Mannich aminomethylation of flavonoids and anti-proliferative activity against breast cancer cell

Article abstract of DOI:10.1007/s11696-018-0402-1

We herein report Mannich aminomethylation of variously structural flavonoids and their biological evaluation against human breast cancer cell. Mannich reaction showed that substitution at C-6 position depends on amine basicity and C-ring feature of flavon

Full text of DOI:10.1007/s11696-018-0402-1

Chemical Papers
https://doi.org/10.1007/s11696-018-0402-1
ORIGINAL PAPER
Mannich aminomethylation of favonoids and anti‑proliferative
activity against breast cancer cell
T. Kim‑Dung Hoang1,3 · T. Kim‑Chi Huynh1,3 · T. Hong‑Tuoi Do4 · Thanh‑Danh Nguyen2,3
Received: 13 November 2017 / Accepted: 23 January 2018
© Institute of Chemistry, Slovak Academy of Sciences 2018
Abstract
We herein report Mannich aminomethylation of variously structural favonoids and their biological evaluation against human
breast cancer cell. Mannich reaction showed that substitution at C-6 position depends on amine basicity and C-ring feature
of favonoids. All fve favonoid substrates reacted with strong amine bases to aford the bis(6,8-aminomethyl) derivatives,
while with weak amines, the diferent products were obtained dependently on structural characteristic of favonoid. 3-OH
and 3-O-substituted groups on the C-ring exhibited the deactivated aminomethylation at C-6 position, whereas substitution
at this position was independent on bond feature at C-2 and C-3 on the C-ring. Screening anti-proliferative activity showed
six favonoids possessed activity against breast cancer cell, MDA-MB-231. Among them, the favonoids, luteolin (2) and
3′,4′,5,7-tetrahydroxy-6,8-bis(pyrrolidin-1-ylmethyl)-3-rutinosylfavone (3a), displayed the highest anti-proliferative activity
with the lowest IC50 values.
Keywords Mannich aminomethylation · Flavonoid · Synthesis · Anti-proliferative · MDA-MB-231
Introduction
of substitution of the pyran ring (e.g., quercetin, a favonol,
and luteolin, a favone, bearing diferent substitution groups
at C-3 position) (Fig. 1). Individual compounds within a
class difer in the pattern of substitution of the benzene rings
(Kumar and Pandey 2013). Biological activities of favo-
noids depend remarkably on their structural characteristics
such as degree of hydroxylation and substitution. A small
alteration in the chemical structure may lead to signifcant
changes in biological activities. Hence, the favonoids pos-
sess a wide range of bioactivities such as antioxidant (Rice-
Evans et al. 1996; Rice-Evans 2001; Pietta 2000), antifungal
(Tempesti et al. 2012), antiviral (Ng et al. 1997; Orhana
et al. 2010; Kaul et al. 1985), anti-proliferative (Chahar et al.
2011; Kanadaswami et al. 2005; Ravishankar et al. 2013),
and anti-infammatory agent (Pan et al. 2010; Cottiglia et al.
2005; Candiracci et al. 2012).
Flavonoid structures are based upon a 15-carbon skel-
eton consisting of two benzene rings linked via a hetero-
cyclic pyran ring. They can be divided into a variety of
classes which difer in the level of oxidation and pattern
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-018-0402-1) contains
supplementary material, which is available to authorized users.
* T. Kim-Dung Hoang
htkdung@ict.vast.vn
* Thanh-Danh Nguyen
danh5463bd@yahoo.com
1
Graduate University of Science and Technology, Vietnam
In recent years, numerous studies have been conducted
on the potential antimetastasis activity of natural favo-
noids in various tumor cell lines. For instance, querce-
tin has been shown to reduce the human prostate cancer,
PC-3 cells (Vijayababu et al. 2006), and kaempferol, gen-
istin, and daidzein revealed to inhibit human breast can-
cer, MDA-MB-231 cells (Lee et al. 2008; Phromnoi et al.
2009). Among of the favonoid derivatives, aminometh-
ylated favonoids are particularly valued due to potential
Academy of Science and Technology, 18 Hoang Quoc Viet,
Cau Giay, Hanoi, Vietnam
2
Institute of Research and Development, Duy Tan University,
Da Nang, Vietnam
3
Institute of Chemical Technology, Vietnam Academy
of Science and Technology, 1 Mac Dinh Chi Street, District
1, Ho Chi Minh, Vietnam
4
Department of Pharmacology, Faculty of Pharmacy,
University of Medicine and Pharmacy, Ho Chi Minh City, 41
Dinh Tien Hoang Street, District 1, Ho Chi Minh, Vietnam
Vol.:(0123456789)
1 3

Chemical Papers
Results and discussion
OH
3'
4'
5'
OH
2'
1'
1
Chemistry
8
B
7
9
HO
O
2
The general synthetic routes are illustrated in Scheme 1.
Our strategy for the synthesis of the aminomethylated
derivatives at C-6 and C-8 of A-ring of favonoids relied
upon electrophilic substitution via the Mannich reaction
carried out between favonoids with formaldehyde and
secondary cyclic amines at room temperature in ethanol.
Three cyclic amines with diferent basicity, pyrrolidine
(pKa = 11.27), 1-methylpiperazine (pKa = 9.14), and
morpholine (pKa = 8.36) were converted in situ into the
respective electrophilic iminium ions. To get an insight
into infuence of favonoid structures on the substitution
at various positions, fve natural favonoids with structural
variations were used as the substrates.
6'
A
C
3
6
10
R
4
5
OH
O
R = H: luteolin
R = OH: quercetin
Fig. 1 Structures of favonoids
activity against human cancer cells (Ha et al. 2016; Nguyen
et al. 2017; Chen et al. 2015; Tugrak et al. 2015). Although
Mannich aminomethylation of favonoids has been known,
most of the reported studies used C-6-substituted favo-
noids as substrates (Zhang et al. 2008; Babu et al. 2008;
Yusakul et al. 2016) or cyclic imines were used as the elec-
trophilic reagents (Nguyen et al. 2011). Recently, there are
several studies on the aminomethylation of natural favo-
noids using in situ generated iminium ions that proceeded
with formation of C-6 (Frasinyuk et al. 2015; Kukhareva
et al. 2004) or C-8-monosubstituted (Buravlev et al. 2017;
Gorbunov et al. 2016) or disubstituted favonoids (Nguyen
et al. 2015b; Zhurakulov et al. 2015; Nifant’ev et al. 2013).
Helgen et al. (2015) have aminomethylated quercetin to
aford only a 8-monoaminomethyl derivative when using
an imine salt made from 1-methylpiperazine while only a
6,8-diaminomethyl derivative was produced with the imine
salts of piperidine. All cases focused on the investigation
of bioactivity and no systematic studies were carried out
for investigating the infuence of amine and iminium ions
reactivity and features of favonoid skeleton driving the
Mannich aminomethylation.
The aminomethylation of quercetin with these bases and
aqueous formaldehyde aforded products 1a–1c in over
60% yields. The result showed that aminomethylation of
favonoids was strongly afected by basicity of amines. The
substitution with pyrrolidine occurred at both C-6 and C-8
positions. This is evident from 1H NMR of 1a which pro-
tons at C-6 (δ 6.20 ppm) and C-8 (δ 6.40 ppm) positions of
quercetin disappeared. Interestingly, only C-8 monosubsti-
tuted products were obtained when favonoids were treated
with an excess of 1-methylpiperazine and morpholine to
give 1b and 1c, respectively. This is attributed to the lower
basicity of these amines. Similar results were reported by
Lis and Marisca (1987) for the other substrates under the
same condition.
The position of the aminomethyl groups in products
1b and 1c was unambiguously determined by HMBC
NMR spectroscopy. The aminomethylene protons of 1b
and 1c (δ 3.87 and 3.82 ppm, respectively), which were
well resolved, were coupled long-range to respective C-9
carbons at δ 153.63 and 153.94 ppm. This allows for the
assignment of the aminomethyl group to the C-8 position
of the quercetin derivatives.
In connection with our ongoing research program
involving the efect of natural product structures on the
synthesis (Dang et al. 2014, 2017; Nguyen et al. 2015a,
2016; Hoang et al. 2015), we have showed that alkyla-
tion and acylation of 5-hydroxyfavonoids signifcantly
depended on reagents as well as structural characteristic
of ring C of favonoids, and these derivatives also exhib-
ited especial bioactivities (Hoang et al. 2015). The pre-
viously reported results prompted us to investigate this
efect on the aminomethylation of favonoids as well as
their bioactive evaluation. Herein, we report that the Man-
nich reaction of various favonoid substrates with iminium
salts generated in situ from secondary cyclic amines and
aqueous formaldehyde can provide either 8-monosubsti-
tuted or 6,8-disubstituted derivatives depending on amine
reagents and substrate features.
To determine infuence of 3-OH group in favonoids, lute-
olin (2) was used in comparison with quercetin. As expected,
the aminomethylation of 2 with the weakest base aforded
the bis(6,8-morpholin) product (2a) in 63% yield. In com-
parison with formation of 1c, this result refected that 3-OH
group obstructed to electrophilic substitution at C-6 in the
favonoid skeleton. Although reason of the efect has not
been described previously, it may be because the electron
pair of oxygen at C-3 position stabilized the non-covalent
six-membered ring (Hoang et al. 2015) which is attributed
to reduction of electron density at C-6 position of quercetin.
In a similar fashion, rutin (3) was treated with all bases.
Unfortunately, conversion occurred in very poor yields and
1 3

Chemical Papers
Scheme 1 Synthetic routes of
favonoids. Reagents. (i) amines
(10 equiv.), HCHO (37%, 10
equiv.), ethanol, rt
OH
OH
OR1
OR1
X2
O
O
O
O
HO
X1
HO
i
R2
R2
OH
OH
1a: R1 = H, R2 = OH, X1 = X2
=
N
CH2
N
1: R1 = H, R2 = OH
2: R1 = R2 = H
3: R1 = H
CH2
N
H3C
1b: R1 = H, R2 = OH, X1 = H, X2
1c: R1 = H, R2 = OH, X1 = H, X2
=
=
OH
O
N CH2
HO
OH
R2
=
O
O
CH3
OH
2a: R1 = R2 = H, X1 = X2
3a: R1 = H, X1 = X2
=
O
O
O
CH2
N
HO
OH
=
N CH2
4: R1 = CH3, R2 = H
OH
HO
O
OH
R2
=
O
O
CH3
O
HO
OH
OH
4a: R1 = CH3, R2 = H, X1 = X2
=
O
N
CH2
OH
OH
OCH3
OCH3
X2
O
HO
X1
O
O
HO
i
OH
O
=
OH
N
CH2
5
5a: X1 = X2
5b: X1 = X2
N
CH2
=
O
no pure aminomethylation products were obtained when
treating with either 1-methylpiperazine or morpholine
under the same reaction conditions. However, the amino-
methylation of 3 with pyrrolidine was observed more eas-
ily to aford bis(6,8-pyrrolidine) product (3a) in 54% yield,
indicating that 3-O-substituted group is also an important
factor in this transformation.
5a, and 5b, were determined physical properties and identi-
fed the structure by HRMS and NMR spectra.
Anti‑proliferative activity
The anti-proliferative activity of all favonoid derivatives
was tested in the breast cancer cell line MDA-MB-231
(Denizot and Lang 1986). A screening program revealed
that several favonoids exhibited anti-proliferative activity at
100 μM, as depicted in Table 1. Six compounds 1, 1c, 2, 2a,
3a, and 5a showed good anti-proliferative activity (inhibition
more than 50%) which would be further used to test IC50
values. It is noteworthy that the disubstituted derivatives of
rutin (3a) and hesperetin (5c) possessed the activity greater
than the parent favonoids, while all derivatives of quercetin
exhibited the lower activity than the parent compounds.
IC50 values against MDA-MB-231 cell are shown in
Table 2. Although the IC50 values of the favonoids are
Finally, to understand the infuence of the double bond
of C-ring on the favonoid aminomethylation, hesperetin (4)
was employed in comparison with substitution of diosmetin
(5). Similar to luteolin, the reaction of 4 with morpholine
was converted into bis(6,8-morpholine) derivative (4a) in
a good yield (81%). In addition, conversion of 5 with both
pyrrolidine and morpholine gave diaminomethylated deriva-
tives 5a and 5b, respectively in over 60% yields. It showed
that bond feature at C-2 and C-3 was independent on the
Mannich aminomethylation at the C-6 position. All the syn-
thesized favonoids, including new derivatives 1a, 2a, 3a, 4a,
1 3

Chemical Papers
Table 1 Percent inhibition
of MDA-MB-231 cell by all
favonoids
Experimental
Sub.
Inhibition at
100 μM (%)
Chemistry
1
78.4 7.4
1a
1b
1c
2
0
0.5
All the materials were purchased from Merck (Germany)
or Aldrich. The other solvents were purchased from Fluka
and used without further purifcation. Melting points were
measured with an Electrothermal Model 9200 (England).
UV–Vis spectra were measured using a Shimadzu, Model
UV-1650PC spectrophotometer, and reported as λmax in
nm (abs.). FTIR spectra were obtained with an Equi-
nox 55 IR—Bruker (Germany) spectrometer; absorption
bands are recorded in wave number (cm−1). The ESI–MS
were performed on a VG Zab Spec (70 eV) instrument.
1H (500 MHz) and 13C (125 MHz) NMR were recorded
on a BRUKER AVANCE 500 NMR spectrometer using
DMSO-d6 as solvent and tetramethylsilane (TMS) as an
internal standard. Chemical shifts are reported in δ rela-
tive to TMS.
12.9 0.2
59.5 2.3
82.5 10.2
77.6 5.7
6.1 0.1
88.6 6.9
8.2 0.2
9.2 0.3
31 0.6
2a
3
3a
4
4a
5
5a
5b
77.4 9.1
11.6 0.6
Table 2 IC50 value of
selected favonoids against
MDA-MB-231 cell
Sub.
IC50 (μmol/mL)
1
25.30 0.66
38.67 4.52
7.29 0.08
44.74 5.01
7.57 0.40
58.61 4.02
0.12 0.00
1c
General procedure for aminomethylation of favo‑
noids
2
2a
3a
To a stirred solution of favonoid (3.31 mmol) in ethanol
(50 mL) was slowly added amines (33.3 mmol) and a 37%
formaldehyde solution (2.48 g, 35 mmol). The mixture
was stirred at room temperature for 4 h, monitoring by
TLC. The residue was fltered of, washed with 50 mL of
ethanol, and then dried in the vacuum.
5a
Paclitaxel
higher than that of the standard drug (paclitaxel), most of the
compounds displayed a signifcant anti-proliferative activity
against MDA-MB-231 cell. Among them, favonoids 2 and
3a exhibit the highest anti-proliferative activity with IC50 of
7.29 and 7.57 μmol/mL, respectively.
2‑(3,4‑Dihydroxyphenyl)‑3,5,7‑trihy‑
droxy‑6,8‑bis(pyrrolidin‑1‑ylmethyl)‑4H‑chromen‑4‑one
(1a)
Yellow powder, m.p. 223–225 °C. Yield 68%. FTIR (KBr)
ν/cm−1 3376, 2968, 1651, 1560, 1260, 1069. UV–Vis λmax
(nm) (abs.) 238 (0.15), 246 (0.16), 287 (0.09), 321 (0.11),
Conclusions
1
Our studies on Mannich aminomethylation of favonoids
were suggested as follows: (1) amine basicity was criti-
cal to design the aminomethylated products and iminium
ions prepared from strong bases attracted to both C-6 and
C-8 positions of favonoid. (2) With weak bases, 3-OH and
3-O-substituted favonoids could obstruct electrophilic sub-
stitution at C-6 of favonoid. (3) Bond feature at C-2 and
C-3 did not almost afect the substitution at C-6 even the
weak bases.
341 (0.10), 268 (0.12). H NMR (500 MHz, DMSO-d6,
δ, ppm): 7.69 (1H, d, J 2.0 Hz, H-2′),7.44 (1H, dd, J 8.5,
2.0 Hz, H-6′), 6.85 (1H, d, J 8.5 Hz, H-5′), 3.92 (2H, s,
C-8-CH2), 3.88 (2H, s, C-6-CH2), 2.72–2.62 (8H, m,
N(CH2CH2)2), 1.76–1.67 (8H, m, N(CH2CH2)2). 13C NMR
(125 MHz, DMSO-d6, δ, ppm): 175.35 (C-4), 168.00 (C-7),
156.92 (C-5), 153.52 (C-9), 147.61 (C-4′), 145.50 (C-2),
145.14 (C-3′), 135.26 (C-3), 122.42 (C-1′), 119.67 (C-6′),
115.56 (C-5′), 114.89 (C-2′),103.83 (C-10), 100.85 (C-6),
100.30 (C-8), 52.82 (C-8-CH2), 52.73 (C-6-CH2), 48.68
(N(CH2CH2)2), 46.90 (N(CH2CH2)2), 23.12 (N(CH2CH2)2),
23.05 (N(CH2CH2)2). HRMS (m/z): 469.1965 [M+H]+;
469.1975 calcd [M+H]+ for C25H29N2O7.
All the compounds were evaluated for cytotoxicity of
breast cancer cell, MDA-MB-231. Six compounds 1, 1c,
2, 2a, 3a, and 5a showed good anti-proliferative activity.
Among them, the favonoids 2 and 3a displayed the lowest
IC50 values.
1 3

Chemical Papers
2‑(3,4‑Dihydroxyphenyl)‑3,5,7‑trihydroxy‑8‑((4‑methylpip‑
erazin‑1‑yl)methyl)‑4H‑chromen‑4‑one (1b)
(C-3′), 121.75 (C-1′), 118.97 (C-6′), 113.43 (C-5′), 116.06
(C-2′), 103.71 (C-10), 102.76 (C-6), 102.55 (C-3), 100.90
(C-8), 66.15 (N-(CH2CH2)2O), 65.97 (N-(CH2CH2)2O),
52.78 (N-(CH2CH2)2O), 52.36 (N-(CH2CH2)2O), 51.43
(C-6-CH2), 50.36 (C-8-CH2). HRMS (m/z): 485.1920
[M+H]+; 485.1924 calcd [M+H]+ for C25H29N2O8.
Yellow powder, m.p. 233–235 °C (233–235 °C, Helgen et al.
2015). Yield 66%. FTIR (KBr) ν/cm−1 3296, 2975, 1655,
1555, 1201, 1043. UV–Vis λmax (nm) (abs.) 229 (0.78),
1
246 (1.09), 277 (0.36), 363 (1.16). H NMR (500 MHz,
DMSO-d6, δ, ppm) (Zhang et al. 2008; Helgen et al. 2015):
12.52 (1H, brs, OH) 7.70 (1H, d, J 2.0 Hz, H-2′), 7.57 (1H,
dd, J 8.5, 2.0 Hz, H-6′), 6.89 (1H, d, J 8.5 Hz, H-5′), 6.17
(1H, s, H-6), 3.87 (2H, s, C-8-CH2), 2.63–2.58 (4H, m,
CH3-N-(CH2CH2)2), 2.38–2.36 (4H, m, CH3-N-(CH2CH2)2),
2.16 (3H, s, N-CH3). 13C NMR (125 MHz, DMSO-d6, δ,
ppm): 175.90 (C-4), 164.15 (C-7), 159.54 (C-5), 153.63
(C-9), 147.66 (C-4′), 146.45 (C-2), 145.08 (C-3′), 135.62
(C-3), 122.17 (C-1′), 119.89 (C-6′), 115.61 (C-5′), 114.97
(C-2′), 102.73 (C-8), 99.74 (C-10), 98.12 (C-6), 54.43
(CH3-N-(CH2CH2)2), 51.95 (CH3-N-(CH2CH2)2), 51.18
(C-8-CH2), 45.45 (N-CH3). HRMS (m/z): 415.1471
[M+H]+; 415.1505 calcd [M+H]+ for C21H23N2O7.
2‑(3,4‑Dihydroxyphenyl)‑5,7‑dihy‑
droxy‑6,8‑bis(pyrrolidin‑1‑ylme‑
thyl)‑3‑(((2S,3R,4S,5S,6R)‑3,4,5‑trihydroxy‑6‑((((2R,
3R,4R,5R,6S)‑3,4,5‑trihydroxy‑6‑methyltetrahy‑
dro‑2H‑pyran‑2‑yl)oxy)methyl)tetrahydro‑2H‑pyran‑2‑yl)
oxy)‑4H‑chromen‑4‑one (3a)
Orange powder, m.p. 154–156 °C, yield 57%. FTIR (KBr)
ν/cm−1 3479, 2983, 1754, 1629, 1503, 1218, 1073. UV–Vis
λmax (nm) (abs.) 229 (0.56), 243 (0.64), 269 (0.31), 299
(0.53). 1H NMR (500 MHz, DMSO-d6, δ, ppm): 7.62–7.60
(2H, m, H-5′,6′), 6.83 (1H, d, J 9.0 Hz, H-2′), 5.30 (1H, d,
J 7.5 Hz, H-1-glucose), 4.33 (1H, s, H-1-rhamnose), 3.94
(2H, s, C-8-CH2), 3.84 (2H, s, C-6-CH2), 3.70 (2H, d, J
10.5 Hz, H-6-glucose), 3.30–3.19 (5H, m, H-2,3,4,5-glu-
cose, H-5-rhamnose), 3.09–3.02 (3H, m, H-2,3,4-rhamnose),
2.81 (2H, m, N(CH2CH2)2), 2.69 (2H, m, N(CH2CH2)2),
1.81 (2H, m, N(CH2CH2)2), 1.74 (2H, m, N(CH2CH2)2),
1.00 (3H, d, J 6.5 Hz, H-6-rhamnose). 13C NMR (125 MHz,
DMSO-d6, δ, ppm): 176.64 (C-4), 162.30 (C-7), 157.50
(C-2), 155.34 (C-9), 153.98 (C-5), 148.79 (C-3), 148.65
(C-4′), 144.85 (C-3′), 132.75 (C-1′), 121.44 (C-6′), 116.15
(C-5′), 115.15 (C-2′), 103.88 (C-10), 103.19 (C-6), 101.46
(C-1-glucose), 100.94 (C-1-rhamnose), 100.73 (C-8),
76.57 (C-3-glucose), 75.93 (C-5-glucose), 74.05 (C-2-glu-
cose), 71.90 (C-2-rhamnose), 70.52 (C-4-rhamnose), 70.25
(C-4-glucose, C-3-rhamnose), 68.21 (C-5-rhamnose), 66.90
(C-6-glucose), 53.25 (N(CH2CH2)2), 52.70 (N(CH2CH2)2),
48.93 (C-8-CH2), 47.03 (C-6-CH2), 23.10 (N(CH2CH2)2),
23.04 (N(CH2CH2)2), 17.72 (C-6-rhamnose). HRMS
(m/z): 777.3099 [M+H]+; 777.3082 calcd [M+H]+ for
C37H49N2O16.
2‑(3,4‑Dihydroxyphenyl)‑3,5,7‑trihy‑
droxy‑8‑(morpholinomethyl)‑4H‑chromen‑4‑one (1c)
Yellow powder, m.p. 253–257 °C, yield 65%. FTIR (KBr)
ν/cm−1 3320, 2970, 1654, 1552, 1201, 1072. UV–Vis λmax
(nm) (abs.) 229 (0.20), 246 (0.28), 276 (0.09), 363 (0.30).
1H NMR (500 MHz, DMSO-d6, δ, ppm) (Zhang et al. 2008):
7.73 (1H, d, J 2.0 Hz, H-2′), 7.61 (1H, dd, J 8.5, 2.0 Hz,
H-6′), 6.91 (1H, d, J 8.5 Hz, H-5′), 6.22 (1H, s, H-6), 3.82
(2H, C-8-CH2), 3.59 (4H, m, N-(CH2CH2)2O), 2.53 (4H,
m, N-(CH2CH2)2O). 13C NMR (125 MHz, DMSO-d6, δ,
ppm): 175.95 (C-4), 163.48 (C-7), 159.54 (C-5), 153.94
(C-9), 147.68 (C-2), 146.58 (C-4′), 145.08 (C-3′), 135.64
(C-3), 122.21 (C-1′), 119.93 (C-6′), 115.60 (C-5′), 115.05
(C-2′), 102.87 (C-10), 100.17 (C-8), 97.94 (C-6), 66.08
(N-(CH2CH2)2O), 52.70 (N-(CH2CH2)2O), 50.96 (C-8-CH2).
HRMS (m/z): 402.1189 [M+H]+; 402.1189 calcd [M+H]+
for C20H20NO8.
2‑(3,4‑Dihydroxyphenyl)‑5,7‑dihydroxy‑6,8‑bis(morpholino
5,7‑Dihydroxy‑2‑(3‑hydroxy‑4‑methoxyphenyl)‑6,8‑bis(mor
methyl)‑4H‑chromen‑4‑one (2a)
pholinomethyl)‑4H‑chromen‑4‑one (4a)
Yellow powder, m.p. 230–233 °C, yield 63%. FTIR (KBr)
ν/cm−1 3260, 2963, 1649, 1583, 1213, 1072. UV–Vis λmax
(nm) (abs.) 236 (0.65), 247 (0.68), 252 (0.68), 289 (0.38).
1H NMR (500 MHz, DMSO-d6, δ, ppm): 7.44 (1H, dd, J
9.0, 2.0 Hz, H-6′), 7.42 (1H, d, J 2.0 Hz, H-2′), 6.91 (1H, d,
J 9.0 Hz, H-5′), 6.68 (1H, s, H-3), 3.78 (2H, s, C-8-CH2),
3.73 (2H, s, C-6-CH2), 3.60–3.56 (8H, m, N-(CH2CH2)2O),
2.54–2.49 (8H, m, N-(CH2CH2)2O). 13C NMR (125 MHz,
DMSO-d6, δ, ppm): 181.95 (C-4), 164.37 (C-7), 163.61
(C-2), 158.16 (C-5), 154.43 (C-9), 149.75 (C-4′),145.79
Pale yellow powder, m.p. 278–280 °C, yield 81%. FTIR
(KBr) ν/cm−1 3420, 2961, 1649, 1513, 1217, 1069.
UV–Vis λmax (nm) (abs.) 250 (0.29), 262 (0.34), 294
(0.18), 343 (0.31). 1H NMR (500 MHz, DMSO-d6, δ, ppm):
7.57 (1H, dd, J 8.5, 2.5 Hz, H-6′), 7.47 (1H, d, J 2.5 Hz,
H-2′), 7.12 (1H, d, J 8.5 Hz, H-5′), 6.76 (1H, s, H-3), 3.87
(3H, s, OCH3), 3.79 (2H, s, C-6-CH2), 3.75 (2H, s, C-8-
CH2), 3.62–3.57 (8H, m, N-(CH2CH2)2O), 2.54–2.50 (8H,
m, N-(CH2CH2)2O). 13C NMR (125 MHz, DMSO-d6, δ,
ppm): 181.97 (C-4), 164.45 (C-7), 163.21 (C-2), 158.12
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Chemical Papers
(C-5), 154.46 (C-9), 151.17 (C-4′), 146.84 (C-3′), 123.22
(C-1′), 118.64 (C-6′), 112.99 (C-2′), 112.22 (C-5′),103.68
(C-6), 103.19 (C-10), 102.80 (C-3), 100.99 (C-8), 66.14
(N-(CH2CH2)2O), 65.94 (N-(CH2CH2)2O), 55.77 (OCH3),
52.81 (N-(CH2CH2)2O), 52.33 (N-(CH2CH2)2O), 51.47
(C-8-CH2), 50.33 (C-8-CH2). HRMS (m/z): 499.2077
[M+H]+; 499.2080 calcd [M+H]+ for C26H31N2O8.
Cell Culture
Human breast adenocarcinoma (MDA-MB-231) cell line
(ATCC® HTB-26™) was provided by School of Medicine,
Sungkyunkwan University, Korea. MDA-MB-231 cells were
cultured in DMEM (Gibco, USA), containing 10% FCS
(Gibco, USA), 2 mM l-glutamine, 100 IU/mL penicillin,
and 100 µg/mL streptomycin (Gibco, USA) in a humidifed
atmosphere of 5% CO2 at 37 °C to attain confuency.
5,7‑Dihydroxy‑2‑(3‑hydroxy‑4‑methoxyphenyl)‑6,8‑bis(pyrr
olidin‑1‑ylmethyl)chroman‑4‑one (5a)
Anti‑proliferative activity assay
Pale yellow powder, m.p. 178–180 °C, yield 64%. FTIR
(KBr) ν/cm−1 3418, 2958, 1643, 1537, 1257, 1060.
UV–Vis λmax (nm) (abs.) 239 (0.20), 248 (0.21), 259
The samples were dissolved in DMSO at 10 mM and then
diluted with culture medium to get tested concentrations.
Paclitaxel (Anzatax®, Mayne Pharma, New Zealand) was
used as the reference compound. DMSO at diferent con-
centrations was used as a blank control. The cells were
then trypsinized, harvested, and counted using trypan blue
(Sigma-Aldrich, USA), and seeded in 96-well plates at 104
cells/well. After 24 h incubation at 37 °C, 5% CO2, cells
were treated with culture medium containing tested com-
pounds at 100 µM for 72 h. After 72 h treatment, cell viabil-
ity was evaluated as mitochondrial succinate dehydrogenase
(SDH) activity, a marker of viable cells using MTT test as
described by Denizot and Lang (1986).
1
(0.19), 283 (0.27). H NMR (500 MHz, DMSO-d6, δ,
ppm): 6.97 (1H, d, J 2.0 Hz, H-2′), 6.89 (1H, dd, J 8.0,
2.0 Hz, H-6′), 6.94 (1H, d, J 8.0 Hz, H-5′), 5.38 (1H, dd,
J 12.0, 3.0 Hz, H-2), 4.01 (2H, s, C-8-CH2), 3.98 (2H,
s, C-6-CH2), 3.77 (3H, s, OCH3), 3.17–3.12 (8H, m,
N-(CH2CH2)2), 3.01 (2H, dd, J 17.0, 12.0 Hz, H-3a),
2.65 (1H, dd, J 17.0, 3.0 Hz, H-3b), 1.90–1.87 (8H, m,
N-(CH2CH2)2). 13C NMR (125 MHz, DMSO-d6, δ, ppm):
191.65 (C-4), 177.57 (C-7), 163.11 (C-5), 160.91 (C-9),
147.68 (C-4′), 146.54 (C-3′), 131.85 (C-1′), 117.20
(C-6′), 113.88 (C-2′), 112.03 (C-5′), 98.88 (C-6), 98.64
(C-8), 96.03 (C-10), 77.64 (C-2), 55.67 (OCH3), 52.50
(N-(CH2CH2)2), 52.31 (N-(CH2CH2)2), 47.83 (C-8-CH2),
47.69 (C-6-CH2), 41.37 (C-3), 22.68 (N-(CH2CH2)2).
HRMS (m/z): 469.2345 [M+H]+; 469.2339 calcd [M+H]+
for C26H33N2O6.
Briefy, SDH activity was detected after 3 h incubation
in culture medium without serum containing 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
which was converted into formazan crystals dissolved in
acidifed isopropanol. The produced purple solution was
spectrophotometrically measured at 570 nm on Multikan™
microplate reader. Each tested concentration was performed
in triplicate. The percentage of proliferation inhibition was
calculated as follows:
5,7‑Dihydroxy‑2‑(3‑hydroxy‑4‑methoxyphenyl)‑6,8‑bis(mor
pholinomethyl)chroman‑4‑one (5b)
% Proliferation inhibition = 100 − [(Asample∕Acontrol) × 100].
Pale yellow powder, m.p. 175–180 °C, yield 62%. FTIR
(KBr) ν/cm−1 3450, 2948, 1622, 1535, 1259, 1027. UV–Vis
λmax (nm) (abs.) 244 (0.21), 279 (0.86). 1H NMR (500 MHz,
DMSO-d6, δ, ppm): 13.26 (1H, br.s, OH), 9.15 (1H, br.s,
OH), 6.96 (1H, m, H-2′), 6.94 (1H, m, H-5′), 6.91 (1H, dd,
J 8.0, 2.0 Hz, H-6′), 5.46 (1H, dd, J 12.0, 3.0 Hz, H-2),
4.04 (2H, s, C-8-CH2), 4.00 (2H, s, C-6-CH2), 3.78 (3H, s,
OCH3), 3.78 (8H, m, N-(CH2CH2)2O), 3.14 (8H, dd, J 17.0,
12.0 Hz, H-3a), 3.07 (8H, m, N-(CH2CH2)2O), 2.75 (1H,
dd, J 17.0, 3.5 Hz, H-3b). 13C NMR (125 MHz, DMSO-d6,
δ, ppm): 193.91 (C-4), 162.76 (C-5), 161.08 (C-9), 147.79
(C-4′), 146.53 (C-3′), 131.28 (C-1′), 117.37 (C-6′), 113.95
(C-2′), 112.04 (C-5′), 98.21 (C-6), 97.47 (C-8), 89.16
(C-10), 78.05 (C-2), 63.81–63.13 (N-(CH2CH2)2O), 55.64
(OCH3), 50.96–50.85 (N-(CH2CH2)2O), 50.62 (C-8-CH2),
50.16 (C-6-CH2), 41.37 (C-3). HRMS (m/z): 501.2234
[M+H]+; 501.2237 calcd [M+H]+ for C26H33N2O8.
The compounds with signifcant potential against MDA-
MB-231 cells (percent of proliferation inhibition at 100 μM
more than 50%) were then tested for their toxicity at fve
diferent concentrations (2–100 μM). Concentrations induc-
ing a 50% inhibition of cell growth (IC50) were deduced by
exponential regression of the inhibitory percentage − tested
concentration. Values were mean SD from three independ-
ent experiments.
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