Synthesis and cytotoxic evaluation of halogenated furanones

Article abstract of DOI:10.1007/s00706-020-02708-0

Abstract: The objective of the current study is to evaluate the potency of halogen-furan-2(5H)-one-type derivatives against human cancer cell lines. Four known bromofuran-2(5H)-one-type derivatives, as well as five new and two known bromo-4-(phenylamino)furan-2(5H)-one-type compounds and six novel and two known halogen-4-alkyl-5-phenyl-3-(phenylamino)furan-2(5H)-one-type derivatives, were synthesized and evaluated for their anticancer activity against prostate (PC-3) and colon (HCT-116) human cancer cell lines. The results showed that only the bromofuran-2(5H)-ones were cytotoxic in both cell lines. Three of these displayed particularly useful antiproliferative activities, in both cancer cells evaluated. (E)-5-(Bromomethylene)furan-2-(5H)-one was the most active against PC-3 (IC₅₀ 0.93 ± 0.02?μM) while 3,4-dibromofuran-2(5H)-one was the most active against HCT-116 (IC₅₀ 0.4 ± 0.04?μM). Furthermore, flow cytometry studies revealed that the bromofuran-2(5H)-ones induced cell death by apoptosis. Also, it was found that the cytotoxic furanones induced lipid peroxidation, determined by TBARS assay. Thus, cytotoxicity of the active compounds could be associated with ROS production. Additionally, it must be taken into account that all cytotoxic compounds contain an electrophilic carbon atom in position 4, which can explain, through a non-specific reactivity with nucleophiles, the cytotoxic activity of these compounds. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-020-02708-0

Monatshefte für Chemie - Chemical Monthly (2020) 151:1841–1849
https://doi.org/10.1007/s00706-020-02708-0
ORIGINAL PAPER
Synthesis and cytotoxic evaluation of halogenated furanones
Víctor A. Castro‑Torres1 · Nadia J. Jacobo‑Herrera2 · Lidia Díaz‑Sánchez1 · Leticia Rocha‑Zavaleta3 ·
Patricia García‑López4 · Mariano Martínez‑Vázquez1
Received: 10 August 2020 / Accepted: 22 October 2020 / Published online: 5 November 2020
© Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
The objective of the current study is to evaluate the potency of halogen-furan-2(5H)-one-type derivatives against human cancer
cell lines. Four known bromofuran-2(5H)-one-type derivatives, as well as fve new and two known bromo-4-(phenylamino)-
furan-2(5H)-one-type compounds and six novel and two known halogen-4-alkyl-5-phenyl-3-(phenylamino)furan-
2(5H)-one-type derivatives, were synthesized and evaluated for their anticancer activity against prostate (PC-3) and colon
(HCT-116) human cancer cell lines. The results showed that only the bromofuran-2(5H)-ones were cytotoxic in both
cell lines. Three of these displayed particularly useful antiproliferative activities, in both cancer cells evaluated. (E)-5-
(Bromomethylene)-furan-2-(5H)-one was the most active against PC-3 (IC50 0.93 0.02 μM) while 3,4-dibromofuran-
2(5H)-one was the most active against HCT-116 (IC50 0.4 0.04 μM). Furthermore, fow cytometry studies revealed that
the bromofuran-2(5H)-ones induced cell death by apoptosis. Also, it was found that the cytotoxic furanones induced lipid
peroxidation, determined by TBARS assay. Thus, cytotoxicity of the active compounds could be associated with ROS pro-
duction. Additionally, it must be taken into account that all cytotoxic compounds contain an electrophilic carbon atom in
position 4, which can explain, through a non-specifc reactivity with nucleophiles, the cytotoxic activity of these compounds.
Graphic abstract
Keywords Halofuranones · Antitumor agents · Cytotoxic compounds · Drug research
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-020-02708-0) contains
supplementary material, which is available to authorized users.
* Nadia J. Jacobo-Herrera
Belisario Domínguez Secc 16, Tlalpan, 14080 Mexico City,
Mexico
nadia.jacobo@gmail.com
3
4
* Mariano Martínez-Vázquez
Instituto de Investigaciones Biomédicas, Universidad
Nacional Autónoma de México (UNAM), Circuito, Mario de
La Cueva s/n, C.U., Coyoacán, 04510 Mexico City, Mexico
marvaz@unam.mx
1
Instituto de Química, Universidad Nacional Autónoma de
Instituto Nacional de Cancerología, Av. San Fernando 22,
Belisario Domínguez Secc 16, Tlalpan, 14080 Mexico City,
Mexico
México (UNAM), Circuito Exterior S/N Circuito Exterior,
C.U., Coyoacán, 04510 Mexico City, Mexico
2
Unidad de Bioquímica, Instituto Nacional de Ciencias
Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15,
Vol.:(0123456789)
1 3

1842
V. A. Castro-Torres et al.
atoms in the structure of furanones [6]. Therefore, it was
decided to synthesize and evaluate the cytotoxicity against
PC-3 (prostate) and HCT-116 (colon) human cancer cell
lines of three series of halogenated furanones. The frst
consisted of four known bromofuran-2-(5H)-one-type
derivatives (1–4). The second consisted of fve original
(7–11) and two known (5, 6) bromo-4-(phenylamino)-
furan-2-(5H)-one-type compounds and the third one
of six novel (15–19) and two known (12, 13) halogen-
4-alkyl-5-phenyl-3-(phenylamino)furan-2-(5H)-one-type
derivatives.
Introduction
With more than 18 million new cases in 2018, cancer
continues to be a disease of global concern. Colon and
prostate cancers have been reported to be among the top
fve most frequent cancers, excluding skin cancer that is
not caused by melanomas [1]. Although new therapies and
novel drugs have been used against cancer, medications
with potent antitumor activity without aggressive side
efects have yet to be discovered. There are a large num-
ber of biologically active natural and synthetic compounds
that incorporate a furanone skeleton into their structures.
Biological activities include antitumor, antibacterial, anti-
fungal, antiviral, anti-infammatory, and antioxidant. For
instance, it has been reported that natural cardiac glyco-
sides containing a furanone ring, that are in clinical use to
treat heart failure and some types of cardiac arrhythmias,
induce selective anticancer efects [2–4]. On the other
hand, it is known that synthetic 4-(4-substituted amin-
obenzyl)furan-2(5H)-one derivatives, with an exocyclic
double bond, exhibited cytotoxicity and topoisomerase I
inhibitory activity against three human cancer cell lines
[5]. Recently, it was reported that the inclusion of halo-
gens in the structure of 2(5H)-furanones increased their
cytotoxicity activity against human cancer cell lines, thus,
highlighting the importance of the presence of halogen
Results and discussion
Chemistry
The synthesis of derivative 1 was done by reacting
3,5-dibromo-4-oxopentanoic acid with sulfuric acid [7].
While the compound 2 was obtained from the 5-bromo-4-ox-
opentanoic acid methyl ester [8, 9]. The mucobromic acid
(3) and the 3,4-dibromofuran-2(5H)-one (4) were obtained
from furfural [10, 11] (Scheme 1) (see supplemental fles).
The compounds 5–11 were obtained by reaction of 4 with
the respective mono- or di-halogenated aniline in methanol
(Scheme 2). The compounds 5 and 6 had been previously
synthesized [12].
Scheme 1
1 3

Synthesis and cytotoxic evaluation of halogenated furanones
1843
Scheme 2
As an unexpected result, compound 10 does not have a
bromine atom in position C3, like furanones 5–9 and 11.
an aromatic aldehyde that can also be substituted. Green
chemistry conditions were managed in this reaction since an
oligosaccharide (β-cyclodextrin) was used as catalyst, and
water was the solvent used (Scheme 3) (see supplemental
fles).
1
In the 10 H NMR spectrum, an AA’BB’ system for the
aromatic protons was observed. In addition, a singlet at
5.33 ppm assigned to proton H3 which correlate with a sig-
nal at 85.05 ppm in the 13C NMR spectrum was observed.
Additionally, an interaction between H3 and C2 and C5 was
observed in the 10 HMBC spectrum (see supplemental fles).
It appears that in 4-(phenylamino) furan-2-(5H)-one-type
compounds the bromine atom at C3 is required for cytotoxic
activity since 10, in this series, was the least active. The lack
of the presence of an bromine atom at C3 alter the electro-
philic nature of the furanone ring, which is refected not
just in the lower activity of 10 but also in the null cytotoxic
activity of compounds 12–19.
The furanones 12 and 13 were previously synthetized
[13]. The 1H and 13C 2D spectra of each synthesized com-
pounds are in the supplementary fles. The structures of
known furanones 1–6, 12, 13 were confrmed by comparison
of their 1H and 13C NMR data with those previously pub-
lished [7–11]. The structures of the new compounds 7–11
and 14–19 were deduced by 2D NMR experiments data (see
supplementary material).
Biological assay
The synthesis of furanones 12–19 was conducted using
a one-pot reaction between diethyl acetylene dicarboxylate,
an aromatic amine which can have diferent substituents and
The cytotoxicity of furanones 1–19 was evaluated against
HCT-116 (human colon carcinoma) and PC-3 (human
Scheme 3
1 3

1844
V. A. Castro-Torres et al.
Table 1 Cytotoxic activity of furanones 1–19 against PC3 and HCT-
prostate cancer) cell lines by sulforhodamine assay. Com-
pounds 1–4 were cytotoxic in both lines, especially fura-
nones 1, 2, and 4 were more active than cisplatin. The struc-
tures of the 1, 2 suggest that their cytotoxicity could be by
the typical 1,4 attack of a nucleophilic compound such as
amine or alcohol of an enzyme that could induce cell death
[14]. This proposal is supported by the fact that all cyto-
toxic compounds contain an electrophilic carbon atom in
position 4. On the other hand, it has been published that
some furan-2(5H)-one-type compounds induce cell death by
production of ROS [15]. In our case, there is a positive cor-
relation between the cytotoxic activity of furanones 1–4 and
their ROS production indicated by lipoperoxidation activity,
measured by TBARS assay (Fig. 1). In contrast, furanones
7 and 15 did not show intense lipoperoxidation activity,
and their cytotoxic activity was low. Then compounds 1–4
may induce cell death by production of ROS as well as its
electrophilic characteristics. Separate mention deserves the
compounds 14 and 15 that showed moderate cytotoxicity
against HCT-116. However, the addition of a chlorine or
fuorine atom eliminates the activity as in furanones 16 or
19 (Table 1).
116 expressed as the IC50
Entry
IC50/µM
PC-3
HCT-116
Cisplatin
15.85 0.05
1.13 0.02
0.93 0.02
26.25 0.02
1.31 0.03
56.25 0.01
65.1 0.03
60.0 0.05
45.2 0.02
66.7 0.03
>100 0.01
43.2 0.03
85.3 0.01
62.3 0.03
56.25 0.08
78.75 0.05
>100
5.56 0.06
1.12 0.1
1.5 0.09
14.06 0.08
0.4 0.04
75.0 0.05
74.3 0.09
56.25 0.01
33.2 0.05
43.3 0.07
78.5 0.09
65.3 0.01
>100
55.43 0.03
20.0 0.04
30.0 0.01
>100
>100
>100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
>100
>100
>100
On the other hand, to evaluate the safety of 1–4 com-
pounds, the cytotoxic effect on non-cancerous primary
fbroblast cells (HGF-1) was assessed. 1 and 3 showed a
moderated cytotoxicity while 2 and 4 had a minimum impact
on the growth of this primary cell culture at 50 µM dose
(Table 2).
>100
Experiments were repeated at least in three occasions and results are
expressed in SEM
The low cytotoxicity of 3 (mucobromic acid) in non-
cancer cells had previously been determined in a human
lymphoblastoid TK6 cell line where at the 100 µM dose it
showed marginal activity. However, 3 at a dose of 4000 µM
induced 80% of inhibition of TK6 cells growth [17].
Although furanones 5–11 were less cytotoxic than com-
pounds 1–4, there is some data on the structure–activ-
ity relationship that is worth highlighting. The frst thing
that stands out is that the activity of compounds 5–11
is diferent in each cancer line used. Taking advantage
that 5–9 and 11 derivatives, only difer in C4 substitu-
tion so the variation of the activity of the compounds
will depend on the chemical nature of this residue. In the
PC-3 line (prostate) the most active compound in this
series was the p-phenylazoaniline derivative 11 (IC50
43.2 0.03 µM), followed by the difuoro derivative 8
(IC50 45.2 0.02 µM), a result that contrasts with that
Table 2 Cytotoxic activities of
Entry
Inhibition/% in the
growth of HGF-1
cells
compounds 1–4 against HGF-1
cells, after 48 h of treatment
[16]
50 µM
1
2
3
4
68.95
NC
16.75
NC
Fig. 1 Lipoperoxidation induced by furanones 1–4, 7, and 15
NC non-cytotoxic
1 3

Synthesis and cytotoxic evaluation of halogenated furanones
1845
Table 3 Percentages of apoptosis induced by IC50 of each compound
obtained by the dichloro derivative 9 (IC50 66.7 0.03 µM)
which was the least active. Mono-halogenated compounds
6 (IC50 65.1 0.03 µM) and 7 (IC50 60.0 0.05 µM) were
not more active than 11 despite 7 containing a fuorine
atom. In the HCT-116 (colon) lines, the most active com-
pound was 8 (IC50 33.2 0.05 µM), a result that indicates
the importance of two fuorine atoms for cytotoxicity in
both cell lines. Dichloro derivative 9 (IC50 43.3 0.07 µM)
turned out to be more active in the HCT-116 cell line than
in the prostate line. The other compounds showed similar
activities which indicates that the chemical variation of the
substituent in C4 was not crucial for the cytotoxic activity.
Apoptosis was assessed for compounds 1–4, due to their
cytotoxicity in both cell lines. The apoptosis induction by
1–4 furanones in PC-3 (prostate) and HCT-116 (colon)
cells was determined by double staining with annexin V/
PI. The results showed that in HCT cells, all the com-
pounds evaluated induced principally early apoptosis.
On the other hand, in the PC-3 cells, furanones 1 and 2
induced early apoptosis, and 3 and 4 increased the later
apoptosis (Fig. 2, Table 3).
Apoptotic cells/% 48 h
treatment
HCT-116
PC-3
Vehicle
Cisplatin
1
2
3
4
7.12 0.1
60.84 0.4
48.40 0.2
60.21 0.1
68.53 0.2
57.41 0.08
0.18 0.02
56.09 0.1
35.30 0.4
34.19 0.2
74.9 0.2
65.12 0.2
The numbers represent the average of three independent experi-
ments SD
Conclusion
In this work, the synthesis and cytotoxic evaluation of
19 halogenated furanones are reported. Five (7–11) that
belong to 4-(phenylamino)-furan-2-(5H)-one-type com-
pounds and six (14–19) that belong to 4-alkyl-5-phenyl-3-
(phenylamino)-furan-2-(5H)-one-type derivatives are new
Fig. 2 PC-3 and HCT-116 apoptosis induction by furanones 1–4 IC50 (µM), determined by fow cytometric assay (annexin V/PI)
1 3

1846
V. A. Castro-Torres et al.
structures. The furanones 1–6, 12, 13 have already been
synthesized [7–11].
General procedure for the synthesis of furanones
5–11
The results showed that only furanones 1–4 were active
against the two human cancer cell lines evaluated. These
fndings indicate that a furanone structure with lipoperoxi-
dation activities and electrophilic properties is more critical
to the cytotoxic efect than the presence of halogen atoms in
larger structures. The selectivity of 1–4 against cancer cells
was demonstrated by the low or null cytotoxic activity of
these furanones against non-cancerous primary fbroblast
cells (HGF-1) [16]. The relative ease in the synthesis of the
cytotoxic furanones 1–4 makes these compounds suitable
candidates for further study. Although 1, known as C30,
has been extensively used in quorum sensing (QS) studies,
to our knowledge, this is the frst time that 1 is reported as a
cytotoxic agent [18].
General procedure for the synthesis of furanones 5–11 was
conducted as previously reported with slight modifcations
[12] (Scheme 2). To a solution of 1.0 mmol of 4 in 10 cm3
of dry methanol was added 2.0 mmol of an aromatic amine.
The solution was left 24 h at room temperature. Afterward,
the solvent was evaporated under reduced pressure, then
20 cm3 of CH2Cl2 was added and washed with water (3×5
cm3). The organic layer was dried over anhydrous Na2SO4,
fltrated, and the solvent evaporated under reduced pres-
sure. The crude residue was recrystallized from CHCl3/hex-
ane, afording the respective solid furanones. Compounds
7, 8, and 10 were purifed by open column chromatogra-
phy packed with silica and eluted with hexane/EtOAc 7:3
mixture.
3‑Bromo‑4‑[(4‑chlorophenyl)amino]furan‑2(5H)‑one
(5) Rf = 0.55 (hex/EtOAc 7:3, v/v); yield: 72%; m.p.:
125–126 °C; HPLC purity: 95%. The NMR analysis was
in accordance with previously published data [12] (for 2D
NMR experiments see supplementary material).
Experimental
All reagents were acquired from Sigma-Aldrich, except
tetronic acid which was purchased from Alfa Aesar Chemi-
cals. All solvents (acetone, ethyl acetate, dichloromethane,
hexane, ethanol, methanol) used were previously distilled.
Diisopropyl ether was acquired from Sigma-Aldrich. Deu-
terated chloroform (CDCl3) and dimethylsulfoxide-d6 were
purchased from Cambridge Isotope Laboratories, Inc.
TLC was performed using DC-Fertigfolien ALUGRAMR
Xtra SIL G/UV254 20×20 cm. Normal phase fash column
chromatography was performed using Silica gel 60 Kieselgel
60 0.063–0.2 mm.
3‑Bromo‑4‑[(4‑bromophenyl)amino]furan‑2(5H)‑one
(6) Rf = 0.55 (hex/EtOAc 7:3, v/v); yield: 76%; m.p.:
145–147 °C; HPLC purity: 99%. The NMR analysis was
in accordance with previously published data [12] (for 2D
NMR experiments see supplementary material).
3‑Bromo‑4‑[(4‑fluorophenyl)amino]furan‑2(5H)‑one (7,
C10H8BrFNO2) Rf = 0.6 (hex/EtOAc 7:3, v/v); yield: 81%;
m.p.: 128–130 °C; HPLC purity: 99%; 1H NMR (300 MHz,
CDCl3): δ = 7.13 (s, 2H), 7.12 (s, 2H), 6.77 (s, 1H), 4.81 (s,
2H) ppm; 13C NMR (75 MHz, CDCl3): δ = 169.46, 161.16
(d, J = 124 Hz), 160.65, 133.09, 125.06 (d, J = 8.6 Hz),
117.11 (d, J=23 Hz), 77.83, 67.05 ppm; IR (KBr): ꢀ =3285,
1H, 13C, DEPT-135, HSQC, and HMBC NMR were
recorded on a Bruker Fourier Spectrometer 300 MHz.
Chemical shift values are expressed in δ (ppm) relative to
1
tetramethyl silane (TMS, δ = 0.00 ppm for H NMR and
δ=77.0 ppm for 13C NMR). Data are reported as follows:
chemical shift, multiplicity (s=singlet, d=doublet, t=tri-
plet, q=quartet, m=multiplet), coupling constants (J; Hz),
and integration. The exact mass was calculated by a Jeol
AccuTOF JMST100LC-DART apparatus. Infrared spectra
were acquired in FT-IR Bruker spectrometer (KBr pellet).
HPLC was performed in Agilent Liquid Chromatograph
equipped with a Waters 2996 diode array. A column Synergi
Polar-RP 80A 150×2.0 mm 4 um was used. Methanol was
the initial eluent and water after 30 min. Flow = 0.2 cm3/
min, wavelength 254 or 240 nm, sample solvent: acetonitrile
or methanol.
1723, 1626, 1593, 1189, 506 cm−1; MS (DART): m/z calcd
12
for
C
1H881Br119F114N116O2 ([M + H]+]): 273.97020,
10
found 273.96988 (for 2D NMR experiment see supplemen-
tary material).
3‑Bromo‑4‑[(3,4‑difuorophenyl)amino]furan‑2(5H)‑one (8,
C10H7BrF2NO2) Rf =0.45 (hex/EtOAc 7:3, v/v); yield: 70%;
m.p.: 140–142 °C; HPLC purity: 91%; 1H NMR (300 MHz,
DMSO-d6): δ = 9.62 (s, 1H), 7.5–7.34 (m, 2H), 7.12 (dt,
J=8.4, 4.0 Hz, 1H), 5.08 (s, 2H) ppm; 13C NMR (75 MHz,
DMSO-d6): δ = 169.96, 161.77, 150.11 (dd, J = 174.1,
13 Hz), 146.87 (dd, J=171.8, 13 Hz), 135.59 (d, J=5.5 Hz),
119.92 (dd, J=6.3, 3.3 Hz), 118.26 (d, J=18 Hz), 112.64
(d, J = 19.8 Hz), 75.44, 67.80 ppm; IR (KBr): ꢀ = 3239,
(Z)-4-Bromo-5-(bromomethylene)furan-2-(5H)-one (1)
[7], (E)-5-(bromomethylene)furan-2-(5H)-one (2) [8, 9],
as well as 3,4-dibromo-5-hydroxyfuran-2(5H)-one (3) and
3,4-dibromofuran-2(5H)-one (4) [10, 11] were synthesized
according to literature.
1727, 1632, 1602, 1524 cm−1; MS (DART): m/z calcd for
12
C
1H779Br119F214N116O2 ([M+H]+]): 289.96282, found
10
1 3

Synthesis and cytotoxic evaluation of halogenated furanones
1847
289.96302 (for 2D NMR experiment see supplementary
material).
(4 × 10 cm3). The organic layers were washed with water,
saturated brine solution (2 ×10 cm3) and dried over anhy-
drous Na2SO4. The combined organic layers were evaporated
under reduced pressure and the resulting crude product was
purifed by silica open column chromatography by using
ethyl acetate and hexane (7:3) as eluent to give the respective
furanone derivative.
3‑Bromo‑4‑[(3,4‑dichlorophenyl)amino]furan‑2(5H)‑one (9,
C10H7BrCl2NO2) Rf =0.55 (hex/EtOAc 7:3, v/v); yield: 86%;
m.p.: 163–165 °C; HPLC purity: 99%; 1H NMR (300 MHz,
DMSO-d6): δ = 9.68 (s, 1H), 7.60 (d, J=8.7 Hz, 1H), 7.55
(d, J = 2 Hz, 1H), 7.25 (dd, J = 8.7, 2.1 Hz, 1H), 5.14 (s,
2H) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 169.40,
160.77, 138.42, 131.40, 130.91, 126.73, 123.74, 122.14,
76.08, 67.52 ppm; IR (KBr): ꢀ = 3232, 1727, 1646, 1618,
Ethyl 5‑oxo‑2‑phenyl‑4‑(phenylamino)‑2,5‑dihydro‑
furan‑3‑carboxylate (12) Rf = 0.45 (EtOAc); yield: 80%;
m.p.: 152–153 °C; HPLC purity: 99%. NMR analysis was
in accordance with previously published data [13] (for 2D
NMR experiments see supplementary material).
37
1477 cm−1; MS (DART): m/z calcd for 12C101H781Br135Cl1
Cl114N116O2 ([M+H]+]): 325.89872, found 325.89867 (for
2D NMR experiment see supplementary material).
Ethyl 2‑(4‑chlorophenyl)‑5‑oxo‑4‑(phenylamino)‑2,5‑dihy‑
drofuran‑3‑carboxylate (13) Rf =0.40 (EtOAc); yield: 90%;
m.p.: 165–167 °C; HPLC purity: 99%. NMR analysis was
in accordance with previously published data [13] (for 2D
NMR experiments see supplementary material).
4 ‑ [ ( 4 ‑ I o d o p h e ny l ) a m i n o ] f u r a n ‑ 2 ( 5 H) ‑ o n e ( 1 0 ,
C10H9INO2) Rf = 0.50 (hex/EtOAc 1:1, v/v); yield: 83%;
m.p.: 249–251 °C; HPLC purity: 98%; 1H NMR (300 MHz,
DMSO-d6): δ = 9.79 (s, 1H), 7.66 (d, J=8.2 Hz, 2H), 7.01
(d, J = 8.3 Hz, 2H), 5.33 (s, 1H), 4.85 (s, 2H) ppm; 13C
NMR (75 MHz, DMSO-d6): δ = 175.30, 162.70, 140.47,
138.47, 121.18, 86.82, 85.06, 68.56 ppm; IR (KBr):
Ethyl 4‑[(4‑bromophenyl)amino]‑5‑oxo‑2‑phenyl‑2,5‑di‑
hydrofuran‑3‑carboxylate (14, C19H17BrNO4) Rf = 0.35
(EtOAc); yield 85%; m.p.: 144–147 °C; HPLC purity: 99%;
1H NMR (300 MHz, CDCl3): δ = 7.39 (d, J=8.7 Hz, 2H),
7.36 (d, J = 8.5 Hz, 2H), 7.25 (dd, J = 12.7, 7.6 Hz, 3H),
7.20 (d, J=7.1 Hz, 2H), 5.68 (s, 1H), 4.18 (q, 7.1 Hz, 2H),
1.17 (t, J=6.9 Hz, 3H) ppm; 13C NMR (75 MHz, CDCl3): δ
= 165.06, 162.87, 156.37, 135.46, 134,78, 132.03, 128,74,
127.46, 123.43, 118.94, 113.28, 61.40, 61.35, 13.91 ppm;
IR (KBr): ꢀ = 1676, 1227, 826 cm−1; MS (DART +): m/z
ꢀ =3435, 1698, 1612, 1552 cm−1; MS (DART+): m/z calcd
12
for
C
1H9127I114N116O2 ([M + H]+]): 301.96780, found
10
301.96761 (for 2D NMR experiment see supplementary
material).
(E)‑3‑Bromo‑4‑[(4‑(phenyldiazenyl)phenyl)amino]‑
furan‑2(5H)‑one (11, C16H13BrN3O2) Rf = 0.35 (hex/EtOAc
1:1, v/v); yield: 85%; m.p.: 142–143 °C; HPLC purity:
calcd for 12C19 H1781Br114N116O4 ([M+H]+]): 404.03205,
1
1
99%; H NMR (300 MHz, DMSO-d6): δ = 9.85 (s, 1H),
7.90 (t, J=8.2 Hz, 4H), 7.59 (q, J=6.5 Hz, 3H), 7.57–7.42
(m, 2H), 5.27 (s, 2H) ppm; 13C NMR (75 MHz, DMSO-
d6): δ = 169.86, 160.98, 152.46, 148.75, 141.83, 131.76,
129.92, 124.24, 122.91, 122.21, 77.21, 68.35 ppm; IR
(KBr): ꢀ =3434, 1731, 1638, 1583 cm−1; MS (DART+): m/z
found 404.03405 (for 2D NMR experiment see supplemen-
tary material).
E t hy l 2 ‑ ( 4 ‑ c h l o ro p h e ny l ) ‑ 4 ‑ [ ( 4 ‑ f l u o ro p h e ny l ) ‑
amino]‑5‑oxo‑2,5‑dihydrofuran‑3‑carboxylate (15,
C19H16ClFNO4) Rf = 0.40 (EtOAc); yield: 70%; m.p.: 157–
160 °C; HPLC purity: 96%; 1H NMR (300 MHz, CDCl3): δ
= 7.46–7.31 (m, 2H), 7.29–7.19 (m, 2H), 7.14 (d, J=8.4 Hz,
2H), 6.97 (t, J=8.6 Hz, 2H), 5.65 (s, 1H), 4.20 (q, J=7.1 Hz,
2H), 1.19 (t, J = 7.1 Hz, 3H) ppm; 13C NMR (75 MHz,
CDCl3): δ = 165.03, 162.8, 160.54 (d, J = 246.9 Hz),
156.84, 134.65, 133.69, 132.15 (d, J=2.9 Hz), 129.05 (d,
J=9.5 Hz), 124.45 (d, J=8.2 Hz), 116.15 (d, J=22.7 Hz),
112.82, 61.56, 61.36, 14.12 ppm; IR (KBr): ꢀ =3306, 1722,
1
calcd for 12C16 H1381Br114N316O2 ([M+H]+]): 360.01707,
found 360.01622 (for 2D NMR experiment see supplemen-
tary material).
General procedure for the synthesis of furanones
12–19
β-Cyclodextrin (0.226 g, 0.2 mmol) was dissolved in 30
cm3 distilled water and stirred for 10 min. After aniline
(2.0 mmol) was added and stirred for another fve min more
and then, 0.170 g diethyl acetylene dicarboxylate (2.0 mmol)
was slowly added through an addition funnel and fnally the
respective aromatic aldehyde was also added (2.0 mmol).
The reaction mixture was heated at 60–70 °C until comple-
tion of the reaction as indicated by TLC. The reaction mix-
ture was cooled at room temperature and β-cyclodextrin was
fltered; the aqueous phase was extracted with ethyl acetate
1659, 1510, 1201, 837 cm−1; MS (DART+): m/z calcd for
12
C
1H1635Cl119F114N116O4 ([M+H]+]): 376.07519, found
19
376.07514 (for 2D NMR experiment see supplementary
material).
Ethyl 4‑[(4‑bromophenyl)amino]‑2‑(4‑chlorophenyl)‑5‑oxo‑
2 , 5 ‑ d i h y d r o f u r a n ‑ 3 ‑ c a r b o x y l a t e
( 1 6 ,
C
19H16BrClNO4) Rf = 0.35 (EtOAc); yield: 85%; m.p.:
1 3

1848
V. A. Castro-Torres et al.
1
170–172 °C; HPLC purity: 99%; H NMR (300 MHz,
DMSO-d6): δ = 7.57–7.53 (m, 2H), 7.47 (d, J = 8.9 Hz,
2H), 7.36 (m, 4H), 6.07 (s, 1H), 4.20–3.87 (m, 2H), 1.04
(t, J=7.1 Hz, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): δ
= 164.54, 162.14, 153.89, 136.03, 135.59, 132.36, 131.57,
129.63, 128.23, 124.19, 117.64, 110.80, 59.64, 59.54,
14.01 ppm; IR (KBr): ꢀ = 3295, 1722, 1682, 1660, 1589,
Cytotoxicity assay
Cell culture: Roswell Park Memorial Institute medium
(RPMI-1640), FBS (fetal bovine serum, BIO-S1650-500)
and Trypsin (BIO-L0931-100) were obtained from Bio-
west company). Dimethyl sulfoxide and Cisplatin (cis-
diammineplatinum(II) dichloride) were obtained from
Sigma-Aldrich.
1489, 828 cm−1; MS (DART+): m/z calcd for 12C19 H1681Br-
1
35Cl114N116O4 ([M + H]+]): 437.69534, found 437.69547
The cytotoxicity of compounds 1–19 was evaluated
against HCT-116 (human colon carcinoma) and PC-3
(human prostate cancer) cell lines obtained from the Amer-
ican Type Culture Collection (ATCC). Cells were cul-
tured in RPMI-1640 medium supplemented with 10% fetal
bovine serum, 2 mM L-glutamine, 100 IU/cm3 G-penicilin,
100 μg/cm3 streptomycin, and 0.25 µg/cm3 B-amphotericin
and incubated at 37 °C under a 5% CO2 atmosphere. For
experiments, cells were seeded in 96-well plates (0.7×105
for adherent cells in 100 mm3 medium). After 24 h, fura-
nones 1–20 (0.39–25 μg/cm3) dissolved in DMSO (5%) were
added to each well and incubated for 2 days (48 h) before the
sulforhodamine B. Results are summarized in Table 1; cis-
platin was used as a reference compound. The cytotoxicity
of 1–4 against primary fbroblast cells (HGF-1) was evalu-
ated according to a previous method reported [16].
1
(for 2D NMR experiment see supplementary material).
E t h y l 2 ‑ ( 4 ‑ c h l o r o p h e n y l ) ‑ 4 ‑ [ ( 4 ‑ i o d o p h e n y l ) ‑
amino]‑5‑oxo‑2,5‑dihydrofuran‑3‑carboxylate (17,
C19H17ClNIO4) Rf = 0.30 (EtOAc); yield: 75%; m.p.: 125–
127 °C; HPLC purity: 98%; 1H NMR (300 MHz, DMSO-d6):
δ = 7.61 (d, J=8.6 Hz, 2H), 7.41 (d, J=8.6 Hz, 2H), 7.22
(s, 4H), 5.84 (s, 1H), 4.20–3.66 (m, 2H), 1.04 (t, J=7.1 Hz,
3H) ppm; 13C NMR (75 MHz, DMSO-d6): δ = 166.98,
164.67, 151.93, 137.79, 136.85, 136.63, 132.43, 129.92,
128.52, 124.57, 114.27, 90.34, 59.80, 59.49, 14.53 ppm;
12
MS (DART +): m/z calcd for
C
1H1735Cl1127I114N116O4
19
([M + H]+]): 483.97319, found 483.97370 (for 2D NMR
experiment see supplementary material).
Ethyl 4‑[(4‑iodophenyl)amino]‑5‑oxo‑2‑phenyl‑2,5‑dihydro‑
furan‑3‑carboxylate (18, C19H16INO4) Rf = 0.35 (EtOAc);
yield: 75%; m.p.: 185–187 °C; HPLC purity: 99%; 1H NMR
(300 MHz, CDCl3): δ = 7.71–7.47 (m, 2H), 7.42–7.04 (m,
7H), 5.69 (s, 1H), 4.18 (q, J=7.1 Hz, 2H), 1.17 (t, J=7.2 Hz,
3H) ppm; 13C NMR (75 MHz, CDCl3): δ = 165.20, 162.93,
156.46, 138.09, 136.28, 134.86, 128.87, 127.56, 123.67,
113.44, 90.11, 61.48, 61.35, 31.71, 22.78, 14.03 ppm; IR
TBARS assay
The experiments were performed according to a previously
protocol reported [18]. Adult female rats were used under
approval of NOM-062-ZOO-1999. Animals were euthanized
in CO2 chamber. The whole brain homogenized in PBS
solution (0.2 g of KCl, 0.2 g of KH2PO4, 8 g of NaCl, and
2.16 g of NaHPO4·7 H2O/dm3, pH adjusted to 7.4). It was
used thiobarbituric acid (TBA) 1% in 0.05 N NaOH solu-
tion mixed with 30% trichloroacetic acid in 1:1 proportion.
Protein content in brain supernatant solutions was meas-
ured using the Folin and Ciocalteu’s phenol reagent. Lipid
peroxidation was induced with EDTA (100 µM) according
to protocol, during 5 h at one-hour intervals. 0.5 cm3 of
TBA solution were taken for each tube and cooled on ice for
10 min, centrifuged to 12 000 rpm during 5 min, and fnally
heated at 95 °C for 30 min. The tubes were allowed to reach
room temperature and 200 mm3 of supernatant were taken
for analysis. TBARS content was analyzed using optical
density at λ=540 nm using Bio-Tek EL microplate reader.
Results were analyzed using Origin 2020 software.
(KBr): ꢀ =1711, 1681, 1661, 1584, 1486, 1240, 823 cm−1;
12
MS (DART +): m/z calcd for
C
1H16127I114N116O4
19
([M + H]+]): 449.01240, found 449.01053 (for 2D NMR
experiment see supplementary material).
Ethyl 2‑(4‑chlorophenyl)‑4‑[(3,4‑difluorophenyl)‑
amino]‑5‑oxo‑2,5‑dihydrofuran‑3‑carboxylate (19,
C19H15ClFNO4) Rf = 0.40 (EtOAc), yield 20%; m.p.: 145–
147 °C; HPLC purity: 98%; 1H NMR (300 MHz, CDCl3):
δ = 7.46 (ddd, J = 9.6, 6.9, 2.2 Hz, 1H), 7.30–7.23 (m,
2H), 7.19–7.12 (m, 2H), 7.12–6.98 (m, 2H), 5.64 (s, 1H),
4.23 (q, J = 7.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H) ppm;
13C NMR (75 MHz, CDCl3): δ = 164.81, 162.66, 156.32,
151.68 (d, J = 12.6 Hz), 146.40 (d, J = 12.6 Hz), 134.75,
133.19, 132.46 (d, J = 12.7 Hz), 129.14, 128.74, 117.82,
117.60, 117.36, 113.01, 111.92, 111.77 (d, J = 21.4 Hz),
61.59, 60.94, 13.95 ppm; IR (KBr): ꢀ =1711, 1681, 1661,
Annexin V and propidium iodide assay
1584, 1486, 1240, 823 cm−1; MS (DART+): m/z calcd for
Apoptotic cell death was determined using FITC-Annexin-V
Apoptosis Detection Kit (Sigma Aldrich APOAF-50TST)
in accordance with the manufacturer’s protocol. HCT-116
and PC-3 cells were seeded in 6-well plates at a density
12
C
1H1535Cl119F214N116O4 ([M+H]+]): 394.06577 found,
19
394.05497 (for 2D NMR experiment see supplementary
material).
1 3

Synthesis and cytotoxic evaluation of halogenated furanones
1849
of 4.0 × 105 cells/cm3 in 3 cm3 of cell culture medium.
Cells were treated with concentrations (1.13, 0.73, 0.93,
26.25, 1.31 µM for PC-3 and 1.12, 0.54, 1.5, 14.06, 0.4 µM
for HCT-116) of each furanone (1–5) for 48 h, at 37 °C.
Untreated cells were used as a control. Then, cells were
washed with PBS, resuspended in the binding bufer and
stained with FITC annexin-V and propidium iodide (PI), fol-
lowed by incubation in the dark at 25 °C for 15 min. Finally,
fow cytometry analysis of the cells was performed using
Attune Acoustic Focusing Flow Cytometer (Red-Blue laser)
from Thermo Scientifc in National Flow Cytometry Labora-
tory, 20.000 cells were analyzed. Data were analyzed using
FlowJo Software v10.6.2 (Fig. 1).
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Statistical analysis
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All experiments were assayed in triplicate (n=3). Data are
expressed as means SEM. All statistical analyses were
performed using GraphPad Pro. Prism 5.0 (GraphPad, San
Diego, CA). Student’s t test and two-way ANOVA were
employed to analyze the diferences between sets of data. A
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Acknowledgements Víctor A. Castro-Torres is a doctoral student from
the Programa de Doctorado en Ciencias Biomédicas, Universidad
Nacional Autónoma de México (UNAM) and has received CONACyT
fellowship 267787. This study was supported by Grants from PAPIIT
IN211819 and CONACyT 402838 and 00042. We acknowledge the
technical support of MS Antonio Nieto-Camacho and MS María Teresa
O. Ramírez-Apan from Instituto de Química-UNAM.
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
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