Synthesis of 4,4-Disubstituted 3-Methylidenechroman-2-ones as Potent Anticancer Agents

Article abstract of DOI:10.1002/cmdc.201700080

The synthesis of a new library of 4,4-disubstituted 3-methylidene-3,4-dihydro-2H-chroman-2-ones applying Horner–Wadsworth–Emmons methodology for the construction of an exo-methylidene moiety is reported. Corresponding 3-diethoxyphosphorylchroman-2-ones were synthesized in a three-step reaction sequence consisting of O-methylation of ethyl 2-diethoxyphosphoryl-3-oxoalkanoates, followed by reaction of the obtained 2-diethoxyphosphoryl-3-methoxy-2-alkenoates with phenols or 1-naphthol. The resulting 3-diethoxyphosphorylochromen-2-ones proved to be effective Michael acceptors in reactions with various Grignard reagents. Preliminary biological evaluations showed that many of the synthesized 3-methylidenechroman-2-ones possess very high cytotoxic activity against NALM-6 and HL-60 cancer cell lines (IC₅₀<1.0 μm) as well as high activity against the MCF-7 cancer cell line (IC₅₀<10 μm). Furthermore, two of the highly active 3-methylidenechroman-2-ones with geminal methyl and ethyl substituents at position 4 showed promising therapeutic indexes of 10 and 13 in tests against human umbilical vein endothelial cells (HUVECs).

Full text of DOI:10.1002/cmdc.201700080

Accepted Article
Title: Synthesis of novel 4,4-disubstituted 3-methylidenechroman-2-
ones as potent anticancer agents
Authors: Rafał Jakubowski, Dorota Pomorska, Angelika Długosz,
Anna Janecka, Urszula Krajewska, Marek Różalski, Marek
Mirowski, Tomasz Bartosik, and Tomasz Janecki
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10.1002/cmdc.201700080
ChemMedChem
Synthesis of novel 4,4-disubstituted 3-methylidenechroman-2-ones as potent
anticancer agents
Rafał Jakubowski,[a,b] Dorota K. Pomorska,[c] Angelika Długosz,[c] Anna Janecka,[c] Urszula Krajewska,[d]
Marek Różalski,[d] Marek Mirowski,[d] Tomasz Bartosik,[a] Tomasz Janecki*[a]
The synthesis of new library of 4,4-disubstituted 3-methylidene-3,4-dihydro-2H-chroman-2-ones
applying Horner-Wadsworth-Emmons methodology for the construction of egzo-methylidene moiety
is reported. Corresponding 3-diethoxyphosphorylchroman-2-ones were synthesized in a three step
reaction sequence consisting of O-methylation of ethyl 2-diethoxyphosphoryl-3-oxoalkanoates,
followed by reaction of obtained 2-diethoxyphosphoryl-3-methoxy-2-alkenoates with phenols or 1-
naphthol. Obtained 3-diethoxyphosphorylochromen-2-ones proved to be effective Michael acceptors
in the reaction with various Grignard reagents. Preliminary biological evaluation showed that many
of the synthesized 3-methylidenechroman-2-ones possess very high cytotoxic activity against NALM-
6 and HL-60 cancer cell lines (IC50 < 1.0 M) as well as high activity against MCF-7 cancer cell line (IC50
< 10 M). Furthermore, two of the highly active 3-methylidenechroman-2-ones with geminal methyl
and ethyl substituents in position 4 showed promising therapeutic index of 10 and 13 when tested
against human umbilical vein endothelial cells (HUVEC).
Introduction
Chromen-2-ones 1, commonly known as coumarins belong to one of the most important class of
natural products that display a wide spectrum of biological properties (Figure 1).1 Due to the unique
structure coumarin derivatives interact with many enzymes and receptors and are frequent targets in
the search for new drug candidates. Among various biological activities they often display strong
anticancer activity.2,3 Also chroman-2-ones 2 or 3,4-dihydrocoumarins, although less common in
nature, show various biological activities, and their cytotoxic activity is well recognized.4 For example,
coumarin glycosides 3 isolated from the aerial parts of Diceratella elliptica show high activity against
three human carcinoma cell lines: liver (HEPG2), cervix (HELA) and colon (HCT116). Also, variously
substituted dihydrocoumarin derivatives 4 were found to be sirtuins’ inhibitors (SIRTi - silent
information regulator inhibitors) and showed antiproliferative properties and tubulin
hyperacetylation in MCF-7 breast cancer cells5 as well as high pro-apoptotic and cytodifferentiating
properties in human leukemia cell line (U937).6
[a] R. Jakubowski, T. Bartosik, Prof. T. Janecki
Institute of Organic Chemistry, Lodz University of Technology, Żeromskiego 116, 90-924 Łódź, Poland,
E-mail: tomasz.janecki@p.lodz.pl
[b] R. Jakubowski
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-
363 Łódź, Poland
[c] D. Pomorska, A. DŁugosz, Prof. A. Janecka
Department of Biomolecular Chemistry, Medical University of Łódź, Mazowiecka 6/8, 92-215 Łódź,
Poland
[d] U. Krajewska, Dr. M. Różalski, Prof. M. Mirowski
Department of Pharmacutical Biochemistry and Molecular Diagnostics, Faculty of Pharmacy, Medical
University of Łódź, Muszyńskiego 1, 90-151 Łódź, Poland
Supporting information for this article can be found under:
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10.1002/cmdc.201700080
ChemMedChem
Figure 1 Structures of chromen-2-ones 1 and chroman-2-ones 2-5.
Recently, we have reported on the efficient synthesis of a series of 3-methylidenechroman-2-
ones 5 with a variety of substituents at position 4, using Horner-Wadsworth-Emmons methodology.7
These compounds contain an exo-methylidene moiety conjugated with ester functionality, an
arrangement which is present in a large group of natural and synthetic -alkylidene-- and -lactones
and lactams and is believed to be crucial for their biological activity, such as cytotoxic or
antimicrobial.8,9 Therefore, synthesized 5 can be regarded as molecular hybrids containing two
pharmacophore units – -methylidene--lactone moiety and chroman-2-one skeleton. This
combination, which is not present in natural compounds, proved to be very effective, because
several of the synthesized compounds displayed strong cytotoxic activity against human leukemia
and breast cancer cell lines and have been subjected to further, more detailed, biological studies.10,11
Investigation of the structure-activity relationship in 5 revealed that the most important factor
influencing the cytotoxicity was the nature of the substituent in position 4. In general,
methylidenechromanones 5 containing n-butyl or i-propyl substituent in this position were always
more active than compounds having methyl or vinyl substituent and in some cases this difference
was really significant.7
Encouraged by these results we decided to perform further investigations in order to
broaden the spectrum of available 3-methylidenechroman-2-ones with hope for even more active
compounds. Accordingly, herein we report on the synthesis of 4,4-disubstituted 3-
methylidenechroman-2-ones 15 with various substitution patterns in position 4 and present the
results of preliminary cytotoxic evaluation of the obtained compounds against two human leukemia
cell lines, promyelocytic HL-60 and lymphoblastic NALM-6 as well as against a solid tumor-derived
breast cancer adenocarcinoma MCF-7.
Results and discussion
We started our investigations with the synthesis of crucial intermediates, ethyl 2-
diethoxyphosphoryl-3-alkoxy-2-alkenoates, with various substituents in position 3. Initially we tested
the reaction of ethyl diethoxyphosphorylacetate (6) with triethylorthoacetate (7) in the presence of
Ac2O and ZnCl2 as catalysts. Pleasingly, heating the mixture for 16 hours gave desired ethyl 3-ethoxy-
2-diethoxyphosphoryl-2-butenoate 8 as a mixture of E and Z isomers in 45:55 ratio, respectively.
Distillation of the crude product yielded pure 8 in reasonable, 51% yield (Scheme 1).
2
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10.1002/cmdc.201700080
ChemMedChem
Scheme 1 Reagents and conditions: (a) Ac2O, ZnCl2, 140°C, 16h.
Unfortunately, this procedure was not general and reactions with other orthoesters, such as
triethyl orthopropionate or triethyl orthobenzoate gave complex mixtures of products. Therefore, we
decided to test the methylation reaction of easily available12 ethyl 2-diethoxyphosphoryl-3-
oxoalkanoates 9 which should provide desired ethyl 2-diethoxyphosphoryl-3-methoxy-2-alkenoates
10. Initial experiments in which ethyl 2-diethoxyphosphoryl-3-oxobutanoate 9a was reacted with MeI
or Me2SO4 in the presence of NaH, KH or K2CO3 in DMSO, THF or HMPA gave unreacted 9a and/or
mixtures of C- and O-methylation products with C-methylation product 11a predominating. To our
delight, application of CF3SO3Me/Cs2CO3 system in acetonitrile gave much better results with O- to C-
methylathion products 10a/11a in 76/24 ratio. Fortunately, we were able to separate pure O- and C-
alkylation products 10a and 11a in 61% and 17% yield, respectively, using column chromatography.
Employing this procedure we prepared pure ethyl 2-diethoxyphosphoryl-3-methoxy-2-alkenoates
10a-d in satisfactory yields (Scheme 2,Table 1).
Scheme 2 Reagents and conditions: (a) CF3SO3Me, Cs2CO3, CH3CN, 0oC, then r.t., 1.5h.
Table 1 Ethyl 2-diethoxyphosphoryl-3-methoxy-2-alkenoates 10.
10 R1
10/11 E/Z
Yieldb
ratio a ratio a (%)
76/24 52/48 61
53/47 62/38 60
a
b
c
Me
Et
n-Bu 63/37 63/37 57
Ph 92/8 28/72 83
d
aTaken from 31P NMR spectra of the crude product.
bYield of isolated, purified product, based on 9.
All 10 were formed as mixtures of E and Z isomers and the ratio could be calculated from their 31P
3
NMR spectra. Furthermore, based on very characteristic JPC(R1) coupling constants we were able to
assign E configuration to alkenoates 10a-d in which 3JPC(R1) coupling constants fell in the range of 0.0 -
1.9 Hz and Z configuration to alkenoates 10a-d in which 3JPC(R1) coupling constants fell in the range of
3
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10.1002/cmdc.201700080
ChemMedChem
10.5-12.4 Hz (Figure 2). 3 JPC(R1) coupling constants could be easily determined from the corresponding
13C NMR spectra. Determined E/Z ratios are given in Table 1.
Figure 2 3JPC(R1) coupling constants for (E)- and (Z)-10a-d.
With the synthesis of 10a-d in hand we performed their reaction with phenols 12a,b and 1-
naphthol 12c at room temperature in the presence of trifluoromethanesulfonic acid. The reaction
was monitored by TLC and was completed in 72 hours. As expected, Friedel-Crafts alkylation followed
by intramolecular cyclization took place and position 4-substituted 3-diethoxyphosphorylchromen-2-
ones 13a-j were obtained in moderate to very good yields (Scheme 3, Table 2). Structures of
chromenones 13a-j were fully confirmed by the analysis of their 1H, 31P and 13C NMR spectra.
Scheme 3 Reagents and conditions: (a) CF3SO3H, CH2Cl2, r.t., 72 hours
Table 2 Synthesis of 4-substituted 3-diethoxyphosphorylchromen-2-ones 13a-j.
13 R1
R2 R3
R4
Yielda
(%)
a
b
c
Me
H
H
OMe
H
81
Me
Me
Et
OMe OMe 69
CHCHCHCH
H
H
60
59
d
e
f
H
H
OMe
Et
OMe OMe 47
Et
CHCHCHCH
H
H
26
38
g
h
n-Bu
n-Bu
H
H
OMe
OMe OMe 31
4
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10.1002/cmdc.201700080
ChemMedChem
i
j
Ph
Ph
H
H
OMe
H
85
OMe OMe 70
aYield of isolated, pure product, based on 10.
4-Substituted 3-diethoxyphosphorylochromen-2-ones 13a-j were next used as Michael
acceptors in the reaction with several Grignard reagents (ethyl-, n-buthyl, isopropylmagnesium and
cyclohexylmagnesium chlorides, benzylmagnesium bromide, allylmagnesium iodide) (Scheme 4).
Additions were performed using 5 equivalents of Grignard reagent in the presence of catalytic
amount of CuI. The additions proceeded smoothly in THF at 0oC yielding 4,4-disubstituted 3-
diethoxyphosphoryl-3,4-dihydro-2H-chroman-2-ones 14a-z in very good yields as single trans- or a
mixture of trans- and cis- isomers (Table 3). Configurational assignments were made on the basis of
3
3
3
diagnostic JPC(R5), JPC(R1) and JPC(4a) coupling constants, taken from 13C NMR spectra, which fell in the
range 15.3-19.4 Hz, 1.5-3.2 Hz and 0-2.3Hz, respectively, for trans-14a-z. Similar coupling constants,
characteristic for trans diaxial arrangement of diethoxyphosphoryl group and substituent in position
4 were observed in trans-4-substituted-3-diethoxyphosphorylchroman-2-ones7,13. Furthermore, for
3
3
3
14k,r,v it was possible to determine JPR5, JPR1 and JP4a coupling constants also for cis-isomers which
fell in the range 1.9-4.5 Hz, 15.6-18.4 Hz and 0-1.3Hz, respectively and fully confirm the above
assignments. Interestingly, in all adducts 14 protons of one of the ethoxy groups within
diethoxyphosphoryl moiety absorbed at an unexpectedly low frequency. For example, the methyl
protons of one of the ethoxy groups of 14a had a chemical shift of 0.84 ppm with comparison to 1.28
of the other. This phenomenon was observed earlier in trans-4-substituted-3-
diethoxyphosphorylchroman-2-ones, in which almost antiparallel array of P=O and C=O bonds
minimized interactions between these polar groups13. In this arrangement one of the ethoxy groups
is placed in the shielding area of the carbonyl bond and/or benzene ring. We believe that this
explanation is also valid for 4,4-disubstituted 3-diethoxyphosphoryl-3,4-dihydro-2H-chroman-2-ones
14a-z.
Scheme 4 Reagents and conditions: (a) R5MgX, CuI, THF, 0oC, then r.t., 48 h; (b) (CH2O)n, NaH, THF,
r.t., 90 min; (c) (CH2O)n, t-BuOK, THF, r.t., 16 h; (d) t-BuOK, THF, 50oC, 72 h.
5
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10.1002/cmdc.201700080
ChemMedChem
Table
3
4,4-Disubstituted 3-diethoxyphosphoryl-3,4-dihydro-2H-chroman-2-ones 14a-z, 4,4-
disubstituted 3-methylidene-3,4-dihydro-2H-chroman-2-ones 15a-e,g-z and 3-(but-3-en-1-yl)-7-
methoxy-4-methyl-2H-chromen-2-one 16 prepared.
Compound R1
R2 R3
R4
R5
14
14
15
trans/cis yieldb Procedure,
ratioa
86/14
97/3
[%]
82
77
72
66
77
75
64
76
68
58
66
66
65
63
68
60
76
76
73
74
77
89
84
65
yieldb [%]
(b), 81
(b), 61
(b), 66
(b), 77
(b), 60
(b), 91c
(b), 90
(b), 71
(b), 71
(b), 89
(b), 57
(b), 83
(b), 73
(b), 58
(b), 90
(b), 88
(c), 83
(c), 71
(d), 61
(c), 89
(c), 81
(c), 67
(d), 60
(d), 72
a
b
c
Me
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
OMe
OMe
OMe
OMe
OMe
n-Bu
i-Pr
Bn
98/2
d
e
f
96/4
Cyclohexyl 91/9
allyl
89/11
95/5
g
h
i
OMe OMe Et
OMe OMe n-Bu
OMe OMe i-Pr
OMe OMe Bn
97/3
96/4
j
>99/1
k
l
OMe OMe Cycloxexyl 67/33
CHCHCHCH
CHCHCHCH
CHCHCHCH
CHCHCHCH
CHCHCHCH
H
H
H
H
H
H
H
H
H
Et
86/14
88/12
>99/1
95/5
m
n
o
p
q
r
n-Bu
i-Pr
Bn
Cyclohexyl >99/1
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
Et
-
Et
n-Bu
i-Pr
Bn
52/48
73/27
82/18
-
s
Et
t
Et
u
v
w
x
Et
OMe OMe Et
OMe OMe n-Bu
OMe OMe i-Pr
OMe OMe Bn
Et
53/47
91/9
73/27
Et
Et
6
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10.1002/cmdc.201700080
ChemMedChem
y
z
n-Bu
n-Bu
H
H
OMe
H
n-Bu
-
-
58
82
(c), 69
(c), 83
OMe OMe n-Bu
a Taken from the 31P NMR spectrum of purified products.
b Yield of isolated, purified product, based on 13 or 14, respectively.
c 3-(But-3-en-1-yl)-7-methoxy-4-methyl-2H-chromen-2-one 16 was obtained.
Finally, 4,4-disubstituted chroman-2-ones 14a-z were used in the Horner-Wadsworth-
Emmons olefination of formaldehyde. Thus, treatment of 14a-p with NaH and then with
paraformaldehyde (Scheme 4, procedure b) gave, after purification by column chromatography, 4,4-
disubstituted 3-methylidene-3,4-dihydro-2H-chroman-2-ones 15a-e,g-p in good to excellent yields
(Table 3). Interestingly, when 4-allyl-3-diethoxyphosphoryl-4-methyl-7methoxy-3,4-dihydro-2H-
chroman-2-one (14f) was used as a Horner-Wadsworth-Emmons reagent, 3-(but-3-en-1-yl)-7-
methoxy-4-methyl-2H-chromen-2-one (16) was obtained, instead of the expected 3-
methylidenechroman-2-one 15f. Formation of 16 can be easily rationalized assuming that Cope
rearrangement took place in the reaction conditions. Although applied olefination procedure worked
well with chroman-2-ones 14a-p (R1 = Me), reactions performed with chroman-2-ones 14q-z (R1 = Et
or n-Bu) were inefficient in these conditions. To our delight, replacement of NaH by t-BuOK,
prolonging the reaction time and in some cases rising the reaction temperature to 50oC (Scheme 4,
procedures c and d) gave, after purification by column chromatography, 4,4-disubstituted 3-
methylidene-3,4-dihydro-2H-chroman-2-ones 15q-z in very good yields (Table 3). Spectroscopic data
(1H NMR, 13C NMR, MS) were in full agreement with the structures of all obtained 3-
methylidenechroman-2-ones 15 as well as with chromen-2-one 16.
Biological evaluation
As mentioned in the introduction, methylidenechroman-2-ones are potential anticancer agents.
Therefore, we subjected obtained library of 3-methylidenechroman-2-ones 15a-e,g-z to the in vitro
cytotoxicity screening against two human leukemia cell lines NALM-6 and HL-60 as well as MCF-7
breast cancer cell line. Carboplatin was used as a reference compound.14 Obtained IC50 values are
given in Table 4. These data show that all synthesized compounds display high or very high activity
(IC50 < 10 M or IC50 < 1.0M, respectively) against HL-60 and NALM-6 and the majority of them also
shows high activity against MCF-7 cells. Inspection of the structure-activity relationship indicates
that, as it was observed for methylidenechromenones 5, cytotoxicity of the tested compounds
depends strongly on the nature of substituents in position 4 and, to much smaller extend, on the
structure of the aromatic moiety. In general, cytotoxicity of compounds with different aromatic
fragments containing one or two methoxy groups or an additional ortho-fused benzene ring are
usually similar when substituents in position 4 are the same. For example, compounds 15d,j,o
sharing the same substituents in position 4 (Me and Bn) but varying in the aromatic moiety have very
similar IC50 values for the same cancer cell line, e.g. 0.83, 0.66 and 0.78 M for NALM-6 and 3.86,
2.98 and 4.33 M for MCF-7, respectively. For HL-60 cells corresponding values for 15a,j,o are 3.77,
0.86 and 3.30 M and very high activity of 15j can be attributed to the presence of an additional
methoxy group in the aromatic moiety. However, this relation is not general and no clear relationship
between the structure of aromatic moiety and activity can be made. On the other hand, the
influence of the geminal substituents in position 4 on the activity is much more clear. Compounds
containing methyl or ethyl group (R1) combined with ethyl, n-butyl or benzyl group (R5) usually are
more active than compounds with i-propyl or cyclohexyl group (R5). This relationship is especially
straightforward when cytotoxicity of compounds 15g-t against NALM-6 cell line is considered. All
nine compounds within this group with this substitution pattern display very high cytotoxic activity
7
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10.1002/cmdc.201700080
ChemMedChem
(IC50 < 1.0 M). On the other hand, the presence of two germinal n-butyl groups like in 15y,z does not
produce very active derivatives.
Table 4 In vitro cytotoxic activity of 3-methylidenechroman-2-ones 15a-e,g-z tested on three cancer
cell lines and on normal HUVEC and MCF-10A cells.
Cytotoxicity IC50a (M)
NALM-6 MCF-7 HUVEC
15
R4
R5
HL-60
MCF-10A
a
b
c
H
H
H
H
H
Et
2.39±0.30 1.00±0.10 9.30±0.88
3.69±0.58 2.58±0.34 5.71±0.10
2.51±0.22 3.45±0.43 7.57±0.52
-
-
-
-
-
-
n-Bu
i-Pr
Bn
d
e
g
h
i
3.77±0.32 0.83±0.06 3.86±0.41 3.08±0.45 5.10±1.11
Cyclohexyl 5.45±0.34 4.89±0.22 59.7±0.89
-
-
OMe Et
1.30±0.28 0.63±0.03 4.00±0.50 7.09±0.74 13.8±2.80
OMe n-Bu
OMe i-Pr
OMe Bn
4.92±0.13 0.65±0.06 6.12±0.73
7.06±0.46 4.86±0.34 10.52±0.71
-
-
-
-
j
0.86±0.07 0.66±0.07 2.98±0.15 1.58±0.19 5.52±0.78
k
l
OMe Cycloxexyl 7.40±0.20 6.86±0.35 5.87±0.95
-
-
-
Et
2.27±0.31 0.72±0.05 4.20±0.42 9.13±0.65 9.53±2.41
m
n
o
p
q
r
-
n-Bu
i-Pr
Bn
5.18±0.48 0.83±0.08 5.75±0.62
5.44±0.40 2.57±0.33 7.04±0.85
3.30±0.30 0.78±0.04 4.33±0.35
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Cyclohexyl 7.24±0.18 6.48±0.22 28.63±0.15
H
H
H
H
Et
0.87±0.04 0.75±0.03 5.60±0.32
1.58±0.20 0.93±0.04 5.28±0.95
7.63±0.19 6.50±0.36 9.80±0.62
n-Bu
i-Pr
Bn
s
t
1.06±0.13 0.75±0.03 3.81±0.21 3.47±0.53 3.73±0.36
8
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10.1002/cmdc.201700080
ChemMedChem
u
v
w
x
OMe Et
3.36±0.40 0.85±0.04 4.60±0.11 1.87±0.28 6.07±0.44
OMe n-Bu
OMe i-Pr
OMe Bn
5.38±0.51 5.00±0.21 8.00±0.42
8.42±0.43 8.58±0.80 8.58±0.80
1.93±0.25 0.85±0.05 5.62±0.45
5.25±0.23 6.27±0.38 3.84±0.36
4.64±0.19 5.73±0.39 13.37±0.90
9.70±1.20 2.90±0.10 0.70±0.30
-
-
-
-
-
-
y
z
H
-
-
-
-
OMe
-
-
Carboplatin
25±3.2
8.2±0.91
a IC50, 50% inhibitory concentration determined by MTT assay after 48 h exposure of the cells to the
drugs. IC50 values were calculated from concentration–survival curves using a non-linear estimation
(quasi-Newton algorithm) method. Each data represents the mean from dose response curves of at
least three experiments.
Selected 3-methylidenechroman-2-ones 15 were also tested against healthy cells, human
umbilical vein endothelial cell line (HUVEC) and human mammary gland/breast (MCF10A) cell line
(Table 3). In most cases cytotoxicity of the tested compounds was comparable or 2-4 times higher
than toxicity towards normal cells. However, two chromanones, 15g and 15l displayed much better
therapeutic index of 10 and 13 respectively, against NALM-6 cells. Both compounds have the same
substituents (methyl and ethyl) in position 4. These two chromanones were therefore selected for
more detailed biological studies, which are currently in progress.
Conclusions
Herein, we have presented novel, simple and convenient protocol for the synthesis of biologically
important 4,4-disubstituted 3-methylidene-3,4-dihydro-2H-chroman-2-ones 15 applying Horner-
Wadsworth-Emmons methodology. Furthermore, general method for the synthesis of starting 2-
diethoxyphosphoryl-3-methoxy-2-alkenoates 10 was developed and the relative configuration of
intermediate 4,4-disubstituted 3-diethoxyphosphoryl-3,4-dihydro-2H-chroman-2-ones 14 was
elaborated using NMR analysis. Also, optimization of the Horner-Wadsworth-Emmons olefination
step was performed and procedures effective for the specific substrates were established. Screening
tests against three human cancer cell lines revealed high efficiency of the obtained
methylidenechroman-2-ones 15 as anticancer agents. The most potent compounds had IC50 values
below 1.0 M, against HL-60 and NALM-6 cancer cell lines and below 10 M against MCF-7 cell line.
Also, some very interesting structure-activity relationship, especially for NALM-6 cell line, were
established, what can be very useful in further planning of even more active analogs. Finally, based
on the cytotoxicity test against healthy human HUVEC cells, two chromanones, 15g and 15l, with best
therapeutic index of 10 and 13 respectively, were chosen for more detailed biological studies.
Experimental
General section
NMR spectra were recorded on a Bruker DPX 250 or Bruker Avance II instrument at 250.13 MHz or
700 MHz for 1H, 62.9 MHz or 176 MHz for 13C, and 101.3 MHz for 31P NMR using tetramethylsilane as
internal and 85% H3PO4 as external standard. 31P NMR spectra were recorded using broadband
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10.1002/cmdc.201700080
ChemMedChem
proton decoupling. IR spectra were recorded on a Bruker Alpha ATR spectrophotometer. Melting
points were determined in open capillaries and are uncorrected. Column chromatography was
performed on Aldrich® silica gel 60 (230 - 400 mesh). Thin-layer chromatography was performed with
precoated TLC sheets of silica gel 60 F254 (Aldrich®). The purity of tested compounds was determined
by combustion elemental analyses (CHN, elemental analyzer EuroVector 3018, Elementar
Analysensysteme GmbH). Analyses indicated by the symbols of the elements or functions were
within ±0.4% of the theoretical values. MS spectra were performed on combined Waters 2695-
Waters ZQ 2000 LC/MS apparatus. Reagents and starting materials were purchased from commercial
vendors and used without further purification. All organic solvents were dried over appropriate
drying agents and distilled prior to use. Standard syringe techniques were used for transferring dry
solvents.
Synthesis of compounds 10a-d, 11a-d, 13a-j and 14a-z
See Supporting information for synthesis details and characterization data.
Synthesis of compounds 15a-e,g-z and 16
Procedure a
To a solution of the corresponding chromanone 14a-p (0.5 mmol) in THF (5 mL), NaH (14 mg, 0.6
mmol) was added and the resulting mixture was stirred at rt for 30 min. Then paraformaldehyde (75
mg, 2.5 mmol) was added in one portion. After 90 min., the reaction mixture was quenched with
brine (5 mL), THF was removed under reduced pressure and the aqueous layer was extracted with
CH2Cl2 (3 x 10 mL). The organic layer was dried over MgSO4 and the solvent was evaporated. The
crude product was purified by column chromatography (eluent: CH2Cl2) to afford 15a-e,g-p and 16 in
57-91% yield. See Supporting information for characterization data.
Procedure b
To a solution of the corresponding chromanones 14q,r,t-v,y,z (0.5 mmol) in THF (5 mL), t-BuOK (67
mg, 0.6 mmol) was added and the resulting mixture was stirred at rt for 30 min. Then
paraformaldehyde (75 mg, 2.5 mmol) was added in one portion. After 16 h.., the reaction mixture
was quenched with brine (5 mL), THF was removed under reduced pressure and the aqueous layer
was extracted with CH2Cl2 (3 x 10 mL). The organic layer was dried over MgSO4 and the solvent was
evaporated. The crude product was purified by column chromatography (eluent: CH2Cl2) to afford 15
q,r,t-v,y,z in 67-89% yield. See Supporting information for characterization data.
Procedure c
To a solution of the corresponding chromanones 14q,r,t-v,y,z (0.5 mmol) in THF (5 mL), t-BuOK (67
mg, 0.6 mmol) was added and the resulting mixture was stirred at rt for 30 min. Then
paraformaldehyde (75 mg, 2.5 mmol) was added in one portion and the solution was stirred in 50°C
for 72 h. Then, reaction mixture was quenched with brine (5 mL), THF was removed under reduced
pressure and the aqueous layer was extracted with CH2Cl2 (3 x 10 mL). The organic layer was dried
over MgSO4 and the solvent was evaporated. The crude product was purified by column
chromatography (eluent: CH2Cl2) to afford 15 q,r,t-v,y,z in 67-89% yield. See Supporting inforation for
characterization data.
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10.1002/cmdc.201700080
ChemMedChem
Cell culture and treatment
Two human leukemia cell lines, promyelocytic leukemia (HL-60) and lymphoblastic leukemia (NALM-
6), and a solid tumor-derived human breast adenocarcinoma cell line (MCF-7) were purchased from
the European Collection of Cell Cultures (ECACC). Leukemia cells were cultured in RPMI 1640 plus
GlutaMax I medium (Invitrogen, Grand Island, NY, USA), supplemented with 10% heat-inactivated
FBS (Biological Industries, Beit-Haemek, Israel) and antibiotics (100 μg/mL streptomycin and 100
U/mL penicillin) (Sigma-Aldrich, St. Louis, MO, USA). MCF-7 cells were cultured in Minimum Essential
Medium Eagle (MEME, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated
fetal bovine serum (FBS) (Biological Industries, Beit-Haemek, Israel), 2mM glutamine, Men Non-
essential amino acid solution and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin), all
from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Normal human mammary gland/breast cell
line (MCF10A) and normal human umbilical vein endothelial cells (HUVECs) were purchased from the
American Type Culture Collection (ATCC). MCF-10A cells were cultured using MEGM Mammary
Epithelial BulletKit, while HUVECs using EGM-2 Endothelial Medium BulletKit, both purchased from
Lonza (Lonza, Walkersville, MD, USA). Cells were maintained at 37 °C in a 5 % CO2 atmosphere and
were grown until 80% confluent.
For all experiments tested compounds were dissolved in DMSO (Sigma-Aldrich, Louis, MO, USA) and
further diluted in culture medium to obtain less than 0.1% DMSO concentration. In each experiment
controls without and with 0.1% DMSO were performed. DMSO in this concentration had no effects
on the observed parameters.
Metabolic activity assay (MTT)
The MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay, which measures
activity of cellular dehydrogenases, was performed according to a known procedure.15 Exponentially
growing cells were seeded on 96-well plates (Nunc, Roskilde, Denmark) in 100 µL and left to growth
for 24 h. Subsequently, various concentrations of the tested compounds (freshly prepared in DMSO
and diluted with complete culture medium) were added in 100 µL of culture medium to obtain the
concentration range from 10-7 to 10-3 M and the plates were incubated for 24 or 48 h. Afterwards,
MTT (5 mg/mL in PBS) was added and incubation was continued for 2 h. The metabolically active cells
reduced MTT to blue formazan crystals. Then, the MTT-containing medium was carefully aspirated
and 100 µL DMSO was added to dissolve the crystals. The absorbance of the blue formazan product
was measured at 560 nm using an automated plate reader (iMark Bio-Rad, Hercules, CA, USA) and
compared with control (untreated cells). The IC50 values were calculated from concentration–
response curves.
Acknowledgements
This work was financially supported by the National Science Centre of Poland (project DEC-
2012/07/B/ST5/02006).
References
[1] K. P. Barot, S. V. Jain, L. Kremer, S. Singh and M. D. Ghate, Med. Chem. Res., 2015, 24, 2771-2798.
[2] A. Thakur, R. Singla and V. Jaitak, Eur. J. Med. Chem., 2015, 101, 476-495.
[3] S. Emami and S. Dadashpour, Eur. J. Med. Chem., 2015, 102, 611-630.
11
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10.1002/cmdc.201700080
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[4] D. P. Kamat, S. G. Tilve, V. P. Kamat and J. K. Kirtany, Org. Prep. Proc. Int. 2015, 47, 1-79.
[5] R. C. Neugebauer, U. Uchiechowska, R. Meier, H. Hruby, V. Valkov, E. Verdin, W. Sippl and M.
Jung, J. Med. Chem., 2008, 51, 1203-1213.
[6] D. Rotili, V. Carafa, D. Tarantino, G. Botta, A. Nebbioso, L. Altucci and A. Mai, A., Bioorg. Med.
Chem., 2011, 19, 3659-3668.
[7] J. Modranka, A. Albrecht, R. Jakubowski, H. Krawczyk, M. Różalski, U. Krajewska, A. Janecka, A.
Wyrębska, B. Różalska and T. Janecki, Bioorg. Med. Chem., 2012, 20, 5017-5026.
[8] A. Albrecht, Ł. Albrecht and T. Janecki, Eur. J. Org. Chem., 2011, 15, 2747-2766.
[9] A. Janecka, A. Wyrębska, K. Gach, J. Fichna and T. Janecki, Drug Disc. Today, 2012, 17, 561-572.
[10] A. Wyrębska, K. Gach, U. Lewandowska, K. Szewczyk, E. Hrabec, J. Modranka, R. Jakubowski, T.
Janecki, J. Szymański and A. Janecka, Basic Clin. Pharm. Toxicol., 2013, 113, 391-400.
[11] K. Gach, J. Szymański, D. Pomorska, A. Długosz, J. Modranka, T. Janecki and A. Janecka, Tumor
Biol., 2015, 36, 5971-5977.
[12] B. Corbel, I. L’Hostis-Kervella and J. P. Haelters, Synth. Commun., 1996, 26, 2561-2568.
[13] T. Janecki and T. Wąsek, Tetrahedron, 2004, 60, 1049-1055.
[14] E. Wong, E. and C. M. Giandomenico, Chem. Rev., 1999, 99, 2451-2466.
[15] T. Mosmann, J. Immunol. Methods, 1983, 65, 55-63.
Keywords
Phosphorylated heterocycles, 3-methylidenechroman-2-ones, Michael addition, Horner-Wadsworth-
Emmons olefination, cytotoxicity
Table of Contents text
The efficient synthesis of new library of 4,4-disubstituted 3-methylidene-3,4-dihydro-2H-chroman-2-
ones using Horner-Wadsworth-Emmons methodology is described. These compounds can be
regarded as molecular hybrids containing two pharmacophore units – -methylidene--lactone
moiety and chroman-2-one skeleton. Cytotoxicity tests revealed, that many of the synthesized
chroman-2-ones possess very high cytotoxic activity against NALM-6 and HL-60 cancer cell lines (IC50
< 1.0 M) as well as high activity against MCF-7 cancer cell line (IC50 < 10 M).
12
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