the identification
of a group of genes that regulated the susceptibility of breast
cancer cells to antineoplastic drugs.42 Whether these genes also
regulate the susceptibility towards 4c, 4d, and 4e remains an
interesting point for future studies.
Our data further show that 4c, 4d, and 4e differentially affected
the androgen-dependent and androgen-independent prostate can-
cer cell lines LnCaP and PC-3 in that only LnCaP were sensitive to
the compounds and responded with increased PARP-1 cleavage
and apoptosis. These two cell lines are known to exhibit different
capabilities in angiogenesis and tumor aggressiveness, and espe-
cially the PC-3 cell line has acquired a more aggressive phenotype
that is highly angiogenic.42 Molecular profiling of the two cell lines
revealed that several thousand genes are differentially expressed
which may account for the aggressive and drug-resistant pheno-
type of the PC-3 cells.43
5. Conclusions
This study has revealed three novel coumarin derivatives with
hydrazinylidene-chromandione structure which exert more potent
anti-proliferative and pro-apoptotic effects on various cancer cell
lines than the reference compound 4-hydroxycoumarine. These
novel compounds may therefore represent interesting novel lead
compounds for further anti-cancer studies.
4. Discussion
Some coumarins including the 4-hydroxycoumarin derivative
warfarin are well-known anticoagulants that act as a vitamin K
antagonists. However, many other biological activities have also
been appointed to coumarins and certain derivatives such as
anti-bacterial, anti-inflammatory, and anti-viral effects.
Acknowledgments
Their anti-coagulant property has proven useful also in cancer
patients to reduce venous thromboembolism which is a frequent
complication of cancer.17,33 In addition to this beneficial effect in
cancer patients, a direct anti-tumor and anti-metastatic activity
has also been described.33,34 The potential mechanisms of action
may occur on multiple levels. Thus, it has been reported that cou-
marins may interfere with proper cell cycle progression and trigger
cell cycle arrest and subsequent apoptosis. In a human cervical
cancer cell line (HeLa), coumarin derivatives were shown to induce
cell-cycle arrest and apoptosis by downregulating the anti-apopto-
tic factors Bcl-2 and Bcl-xL, and upregulating the pro-apoptotic fac-
tor Bax.35 Additionally, coumarins are prodrug inhibitors of the
metalloenzyme carbonic anhydrase (CA).36 This enzyme efficiently
hydrates carbon dioxide to protons and bicarbonate and thereby
promotes acidification of solid tumors. 15 different subtypes of
CA exist and certain subtypes such as CA IX and XII were reported
to be overexpressed in many tumor types and to be involved in
cancer progression and metastases formation.37 Coumarins were
also shown to directly inhibit sulfatase and aromatase activities,
two enzymes taking part in estrogen synthesis38,39 and to interfere
and modulate estrogen receptor activation.40 Moreover, it was re-
cently proposed that the cytochrome P450 1 (CYP1) enzymes are
targets of coumarins. These enzymes take part in the metabolic
activation of procarcinogens and deactivation of certain anticancer
drugs22 and therefore, inhibition of these enzymes may exert an
anti-cancer effect.
We wish to thank the South-East European Pact for stabilization
in the frame of the ‘Deutscher Akademischer Austauschdienst’
(DAAD) for financial support. We also thank Prof. Dr. Evamarie
Hey-Hawkins and Dr. Alexandra Hildebrand from the University
of Leipzig for helpful discussions.
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