Synthesis and Cytotoxic Evaluation of 3-Methylidenechroman-4-ones

Article abstract of DOI:10.3390/molecules24101868

In the search for new anticancer agents, a library of variously substituted 3-methylidenechroman-4-ones was synthesized using Horner–Wadsworth–Emmons methodology. Acylation of diethyl methylphosphonate with selected ethyl salicylates furnished 3-diethoxyphosphorylchromen-4-ones which were next used as Michael acceptors in the reaction with various Grignard reagents. The adducts were obtained as the mixtures of trans and cis diastereoisomers along with a small amount of enol forms. Their relative configuration and preferred conformation were established by NMR analysis. The adducts turned up to be effective Horner–Wadsworth–Emmons reagents giving 2-substituted 3-methylidenechroman-4-ones, which were then tested for their possible cytotoxic activity against two leukemia cell lines, HL-60 and NALM-6, and against MCF-7 breast cancer cell line. All new compounds (14a–o) were highly cytotoxic for the leukemic cells and showed a moderate or weak effect on MCF-7 cells. Analog 14d exhibited the highest growth inhibitory activity and was more potent than carboplatin against HL-60 (IC₅₀ = 1.46 ± 0.16 μM) and NALM-6 (IC₅₀ = 0.50 ± 0.05 μM) cells. Further tests showed that 14d induced apoptosis in NALM-6 cells, which was mediated mostly through the extrinsic pathway.

Full text of DOI:10.3390/molecules24101868

molecules
Article
Synthesis and Cytotoxic Evaluation
of 3-Methylidenechroman-4-ones
Jacek Ke˛dzia 1, Tomasz Bartosik 1, Joanna Drogosz 2, Anna Janecka 2, Urszula Krajewska 3
and Tomasz Janecki 1,
*
1
˙
Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Łódz´, Poland;
jacek.kedzia@p.lodz.pl (J.K.); tomasz.bartosik92@gmail.com (T.B.)
2
3
Department of Biomolecular Chemistry, Medical University of Ł
ó
dz´, Mazowiecka 6/8, 92-215 Ł
ó
dz´, Poland;
joanna.drogosz@studumed.lodz.pl (J.D.); anna.janecka@umed.lodz.pl (A.J.)
Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Faculty of Pharmacy,
Medical University of Łódz´, Muszyn´skiego 1, 90-151 Łódz´, Poland; ukrajewska@tlen.pl
Correspondence: tomasz.janecki@p.lodz.pl; Tel.: +48-426313220
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Carla Boga and Gabriele Micheletti
Received: 26 April 2019; Accepted: 11 May 2019; Published: 15 May 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Inthesearch for newanticancer agents, alibraryof variouslysubstituted 3-methylidenechroman-
4-ones was synthesized using Horner–Wadsworth–Emmons methodology. Acylation of diethyl
methylphosphonate with selected ethyl salicylates furnished 3-diethoxyphosphorylchromen-4-ones
which were next used as Michael acceptors in the reaction with various Grignard reagents. The adducts
were obtained as the mixtures of trans and cis diastereoisomers along with a small amount of enol
forms. Their relative configuration and preferred conformation were established by NMR analysis.
The adducts turned up to be effective Horner–Wadsworth–Emmons reagents giving 2-substituted
3-methylidenechroman-4-ones, which were then tested for their possible cytotoxic activity against
two leukemia cell lines, HL-60 and NALM-6, and against MCF-7 breast cancer cell line. All new
compounds (14a
effect on MCF-7 cells. Analog 14d exhibited the highest growth inhibitory activity and was more
potent than carboplatin against HL-60 (IC50 = 1.46 0.16 M) and NALM-6 (IC50 = 0.50 0.05 M)
–o) were highly cytotoxic for the leukemic cells and showed a moderate or weak
±
µ
±
µ
cells. Further tests showed that 14d induced apoptosis in NALM-6 cells, which was mediated mostly
through the extrinsic pathway.
Keywords: 3-methylidenechroman-4-ones; Michael addition; Horner–Wadsworth–Emmons
olefination; cancer cell lines; apoptosis
1. Introduction
Chroman-4-one skeleton
which possess many desirable biological activities including anticancer, antibacterial, and antioxidant
properties [ ]. One relatively small subgroup of flavonoids are homoisoflavonoids which are
characterized by the presence of arylmethyl or arylidene group in position 3 (Figure 1). A special
place within this group belongs to 3-arylidenechroman-4-ones which were found in many plants.
For example, Bonducellin was isolated from Caesalpina bonducella [ ] and Eucomin from Eucomis
1 is a core structure for a large group of plant metabolites called flavonoids,
1,
2
2–4
4
5
3
6
bicolor BAK (Liliaceae) [4].
Both natural and synthetic 3-arylidenechroman-4-ones
are potent and selective monoamine oxidase-B (MAO-B) inhibitors [
4
display valuable biological activities. They
] and possess anti-cholinesterase
], what makes them good candidates for the treatment of various neurological diseases
such as Alzheimer’s or Parkinson’s disease. Furthermore, they show significant cytotoxicity for several
cancer cell lines [ 10] and are cytochrome P450 aromatase inhibitors [11] being used for the treatment of
5,6
activity [7,8
9,
Molecules 2019, 24, 1868; doi:10.3390/molecules24101868
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1868
2 of 14
advanced breast cancer. Also, their antifungal [12
activity was reported.
,13], antioxidant [14,15], and anti-inflammatory [16]
Figure 1. Structure and representative examples of homoisoflavonoids.
On the other hand, 3-methylidenechroman-4-ones
3-arylidenechroman-4-ones , have not been found in nature. Nevertheless, several syntheses of these
compounds were reported. 2-Aryl-3-methylidenechroman-4-ones were obtained by Mannich reaction
with 2-arylchroman-4-ones [17 18] and 2-alkyl(aryl)-3-methylidenechroman-4-ones were prepared by
palladium-catalyzed tandem carbonylation-allene insertion reaction [19 20]. 3-Methylidenechroman-4-
one was prepared by direct -methylidenation mediated by diisopropylammonium trifluoroacetate [21
7, which are structurally closely related to
4
,
,
α
]
or by dehydration of 3-hydroxymethylchromen-4-one in the presence of methanesulfonylchloride [22].
Finally, 2-alkoxycarbonylmethyl-3-methylidenechroman-4-ones were obtained from enol silyl ethers of
the corresponding chroman-4-ones [23,24]. Unfortunately, most of these methods have a very limited
scope and/or are inefficient. For example, yields of the Mannich reactions were very low (3–22%) [17
]
or were not given because the obtained, crude 3-methylidenechroman-4-ones were used in further
transformations [18]. On the other hand, palladium-catalyzed insertion reactions were more effective
(23–90% yield) but required not readily available allenes as substrates. In turn the scope of direct
α
-methylidenation, dehydration of 3-hydroxymethylchromen-4-one or use the enol silyl ethers was
limited to a single example in each case.
In contrast to 3-arylidenechroman-4-ones
4
, biological activity of 3-methylidenechroman-4-ones
7
is poorly recognized. There is only one report describing their significant bacteriostatic activity
against Gram-positive microorganisms [17]. However, an exo-cyclic methylidene bond conjugated
with a carbonyl group present in 3-methylidenechroman-4-ones is a structural motif found also
in a large number of natural products, such as -methylidene- - and -lactones [25 26] which react
by the Michael-type addition with various bionucleophiles, disrupting key biological processes
and are considered promising anticancer agents [27 28]. Consequently, we reasoned that also
7
α
γ
δ
,
,
3-methylidenechroman-4-ones 7 might have considerable cytotoxic activity.
In this report, we present a new, general synthetic method for obtaining variously
substituted 3-methylidenechroman-4-ones 14, based on the (well-recognized in our laboratory)
Horner–Wadsworth–Emmons approach for the construction of exo-methylidene bond [29–31].
All obtained 3-methylidenechroman-4-ones 14 were evaluated in terms of their cytotoxic activity against
three human cancer cell lines: promyelocytic leukemia HL-60, NALM-6, and breast adenocarcinoma
cell line MCF-7. The most cytotoxic compound, 14d was selected for further experiments and its effect
on the induction of apoptosis was investigated.

Molecules 2019, 24, 1868
3 of 14
2. Results and Discussion
2.1. Chemistry
The first step was the synthesis of 3-diethoxyphosphorylchromen-4-ones 12a
–
c, which are crucial
intermediates in our methodology. Literature search revealed that there is no efficient method
for obtaining 2-unsubstituted 3-phosphorylchromen-4-ones. In the only report we have found,
3-diethoxyphosphorylchromen-4-one was formed in 5% yield, as a side product in free radical
phosphorylation of chromen-4-one [32]. Therefore, we worked out a two-step procedure, which starts
with the reaction of commercially available ethyl salicylates 8a–c with diethyl methylphosphonate 9
in the presence of three equivalents of LDA (Scheme 1). Using this stoichiometry, protection of the
hydroxyl group is avoided and we believe this is the major improvement over the reported acylation of
dimethyl methylphosphonate using benzyl protected methyl salicylate [33]. The standard work-up and
column chromatography purification gave 2-(2-hydroxyphenyl)-2-oxoethylphoshonates 10a
–c in good
yields (Table 1). In the next step, reaction between phophonates 10a and dimethylformamide dimethyl
–c
acetal 11 gave, after purification by column chromatography, 3-diethoxyphosphorylchromen-4-ones
12a–c in high yields (Table 1 and Supplementary Materials).
Scheme 1. Synthesis of 3-diethoxyphosphorylchromen-4-ones 12a–c.
Table 1. Yields of phosphonates 10a-c and 3-diethoxyphosphorylchromen-4-ones 12a–c.
R1,R1
R2
10 Yield [%] 1
12 Yield [%] 1
Compound
a
b
c
H,H
H,H
H
Me
H
80
75
80
92
89
78
CH=CH-CH=CH
1
Yield of pure, isolated product, based on 8 or 10, respectively.
With 3-diethoxyphosphorylchromen-4-ones 12a
various Grignard reagents (Scheme 2). In all cases, after standard work-up, we received adducts
13a , which were purified by column chromatography with yields given in Table 2. Interestingly,
examination of the 1H, 13C and 31P NMR spectra revealed that all adducts 13 were formed as mixtures of
–c in hand, we performed their reactions with
–
o
trans and cis diastereoisomers (trans- or cis-13a–o), along with small amount of enol form (enol-13a–o),
with trans diastereoisomers strongly predominating. Ratios, determined from the 31P NMR spectra of
the crude reaction mixtures, are given in Table 2.

Molecules 2019, 24, 1868
4 of 14
Scheme 2. Synthesis of Michael adducts 13a–o.
Table 2. Adducts 13a-o and methylidenechroman-4-ones 14a–o obtained.
13
R1,R1
14 Yield [%] 2
R2
R3
Compound
trans/cis/enol 1
Yield [%] 2
a
b
c
d
e
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H,H
H
H
H
H
H
Me
Me
Me
Me
Me
H
Me
Et
n-Bu
iPr
Ph
Me
Et
n-Bu
iPr
Ph
Me
Et
n-Bu
iPr
Ph
67/30/3
71/25/4
76/20/4
71/26/3
81/10/9
73/24/3
75/22/3
72/25/3
72/26/2
83/11/6
79/14/7
89/4/7
84
68
85
66
64
74
76
83
70
84
81
64
53
66
76
68
71
59
53
84
64
59
52
53
59
66
67
60
48
70
f
g
h
i
j
k
l
m
n
o
1
H,H
H,H
CH=CH-CH=CH
CH=CH-CH=CH
CH=CH-CH=CH
CH=CH-CH=CH
CH=CH-CH=CH
H
H
H
H
73/16/11
81/15/4
65/6/29
2
Ratios taken from 31P NMR spectra of the crude mixtures. Yield of pure, isolated product, based on 12 or
13, respectively.
Careful analysis of the NMR spectra showed also that both trans- and cis-13a–o exist in the
half-chair conformation and diethoxyphosphoryl group occupies the axial position (Figure 2). Simple
application of Karplus correlation between corresponding dihedral angles and coupling constants
3
3
1
3JH2-H3, JH2-P and JC(R3)-P, determined from the H and 13C NMR spectra of trans- and cis-13a
–o
shows full agreement with the proposed configurations and conformations. Corresponding coupling
constants are given in Figure 2. Similar half-chair conformation with diethoxyphosphoryl group in
axial position was reported for 3-diethoxyphosphorylchroman-2-ones [34,35].
Figure 2. Half-chair conformation of trans- and cis-13a–o
and characteristic 3JH2-H3, 3JH2-P and 3JC(R3)-P
coupling constants.
On the other hand, 1H NMR spectra of enols-13a
–
o
revealed very characteristic doublets
4
with coupling constant JP-H ~ 1 Hz and chemical shift in the range of 11–12 ppm, which can
be assigned to the proton of the hydroxyl group. Coupling between this proton and phosphorus
indicates the presence of resonance-assisted hydrogen bond (RAHB) [36] and can be visualized by
resonance structures shown in Figure 3. Recently, we have reported on the existence of RAHB in

Molecules 2019, 24, 1868
5 of 14
3-(dimenthoxyphosphoryl)-2-phenyl-1,2-dihydroquinolin-4-ol [37], what was the first example of this
phenomenon in organophosphorus compounds. Now, we can confirm the existence of RAHB also in
3-diethoxyphosphoryl-2H-chromen-4-oles.
Figure 3. Two main resonance structures involved in the resonance-assisted hydrogen bond (RAHB) in
3-diethoxyphosphoryl-2H-chromen-4-oles.
Finally, all adducts 13a-o were transformed into 3-methylidenechroman-4-ones 14a–o performing
Horner–Wadsworth–Emmons olefination of formaldehyde. The best results were obtained with
K2CO3 used as a base and formalin as a source of formaldehyde (Scheme 3). Standard work-up and
purification by column chromatography furnished 3-methylidenechroman-4-ones 14a–o in moderate
to good yields (Table 2).
Scheme 3. Synthesis of 3-methylidenechroman-4-ones 14a–o.
2.2. Biology
2.2.1. In Vitro Cytotoxicity of New Analogs Against Three Cancer Cell Lines
All obtained 3-methylidenechroman-4-ones 14a–o were evaluated for their possible cytotoxic activity
against three human cancer cell lines: leukemia HL-60 and NALM-6 and breast adenocarcinoma MCF-7
using the MTT assay (after 48 h incubation) (Table 3). Carboplatin served as a reference compound.
Analysis of the structure–activity relationship revealed that chromanones 14a–j were, in general,
more potent than benzochromanones 14k
–
o, containing additional benzene ring ortho-fused with a
chromanone skeleton. All compounds (14a
–
o
) were much more cytotoxic for leukemia cells than for
the solid tumor MCF-7 cells. In both series of chromanones, 14a–e –j
(R2 = H) and 14f (R2 = Me), the
most potent compounds against leukemic HL-60 and NALM-6 cells were these containing an i-propyl
substituent in position 2, i.e., 14d and 14i, respectively. For HL-60 cells only 14d was more cytotoxic
than the reference carboplatin. The highest cytotoxicity was observed against NALM-6 cells, with three
analogs 14b, 14d, and 14i exhibiting lower half maximal inhibitory concentration values (IC50) than
carboplatin. Analog 14d was the most cytotoxic chromanone for NALM-6 cells (IC50 of 0.5 ± 0.05 µM
)
and was selected for further investigation of its potential antineoplastic properties.

Molecules 2019, 24, 1868
6 of 14
Table 3. Tumor cell growth inhibitory activity of 14a–o on three cancer cell lines.
O
R1
R1
O
R3
R2
14
IC50 [µM] 1
R1, R1
R2
R3
14
HL-60
NALM-6
MCF-7
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H
H
H
H
H
Me
Me
Me
Me
Me
H
Me
Et
n-Bu
iPr
Ph
Me
Et
n-Bu
iPr
Ph
Me
Et
n-Bu
iPr
5.91 ± 0.32
5.24 ± 0.52
8.79 ± 0.81
1.46 ± 0.16
23.86 ± 2.30
7.52 ± 0.81
20.87 ± 1.73
32.16 ± 3.17
3.45 ± 0.34
30.51 ± 3.62
6.96 ± 0.42
47.00 ± 4.11
8.64 ± 0.53
10.47 ± 2.04
49.96 ± 3.89
2.9 ± 0.1
2.13 ± 0.04
0.60 ± 0.02
4.23 ± 0.46
0.50 ± 0.05
5,76 ± 0.23
3.28 ± 0.35
4.83 ± 0.45
6.36 ± 0.36
0.58 ± 0.05
6.09 ± 0.61
4.31 ± 0.36
10.91 ± 2.8
6.00 ± 0.23
5.52 ± 0.25
6.59 ± 0.41
0.7 ± 0.3
9.70 ± 0.80
12.50 ± 0.71
16.00 ± 0.20
19.40 ± 0.80
17,10 ± 0.30
10.50 ± 0.34
11.60 ± 0.07
13.50 ± 0.60
8.48 ± 0.93
30.00 ± 1.90
13.50 ± 1.00
34.60 ± 4.50
17.40 ± 2.50
15.70 ± 0.70
71.0 ± 7.50
3.8 ± 0.45
H, H
CH=CH-CH=CH
CH=CH-CH=CH
CH=CH-CH=CH
CH=CH-CH=CH
CH=CH-CH=CH
H
H
H
H
Ph
Carboplatin
1
Compound concentration required to inhibit metabolic activity by 50%. The cells were incubated with the analogs
for 48 h. Values are expressed as mean SEM from the concentration-response curves of at least three experiments
using a nonlinear estimation (quasi-Newton algorithm) method.
±
2.2.2. Apoptotic Cell Death Determination
It is now well documented that most anticancer drugs induce apoptosis. One of the main
characteristics of apoptosis, phosphatidylserine (PS) translocation to the outer surface of the cellular
membrane, was investigated by double-staining with Annexin V and propidium iodide (PI). Annexin V
is a protein exerting high affinity to PS exposed on the outer surface of the plasma membrane, enabling
detection of even an early stage apoptosis. The late stage of apoptosis is characterized by loss of
membrane integrity allowing permeation of a dye such as PI into the cells [38
,
39]. Treatment of
M (2 IC50)
NALM-6 cells for 24 h (Figure 4A) with the selected analog 14d at 1.25 M (IC50) and 2.5
µ
µ
concentrations led to the increase of Annexin V and PI-positive cells from 2.2% to 29.4% and 91.4%,
respectively (Figure 4B,C), showing that 14d induced the late stage of apoptosis in NALM-6 cells.

Molecules 2019, 24, 1868
7 of 14
Figure 4. Effect of 14d on induction of apoptosis in NALM-6 cells. (
on NALM-6 cells after 24 h incubation; ( ) Quantitative analysis of apoptosis by flow cytometry. The
data are presented as mean SEM of three independent experiments. Statistical significance was
determined using one-way ANOVA and a post-hoc multiple comparison Student–Newman–Keuls test.
**** p < 0.0001; *** p < 0.001; ** p < 0.01; ns—not statistically significant. ( ) Representative results of
A) The cytotoxic activity of 14d
B
±
C
cell apoptosis obtained by Annexin V and PI staining using flow cytometry in NALM-6 cells untreated
(control) or treated with 14d at IC50 and 2 IC50 concentrations for 24 h.
Apoptosis occurs when caspases, which have proteolytic activity, cleave specific substrates,
causing cell death. Depending on the initiator caspase involved in this process, the apoptosis may
be mediated by the intrinsic (caspase 9) or extrinsic (caspase 8) pathway [40]. To investigate which
caspases were involved in the apoptosis inducted by analog 14d in NALM-6 cells, the cells were treated
with 14d at 1.25 µM and 2.5 µM concentrations for 6 h. Then, the activity of executioner caspase 3 and
initiator caspases 8 and 9 was quantified using fluorogenic indicators. Results presented in Figure 5
indicate that the levels of caspase 3, 8 and 9 were significantly increased: 2.7- and 3.7-fold for caspase 3,
4.9- and 11.3-fold for caspase 8, and 1.25- and 5.7-fold for caspase 9 after treatment of the cells with
1.25 µM and 2.5 µM concentration of 14d, respectively. Activation of the extrinsic pathway was more
prominent than the intrinsic pathway.
Presented results indicate that compound 14d is a potent cytotoxic agent that significantly inhibits
metabolic activity of NALM-6 cells with IC50 value as low as 0.5 µM. Analog 14d also promotes
apoptosis in the investigated cell line, which is mediated by the extrinsic and in a much lesser extend
intrinsic pathway.

Molecules 2019, 24, 1868
8 of 14
**
***
C ontrol
14
12
10
8
14d IC 50
14d 2IC 50
**
**
6
*
*
4
*
2
0
3
8
9
-
-
-
e
e
e
s
s
s
a
a
a
p
p
p
s
s
s
a
a
a
c
c
c
Figure 5. Activity of caspase 3, 8 and 9 in HL-60 cells after 6 h treatment with analog 14d at 1.25
µM
(IC50) and 2.5 M (2IC50) concentrations. Results are expressed as mean SEM of triplicate experiment.
µ
±
Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison
Student–Newman–Keuls test; *** p < 0.001; ** p < 0.01; * p < 0.05.
3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
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 with tetramethylsilane used
as an internal and 85% H3PO4 as an external standard. 31P NMR spectra were recorded using broadband
proton decoupling. IR spectra were recorded on a Bruker Alpha ATR spectrophotometer. Melting
points were determined in open capillaries and are uncorrected. Optical rotations were measured
on a Perkin-Elmer 241 polarimeter. The [
α
]
values are given in deg
·
cm2
·
g−1 and concentration c
D
in g
·
(100 mL)−1. Column chromatography was performed on silica gel 60 (230–400 mesh) (Aldrich,
Steinheim, Germany). Thin-layer chromatography was performed on the pre-coated TLC sheets of
silica gel 60 F254 (Aldrich, Steinheim, Germany). The purity of the synthesized compounds was
confirmed by the combustion elemental analyses (CHN, elemental analyzer EuroVector 3018, Elementar
Analysensysteme GmbH (Langenselbold, Germany). MS spectra were recorded on Waters 2695-Waters
ZQ 2000 LC/MS apparatus (Waters Corporation, Milford, MA, USA). All reagents and starting materials
were purchased from commercial vendors and used without further purification. Organic solvents were
dried and distilled prior to use. Standard syringe techniques were used for transferring dry solvents.
3.1.2. General Procedure for the Synthesis of 2-Substituted 3-Metylidenechroman-4-ones 14a–o.
To the vigorously stirred solution of 2-substituted 3-diethoxychroman-4-on 13a–o (0.15 mmol)
in THF (1.5 mL), formaldehyde (36–38% solution in water, 0.125 mL, ca. 1.50 mmol) was added at
0 ◦C, followed by addition of K2CO3 (41 mg, 0.30 mmol) in water (0.4 mL). The resulting mixture was
stirred vigorously at 0 ◦C for 3 h. Next Et2O (5 mL) was added and layers were separated. The water
fraction was washed with Et2O (5 mL). Organic fractions were combined, washed with brine (5 mL)
and dried over MgSO4. The solvents were evaporated under reduced pressure and the resulting crude
product was then purified by column chromatography (eluent CH2Cl2).

Molecules 2019, 24, 1868
9 of 14
2-Methyl-3-methylidenechroman-4-one (14a) (17.7 mg, 68%). Colourless oil. 1H NMR (700 MHz,
Chloroform-d) 1.63 (d, J = 6.4 Hz, 3H), 5.03–5.14 (m, 1H), 5.55 (dd, J = 2.0, 0.8 Hz, 1H), 6.32 (dd, J = 1.6,
0.8 Hz, 1H), 6.95 (dd, J = 8.3, 1.0 Hz, 1H), 7.03 (ddd, J = 8.1, 7.2, 1.1 Hz, 1H), 7.47 (ddd, J = 8.6, 7.1,
δ
1.8 Hz, 1H), 7.97 (dd, J = 7.9, 1.8 Hz, 1H). 13C NMR (176 MHz, Chloroform-d)
δ 19.03, 76.36, 118.30,
121.44, 121.59, 121.82, 128.00, 136.14, 143.68, 161.30, 182.66. ESI-MS [M + H]+ = 175.2. Anal. Calcd for
C11H10O2: C, 75.84%; H, 5.79%. Found: C, 75.61%, H, 5.71%.
2-Ethyl-3-methylidenechroman-4-one (14b) (20.1 mg, 71%). Colourless oil. 1H NMR (700 MHz,
Chloroform-d)
δ 1.04 (t, J = 7.3 Hz, 3H), 1.75–1.89 (m, 1H), 1.91–2.03 (m, 1H), 4.90 (t, J = 7.0 Hz,
1H), 5.50 (s, 1H), 6.33 (s, 1H), 6.96 (d, J = 8.4 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 7.47 (s, 1H), 7.95
(d, J = 7.9 Hz, 1H). 13C NMR (176 MHz, Chloroform-d)
δ 9.83, 26.65, 81.89, 118.47, 121.41, 121.66, 122.33,
127.84, 136.21, 142.36, 160.77, 182.49. ESI-MS [M + H]+ = 189.3. Anal. Calcd for C12H12O2: C, 76.57%;
H, 6.43%. Found: C, 76.41%, H, 6.51%.
2-Butyl-3-methylidenechroman-4-one (14c) (19.1 mg, 59%). Colourless oil. 1H NMR (700 MHz,
Chloroform-d)
δ 0.91 (t, J = 7.3 Hz, 3H), 1.31–1.45 (m, 3H), 1.47–1.55 (m, 1H), 1.71–1.80 (m, 1H),
1.89–2.00 (m, 1H), 4.86–5.10 (m, 1H), 5.50 (t, J = 1.2 Hz, 1H), 6.32 (t, J = 1.0 Hz, 1H), 6.95 (dd, J = 8.4,
1.0 Hz, 1H), 7.02 (ddd, J = 8.1, 7.2, 1.1 Hz, 1H), 7.47 (ddd, J = 8.6, 7.1, 1.8 Hz, 1H), 7.96 (dd, J = 7.8,
1.8 Hz, 1H).13C NMR (176 MHz, Chloroform-d)
δ 14.09, 22.48, 27.50, 33.18, 80.64, 118.48, 121.43, 121.66,
122.17, 127.85, 136.22, 142.65, 160.78, 182.56. ESI-MS [M + H]+ = 217.4. Anal. Calcd for C14H16O2: C,
77.75%; H, 7.46%. Found: C, 77.52%, H, 7.25%.
2-Isopropyl-3-methylidenechroman-4-one (14d) (16.1 mg, 53%). Colourless oil. 1H NMR (700 MHz,
Chloroform-d)
δ 0.90 (d, J = 6.8 Hz, 3H), 1.04 (d, J = 6.6 Hz, 3H), 1.96–2.16 (m, 1H), 4.62 (dt, J = 8.5,
1.1 Hz, 1H), 5.46 (t, J = 1.2 Hz, 1H), 6.36 (t, J = 1.0 Hz, 1H), 6.95 (ddd, J = 8.3, 1.1, 0.5 Hz, 1H), 7.00
(ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 7.47 (ddd, J = 8.3, 7.1, 1.8 Hz, 1H), 7.93 (ddd, J = 7.9, 1.8, 0.5 Hz, 1H).
13C NMR (176 MHz, Chloroform-d)
δ 18.51, 18.83, 30.78, 86.66, 118.41, 121.49, 121.57, 123.80, 127.66,
136.31, 141.26, 160.53, 182.44. ESI-MS [M + H]+ = 203.0. Anal. Calcd for C13H14O2: C, 77.20%; H,
6.98%. Found: C, 77.42%, H, 7.05%.
3-Methylidene-2-phenylchroman-4-one (14e) (29.8 mg, 84%). Yelowish oil. 1H NMR (700 MHz,
Chloroform-d)
δ 5.16–5.23 (m, 1H), 6.02 (d, J = 1.7 Hz, 1H), 6.44 (t, J = 1.2 Hz, 1H), 7.35–7.39
(m, 1H), 7.39–7.45 (m, 4H), 7.50 (ddd, J = 8.7, 7.2, 1.8 Hz, 1H), 7.99 (dd, J = 7.9, 1.7 Hz, 1H). 13C NMR
(176 MHz, Chloroform-d) δ 82.49, 118.47, 121.76, 122.13, 125.09, 127.58, 128.02, 128.78, 128.87, 136.30,
137.27, 142.76, 161.14, 182.22. ESI-MS [M + H]+ = 237.4. Anal. Calcd for C16H12O2: C, 81.34%; H,
5.12%. Found: C, 81.42%, H, 5.23%.
2,8-Dimethyl-3-methylidenechroman-4-one (14f) (18.1 mg, 64%). Yelowish oil. 1H NMR (700 MHz,
Chloroform-d)
δ 1.64 (d, J = 6.5 Hz, 3H), 2.23 (s, 3H), 5.10 (qt, J = 6.4, 1.8 Hz, 1H), 5.54 (dd, J = 2.0,
0.9 Hz, 1H), 6.31 (dd, J = 1.6, 0.9 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H), 7.33 (ddd, J = 7.2, 1.8, 1.0 Hz, 1H), 7.82
(dd, J = 7.9, 1.7 Hz, 1H). 13C NMR (176 MHz, Chloroform-d)
δ
15.72, 19.13, 76.19, 121.08, 121.21
×
2,
125.56, 127.50, 136.91, 143.77, 159.53, 183.04. ESI-MS [M + H]+ = 189.0. Anal. Calcd for C12H12O2: C,
76.57%; H, 6.43%. Found: C, 76.42%, H, 6.55%.
1
2-Ethyl-8-methyl-3-methylidenechroman-4-one (14g) (17.9 mg, 59%). Yelowish oil. H NMR (700 MHz,
Chloroform-d)
4.76 – 5.05 (m, 1H), 5.50 (dd, J = 1.5, 1.0 Hz, 1H), 6.32 (t, J = 1.1 Hz, 1H), 6.92 (t, J = 7.5 Hz, 1H), 7.34
(ddd, J = 7.2, 1.8, 0.9 Hz, 1H), 7.81 (ddd, J = 7.9, 1.8, 0.7 Hz, 1H). 13C NMR (176 MHz, Chloroform-d)
δ 1.07 (t, J = 7.4 Hz, 3H), 1.73 – 1.86 (m, 1H), 1.90–2.03 (m, 1H), 2.25 (t, J = 0.7 Hz, 3H),
δ
10.09, 15.69, 26.69, 81.76, 121.08, 121.11, 121.88, 125.45, 127.66, 136.99, 142.60, 158.94, 182.87. ESI-MS
[M + H]+ = 203.0. Anal. Calcd for C13H14O2: C, 77.20%; H, 6.98%. Found: C, 77.38%, H, 7.09%.
1
2-Butyl-8-methyl-3-methylidenechroman-4-one (14h) (20.0 mg, 52%). Yelowish oil. H NMR (700 MHz,
Chloroform-d)
δ 0.92 (t, J = 7.3 Hz, 3H), 1.29–1.49 (m, 3H), 1.49–1.58 (m, 1H), 1.71–1.80 (m, 1H), 1.89–2.01
(m, 1H), 2.24 (s, 3H), 4.98 (ddt, J = 9.0, 5.1, 1.4 Hz, 1H), 5.50 (t, J = 1.2 Hz, 1H), 6.30 (d, J = 1.2 Hz,

Molecules 2019, 24, 1868
10 of 14
1H), 6.92 (t, J = 7.6 Hz, 1H), 7.31–7.39 (m, 1H), 7.81 (dd, J = 7.8, 1.7 Hz, 1H). 13C NMR (176 MHz,
Chloroform-d)
δ 14.12, 15.73, 22.44, 27.70, 33.12, 80.39, 121.12, 121.73, 125.46, 127.66, 136.99, 142.85,
158.99, 182.96. ESI-MS [M + H]+ = 231.0. Anal. Calcd for C15H18O2: C, 78.23%; H, 7.88%. Found: C,
78.44%, H, 8.05%.
1
2-Isopropyl-8-methyl-3-methylidenechroman-4-one (14i) (17.2 mg, 53%). Yelowish oil. H NMR (700 MHz,
Chloroform-d)
(dd, J = 8.3, 1.3 Hz, 1H), 5.46 (d, J = 1.1 Hz, 1H), 6.35 (t, J = 1.1 Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 7.34
(ddd, J = 7.2, 1.8, 0.9 Hz, 1H), 7.79 (dd, J = 7.9, 1.7 Hz, 1H). 13C NMR (176 MHz, Chloroform-d)
15.71,
δ 0.92 (d, J = 6.7 Hz, 3H), 1.05 (d, J = 6.6 Hz, 3H), 1.95–2.12 (m, 1H), 2.26 (s, 3H), 4.66
δ
18.72, 19.03, 30.58, 86.60, 120.97, 121.17, 123.42, 125.32, 127.48, 137.14, 141.40, 158.69, 182.86. ESI-MS
[M + H]+ = 217.0. Anal. Calcd for C14H16O2: C, 77.75%; H, 7.46%. Found: C, 77.52%, H, 7.27%.
1
8-Methyl-3-methylidene-2-phenylchroman-4-one (14j) (22.1 mg, 59%). Yelowish oil. H NMR (700 MHz,
Chloroform-d)
δ 2.28 (d, J = 0.8 Hz, 3H), 5.23 (dd, J = 1.8, 1.1 Hz, 1H), 6.05 (t, J = 1.7 Hz, 1H), 6.42
(t, J = 1.3 Hz, 1H), 6.95 (t, J = 7.6 Hz, 1H), 7.33 – 7.38 (m, 2H), 7.38–7.45 (m, 4H), 7.83 (ddd, J = 7.9, 1.7,
0.7 Hz, 1H). 13C NMR (176 MHz, Chloroform-d)
δ 15.86, 82.14, 121.45, 121.55, 124.67, 125.61, 127.31,
127.62, 128.71, 128.74, 137.16, 137.57, 142.76, 159.29, 182.71. ESI-MS [M + H]+ = 251.0. Anal. Calcd for
C17H14O2: C, 81.58%; H, 5.64%. Found: C, 81.72%, H, 6.75%.
1
2-Methyl-3-methylidene-2,3-dihydro-4H-benzo[g]chromen-4-one (14k) (22.2 mg, 66%). Yelowish oil. H
NMR (700 MHz, Chloroform-d) δ 1.65 (dd, J = 6.5, 1.2 Hz, 4H), 5.08–5.19 (m, 1H), 5.61 (dt, J = 1.7, 1.1
Hz, 1H), 6.41 (dt, J = 1.5, 0.7 Hz, 1H), 7.33 (d, J = 1.5 Hz, 1H), 7.36 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 7.50
(ddt, J = 8.2, 6.8, 1.4 Hz, 1H), 7.70 (dd, J = 8.4, 1.5 Hz, 1H), 7.89 (dd, J = 8.2, 1.6 Hz, 1H), 8.58 (s, 1H).
13C NMR (176 MHz, Chloroform-d)
δ 19.26, 76.01, 113.14, 121.83, 122.30, 124.74, 126.68, 128.80, 129.23,
129.99, 130.14, 137.88, 143.99, 156.15, 183.18. ESI-MS [M + H]+ = 255.0. Anal. Calcd for C15H12O2: C,
80.34%; H, 5.39%. Found: C, 80.22%, H, 5.27%.
1
2-Ethyl-3-methylidene-2,3-dihydro-4H-benzo[g]chromen-4-one (14l) (23.9 mg, 67%). Yelowish oil. H NMR
(700 MHz, Chloroform-d) δ 1.08 (t, J = 7.4 Hz, 3H), 1.81 (dtd, J = 14.6, 7.5, 1.8 Hz, 1H), 1.97 (dtd, J = 14.2,
7.2, 1.2 Hz, 1H), 4.93 (ddt, J = 8.2, 5.6, 1.2 Hz, 1H), 5.57 (t, J = 1.1 Hz, 1H), 6.42 (d, J = 1.0 Hz, 1H), 7.34 (s,
1H), 7.36 (ddd, J = 8.2, 6.8, 1.2 Hz, 1H), 7.51 (ddd, J = 8.2, 6.7, 1.2 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.89
(d, J = 8.3 Hz, 1H), 8.57 (s, 1H). 13C NMR (176 MHz, Chloroform-d)
δ 9.97, 27.15, 81.74, 113.48, 122.54,
122.75, 124.83, 126.73, 128.89, 129.35, 130.10, 130.15, 138.11, 142.84, 155.70, 183.18. ESI-MS [M + H]+ =
239.2. Anal. Calcd for C16H14O2: C, 80.65%; H, 5.92%. Found: C, 80.43%, H, 6.05%.
2-Butyl-3-methylidene-2,3-dihydro-4H-benzo[g]chromen-4-one (14m) (24.0 mg, 60%). Yelowish oil. 1H
NMR (700 MHz, Chloroform-d) δ 1.36 (tdd, J = 15.8, 8.2, 4.4 Hz, 2H), 1.45 (dddd, J = 13.4, 9.6, 6.6, 4.0
Hz, 1H), 1.51 – 1.59 (m, 2H), 1.75 (ddt, J = 14.0, 10.7, 5.6 Hz, 1H), 1.92–2.00 (m, 1H), 5.00 (dd, J = 8.6, 5.6
Hz, 1H), 5.56 (d, J = 1.3 Hz, 1H), 6.40 (s, 1H), 7.34 (s, 1H), 7.36 (ddd, J = 8.1, 6.8, 1.1 Hz, 1H), 7.51 (ddd,
J = 8.2, 6.8, 1.2 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 8.58 (s, 1H). 13C NMR (176 MHz,
Chloroform-d) δ 14.11, 22.49, 27.62, 33.69, 80.46, 113.49, 122.55, 122.58, 124.83, 126.75, 128.89, 129.35,
130.11, 130.17, 138.13, 143.11, 155.72, 183.26. ESI-MS [M + H]+ = 267.2. Anal. Calcd for C18H18O2: C,
81.17%; H, 6.81%. Found: C, 81.32%, H, 6.95%.
1
2-Isopropyl-3-methylidene-2,3-dihydro-4H-benzo[g]chromen-4-one (14n) (18.2 mg, 48%). Yelowish oil. H
NMR (700 MHz, Chloroform-d) δ 0.91 (d, J = 6.7 Hz, 3H), 1.08 (d, J = 6.4 Hz, 3H), 1.97–2.06 (m, 1H),
4.62 (dd, J = 9.0, 0.9 Hz, 1H), 5.53 (t, J = 1.0 Hz, 1H), 6.45 (q, J = 0.9 Hz, 1H), 7.33 (s, 1H), 7.36 (ddt,
J = 7.7, 6.7, 1.0 Hz, 1H), 7.51 (ddt, J = 7.7, 6.8, 1.0 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 8.3 Hz,
1H), 8.56 (s, 1H). 13C NMR (176 MHz, Chloroform-d)
δ 18.64, 18.92, 31.11, 86.57, 113.41, 122.73, 124.26,
124.78, 126.70, 128.79, 129.34, 129.93, 130.16, 138.19, 141.67, 155.47, 183.19. ESI-MS [M + H]+ = 253.2.
Anal. Calcd for C17H16O2: C, 80.93%; H, 6.39%. Found: C, 81.02%, H, 6.29%.

Molecules 2019, 24, 1868
11 of 14
1
3-Methylidene-2-phenyl-2,3-dihydro-4H-benzo[g]chromen-4-one (14o) (30.1 mg, 70%). Yelowish oil. H
NMR (700 MHz, Chloroform-d) δ 5.32 (dd, J = 1.7, 0.9 Hz, 1H), 6.07 (d, J = 1.7 Hz, 1H), 6.54 (t, J = 1.2
Hz, 1H), 7.36 (ddd, J = 7.4, 3.4, 2.1 Hz, 2H), 7.38 – 7.41 (m, 4H), 7.44–7.48 (m, 2H), 7.51 (ddd, J = 8.2,
6.7, 1.2 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 8.59 (s, 1H). 13C NMR (176 MHz,
Chloroform-d)
δ
82.20, 113.65, 122.64, 125.00, 125.42, 126.88, 127.62, 128.82 2, 129.05, 129.44, 130.16,
×
130.36, 137.62, 138.05, 143.06, 156.14, 183.00. ESI-MS [M + H]+ = 287.2. Anal. Calcd for C20H14O2: C,
83.90%; H, 4.93%. Found: C, 83.72%, H, 5.05%.
3.2. Biology
3.2.1. Cell Lines
HL-60, NALM-6, MCF-7 cell lines were purchased from the European Collection of Cell Cultures
(ECACC). HL-60 and NALM-6 cells were cultured in RPMI 1640 Glutamax medium (Gibco/Life
Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum, penicillin and
streptomycin. MCF-7 cells were maintained in EMEM growth medium (Sigma-Aldrich, St. Louis, MO,
USA), supplemented with 2 mM glutamine, 10% fetal bovine serum, 1% NEAA and gentamycin.
3.2.2. In Vitro Cytotoxicity Assay
The MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was used to
investigate the cytotoxicity of new analogs in HL-60, NALM-6 and MCF-7 cell lines. Briefly, cells
were seeded in a 24-well plate at a concentration of 8
(20 h; 37 ◦C), cells were treated with various concentrations of the analogs for 48 h. Then, cells were
incubated for 1.5 h with MTT solution (100 L/well; 5 mg/mL of PBS). The plates were centrifuged (3000
×
104 cells/mL. Following initial incubation
µ
rpm, 5 min.) and the supernatant was discarded. The formazan product was dissolved by addition
of DMSO (1 mL/well). The absorbance was measured using FlexStation 3 Multi-Mode Microplate
Reader (Molecular Devices, LLC) at 560 nm. Assay was performed in triplicate. Cell viability rate was
calculated by dividing mean sample absorbance by mean control absorbance.
3.2.3. Annexin V and Propidium Iodide Assay
Apoptotic cell death was determined using FITC Annexin V Apoptosis Detection Kit I (BD
Biosciences, San Jose, CA, USA) in accordance with the manufacturer’s protocol. NALM-6 cells were
seeded in 6-well plates at a density of 4.0
treated with 14d at IC50 and 2 IC50 concentrations (1.25
×
105 cells/mL in 2 mL of cell culture medium. Cells were
µ
M and 2.5 µ
M, respectively) for 24 h, at 37 ◦C.
Untreated cells were used as a control. Then, cells were washed with PBS, resuspended in the binding
buffer and stained with FITC Annexin V and propidium iodide (PI), followed by incubation in the
dark at RT for 15 min. Finally, flow cytometry analysis of the cells was performed using CytoFLEX
(Beckman Coulter, Inc., Brea, CA, USA). Data were analyzed using CytExpert Software (2.3, Beckman
Coulter, Inc, Brea, CA, USA).
3.2.4. Caspase 3, Caspase 8, and Caspase 9 Activity
Activity of the key caspases involved in apoptosis was simultaneously quantified using a
fluorometric Caspase 3, Caspase 8 and Caspase 9 Multiplex Activity Assay Kit (Abcam, Cambridge,
UK), according to the manufacturer’s protocol. Briefly, NALM-6 cells were seeded in a 96-well plate
at a density of 2.0
800 rpm for 2 min. and the cells were treated with 14d at 1.25
×
105 cells/90
µ
l of cell culture medium per well. The plates were centrifuged at
µ
M and 2.5 M concentrations. Medium
µ
without cells and untreated cells were used as controls. The cell plate was incubated for 6 h, 37 ◦C, 5%
CO2. Caspase-9, -3, and -8 substrates were added to each well and the cell plate was incubated for
60 min. at RT, protected from light. The plate was centrifuged at 800 rpm for 2 min., followed by a
fluorescence readout using FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, LLC) at
specific wavelengths. The assay was performed in triplicate; blank readings were subtracted from

Molecules 2019, 24, 1868
12 of 14
all measurements and the fold change of fluorescent intensity between control and the treated cells
was calculated.
4. Conclusions
A simple and efficient synthesis of 3-methylidenechroman-4-ones 14a
Horner–Wadsworth–Emmons methodology was described. Furthermore,
–
o
,
applying
relative
configuration of the crucial intermediates for this methodology, trans- or cis- 2-substituted
3-diethoxyphosphorylchroman-4-ones 13a , was elaborated using NMR analysis. The obtained
library of 3-methylidenechroman-4-ones 14a was evaluated for the anti-proliferative activity
–
o
–
o
against leukemia and breast cancer cell lines. Several members of this library were determined
to be capable of killing leukemia cells with improved activity compared to carboplatin used as
a reference compound, exhibiting IC50 values in the low micromolar range. The most potent
2-isopropyl-3-methylidenechroman-4-one 14d was then evaluated for its possible apoptotic activity
against NALM-6 cells. Compound 14d promoted apoptosis mediated by caspase 3/8 and in a much
lesser extend by caspase 3/9 induction, which indicated that mostly the extrinsic pathway was engaged
in the programmed cell death caused by this analog. These results show that compound 14d is a good
lead in a search for new anticancer agents.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/10/1868/s1,
General procedures and characterization data for diethyl (2-(2-hydroxyarylo)-2-oxoethyl)phosphonates (10a
–
c
),
diethoxyphosphorylchromen-4-ones (12a
–c) and 3-diethoxyphosphoryl-2-substituted chroman-4-ones (13a–o
).
Copies of 1H, 13C and 31P NMR spectra of all obtained compounds.
Author Contributions: Conceptualization, T.J., J.K., and A.J.; Data curation, J.K. and J.D.; Formal analysis, J.K.
and J.D.; Funding acquisition, T.J. and A.J.; Investigation, J.K., T.B., J.D., and U.K.; Methodology, T.J., J.K., J.D. and
A.J.; Project administration, T.J. and A.J.; Resources, J.K. and J.D.; Supervision, T.J. and A.J.; Validation, J.K. and
J.D.; Visualization, J.K. and J.D.; Writing—review and editing, T.J. and A.J.
Funding: This research was funded by the National Science Centre of Poland (project DEC-2012/07/B/ST5/02006)
and by a grant from the Medical University of Łódz´ No. 503/1-156-02/503-11-02.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study, in the collection, analyses or interpretation of data, in the writing of the manuscript, or in the decision to
publish the results.
References
1.
Nibbs, A.E.; Scheidt, K.A. Asymmetric methods for the synthesis of flavanones, chromanones, and
azaflavanones. Eur. J. Org. Chem. 2012, 449–462. [CrossRef] [PubMed]
2.
Huang, S.; Zhao, Y.; Zhou, X.; Wu, Y.; Wu, P.; Liu, T.; Yang, B.; Hu, Y.; Dong, X. Design, synthesis and
biological evaluation of 3-benzylideneflavanone derivatives as cytotoxic agents. Med. Chem. Res. 2012, 21,
4150–4157. [CrossRef]
3.
4.
5.
Sidwell, W.T.L.; Tamm, C. The homo-isoflavones II. Isolation and structure of 40-O-methyl-punctatin,
autumnalin and 3, 9-dihydro-autumnalin. Tetrahedron Lett. 1970, 475–478. [CrossRef]
Böhler, P.; Tamm, C. The homo-isoflavones, a new class of natural product. Isolation and structure of eucomin
and eucomol. Tetrahedron Lett. 1967, 8, 3479–3483. [CrossRef]
Desideri, N.; Bolasco, A.; Fioravanti, R.; Monaco, L.P.; Orallo, F.; Yañez, M.; Ortuso, F.; Alcaro, S.
Homoisoflavonoids: Natural scaffolds with potent and selective monoamine oxidase-B inhibition properties.
J. Med. Chem. 2011, 54, 2155–2164. [CrossRef]
6.
7.
Desideri, N.; Monaco, L.P.; Fioravanti, R.; Biava, M.; Yañez, M.; Alcaro, S.; Ortuso, F.
(E)-3-Heteroarylidenechroman-4-ones as potent and selective monoamine oxidase-B inhibitors. Eur. J.
Med. Chem. 2016, 117, 292–300. [CrossRef]
Pourshojaei, Y.; Gouranourimi, A.; Hekmat, S.; Asadipour, A.; Rahmani-Nezhad, S.; Moradi, A.; Nadri, H.;
Moghadam, F.H.; Emami, S.; Foroumadi, A.; et al. Design, synthesis and anticholinesterase activity of novel
benzylidenechroman-4-ones bearing cyclic amine side chain. Eur. J. Med. Chem. 2015, 97, 181–189. [CrossRef]
[PubMed]

Molecules 2019, 24, 1868
13 of 14
8.
9.
Li, Y.; Qiang, X.; Luo, L.; Yang, X.; Xiao, G.; Zheng, Y.; Cao, Z.; Sang, Z.; Su, F.; Deng, Y. Multitarget
drug design strategy against Alzheimer’s disease: Homoisoflavonoid mannich base derivatives serve
as acetylcholinesterase and monoamine oxidase B dual inhibitors with multifuncional properties.
Bioorg. Med. Chem. 2017, 25, 714–726. [CrossRef]
Perj
ési, P.; Das, U.; De Clercq, E.; Balzarini, J.; Kawase, M.; Sakagami, H.; Stables, J.P.; Lorand, T.;
Rozmer, Z.; Dimmock, J.R. Design, synthesis and antiproliferative activity of some 3-benzylidene-2,
3-dihydro-1-benzopyran-4-ones which display selective toxicity for malignant cells. Eur. J. Med. Chem. 2008
,
43, 839–845. [CrossRef]
10. Pordeli, P.; Nakhjiri, M.; Safavi, M.; Ardestani, S.K.; Foroumadi, A. Anticancer effects of synthetic
hexahydrobenzo[g]chromen-4-one derivatives on human breast cancer cell lines. Breast Cancer 2017
24, 299–311. [CrossRef]
,
11. Pouget, C.; Fagnere, C.; Basly, J.-P.; Habrioux, G.; Chulia, A.-J.; New aromatase inhibitors. Synthesis and
inhibitory activity of pyridinyl-Substituted flavanone derivatives. Bioorg. Med. Chem. Lett. 2002, 12,
1059–1061. [CrossRef]
12. Basavaiah, D.; Bakthadoss, M.; Pandiaraju, S. A new protocol for the syntheses of (E)-3-benzylidenechroman-
4-ones: A simple synthesis of the methyl ether of bonducellin. Chem. Commun. 1998, 16, 1639–1640.
[CrossRef]
13. AI Nakib, T.; Bezjak, V.; Meegan, M.J.; Chandy, R. Synthesis and antifungal activity of some
3-benzylidenechroman-4-ones, 3-benzylidenethiochroman-4-ones and 2-benzylidene-1-tetralones. Eur. J.
Med. Chem. 1990, 25, 455–462. [CrossRef]
14. Takao, K.; Yamashita, M.; Yashiro, A.; Sugita, Y. Synthesis and Biological Evaluation of 3-Benzylidene-4-
chromanone Derivatives as Free Radical Scavengers and α-Glucosidase Inhibitors. Chem. Pharm. Bull. 2016,
64, 1203–1207. [CrossRef] [PubMed]
15. Siddaiah, V.; Rao, C.V.; Venkateswarlu, S.; Krishnarajub, A.V.; Subbaraju, G.V. Synthesis, stereochemical
assignments, and biological activities of homoisoflavonoids. Bioorg. Med. Chem. 2006, 14, 2545–2551.
[CrossRef] [PubMed]
16. Hung, T.M.; Cao Van, T.; Nguyen Tien, D. Homoisoflavonoid derivatives from the roots of Ophiopogon
japonicus and their in vitro anti-inflammation activity. Bioorg. Med. Chem. Lett. 2010, 20, 2412–2416.
[CrossRef]
17. Ward, F.E.; Garling, D.L.; Buckler, R.T. Antimicrobial 3-Methylene flavanones. J. Med. Chem. 1981, 24,
1073–1077. [CrossRef]
18. Wallén, E.A.A.; Dahlén, K.; Grøtli, M.; Luthman, K. Synthesis of 3-Aminomethyl-2-aryl-8-bromo-6-
chlorochromones. Org.Lett. 2007, 9, 389–391.
19. Okuro, K.; Alper, H. Palladium-Catalyzed Carbonylation ofo-Iodophenols with Allenes. J. Org. Chem. 1997
62, 1566–1567. [CrossRef]
,
20. Grigg, R.; Liu, A.; Shaw, D.; Suganthan, S.; Woodalla, D.E.; Yoganathan, G. Synthesis of quinol-4-ones and
chroman-4-ones via a palladium-catalysed cascade carbonylation–allene insertion. Tetrahedron Lett. 2000, 41,
7125–7128. [CrossRef]
21. Bugarin, A.; Jones, K.D.; Connell, B.T. Efficient, direct
α-methylenation of carbonyls mediated by
diisopropylammonium trifluoroacetate. Chem. Commun. 2010, 46, 1517–1717. [CrossRef] [PubMed]
22. Vaidya, V.V.; Wankhede, K.S.; Salunkhe, M.M.; Trivedi, G.K. Synthesis of Functionalized Benzopyrans via
Intramolecular 1,3-Dipolar Cycloaddition of Nitrile Oxides. Synth. Commun. 2008, 38, 2392–2403. [CrossRef]
23. Iwasaki, H.; Kume, T.; Yamamoto, Y.; Akiba, K. Reaction of 4-t-butyldimethylsiloxy-1-benzopyrylium salt
with enol silyl ethers and active methylenes. Tetrahedron Lett. 1987, 28, 6355–6358. [CrossRef]
24. Lee, Y.-G.; Ishimaru, K.; Iwasaki, H.; Ohkata, K.; Akiba, K. Tandem Reactions In 4-Siloxy-1-benzopyrylium
Salts: Introduction of Substituents and Cyclohexene and Cyclopentane Annulation in Chromones.
J. Org. Chem. 1991, 56, 2058–2066. [CrossRef]
25. Kitson, R.R.A.; Millemaggi, A.; Taylor, R.J.K. The Renaissance of
Synthetic Approaches. Angew. Chem. Int. Ed. 2009, 48, 9426–9452. [CrossRef] [PubMed]
26. Albrecht, A.; Albrecht, Ł.; Janecki, T. Recent Advances in the Synthesis of -Alkylidene-Substituted
γ-Lactams and δ-Lactams. Eur. J. Org. Chem. 2011, 2011, 2747–2766. [CrossRef]
α-Methylene-γ-butyrolactones: New
α
δ-Lactones,
27. Lagoutte, R.; Winssinger, N. Following the Lead from Nature with Covalent Inhibitors. CHIMIA 2017, 71,
703–711. [CrossRef]

Molecules 2019, 24, 1868
14 of 14
28. Lagoutte, R.; Pastor, M.; Berthet, M.; Winssinger, N. Rapid and scalable synthesis of chiral bromolactones
as precursors to -exo-methylene- –butyrolactone-containing sesquiterpene lactones. Tetrahedron 2018, 74,
6012–6021. [CrossRef]
29. Janecki, T. Organophosphorus reagents as versatile tool in the synthesis of
and -alkylidene- -butyrolactams. In Targets in Heterocyclic Systems; Italian Society of Chemistry: Rome,
α
γ
α
-alkylidene-γ-butyrolactones
α
γ
Italy, 2006; pp. 301–320.
30. Modranka, J.; Albrecht, A.; Janecki, T. A Convenient Entry to 3-Methylidenechroman-2-ones and
2-Methylidenedihydrobenzochromen-3-ones. Synlett 2010, 19, 2867–2870.
31. Pie˛ta, M.; Ke˛dzia, J.; Wojciechowski, J.; Janecki, T. Asymmetric synthesis of 1,4-disubstituted
3-methylidenedihydroquinolin-2(1H)-ones. Tetrahedron Asymm. 2017, 28, 567–576. [CrossRef]
32. Zhou, P.; Jiang, Y.-J.; Zou, J.-P.; Zhang, W. Manganese(III) Acetate Mediated Free-Radical Phosphonylation of
Flavones and Coumarins. Synthesis 2012, 44, 1043–1050. [CrossRef]
33. Somu, R.V.; Boshoff, H.; Qiao, C.; Bennett, E.M.; Barry III, C.E.; Aldrich, C.C. Rationally Designed Nucleoside
Antibiotics That Inhibit Siderophore Biosynthesis of Mycobacterium tuberculosis. J. Med. Chem. 2006, 49, 31–34.
[CrossRef] [PubMed]
34. Janecki, T.; Wa˛sek, T. A novel route to substituted 3-methylidenechroman-2-ones and 3-methylchromen-2-ones.
Tetrahedron 2004, 60, 1049–1050. [CrossRef]
35. Koleva, A.I.; Petkova, N.I.; Nikolova, R.D. Ultrasound-Assisted Conjugate Addition of Organometallic
Reagents to 3-Diethylphosphonocoumarin. Synlett 2016, 27, 2676–2680.
36. Mahmudov, K.T.; Pombeiro, A.J.L. Resonance-Assisted Hydrogen Bonding as a Driving Force in Synthesis
and a Synthon in the Design of Materials. Chem. Eur. J. 2016, 22, 16356–16398. [CrossRef] [PubMed]
37. Koszuk, J.; Bartosik, T.; Wojciechowski, J.; Wolf, W.M.; Janecka, A.; Drogosz, J.; Długosz, A.; Krajewska, U.;
Mirowski, M.; Janecki, T. Synthesis of 3-Methylidene-1-tosyl-2,3-dihydroquinolin-4(1H)-ones as Potent
Cytotoxic Agents. Chem. Biodivers. 2018, 15, e1800242. [CrossRef]
38. Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutellingsperger, C. A novel assay for apoptosis flow cytometric
detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V.
J. Immunol. Methods 1995, 184, 39–51. [CrossRef]
39. Van Engeland, M.; Nieland, L.J.; Ramaekers, F.C.; Schutte, B.; Reutelingsperger, C.P. Annexin V-affinity assay:
A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998, 31, 1–9.
[CrossRef]
40. Pistritto, G.; Trisciuoglio, D.; Ceci, C.; Garufi, A.; D’Orazi, G. Apoptosis as anticancer mechanism: Function
and dysfunction of its modulators and targeted therapeutic strategies. Aging 2016, 8, 603–619. [CrossRef]
Sample Availability: Samples of the compounds 10a
–c,12a–c and 13a,c–d, f-h,k–l are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).