Synthesis, X-ray crystallographic study, and biological evaluation of coumarin and quinolinone carboxamides as anticancer agents

Article abstract of DOI:10.1007/s00706-013-0986-7

A series of coumarin and quinolinone-3-aminoamide derivatives was synthesized and evaluated for its potency in inhibition of cancer cell growth. The structure of N-[2-(dimethylamino) ethyl]-4-hydroxy-2-oxo-1-phenyl-1,2- dihydroquinoline-3-carboxamide was

Full text of DOI:10.1007/s00706-013-0986-7

Monatsh Chem (2013) 144:1063–1069
DOI 10.1007/s00706-013-0986-7
ORIGINAL PAPER
Synthesis, X-ray crystallographic study, and biological evaluation
of coumarin and quinolinone carboxamides as anticancer agents
•
Dimitris Matiadis Valentina Stefanou Giorgos Athanasellis
•
•
•
•
•
Stylianos Hamilakis Vickie McKee Olga Igglessi-Markopoulou
John Markopoulos
Received: 16 December 2012 / Accepted: 27 March 2013 / Published online: 26 April 2013
Ó Springer-Verlag Wien 2013
Abstract A series of coumarin and quinolinone-3-ami-
noamide derivatives was synthesized and evaluated for its
potency in inhibition of cancer cell growth. The structure
of N-[2-(dimethylamino) ethyl]-4-hydroxy-2-oxo-1-phe-
nyl-1,2-dihydroquinoline-3-carboxamide was unambigu-
ously confirmed by X-ray diffraction analysis, which
revealed the cis conformation of the amide bond resulting
from the presence of two intramolecular hydrogen bonds.
in vivo experiments and clinical studies, was published a
few years ago [1]. So far, the results for different coumarins
with various tumor lines are often contradictory. This fact
indicates that there is still a long way to go until researchers
reach a consensus about which cytotoxic agents will be suit-
able for clinical treatment.
Natural or synthetic coumarins with simple structures,
such as esculetin, osthole, and the synthetic 7-hydroxy-6-
nitrocoumarin (Fig. 1) are well-known to exhibit notable
anticancer activities [2–6]. In particular, osthole has proved
to be very effective against liver tumors. Moreover, the
widely known warfarin, marketed for more than 50 years
as an anticoagulant, has been reported [7] to improve the
survival of patients with small-cell lung cancer even
though this has not been confirmed, and metal complexes
of niffcoumar with lanthanides have proven to exhibit
anticancer activity [8, 9].
Keywords Heterocycles Á Antitumor agents Á
X-ray structure determination Á Aminoamides
Introduction
Coumarins comprise a vast group of natural and synthetic
compounds which have proven to possess diverse biological
and pharmacological activities. Among these, cytotoxicity
has made them ‘‘targeted molecules’’ in the research of
anticancer agents. A review presenting the broad range of
effects of coumarins on tumors, as shown by in vitro and
The presence of different substituents enhances some
biological abilities of these compounds. In particular,
coumarins bearing an amide bond at C-3 of the heterocy-
clic nucleus have been identified as potent tumor-specific
cytotoxic agents [10]. In a previous work, the growth
inhibitory properties of novel coumarin sulfonamides were
described using a panel of cancer cell lines [11]. In addi-
tion, the natural product novobiocin, a 3-amidocoumarin,
has attracted the interest of scientists due to its antitumor
activity [12] through inhibition of the heat-shock protein 90
(hsp90).
D. Matiadis Á V. Stefanou Á G. Athanasellis Á S. Hamilakis Á
O. Igglessi-Markopoulou
Laboratory of Organic Chemistry, School of Chemical
Engineering, National Technical University of Athens,
Zografou Campus, 15773 Athens, Greece
N-substituted quinolinone analogues also constitute an
important source for pharmaceutical and agrochemical
industries. Vesnarinone, a 3,4-dihydroquinolinone used as
a cardiotonic agent, was found to be a unique antiprolif-
erative, differentiation-inducing and apoptosis-inducing
drug against several human malignancies, including leu-
kemia and several solid tumors [13–15].
V. McKee
Chemistry Department, Loughborough University,
Leicestershire LE113TU, UK
J. Markopoulos (&)
Laboratory of Inorganic Chemistry, Department of Chemistry,
University of Athens, Panepistimiopolis, 15771 Athens, Greece
e-mail: jmmarko@chem.uoa.gr
123

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D. Matiadis et al.
O
Results and discussion
X
OH
O
Chemistry
HO
O
O
O
Y
The well known anticancer activities of coumarin and
quinolinone-3-carboxamides motivated us to prepare a
compound library to examine their potency as antitumor
agents. Particular attention was paid to the side chain at
C-3 position of the heterocyclic nucleus. Diamines bearing
two free amino groups (ethylenediamine, hexamethylen-
edimine, 1,8-diamineoctane, and o-phenylenediamine)
were used, as well as diamines with tertiary amine groups
(N,N-dimethyethylenediamine and 1-(2-aminoethyl)piperi-
dine). Concerning the heterocyclic ring, N-phenyl-, N-
methyl-4-hydroxyquinolinones, and 4-hydroxycoumarins
were used.
Esculetin (X = OH)
Warfarin (Y = H)
Niffcoumar (Y = NO2)
7-Hydroxy-6-nitrocoumarin (X = NO2)
X
O2
S
N
H
O
O
O
O
O
Y
(N-Aryl)-coumarin-3-sulfonamides
Osthole
OH
OCONH2
OH
H
N
MeO
The starting materials, methyl coumarin-3-carboxylate
and quinolinones were synthesized following published
methodologies. The first were prepared using the N-
hydroxysuccinimide protocol [18] and the latter using the
HOBt method [19].
O
O
O
O
O
Novobiocin
Fig. 1 Coumarin derivatives known as anticancer agents
Schemes 1 and 2 illustrate the approach to the synthesis
of the aminoamides. In the case of free aminoamides, the
diamine and the heterocycle were dissolved in toluene and
heated to produce the target molecules as insoluble solids
[20, 21]. The products were obtained in high yields and
with very good purities after typical filtration and washing
procedures using toluene and diethyl ether. The amines
were used in three fold excess to avoid formation of
unwanted diamides.
CF3
OH
N
O
O
OH
N
O
O
N
N
Linomide
Tasquinimod
However, in the case of substituted aminoamides this
process was unsuccessful. These products were obtained by
the reaction of the reagents in polar protic solvent (meth-
anol) or in solvent free conditions, depending on the
substituents. After careful treatment with the appropriate
solvent, the final products were obtained as pure solids or
crystals.
H
O
N
OMe
OMe
N
N
O
Vesnarinone
Fig. 2 Quinolinone derivatives known as anticancer agents
Structure elucidation
On the other hand, quinolinone carboxamides have been
more extensively examined (Fig. 2). As an example, lino-
In order to elucidate the structures and the conformation
1
of the molecules, we studied them by means of H and
13C NMR spectroscopy and X-ray single crystal crystal-
lography. The NMR spectra showed that the 4-hydroxy
was the predominant tautomer in chloroform as sol-
vent, as only one enolic hydrogen was observed for all
compounds.
mide (or roquinimex) is
a quinoline-3-carboxamide
molecule which has been proven to inhibit the process of
angiogenesis, which is a vital step in tumor development
[16].
Second generation quinoline-3-carboxamide compounds
were synthesized and investigated as to whether they could
maintain their anti-angiogenesis abilities without induce-
ment of a proinflammatory response. The compound
tasquinimod was proven to be 30–60 times more potent
than linomide in rat prostate cancer models and to exhibit a
therapeutic effective oral dose as low as 0.5–1 lM [17].
Crystals of compound 6 were obtained by adding etha-
nol to the crude oily product, without recrystallization.
Parameters for X-ray crystallography data collection and
refinement are summarized in Table 1.
The molecular structure, along with the numbering
scheme, is shown in Fig. 3. Concerning the heterocyclic
123

Synthesis, X-ray crystallographic study, and biological evaluation
1065
Scheme 1
OH
O
O
H2N
NH2
OH
X
O
O
Y
Y
OMe
Reflux, 3-4 h
N
NH2
H
PhMe
X
1-3
4a: X = O, Y = (CH2)2 5a: X = NMe, Y = (CH2)2
4b: X = O, Y = (CH2)6 5b: X = NMe, Y = (CH2)6
4c: X = O, Y = (CH2)8 5c: X = NMe, Y = o-C6H4
4d: X = O, Y = o-C6H4
Scheme 2
OH
O
O
H2N
OH
X
O
O
NR2
NR2
130-140 oC, 20 min
OMe
N
H
X
Solvent free or MeOH
1-3
4e: X = O,
4f: X = O,
R2 = Me, Me
R2 = piperidinyl
6: X = NPh, R2 = Me, Me
part of the molecule, it is noteworthy that the bond C(2)–
˚
O(26) is double (1.240(2)A) and the bond N(1)–C(2) has a
˚
partial double bond character (1.394(2)A). The double
˚
bond character of the bonds C(3)–C(4) (1.386(3)A) and
˚
C(7)–O(8) (1.263(2)A) and the longer than double bond
examined are amides at position 3 of quinolinone and
coumarin compounds bearing functional groups on the
amide chain, and a study of their traits reveals two things:
(a) the relationship of nitrogen (quinolinone) or oxygen
(coumarin) atom on the heterocyclic nucleus versus anti-
cancer activity and (b) the relevance of the amide chain
bearing different functionalities to anticancer activity of the
molecule.
˚
length of the C(4)–O(15) (1.320(2)A) strongly proves that
the 4-hydroxy is also the dominant tautomer in a solid
˚
state. Finally, the bond C(7)–N(9) (1.323(3)A) has obvious
double bond character, as expected from an amide bond
(Table 2).
The cancer cell lines that we applied were DU 145 and
PC3 for prostate cancer, HCT-15 for colon cancer, MCF 7
for breast cancer, IGROV-1 for ovarian cancer, SKHep1
for liver cancer, and HL-60 (TB) for leukemia cancer. The
results are presented in Table 4.
The benzene ring is tilted at 72.48(2)° to the fused rings
(which are close to planar). The amide bond has the cis
conformation, due to the existence of two intramolecular
H-bonds as shown in the picture. There is possibly also a
third (N9–N12); the N–H–N angle is quite acute but it
accounts for the orientation of the pendant amine
(Table 3). Intermolecular interactions including p-p
stacking of the fused rings (average interplanar distance
From these results it is apparent that there are a few
compounds that exhibit GI50 values below 50 % (com-
pound 4c for prostate cancer cell line PC3 and liver cancer
cell line SKHep1, compound 5b for colon cancer cell line
HCT 15), or around 50 % (compound 4b for liver cancer
cell line SKHep1, compound 4e for prostate cancer cell
line DU 145, compound 5a for prostate cancer cell line
PC3); furthermore, compound 4c exhibits very good
activity against prostate cancer cell line DU 145 with a
value of GI50 of 8.14 lL. The next stages in this project
will be: (a) synthesis of novel functionalized derivatives of
coumarins and quinolinones and their in vitro biological
evaluation and (b) synthesis of a larger quantity of com-
pound 4c in order to perform further in vitro and in vivo
experiments (e.g. xenografts).
˚
3.63 A, symmetry operation x-1, y, z) and C–H ÁÁÁ p
interaction involving the phenyl ring (C18–H ÁÁÁ centroid
˚
2.99 A under symmetry operation -1-x, 1-y, -z) are
observed (Fig. 4).
Biological evaluation
The biological evaluation of the investigated compounds
was based on their anticancer activity according to in vitro
NCI standards (GI50, TGI, or LC50). The molecules
123

1066
D. Matiadis et al.
Table 1 Crystal data and structure refinement for N-[2-(dimethylamino)
ethyl]-4-hydroxy-2-oxo-1-phenyl-1,2-dihydroquinoline-3-carboxamide (6)
Parameter
Empirical formula
Formula weight
Temperature/K
C20H21N3O3
351.40
150 (2)
˚
Wavelength/A
0.71073
Crystal system
Space group
Triclinic
P-1
˚
Unit cell dimensions/A
a = 5.7259 (5)
b = 10.9384 (10)
c = 14.0120 (13)
861.21 (14)
2
3
˚
Volume/A
Z
Density (calculated)/Mg m-3
Absorption coefficient/mm-1
F(000)
1.355
0.093
372
Crystal size/mm3
Crystal description
Theta range for data collection
Index ranges
0.39 9 0.10 9 0.04
Pale yellow lath
1.89° to 26.00°
-7 \=h \=7, -13 \=k \=13,
-17 \=l \=17
9,939
Fig. 4 Crystal packing diagram of 6, viewed down the b axis
Table 2 Bond lengths for 6
˚
Length/A
˚
Length/A
Bond
Bond
Reflections collected
N(1)–C(2)
N(1)–C(6)
N(1)–C(20)
C(2)–O(26)
C(2)–C(3)
C(3)–C(4)
C(3)–C(7)
C(4)–O(15)
C(4)–C(5)
C(5)–C(6)
C(5)–C(16)
C(6)–C(19)
C(7)–O(8)
C(7)–N(9)
1.394 (2)
1.400 (2)
1.448 (2)
1.240 (2)
1.446 (3)
1.386 (3)
1.471 (3)
1.320 (2)
1.443 (3)
1.399 (3)
1.405 (3)
1.398 (3)
1.263 (2)
1.323 (3)
N(9)–C(10)
C(10)–C(11)
C(11)–N(12)
N(12)–C(13)
N(12)–C(14)
C(16)–C(17)
C(17)–C(18)
C(18)–C(19)
C(20)–C(21)
C(20)–C(25)
C(21)–C(22)
C(22)–C(23)
C(23)–C(24)
C(24)–C(25)
1.453 (3)
1.510 (3)
1.448 (3)
1.450 (3)
1.454 (3)
1.375 (3)
1.387 (3)
1.375 (3)
1.382 (3)
1.383 (3)
1.395 (3)
1.380 (3)
1.383 (3)
1.386 (3)
Independent reflections
Completeness to theta = 26.00°
Absorption correction
3,372 [R(int) = 0.0425]
99.9 %
Semi-empirical from equivalents
0.9963 and 0.9647
Full-matrix least-squares on F2
3,372/0/241
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
1.041
Final R indices [I [ 2sigma(I)]
R indices (all data)
R1 = 0.0476, wR2 = 0.1084
R1 = 0.0792, wR2 = 0.1230
0.179 and -0.197
-3
˚
Largest diff. peak and hole/e A
Table 3 Hydrogen bonds for 6
Bond D–HÁÁÁA
Length
˚
D-H/A
Length
Length
Angle
DHA/°
˚
˚
HÁÁÁA/A DÁÁÁA/A
N(9)–H(9)ÁÁÁO(26)
0.90 (2) 1.97 (2) 2.669 (2)
134 (2)
156 (2)
O(15)–H(15)ÁÁÁO(8) 0.99 (3) 1.51 (3) 2.445 (2)
N(9)–H(9)ÁÁÁN(12)
0.90 (2) 2.40 (2) 2.784 (3) 106.4 (18)
Conclusion
Fig. 3 Molecular structure of N-[2-(dimethylamino)ethyl]-4-hydroxy-
2-oxo-1-phenyl-1,2-dihydroquinoline-3-carboxamide (6). Displace-
ment ellipsoids for nonhydrogen atoms are drawn at the 50 %
probability level. The intramolecular hydrogen bonds are shown dashed
In summary, tertiary and primary coumarin and quinoli-
none-3-carboxamide derivatives were synthesized and
characterized. The structure of compound
6
was
123

Synthesis, X-ray crystallographic study, and biological evaluation
1067
Table 4 In vitro screening table: summary results from the in vitro drug screening
Compound
Stock conc./mM
20
Values
HCT-15
DU 145
PC3
MCF 7
IGROV-1
SKHep1
HL-60 (TB)
4a
LC50
TGI
[100.0
[100.0
[100.0
[100.0
[100.0
86.6
[100.0
[100.0
[100.0
[100.0
[100.0
71.1
[100.0
[100.0
[100.0
[100.0
[100.0
86.8
[100.0
[100.0
[100.0
[100.0
[100.0
55.4
GI50
LC50
TGI
4b
4c
4d
4e
5a
5b
4f
20
20
20
20
20
20
20
20
20
GI50
LC50
TGI
47.5
[100.0
[100.0
41.8
[100.0
[100.0
77.0
[100.0
[100.0
38.2
[100.0
[100.0
[100.0
20.5
GI50
LC50
TGI
8.14
[100.0
[100.0
93.5
[100.0
[100.0
75.7
[100.0
[100.0
[100.0
100.0
[100.0
[100.0
71.3
GI50
LC50
TGI
[100.0
[100.0
[100.0
[100.0
[100.0
[100.0
[100.0
[100.0
39.2
[100.0
[100.0
53.8
[100.0
[100.0
[100.0
[100.0
[100.0
53.6
[100.0
[100.0
[100.0
[100.0
[100.0
97.6
[100.0
[100.0
100.0
100.0
GI50
LC50
TGI
100.0
[100.0
[100.0
[100.0
[100.0
[100.0
65.8
GI50
LC50
TGI
[100.0
[100.0
83.2
[100.0
[100.0
61.1
GI50
LC50
TGI
[100.0
[100.0
[100.0
[100.0
100.0
[100.0
[100.0
[100.0
[100.0
100.0
[100.0
[100.0
[100.0
[100.0
100.0
GI50
LC50
TGI
6
GI50
LC50
TGI
79.7
100.0
79.5
5c
100.0
100.0
100.0
100.0
100.0
83.0
100.0
100.0
GI50
61.9
For each experimental agent: LC50 is the concentration of drug resulting in 50 % reduction in measured protein, TGI the concentration resulting
in total growth inhibition, and GI50 the concentration resulting in growth inhibition of 50 %
determined by X-ray crystal structural analysis. In the solid
state this compound demonstrated an edge-to-face inter-
action involving the phenyl ring and p-p stacking
interactions of the fused rings, thus forming chains. One
N–H ÁÁÁ O hydrogen bond and one O–H ÁÁÁ O hydrogen
bond, both intramolecular, favoring the cis conformation of
amide bond were observed as expected.
Experimental
All reagents were purchased from Aldrich, Fluka, and Acros
and used without further purification. Dry tetrahydrofuran
(THF) was distilled from Na/Ph2CO. Melting points were
determined with a Gallenkamp MFB-595 melting point
apparatus. NMR spectra were recorded with a Varian
Gemini-2000 300 MHz spectrometer operating at 300 MHz
(1H) and 75 MHz (13C). Chemical shifts are reported in ppm
relative to CDCl3 (1H: d = 7.26 ppm, 13C: d = 77.16 ppm).
Elemental analyses were obtained on a Euro EA3000 Series
Euro Vector CHNS Elemental Analyzer. Compounds 4a–4f
[21], 4e, 4f [24], and 5a–5c [20], were prepared using slightly
modified literature methods.
Compounds were evaluated for cytostatic activities
against various cancer cell lines. Compound 4c exhibited
higher inhibitory effects against DU 145 prostate cancer
cell line (GI50 \ 10 lL).
Our future plans include synthesis of novel functional-
ized derivatives of coumarins and quinolinones and their
in vitro biological evaluation and synthesis of a larger
quantity of compound 4c, in order to perform further
in vitro and in vivo experiments.
Cell viability was assessed at the beginning of each
experiment by the trypan blue dye exclusion method, and
123

1068
D. Matiadis et al.
Ar), 10.23 (br, 1H, NH), 17.15 (br, 1H, OH) ppm; 13C
(75 MHz, CDCl3): d = 36.5 (CH2CH2NCH3), 45.0 (NCH3),
57.8 (CH2CH2NCH3), 97.0 (C-3), 116.2, 122.8, 125.4, 129.2,
129.3, 130.4, 133.4, 137.3, 141.0 (Ar), 160.1 (C-2), 171.5
(CONH), 172.5 (C-4) ppm.
was always greater than 95 %. Cells were seeded into
96-well microtiter plates in 100 mm3 of medium at the
corresponding density (3,500–30,000 cells per well) and
subsequently, the plates were incubated at standard con-
ditions for 24 h to allow the cells to resume exponential
growth prior to addition of the agents to be tested. Then in
order to measure the cell population, cells in one plate were
fixed in situ with trichloroacetic acid (TCA) followed by
sulforhodamine B solution (SRB) staining, as described
elsewhere. To determine the activity, each compound was
dissolved in dimethyl sulfoxide (DMSO) and then was
added at 10-fold dilutions (from 100 to 0.01 lM), and
incubation continued for an additional period of 48 h. The
assay was terminated by addition of cold TCA followed by
SRB staining and absorbance measurement at 540 nm in a
DAS plate reader, to determine (1) the GI50, the concen-
tration required in the cell culture to inhibit cell growth by
50 %, (2) TGI, the concentration that is required to com-
pletely inhibit cell growth, and (3) the LC50, the
concentration that is needed in culture to kill 50 % of the
cellular population as described [23].
Diffraction data were collected at 150 (2) K on a Bruker
Apex II CCD diffractometer using MoKa radiation
˚
(k = 0.71073 A). All the non-hydrogen atoms were refined
using anisotropic atomic displacement parameters, and
hydrogen atoms bonded to carbon were inserted at
calculated positions using a riding model. Hydrogen atoms
bonded to O or N were located from difference maps and
their coordinates refined [22].
Crystallographic data have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC 856973. Copies of the data can be
obtained, free of charge, through application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: ?44-(0)1223-
336033 or e-mail: deposit@ccdc.cam.ac.uk).
Acknowledgments D. M. gratefully acknowledges the Committee
of Research of the National Technical University of Athens for a
doctoral assistantship. J. M. would like to thank the National and
Kapodistrian University of Athens for financial support (special
account for research Grant No. 70/4/3337). The authors are thankful
to Dr. Alexander Vamvakides for performing the in vitro biological
evaluation of the compounds at the Laboratories of ANAVEX Life
Sciences at Pallini, Attiki, Greece.
The standard experimental procedure comprises of the
following steps: (a) the experimental compounds were
dissolved in DMSO, purchased from Sigma-Aldrich, at a
final stock concentration of 20 mM. (b) Dilutions of
20 mm3 of the final stock solution with the medium to final
concentrations of 100, 10, and 1 lM followed in order to
examine the cytotoxicity effect. (c) In parallel, DMSO
control studies were performed by preparing DMSO con-
trol solutions of 20 mm3 of DMSO diluted with the
medium to final concentrations of 100, 10, and 1 lM,
concentrations at which DMSO did not show any cellular
cytotoxicity.
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