The rearrangement of tosylated flavones to 1′-(alkylamino)aurones with primary amines

Article abstract of DOI:10.1016/j.tet.2015.09.062

A rearrangement of 3-tosylflavone to the corresponding 1′-(alkylamino)aurone proceeds under mild conditions in the presence of primary amines in high yields. The reaction is applicable to different substituted 3-tosylflavones and alkyl amines and the respective aurones were isolated as a mixture of E/Z isomers. Further conversion of 1′-(methylamino)aurone to the corresponding thionated compound was performed by reaction with Lawesson's reagent and only the E isomer was obtained. This observation can be explained by the weaker exocyclic double bond, which facilitates rotation and the formation of the thermodynamically preferred E form. The characterization, preliminary biological investigations of the synthesized compounds and lipophilicity studies are discussed.

Full text of DOI:10.1016/j.tet.2015.09.062

Accepted Manuscript
The rearrangement of tosylated flavones to 1’-(alkylamino)aurones with primary
amines
Wolfgang Kandioller, Mario Kubanik, Anna K. Bytzek, Michael A. Jakupec, Alexander
Roller, Bernhard K. Keppler, Christian G. Hartinger
PII:
S0040-4020(15)30090-9
10.1016/j.tet.2015.09.062
TET 27159
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 7 June 2015
Revised Date: 17 September 2015
Accepted Date: 25 September 2015
Please cite this article as: Kandioller W, Kubanik M, Bytzek AK, Jakupec MA, Roller A, Keppler BK,
Hartinger CG, The rearrangement of tosylated flavones to 1’-(alkylamino)aurones with primary amines,
Tetrahedron (2015), doi: 10.1016/j.tet.2015.09.062.
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The rearrangement of tosylated flavones to
1’-(alkylamino)aurones with primary amines
Leave this area blank for abstract info.
Wolfgang Kandioller, Mario Kubanik, Anna K. Bytzek, Michael A. Jakupec, Alexander Roller, Bernhard K.
Keppler, Christian G. Hartinger
University of Vienna, Faculty of Chemistry, Institute of Inorganic Chemistry
University of Auckland, School of Chemical Sciences
University of Vienna, Research Platform Translational Cancer Therapy Research

1
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Tetrahedron
journal homepage: www.elsevier.com
The rearrangement of tosylated flavones to 1’-(alkylamino)aurones with primary
amines
Wolfgang Kandioller,a,† Mario Kubanik,b,† Anna K. Bytzek,a Michael A. Jakupec,a,c Alexander Roller,a
Bernhard K. Keppler,a,c Christian G. Hartingera-c,*
a University of Vienna, Faculty of Chemistry, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria.
b University of Auckland, School of Chemical Sciences, Private Bag 92019, Auckland 1142, New Zealand. E-mail: c.hartinger@auckland.ac.nz.
c University of Vienna, Research Platform Translational Cancer Therapy Research, Waehringer Str. 42, A-1090 Vienna, Austria.
†
These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A rearrangement of 3-tosylflavone to the corresponding 1’-(alkylamino)aurone proceeds under
mild conditions in the presence of primary amines in high yields. The reaction is applicable to
different substituted 3-tosylflavones and alkyl amines and the respective aurones were isolated
as a mixture of E/Z isomers. Further conversion of 1’-(methylamino)aurone to the corresponding
thionated compound was performed by reaction with Lawesson’s reagent and only the E isomer
was obtained. This observation can be explained by the weaker exocyclic double bond, which
facilitates rotation and the formation of the thermodynamically preferred E form. The
characterization, preliminary biological investigations of the synthesized compounds and
lipophilicity studies are discussed.
Available online
Keywords:
Anticancer activity
Aurones
2009 Elsevier Ltd. All rights reserved
.
Flavones
Rearrangement
1. Introduction
In contrast to 3-hydroxyflavones, the amino analogues are less
explored, though they were shown to exhibit interesting
biological features.21,22 This is even more astonishing since amino
groups located at the A or B ring of flavonoids appear to have in
some cases a beneficial influence on the antiproliferative activity,
which might be related to ATP-competitive inhibition of some
protein-tyrosine kinases.23-25
3-Hydroxyflavones (3-HFs) are polyphenolic compounds and
belong to the class of flavonoids, frequently found, e.g., in plants,
fruits and vegetables.1 They are involved in photosensitization,
energy transfer, plant growth and morphogenesis and are often
conjugated to sugar moieties, preferably to
D-glucose, D-
rhamnose and rutinose.2
A few strategies for the synthesis of 3-aminoflavones have
been reported and they are often based on multi-step syntheses.
Dauzonne and colleagues built up the entire flavone backbone in
a 5-step synthesis starting from benzaldehyde derivatives and
bromonitromethane.26,27 Patonay et al. obtained a mixture of 4
compounds from a reaction of 2’-hydroxychalcone dibromides
with sodium azide, out of which only one can be used in a
cyclization reaction to yield the desired 3-aminoflavone.28
Similar to Patonay, De Meyer and co-workers prepared in a
multistep approach an azide, which was reduced to the amine.29
Introduction of an azide functionality was also used in the
conversion of a flavanol into the respective aminoflavone.30
O’Brien et al. have shown that the reduction of 3-
oximoflavanones with stannous chloride/hydrochloric acid leads
to the formation of 3-aminoflavones.31
Flavonoids are well known for their biological properties,
including antiradical, antioxidant, anti-inflammatory,
antimicrobial and anticancer activity.3 For example, the 3-
hydroxyflavone quercetin exhibits anticancer activity, is able to
interact with lipids and proteins of the cell membrane and
prevents oxidative damage by reactive oxygen species (ROS).4 In
vitro studies have revealed an inhibitory effect of the compound
on the proliferation of human colon cancer and breast cancer
cells at micro- to nanomolar concentrations. Flavopiridol, a
synthetic flavone, has entered phase 2 clinical trials for the
treatment of different tumor types. It inhibits cyclin-dependent
kinases (CDKs) by blockage of the ATP binding site of the
protein.5 3-Hydroxyflavones feature the same metal binding sites
as pyr(id)ones, of which Ru(II)–arene complexes are known for
their anticancer activity.6-15 Similarly, 3-hydroxyflavones form
metal complexes with metal ions, such as Al(III), Fe(III), Pb(II),
Zn(II) or Cu(II), and metal complexes were found to possess high
stability, intrinsic fluorescence and biocompatibility.16-20
The most straight-forward approach was reported by Sasaki
and colleagues.32 They used 3-hydroxyflavones as starting
material, which are easily accessible from commercial sources or
by a simple 2-step synthesis from 2-hydroxoacetophenone and
substituted benzaldehydes. Conversion into mesylates or

2
Tetrahedron
ACCEPTED MANUSCRIPT
tosylates, followed by reaction with ammonia or primary
Interestingly, performing the same reaction with derivatives
amines, leads to the desired 3-aminoflavones.30,32 They suggested
that the reaction mechanism involves an attack of the flavone
moiety in position 2 by methylamines in a Michael addition
manner, followed by intramolecular formation of an aziridine
ring, cleavage of the tosyl group and rearrangement to the 3-
aminoflavone.
featuring substituents in ortho position of the B ring or with
dimethylamine as the amine source gave no conversion, most
probably due to steric effects. Attempts to prepare the oxo and
thio derivatives by using hydroxylamine, 1.2-ehandithiol and L-
cysteinium chloride were unsuccessful; however, the conversion
of 3a to the thioflavone 4a was performed with Lawesson’s
reagent in high yield (Scheme 2; yield 88%).
Herein, we report attempts to obtain 3-aminoflavones,
following the synthetic strategy introduced by Sasaki and co-
workers, with the aim to use them as ligands for the preparation
of tumor-inhibiting metal complexes. However, a rearrangement
reaction was observed to yield 1’-(alkylamino)aurones under the
applied reaction conditions. The obtained compounds were
assayed for their in vitro anticancer activity to elucidate potential
biological activity.
O
S
NHR'
NHMe
Lawesson's
reagent
R
O
O
3a-i
(81 ꢀ 93%)
4a
(88%)
Scheme 2. Conversion of 3a to the thionated compound 4a.
Single crystals for 3a and 4a were obtained from cyclohexane
and methanol, respectively, which were suitable for X-ray
diffraction analysis. The X-ray structures revealed the formation
of 1’-(methylamino)aurone 3a and 1’-(methylamino)thioaurone
4a (Figure 1) instead of the expected 3-aminoflavone, which is
not immediately obvious from spectroscopic data. However,
recently Patonay et al. identified the ring contraction to the
aurone structure based on 2D NMR spectroscopy through
comparison with a synthesized 3-aminoflavone.35
2. Results and Discussion
In a first step, 3-hydroxy-2-phenyl-chromen-4-one 1 was
synthesized according to literature procedures (Scheme 1). 2-
Hydroxyacetophenone was initially reacted with benzaldehyde to
give the corresponding chalcone. This intermediate was used
without further purification for the oxidative cyclization (Algar-
Flynn-Oyamada reaction), yielding 1a–g.33 The flavones 1a–g
were treated with tosyl chloride in the presence of triethylamine
and compounds 2a–g were obtained in good yields (70–80%).
The subsequent reaction to the aminoflavones was reported by
Sasaki et al.32,34 to proceed in excellent yields (91%) by using an
excess of methylamine (10 eq) at room temperature and a
reaction period of 18 h. This procedure was followed employing
methyl- (a, phenyl; b, m-methyl; c, p-methyl; d, m-fluoro; e, p-
fluoro; f, m-chloro; g, p-chloro), ethylamine (h, phenyl) and
propylamine (i, phenyl) in the reaction with various tosylated 3-
HFs in high yields (81–93%).
In the molecular structure of 3a, an intramolecular hydrogen
bond between the amine functionality and the carbonyl of the
benzofuranone moiety is evident, yielding a thermodynamically
stable 6-membered ring. The bond length C2–C10 is significantly
shorter in 3a (1.372(3) Å) than in 4a (1.403(2) Å) and indicates
the presence of an exocyclic double bond in 3a. For 4a a partial
Figure 1. Molecular structures of 1’-(methylamino)aurone 3a (top) and
1’-(methylamino)thioaurone 4a (bottom); drawn at 50% probability level.
The phenyl ring in 3a is disordered over two positions with site occupation
factors 0.5 : 0.5 (only one position is shown for clarity). Key bond lengths
and angles for 3a: C2–C10 1.372(3), C3–O2 1.259(3), C2–C3 1.423(3), C10–
N1 1.335(3), C2–O1 1.406(3) Å, C3–C2–C10–N 3.1(4)°; 4a: C2–C10
1.403(3), C3–S1 1.6856(18), C2–C3 1.399(2), C10–N1 1.315(2), C2–O1
1.406(2) Å, C3–C2–C10–N 0.7(3)°.
Scheme 1. Synthetic pathway to 3a–i; i) NaOH, benzaldehyde; ii) H2O2;
iii) TsCl, N(Et)3; iv) R’-NH2, THF, rt, 18 h.

3
delocalization of electron density occurs over the furan moiety of
the heterocycle (compare bond lengths in caption of Figure 1).
The distance of 1.6856(18) Å between C3 and the sulfur atom S1
in 4a was also slightly longer than found, for example, in the case
of thiomaltol (1.677 Å).36 Like in 3a, the amine proton in 4a
forms a hydrogen bond to the sulfur atom.
quantum chemical calculation were performed using
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GAUSSIAN 09W to identify the difference in the rotational
barrier energy (Figure 3).37 The molecule was rotated along the
C2–C10 axis in 10° steps. The obtained energy difference
between the lowest and highest energy states was 53.3 kcal/mol
for 3a and 39.5 kcal/mol for 4a. For both molecules, the starting
configuration with the lowest energy has the benzylidene residue
almost in plane with the benzofuran backbone and the benzene
ring E to the carbonyl or thiocarbonyl groups. The rotational scan
for 3a and 4a shows two maxima where the phenyl group is
almost perpendicular to the heterocyclic ring system. Substitution
of the carbonyl oxygen with sulfur led to a 13.8 kcal/mol lower
rotation barrier along the C2-C10 axis. This can be explained by
the weakened double bond and the larger distance between the
N1 proton and the sulfur atom. The lower energy barrier could
also be the reason for the formation of just one isomer in the case
of 4a, while for 3a both isomers were obtained. The Z
configurations of both 3a and 4a gave local minima (Figure 3)
with the energy of the minimum for the thioaurone being 3.5
kcal/mol higher and therefore less stable than the Z configuration
of the aurone. In addition the difference in energy states between
this local minimum and the lower energy maximum is
29.0 kcal/mol while it is 42.8 kcal/mol for 3a.
The 1H NMR spectra of compounds 3a–i comprise two sets of
signals (e.g., for 3a at a ratio of approximately 4 : 1; Figure 2),
indicating the presence of two different species. Attempts to
separate these two compounds by column chromatography or
recrystallization from different solvents failed. In addition, NMR
1
measurements at temperatures up to 70 °C did not alter the H
NMR spectrum significantly (Figures 2a and 2b). Therefore, we
conclude that a mixture of E and Z isomers has been obtained by
this rearrangement reaction. The thermodynamically more stable
E isomer should be the preferably formed species and was also
1
found in the molecular structure of 3a. Furthermore, H NMR
spectroscopy studies of the thionated product 4a confirmed the
formation of a single isomer (Figure 2c), as observed in the
molecular structure. Note that no signals for C2 were observed in
the 13C{1H} NMR spectra of 3a–i, probably due to overlaps with
other signals.
In order to estimate the lipophilicity of 3a and 4a for
biological testing, a mixture of both compounds was analyzed by
microemulsion electrokinetic chromatography (MEEKC)
Figure 2. 1H NMR spectra in d6-DMSO of a) aurone 3a, b) 3a after 18 h
at 100 °C and c) thioaurone 4a.
Figure 4. Separation of 3a and 4a by MEEKC. DMSO and
dodecanophenone (D) were used as markers for the electroosmotic flow and
the microemulsion droplets.
The free rotation of the C2-C10 bond in the last step of the
reaction mechanism, as proposed by Kónya et al.,35 allows
formation of E and Z isomers. The transformation into the thione
increases the rotational freedom and the E isomer is exclusively
obtained in the case of 4a. To investigate this observation further,
Figure 3. Rotational barrier calculation for 3a (left) and 4a (right) based on the lowest energy state of either molecule set to 0. The structures show the
configurations observed for both compounds at the energy minima and maxima of the curve.

4
Tetrahedron
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(Figure 4). This method was demonstrated to be very versatile
Since the coordination of metal centers to organic molecules is
for such studies in a medicinal chemistry setting.38,39 The thio
compound 4a is significantly more lipophilic (capacity factor
log k = 1.186 ± 0.021) than 3a (log k = 0.961 ± 0.006), which is
often advantageous for the cellular uptake of drug compounds.7
These experimental results are supported by clogP values
calculated with ChemDraw, Molinspiration and ALOGPS 2.1
(Table 1). While the values obtained from the different software
solutions are varying, the trend is very similar in each set of
calculations with the thio derivative being more lipophilic than
1’-(methylamino)aurone 3a. As found by NMR spectroscopy, 3a
is a mixture of the E and Z isomers. However, no separation of
the isomers was achieved using MEEKC, probably due to too
similar capacity factors. Also the clogP determinations did not
give different values for the two isomers.
known to alter their chemical and biological properties, studies
on the complexation of 3a to Ru(η6-p-cymene) moieties were
initiated. However, none of the attempts were successful, most
probably because of the presence of the hydrogen bond between
the NH as donor and the carbonyl-oxygen as the acceptor, as
revealed in the molecular structure (Figure 1).
3. Conclusions
In this paper, we report the conversion of 3-tosylflavone with
primary amines to yield Z- and E-1’-(alkylamino)aurones instead
of the expected 3-(alkylamino)flavones, as demonstrated by
single crystal X-ray diffraction analysis. The conversion of the
obtained aurones to the corresponding thioaurone with
Lawesson’s reagent gave however exclusively the E isomer. This
can be explained by a weaker exocyclic double bond and a
rotation barrier along the C2-C10 axis which is 13.7 kcal/mol
lower than for the aurone.
Table 1. Comparison of the clogP values obtained from
ChemDraw, Molinspiration and ALOGPS 2.1, as well as log k
values obtained from MEEKC studies.
clogP
log k
MEEKC
Since aurones are known to exhibit biological activity,
preliminary in vitro anticancer assays were performed for 3a and
the lipophilicity was estimated by MEEKC studies. The
compound shows moderate cytotoxic activity in the tested cell
lines.
ChemDraw
Molinspir- ALOGPS 2.1
ation
2.05
2.59
3.08
3.42
3.10
3.44
0.96 ± 0.01
1.19 ± 0.02
3a
4a
Since flavones and derivatives are anticancer active
compounds, preliminary biological tests for the 1’-
(methylamino)aurone 3a were conducted in the human ovarian
teratocarcinoma [CH1(PA-1)], colon carcinoma (SW480) and
non-small lung carcinoma (A549) cells by means of the
colorimetric MTT assay (for concentration-effect curves see
Figure 5) and IC50 values of 78 ± 15, 128 ± 9 and 118 ± 7 ꢁM
were determined, respectively. The chemosensitive cell line
CH1(PA-1) responded the most to the treatment with 3a. The
solubility of compound 4a was insufficient for the determination
of its cytotoxic activity. However, the IC50 values in the normally
less sensitive cell lines were in the same dimension. Flavonol
derivatives tested in the same cancer cell lines exhibited much
more pronounced growth inhibitory effects with IC50 values
ranging from 0.60–2.1 µM in CH1(PA-1), 3.7–11 µM in SW480
and 7.9–81 µM in A549 cells.40 In order to evaluate the full
potential as anticancer agents of the aminoaurones, more
biological investigations are required.
4. Experimental
Materials and methods
All reactions were carried out in dry solvents under an inert
atmosphere.
2-Hydroxyacetophenone,
benzaldehyde,
p-
tosylchloride, methylamine (40% in water), ethylamine (70% in
water), Lawesson’s reagent, sodium dodecyl sulfate (SDS),
dodecanophenone,
1-butanol,
heptane,
sodium
dihydrogenphosphate and sodium monohydrogenphosphate were
obtained from commercial suppliers and used as received. The 3-
hydroxyflavones (1a–g) and their tosylated analogues (2a–g)
were synthesized according to literature procedures.30 1H,
31P{1H} and 13C{1H} NMR spectra were recorded at 25 °C at
500.10 (1H) and 125.75 MHz (13C{1H}), 2D NMR data were
collected in a gradient-enhanced mode. The chemical shifts for
the compounds 3a–i are given for the main isomer. No signals for
C2 were observed in the 13C{1H} NMR spectra of 3a–i, probably
due to overlaps with other signals.
X-ray diffraction measurements of single crystals of 3a and 4a
were performed at 298 K. Crystal data, data collection
parameters, and structure refinement details are given in Table 2.
The crystals were positioned at 35 mm from the detector and
1554 and 3073 frames were measured for 80 and 20 sec over 1°
scan width for 3a and 4a, correspondingly. The data were
processed using the SAINT software package.41 The structures
were solved by direct methods and refined by full-matrix least-
squares techniques. Non-hydrogen atoms were refined with
anisotropic displacement parameters. H atoms were refined as
free rotating group for the idealized Me parts and inserted with
riding coordinates for the remaining. The following computer
programs were used: structure solution and refinement,
SHELX97, OLEX2 and SHELXle software packages.42-44
Molecular structures were produced using Mercury 3.5.1. Crystal
structures were deposited at the Cambridge Crystallographic Data
Centre with deposition numbers 1404162 (3a) and 1404163 (4a).
Figure 5. Concentration-effect curves of compound 3a in the human
cancer cell lines A549, CH1(PA-1) and SW480.

5
3
Table 2. Crystallographic data of 3a and 4a
7.54–7.64 (m, 5H, Hphenyl), 7.30 (d, J(H,H) = 8Hz, 1H, H4),
ACCEPTED MANUSCRIPT
9.60–9.95 (brs, 1H, NH); 13C{1H} NMR (125.75 MHz, d6-
DMSO, 25 °C): δ = 31.5 (N-CH3), 113.1 (C7), 122.3 (C5), 122.9
(C4), 124.8 (C3a), 129.2 (Cphenyl), 129.4 (Cphenyl), 130.8 (Cphenyl),
131.3 (C1’), 132.8 (C6), 152.6 (C-N), 160.6, (C7a) 176.4 (C3).
Elemental analysis calcd. (%) for C16H13NO2: C 76.48, H 5.21, N
5.57; found C 76.10, H 4.92, N 5.55.
3a
4a
C16H13NOS
267.35
Chemical formula
M (g mol-1)
Temperature (K)
Crystal size
Crystal color, shape
Crystal system
Space group
a (Å)
C16H13NO2
251.28
298(2)
298(2)
0.42 × 0.10 × 0.03
yellow, needles
monoclinic
P21/n
0.25 × 0.22 × 0.18
red, blocks
monoclinic
C2/c
2-[1-Methylamino-1-(3-methylphenyl)-
methylidene] -benzofuran-3-one (3b)
The reaction was performed according to the general
procedure, using 2b (3.43 g, 8.44 mmol, 1 eq) and methylamine
(41% in water, 6.39 g, 84.4 mmol, 10 eq) in THF (50 mL). Yield:
10.4969(9)
4.9364(4)
24.499(2)
91.136(6)
1269.22(18)
4
23.2157(9)
11.3915(4)
10.6385(4)
109.542(3)
2651.41(17)
8
b (Å)
c (Å)
1
2.05 g highly viscous yellow oil (92%). H NMR (500.13 MHz,
ß (°)
V (Å3)
d6-DMSO): δ = 2.42 (s, 3H, CH3), 2.88 (s, 3H, N–CH3), 7.12–
7.20 (m, 1H, H5), 7.29–7.51 (m, 4H, H7/H2’/H4’–H6’), 7.72 (d,
3J(H,H) = 8Hz, 1H, H4), 9.60–9.95 (brs, 1H); 13C{1H} NMR
(125.75 MHz, d6-DMSO, 25 °C): δ = 21.4 (CH3) 31.5 (N–CH3),
113.2 (C7), 122.3 (C5), 122.9 (C4), 126.9 (C1’), 124.9 (C3a),
129.2 (Cphenyl), 129.6 (Cphenyl), 129.8 (Cphenyl), 131.4 (Cphenyl),
132.7 (C6), 139.7 (C3’), 152.9 (C–N), 160.5 (C7a), 176.3 (C3).
Elemental analysis calcd. (%) for C17H15NO2·0.25H2O: C 75.68,
H 5.79, N 5.19; found C 75.59, H 5.77, N 5.17.
Z
Dc (g cm-3)
ꢁ (cm-1)
1.315
1.339
0.87
2.34
F(000)
528
1120
θ range (°)
h range
1,66 – 25.43
-12/12
3.08 – 30.10
-32/32
k range
-5/5
-16/16
l range
-29/29
-15/14
2-[1-Methylamino-1-(4-methylphenyl)-
methylidene] -benzofuran-3-one (3c)
No. refls. used
No. parameters
Rint
2290
3875
210
173
0.1180
0.0728
The reaction was performed according to the general
procedure, using 2c (6.53 g, 16.1 mmol, 1 eq) and methylamine
(41% in water, 12.2 g, 161 mmol, 10 eq) in THF (100 mL).
Yield: 3.96 g highly viscous yellow oil (93%). 1H NMR
(500.13 MHz, d6-DMSO): δ = 2.43 (s, 3H, CH3), 2.89 (s, 3H, N–
CH3), 7.12–7.20 (m, 1H, H5), 7.27–7.35 (m, 1H, H7), 7.41–7.43
a
R1
0.0482
0.0466
b
wR2
GOFc
0.1320
0.1285
1.020
1.039
residuals, e- Å-3
0.15, -0.14
0.18, -0.34
a R1 = Σ||Fo| - |Fc||/Σ|Fo|.
b wR2 = {Σ[w(Fo2 – Fc2)2]/wΣ(Fo )2]}1/2
.
2
(d, 3J(H,H)
= 8Hz, 1H, H2’/H6’), 7.47–7.62 (m, 3H,
c GOF = {Σ[w(Fo – Fc2)2] /(n – p)}1/2, where n is the number of reflections and p is
3
2
H6/H3’/H5’), 7.72 (d, J(H,H) = 8Hz, 1H, H4), 9.60–9.80 (brs,
1H); 13C{1H} NMR (125.75 MHz, d6-DMSO, 25 °C): δ = 21.5
(CH3) 31.5 (N–CH3), 113.1 (C7), 122.3 (C5), 122.9 (C4), 126.9
(C1’), 124.9 (C3a), 129.4 (C3’/C5’), 129.7 (C2’/C6’), 132.7
(C6), 140.6 (C4’), 152.6 (C–N), 160.6 (C7a), 176.4 (C3).
Elemental analysis calcd. (%) for C17H15NO2·0.25H2O: C 75.68,
H 5.79, N 5.19; found C 75.64, H 5.71, N 5.17.
the total number of parameters refined.
Synthesis
General procedure for the tosylation of 3-hydroxyflavones:
p-Tosylchloride (2 eq) was dissolved in dichloromethane and
filtered to remove traces of sulfonic acid. The filtrate was added
dropwise to the 3-hydroxyflavone (1 eq) and Et3N (2 eq) in
CH2Cl2 under an argon atmosphere at 4 °C. The reaction mixture
was stirred for 18 h at ambient temperature. The obtained clear
solution was washed with water, dried over anhydrous sodium
sulfate and concentrated to dryness. The residue was
recrystallized from isopropanol.
2-[1-Methylamino-1-(3-fluorphenyl)-methylidene]-
benzofuran-3-one (3d)
The reaction was performed according to the general
procedure, using 2d (0.244 g, 0.59 mmol, 1 eq) and methylamine
(41% in water, 0.450 g, 5.9 mmol, 10 eq) in THF (4 mL). Yield:
0.128 g yellow needles (81%). Mp.: 113–115 °C. 1H NMR
(500.13 MHz, d6-DMSO): δ = 2.88 (s, 3H, N–CH3), 7.12–7.22
(m, 1H, H5), 7.25–7.33 (m, 1H, H7), 7.35–7.74 (m, 6H,
H4/H6/H2’/H4’–H6’), 9.55–9.75 (brs, 1H, NH); 13C{1H} NMR
(125.75 MHz, d6-DMSO, 25 °C): δ = 31.6 (N–CH3), 113.2 (C7),
General procedure for the synthesis of 1’-
(alkylamino)aurones: Tosylated compound 2 (1 eq) was reacted
with an excess of alkylamine (10 eq) in THF at room temperature
for 24 h. The reaction mixture was concentrated, extracted with
diethyl ether and the remaining tosyl salt was separated by
filtration. The filtrate was evaporated and the obtained solid was
recrystallized from cyclohexane. Traces of alkylammonium
tosylate were removed by column chromatography (Silica 60,
eluent: diethyl ether : ethyl acetate 5 : 1).
2
116.5 (d, 2J(C,F) = 22 Hz, C4’), 117.7 (d, J(C,F) = 22 Hz, C2’),
4
122.4 (C5), 123.0(C4), 124.7 (C7a), 125.8 (d, J(C,F) = 4Hz,
3
C6’), 131.1 (C1), 131.5 (d, J(C,F) = 9Hz, C5’), 133.0 (C6),
150.7 (C-N), 160.8 (C3a), 162.4 (d, 1J(C,F) = 248Hz, C3’), 176.7
(C3). Elemental analysis calcd. (%) for C16H12FNO2·0.1H2O: C
70.89, H 4.54, N 5.17; found C 70.93, H 4.80, N 5.17.
2-[1-Methylamino-1-(4-fluorphenyl)-methylidene]-
2-[1-Methylamino-1-phenyl-methylidene] -
benzofuran-3-one (3e)
benzofuran-3-one (3a)
The reaction was performed according to the general
procedure, using 2e (3.36 g, 8.19 mmol, 1 eq) and methylamine
(41% in water, 6.20 g, 81.9 mmol, 10 eq) in THF (50 mL). Yield:
1.96 g yellow needles (89%). Mp.: 117–119 °C. 1H NMR
(500.13 MHz, d6-DMSO): δ = 2.89 (s, 3H, N–CH3), 7.12–7.21
The reaction was performed according to the general
procedure, using 2a (0.500 g, 1.27 mmol, 1 eq) and methylamine
(41% in water, 0.960 g, 12.7 mmol, 10 eq) in THF (4 mL). Yield:
0.290 g yellow needles (91%). Mp.: 120–121 °C. 1H NMR
(500.13 MHz, d6-DMSO): δ = 2.88 (s, 3H, N-CH3), 7.12–7.22
(m, 1H, H5), 7.27–7.36 (m, 1H, H7), 7.40–7.51 (m, 1H, H6),
3
(m, 1H, H5), 7.27–7.36 (m, 1H, H7), 7.45 (dd, J(H,F) = 8Hz,
3J(H,H) = 8Hz, 2H, H3’/H5’), 7.49–7.59 (m, 1H, H6), 7.67 (dd,

6
Tetrahedron
ACCEPTED MANUSCRIPT
3J(H,H) = 8Hz, 3J(H,F) = 5Hz, 2H, H2’/H6’), 7.73 (d, 3J(H,H) =
1H, H5), 7.30–7.37 (m, 1H, H7), 7.41–7.51 (m, 1H, H6), 7.52–
8Hz, 1H, H4), 9.60–9.95 (brs, 1H, NH);13C{1H} NMR
(125.75 MHz, d6-DMSO, 25 °C): δ = 31.6 (N–CH3), 113.2 (C7),
116.3 (d, J(C,F) = 20Hz, C3’/5’) 122.4 (C5), 122.9 (C4), 124.8
7.64 (m, 5H, Hphenyl), 7.72–7.82 (m, H4), 9.80–10.10 (brs, 1H,
NH); 13C{1H} NMR (125.75 MHz, d6-DMSO, 25 °C): δ = 11.4
(CH3), 23.9 (-CH2), 45.9 (N-CH2), 113.2 (C7), 122.4 (C5), 123.0
(C4), 124.7 (C7a), 129.3 (Cphenyl), 129.4 (Cphenyl), 130.8 (Cphenyl),
131.3 (Cphenyl), 132.9 (C6), 151.1 (C-N), 160.7, (C3a), 176.7
(C3). Elemental analysis calcd. (%) for C18H17NO2·0.5H2O: C
74.98, H 6.29, N 4.86; found C 74.90, H 6.25, N 4.89.
2
3
(C7a), 131.4 (C1’), 132.0 (d, J(C,F) = 9Hz, C2’/C6’), 132.9
(C6), 151.5 (C-N), 160.6 (C3a), 163.5 (d, 1J(C,F) = 248Hz, C4’),
176.4
(C3).
Elemental
analysis
calcd.
(%)
for
C16H12FNO2·0.1H2O: C 70.89, H 4.54, N 5.17; found C 70.84, H
4.80, N 5.18.
2-[1-Methylamino-1-phenyl-methylidene] -
benzofuran-3-thione (4a)
2-[1-Methylamino-1-(3-chlorphenyl)-methylidene]-
benzofuran-3-one (3f)
1’-(Methylamino)aurone 3a (1.50 g, 5.97 mmol, 1 eq) was
refluxed with Lawesson’s reagent (1.33 g, 3.28 mmol, 1.1 eq) in
dry THF (100 mL) under argon for 20 h. The reaction mixture
was concentrated and the residue was recrystallized from
methanol. Yield: 1.40 g red cubes (88%). Mp.: >80 °C
(decomp.). 1H NMR (500.13 MHz, DMSO-d6): δ = 3.08 (d,
The reaction was performed according to the general
procedure, using 2f (1.67 g, 3.91 mmol, 1 eq) and methylamine
(41% in water, 2.96 g, 39.1 mmol, 10 eq) in THF (50 mL). Yield:
1.03 g yellow needles (92%). Mp.: 126–127 °C. 1H NMR
(500.13 MHz, d6-DMSO): δ = 2.88 (s, 3H, N–CH3), 7.12–7.21
(m, 1H, H5), 7.31–7.36 (m, 1H, H7), 7.38–7.57 (m, 3H,
H6/H3’/H5), 7.58–7.74 (m, 3H, H4/H2’/H6’), 9.55–9.75 (brs,
1H, NH); 13C{1H} NMR (125.75 MHz, d6-DMSO, 25 °C): δ =
31.6 (N–CH3), 113.2 (C7), 122.4 (C5), 122.9 (C4), 124.7 (C7a),
128.2 (Cphenyl), 129.1 (Cphenyl), 130.7 (Cphenyl), 131.1 (C1’),
131.4(Cphenyl), 133.0 (C6), 134.0 (C3’), 150.6 (C-N), 160.8 (C3a),
176.7 (C3). Elemental analysis calcd. (%) for C16H12ClNO2: C
67.26, H 4.23, N 4.90; found C 67.15, H 4.49, N 4.97.
3
3
3J(H,H) = 6 Hz, 3H), 7.29 (dd, J(H,H) = 8 Hz, J(H,H) = 8 Hz,
1H, H5), 7.43 (d, 3J(H,H) = 8 Hz, 1H, H7), 7.53 (dd, 3J(H,H) = 8
3
Hz, J(H,H) = 8 Hz, 1H, H6), 7.67 (s, 5H, Hphenyl), 7.80 (d,
3J(H,H) = 8 Hz, 1H, H4), 13.77–13.83 (brs, 1H, NH); 13C{1H}
NMR (125.75 MHz, d6-DMSO, 25 °C): δ = 32.1 (N–CH3), 112.7
(C7), 122.9 (C5), 123.2 (C4), 129.1 (Cphenyl), 129.3 (Cphenyl),
131.4 (C6), 133.5 (C1’), 142.6 (C7a), 157.5 (C3a), 159.7 (C-N),
166.7
(C3).
Elemental
analysis
calcd.
(%)
for
C16H13NOS·0.1H2O: C 71.40, H 4.94, N 5.20, S 11.91; found C
71.43, H 4.97, N 5.17, S 11.61.
2-[1-Methylamino-1-(4-chlorphenyl)-methylidene]-
benzofuran-3-one (3g)
The reaction was performed according to the general
procedure, using 2g (1.67 g, 3.91 mmol, 1 eq) and methylamine
(41% in water, 2.96 g, 39.1 mmol, 10 eq) in THF (50 mL). Yield:
1.03 g yellow needles (92%). Mp.: 127–129 °C. 1H NMR
(500.13 MHz, d6-DMSO): δ = 2.88 (s, 3H, N–CH3), 7.12–7.21
(m, 1H, H5), 7.29–7.36 (m, 1H, H7), 7.44–7.79 (m, 6H,
H4/H6/H2’/H6’/H3’/H5’), 9.55–9.75 (brs, 1H, NH); 13C{1H}
NMR (125.75 MHz, d6-DMSO, 25 °C): δ = 31.6 (N–CH3), 113.2
(C7), 122.4 (C5), 122.9 (C4), 124.8 (C7a), 129.4 (C3’/5’), 131.3
(C1’), 131.5 (C2’/6’), 132.9 (C6), 135.6 (C4’), 151.1 (C-N),
160.7 (C3a), 176.6 (C3). Elemental analysis calcd. (%) for
C16H12ClNO2·0.1H2O: C 66.84, H 4.28, N 4.87; found C 66.79, H
4.26, N 4.93.
In vitro anticancer activity
Cell lines and culture conditions. CH1(PA-1), SW480 and
A549 cells were grown in 75 cm² culture flasks as adherent
monolayer cultures in Minimal Essential Medium (MEM)
supplemented with 10% heat-inactivated fetal calf serum, 1 mM
sodium pyruvate, 4 mM L-glutamine and 1% non-essential amino
acids (100×). Cultures were maintained at 37 °C in a humidified
atmosphere containing 95% air and 5% CO2.
MTT assay conditions. Cytotoxicity was determined by the
colorimetric MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide) microculture assay. For this purpose,
cells were harvested from culture flasks by trypsinization and
seeded in 100 µL aliquots into 96-well microculture plates. Cell
densities of 1.5 × 103 cells/well [CH1(PA-1)], 2.5 × 103 cells/well
(SW480) and 4 × 103 cells/well (A549) were chosen in order to
ensure exponential growth of untreated controls throughout the
experiment. Cells were allowed to settle and resume exponential
growth in drug-free complete culture medium for 24 h. A stock
of the test compound in DMSO was appropriately diluted in
complete culture medium such that the maximum DMSO content
did not exceed 1%. The dilution was added in 100 µL aliquots to
the microcultures (due to limited solubility, the maximum
concentration tested was added in 200 µL aliquots after removal
of the pre-incubation medium), and cells were exposed to the test
compounds for 96 hours. At the end of exposure, all media were
replaced by 100 µL/well RPMI1640 culture medium
(supplemented with 10% heat-inactivated fetal bovine serum)
plus 20 µL/well MTT solution in phosphate-buffered saline
(5 mg/mL). After incubation for 4 h, the supernatants were
removed, and the formazan crystals formed by viable cells were
dissolved in 150 µL DMSO per well. Optical densities at 550 nm
were measured with a microplate reader, using a reference
wavelength of 690 nm to correct for unspecific absorption. The
quantity of viable cells was expressed in terms of T/C values by
comparison to untreated control microcultures, and 50%
2-[1-Ethylamino-1-phenyl-methylidene] -
benzofuran-3-one (3h)
The reaction was performed according to the general
procedure, using 2a (0.50 g, 1.27 mmol, 1 eq) and ethylamine
(70% in water, 0.82 g, 12.7 mmol, 10 eq) in THF (4 mL). Yield:
0.29 g yellow needles (86%). Mp.: 109–110 °C. 1H NMR
3
(500.13 MHz, d6-DMSO) δ = 1.13 (t, J(H,H) = 8Hz , 3H, CH3),
3
3.22 (q, J(H,H) = 8Hz, 2H, N-CH2), 7.12–7.22 (m, 1H, H5),
7.27–7.36 (m, 1H, H7), 7.40–7.51 (m, 1H, H6), 7.54–7.64 (m,
5H, Hphenyl), 7.72 (d, 3J(H,H) = 8Hz, 1H, H4), 9.60–9.95 (brs, 1H,
NH); 13C{1H} NMR (125.75 MHz, d6-DMSO, 25 °C): δ = 16.4
(CH3), 39.3 (N-CH2), 113.2 (C7), 122.4 (C5), 122.9 (C4), 124.8
(C7a), 129.3 (Cphenyl), 129.4 (Cphenyl), 130.8 (Cphenyl), 131.3
(Cphenyl), 132.9 (C6), 151.8 (C-N), 160.6, (C3a), 176.6 (C3).
Elemental analysis calcd. (%) for C17H15NO2: C 76.96, H 5.70, N
5.28; found C 76.64, H 5.39, N 5.55.
2-[1-Propylamino-1-phenyl-methylidene] -
benzofuran-3-one (3i)
The reaction was performed according to the general
procedure, using 2a (0.200 g, 0.76 mmol, 1 eq) and propylamine
(0.450 g, 7.64 mmol, 10 eq) in dry THF (4 mL). Yield: 0.185 g
highly viscous yellow oil (87%). 1H NMR (500.13 MHz, d6-
3
DMSO) δ = 0.87 (t, J(H,H) = 8Hz , 3H, CH3), 1.48–1.55 (m,
3
2H, CH2), 3.17 (t, J(H,H) = 8Hz, 2H, N-CH2), 7.12–7.22 (m,

7
6. Lipophilicity
inhibitory concentrations (IC50) were calculated from
ACCEPTED MANUSCRIPT
concentration-effect curves by interpolation. Evaluation is based
on means from at least three independent experiments, each
comprising at least three replicates per concentration level.
ChemBioDrawUltra 12.0 and software tools from Molinspiration
(http://www.molinspiration.com) and VCCLAB (Virtual Computational
Chemistry Laboratory, http://www.vcclab.org, 2005) were used to estimate
the compounds’ lipophilicity based on calculated logarithmic octanol−water
partition coefficients (clogP) for the compounds 3a and 4a.45
MEEKC studies
7. Acknowledgements
Instrumentation. CE separations were carried out on a CE
system equipped with an on-column diode-array detector.
Detection was carried out by UV/vis at 200 nm. For all
measurements, capillaries of 48.5 cm total length (40 cm
effective length; 50 ꢁm ID) were used. Capillary and sample tray
were thermostatted at 37 °C. The sample injection was carried
out by hydrodynamic injection at 10 mbar for 15 s and the
separation voltage was 20 kV. New capillaries were conditioned
with 0.1 M HCl, water, 0.1 M NaOH, and again with water
(10 min each). As a daily routine, the capillary was flushed with
0.1 M NaOH, water, and BGE for 5 min each before the first run.
Before each injection, the capillary was purged with 0.1 M
NaOH, water and the BGE for 2 min each.
We thank the “Hochschuljubiläumsstiftung Vienna”, the
“Johanna Mahlke geb. Obermann-Stiftung”, the University of
Auckland and the University of Vienna for financial support. The
authors wish to acknowledge the contribution of NeSI high-
performance computing facilities to the results of this research.
NZ’s national facilities are provided by the NZ eScience
Infrastructure and funded jointly by NeSI’s collaborator
institutions and through the Ministry of Business, Innovation &
Employment's
Research
Infrastructure
programme
(https://www.nesi.org.nz). Michaela Hejl is gratefully
acknowledged for performing the cytotoxicity tests.
Microemulsion preparation. The following component
ratios (weight%) were used to establish a stable microemulsion:
91.26% sodium phosphate buffer (20 mM, pH 7.4), 0.82%
heptane, 1.44% SDS, and 6.48% 1-butanol. For the preparation
of the MEEKC solutions, surfactant, alcohol, oil, and a small
portion of buffer were mixed by sonication for 5 min. The
residual buffer was then slowly added to the mixture. Afterwards,
the solution was let to stand for 30 min at room temperature
before use.
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