Retro-curcuminoids as mimics of dehydrozingerone and curcumin: Synthesis, NMR, X-ray, and cytotoxic activity

Article abstract of DOI:10.3390/molecules22010033

Curcumin and its derivatives have been extensively studied for their remarkable medicinal properties, and their chemical synthesis has been an important step in the optimization of well-controlled laboratory production. A family of new compounds that mimic the structure of curcumin and curcuminoids, here named retro-curcuminoids (7-14), was synthesized and characterized using 1D ¹H- and ¹³C-NMR, IR, and mass spectrometry; the X-ray structure of 7, 8, 9, 10, 12, 13, and 14 are reported here for the first time. The main structural feature of these compounds is the reverse linkage of the two aromatic moieties, where the acid chloride moiety is linked to the phenolic group while preserving α,β-unsaturated ketone functionality. The cytotoxic screening of 7, 8, 9, and 10 at 50 and 10 μg/mL was carried out with human cancer cell lines K562, MCF-7, and SKLU-1. Lipid peroxidation on rat brain was also tested for compounds 7 and 10. Compounds 7, 8, and 10 showed relevant cytotoxic activity against these cancer cell lines, and 10 showed a protective effect against lipid peroxidation. The molecular resemblance to curcuminoids and analogs with ortho substituents suggests a potential source of useful bioactive compounds.

Full text of DOI:10.3390/molecules22010033

molecules
Article
Retro-Curcuminoids as Mimics of Dehydrozingerone
and Curcumin: Synthesis, NMR, X-ray,
and Cytotoxic Activity
1
1
1
Marco A. Obregón-Mendoza , María Mirian Estévez-Carmona , Simón Hernández-Ortega ,
1
1
2
2
Manuel Soriano-García , María Teresa Ramírez-Apan , Laura Orea , Hugo Pilotzi ,
2
3,
1,
Dino Gnecco , Julia Cassani * and Raúl G. Enríquez *
1
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior,
Ciudad Universitaria, México, D.F. C.P. 04510, Mexico; obregonmendoza@yahoo.com.mx (M.A.O.-M.);
mirianestevezc@gmail.com (M.M.E.-C.); shernandezortega@gmail.com (S.H.-O.);
soriano@unam.mx (M.S.-G.); mtrapan@yahoo.com.mx (M.T.R.-A.)
2
Centro de Química, Benemérita Universidad Autónoma de Puebla, Puebla C.P. 72570, Mexico;
maria.orea@correo.buap.mx (L.O.); iken18@hotmail.com (H.P.); gneccod@yahoo.com.mx (D.G.)
Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Unidad Xochimilco,
3
México, D.F. C.P. 04960, Mexico
*
Correspondence: cassani@correo.xoc.uam.mx (J.C.); enriquezhabib@gmail.com (R.G.E.);
Tel.: +52-55-5483-7255 (J.C.); +52-55-5622-4404 (R.G.E.)
Academic Editor: Derek J. McPhee
Received: 24 November 2016; Accepted: 16 December 2016; Published: 29 December 2016
Abstract: Curcumin and its derivatives have been extensively studied for their remarkable
medicinal properties, and their chemical synthesis has been an important step in the optimization
of well-controlled laboratory production. A family of new compounds that mimic the structure
of curcumin and curcuminoids, here named retro-curcuminoids (
7
–
14), was synthesized and
characterized using 1D H- and C-NMR, IR, and mass spectrometry; the X-ray structure of
10 12 13, and 14 are reported here for the first time. The main structural feature of these
compounds is the reverse linkage of the two aromatic moieties, where the acid chloride moiety is
linked to the phenolic group while preserving -unsaturated ketone functionality. The cytotoxic
screening of , and 10 at 50 and 10 g/mL was carried out with human cancer cell lines K562,
MCF-7, and SKLU-1. Lipid peroxidation on rat brain was also tested for compounds and 10
Compounds , and 10 showed relevant cytotoxic activity against these cancer cell lines, and 10
1
13
7
,
8,
9
,
,
,
α, β
7,
8
,
9
µ
7
.
7, 8
showed a protective effect against lipid peroxidation. The molecular resemblance to curcuminoids
and analogs with ortho substituents suggests a potential source of useful bioactive compounds.
Keywords: retro-curcuminoids; curcuminoid; curcumin; α,β-unsaturated-ketone; dehydrozingerone
1
. Introduction
Curcumin (diferuloylmethane), a natural product used in traditional medicine, has been
extensively studied for its multiple pharmacological effects on biological systems, to understand
its chemical properties, and to improve its poor bioavailability [ ]. A large number of studies have
described the multiple beneficial medicinal properties from different points of view [ ]. Curcumin
1
2
has been used as a naturally-occurring resource for the treatment of numerous inflammatory diseases,
and studies have confirmed that it possesses antiproliferative activity against several tumor cells
in vitro
scavenging activity [
recently as a molecular probe in the diagnostic imaging technique near-infrared fluorescence (NIRF),
[
3]. Previous reports have described curcumin as a potent antioxidant with free radical
4
] and a well-described therapeutic agent against Alzheimer disease [ ] or
5,6
Molecules 2017, 22, 33; doi:10.3390/molecules22010033
www.mdpi.com/journal/molecules

Molecules 2017, 22, 33
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which is useful in the monitoring of amyloid beta species on Alzheimer patients [
7]. Moreover, several
chemical analogs, derivatives, and precursors of curcumin, both natural and synthetic, have shown
such activity and also action against inflammatory diseases [8,9].
In 1973, Roughley and Whiting [10] reported the instability of curcumin in alkaline medium
and explained its alkaline degradation. Later, Sardjiman and co-workers [11] reported that at pH
higher than 6.5, curcumin decomposes into several molecular fragments, mainly ferulic acid and
dehydrozingerone (DHZ), as shown in Scheme 1.
Scheme 1. Degradation products of curcumin.
Ferulic acid, a precursor of the biosynthesis of curcumin, has been described as a compound with
antitumoral, chemo-preventive, and photo-protective biological properties [12]. DHZ is a phenolic compound
structurally related to biosynthesis or degradation of curcumin and is present as a minor component
in the rhizomes of Zingiber officinale Roscoe (ginger). In numerous studies, DHZ has been shown to
have radioprotective, antimicrobial, pro-healing, antioxidant, anti-Parkinson, anti-Alzheimer, antimutagenic,
antitumor, anti-cancer, anti-depressant, anti-platelet, and antidiarrheal pharmacological activities [13–16].
Ferulic acid and DHZ are versatile chemical intermediates in organic synthesis with several
bonding possibilities. We, therefore, decided to explore a modified linkage of such moieties involving
the bond of the phenolic group of DHZ and related compounds, for example, acetylated ferulic acid
[
(E)-4-(3-chloro-3-oxoprop-1-en-1-yl)-2-methoxyphenyl acetate)], via the acid chloride derivative of the
carboxylic functionalities and the corresponding phenolic group of the second moiety. The synthesis
of what we call retro-curcuminoids was achieved by reaction of the corresponding acid chlorides with
the appropriate phenolic moieties. We also explored the inhibition of lipoperoxidation and cytotoxic
activity of the new compounds.
2
. Results and Discussion
2
.1. Structural Features
Retro-curcuminoids
chloro-3-oxoprop-1-en-1-yl)-2-methoxyphenyl acetate (
but-3-en-2-one] ( ), and DHZ analogs (E)-4-(2-hydroxyphenyl)but-3-en-2-one, (E)-4-(3-hydroxyphenyl)
7–
14 were prepared by esterification of cinnamoyl chloride (
1) or (E)-4-(3-
2) with DHZ [(E)-4-(4-hydroxy-3-methoxyphenyl)
6
but-3-en-2-one, and (E)-4-(4-hydroxyphenyl)but-3-en-2-one (3, 4, and 5), with ortho, meta and para
hydroxyl substituents, respectively, as shown in Scheme 2.
Scheme 2. General synthesis.

Molecules 2017, 22, 33
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2
.2. Crystallography
The molecular structures of all seven retro-curcuminoids are formed by two aromatic rings
phenyl-C-; phenyl-O-) connected by a moiety C(sp2)-C(sp2)-C(sp2)-C(sp2)-O(sp3)-C(sp2) chain of
atoms (Figure 1).
(
Compound 7
Compound 8
Compound 9
Compound 10
Figure 1. Cont.

Molecules 2017, 22, 33
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Compound 12
Compound 13
Compound 14
Figure 1. View of the molecular structure of compounds 7, 8, 9, 10, 12, 13, and 14.
The distance between the centers of gravity of phenyl Cg rings in structures 10
7,
8,
9,
,
12
,
13, and
1
4
remain constant independent of the type and location of ring substituents. These values are 8.795 Å,
8.807 Å, 8.795 Å, 8.794 Å, 8.830 Å, 8.747 Å, and 8.400 Å. Table 1 shows the crystal data, collection
parameters and refinements for compounds 7, 8, 9, 10, 12, 13 and 14.
The crystal packing of the molecules in the crystal structures is stabilized by van der Waals
interactions and linked by intermolecular C-H•••O hydrogen interactions. The adjacent molecules are
interconnected by intermolecular weak C-H•••π interactions (from different Cg centers of gravity),
which give additional support to molecular packing stability. Table 2 shows the intermolecular
C-H•••O interactions in all seven crystal structures.

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Table 1. Crystal data, collection parameters, and refinements for compounds 7–10 and 12–14.
Compound
CCDC deposit No.
Crystal size (mm)
Color/shape
Empirical formula
Formula weight
Crystal system
Space group
Unit cell dimensions
a, Å
7
8
1451557
9
10
1451555
12
1451617
13
1451556
14
1452007
1451621
1056387
0.37 × 0.05 × 0.03
Light green/needles
C19H16O3
0.30 × 0.20 × 0.09
Light yellow/needles
C19H16O3
0.39 × 0.19 × 0.13
Colorless/prism
C19H16O3
292.32
0.37 × 0.11 × 0.11
Light green/needles
C22H20O6
0.31 × 0.12 × 0.05
Colorless/prism
C22H20O6
380.38
0.35 × 0.21 × 0.15
Light yellow/needles
C20H18O4
322.34
Not determined
Colorless/prism
C23H22O7
292.32
292.32
380.38
410.40
Orthorhombic
P 212121
Monoclinic
P 21/c
Monoclinic
P 21/c
Monoclinic
P 21/c
Monoclinic
P 21/c
Triclinic
Monoclinic
P 21/c
P-1
5.7012(2)
15.1134
18.1035
90.00
33.3788(14)
5.7488(2)
8.0128(4)
90.00
19.2677(6)
4.10240(10)
21.2526(6)
90.00
6.07840(10)
17.3377(4)
18.3150(5)
90.00
22.6222(13)
7.6843(4)
10.9077(7)
90.00
7.9343(10)
10.0440(14)
11.9990(16)
70.652(4)
71.023(4)
89.865(5)
847.2(2)
2
5.9945(4)
36.2260(19)
9.6239(8)
90.00
b, Å
c, Å
α, deg
β, deg
90.00
95.736(2)
90.00
115.0910(10)
90.00
90.5980(10)
90.00
95.314(2)
90.00
91.083(6)
90.00
γ, deg
Volume, Å3
90.00
1559.88(10)
4
1529.86(11)
4
1521.36
4
1930.03(8)
4
1888.00(19)
4
2089.5(2)
4
Z
Density (calculated), Mg/m3
1.245
1.269
1.276
1.309
1.338
1.264
1.305
Absorption coefficient, mm 1
−
0.084
0.085
0.086
0.095
0.098
0.088
0.806
F(000)
616
616
616
800
800
340
864
◦
Θ range for data collection ( )
2.25 to 25.39
2.45 to 25.31
1.94 to 25.40
2.22 to 25.50
2.71 to 25.32
2.16 to25.33
Not determined
−
6
≤ η ≤ 6,
−
17 ≤ κ ≤ 17,
−
39 ≤ η ≤ 39,
−
5
≤ κ ≤ 5,
−
23 ≤ η ≤ 23,
− ≤ κ ≤ 4, −7 ≤ h ≤ 7, −20 ≤ k ≤ 20, −27 ≤ h ≤ 27, −9 ≤ k ≤ 9, −9 ≤ h ≤ 9, −12 ≤ k ≤ 12, −7 ≤ h ≤ 6, −44 ≤ k ≤ 44,
4
Index ranges
−
21 ≤ λ ≤ 21
−9 ≤ λ ≤ 9
−25 ≤ λ ≤ 25
−22 ≤ l ≤ 22
26,381
−13 ≤ l ≤ 13
22,547
−14 ≤ l ≤ 14
12,126
−11 ≤ l ≤ 11
20,379
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
13,670
20,778
16,473
2857 (Rint = 0.1503)
2857/154/246
0.749
2764 (Rint = 0.1323)
2764/0/200
1.028
2781 (Rint = 0.0559)
2781/162/246
1.052
3585 (Rint = 0.0925)
3585/0/257
0.963
3433 (Rint = 0.1724)
3433/0/256
1.010
3065 (Rint = 0.0631)
3065/0/219
1.046
4128
4128/0/275
1.020
Final R indices
R1 = 0.0472,
wR2 = 0.0486
R1 = 0.0494,
wR2 = 0.1162
R1 = 0.0489,
wR2 = 0.1271
R1 = 0.0461,
wR2 = 0.1072
R1 = 0.0606,
wR2 = 0.1255
R1 = 0.0632,
wR2 = 0.1629
R1 = 0.0620,
wR2 = 0.1162
[
(I) > 2σ(I)]
R1 = 0.2148,
wR2 = 0.0749
R1 = 0.0816,
wR2 = 0.1361
R1 = 0.0676,
wR2 = 0.1422
R1 = 0.0851,
wR2 = 0.1267
R1 = 0.1493,
wR2 = 0.11618
R1 = 0.0888,
wR2 = 0.1831
R1 = 0.1574,
wR2 = 0.1945
R indices (all data)
Largest diff. peak and hole (e/A 3
−
)
0.168 and −0.141
0.140 and −0.171
0.156 and −0.227
0.279 and −0.258
0.195 and −0.185
0.435 and −0.190
0.305 and −0.160

Molecules 2017, 22, 33
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◦
Table 2. Geometry of the hydrogen bonding interactions (Å, ) for compounds 7, 8, 9, 10, 12, 13, and 14.
Compound
7
D-H···A
D-H
H···A
D···A
D-H···A
Symmetry Code
C3-H3•••O3
0.930(5)
0.930(5)
0.930(5)
0.931(11)
2.790(2)
2.749(1)
2.902(3)
2.740(3)
3.486(2)
3.463(2)
3.571(6)
3.356(12)
132.5(4)
134.3(4)
130.0(3)
124.5(7)
−x + 1
−x + 1
x − 1/2
x + 1
+y + 1/2,
+y + 1/2
−y + 1/2 + 1
+y
−z + 1/2
C9-H9•••O3
−z + 1/2
−z
C14-H14•••O2
C17-H17•••O1
+z
C2-H2•••O2
C9-H9•••O2
C15-H15•••O2
0.930(2)
0.929(2)
0.931(2)
2.735(2)
2.957(2)
2.761(2)
3.665(2)
3.590(3)
3.543(2)
179.9(1)
126.7(2)
142.1(1)
x
x
x
+y − 1
−y + 1/2
+z
+z + 1/2
+z − 1/2
−z + 1/2
−z + 1/2
−z + 1/2
−z + 1/2
8
9
−y + 1/2 + 1
C5-H5•••O3
0.930(2)
0.930(2)
0.930(2)
0.930(3)
2.672(5)
2.719(4)
2.634(2)
2.605(1)
3.539(5)
3.588(4)
3.554(3)
3.534(3)
155.3(2)
155.8(1)
169.9(1)
177.2(2)
−x
−x
−x
−x
+y − 1/2
+y − 1/2
+y − 1/2
+y − 1/2
C3-H3•••O3
C12-H12•••O2
C17-H17•••O2
C10-H10A•••O2
C12-H12A•••O6
C17-H17•••O6
C20-H20•••O5
0.981(2)
0.980(2)
0.951(2)
0.951(2)
2.597(1)
2.661(2)
2.410(2)
2.484(2)
3.124(2)
3.414(3)
3.183(3)
3.246(3)
113.8(1)
133.9(2)
138.2(1)
137.1(2)
−x + 2
−y + 2
−y + 1/2+1
+y + 1/2
−z
x − 1
z − 1/2
−z + 1/2
−z + 1/2
1
1
1
1
0
2
3
4
−x + 2
−x + 2
x
−x + 1
−x + 1
−x + 2
−x + 2
+y + 1/2
C18-H18•••O1
0.930(3)
0.931(3)
0.929(3)
0.961(3)
0.959(3)
2.358(2)
2.376(2)
2.714(3)
2.680(2)
2.429(2)
2.846(4)
3.267(4)
3.431(4)
3.531(4)
3.372(4)
112.5(2)
160.2(2)
134.7(2)
147.9(2)
167.6(2)
y
z
C5-H5•••O6
−y
−z + 1
C20-H20•••O1
C12-H12A•••O3
C12-H12B•••O5
−y
−z + 1
+y + 1/2
+y + 1/2
z + 1/2 + 2
z + 1/2 + 2
C6-H6•••O1
C11-H11B•••O4
C18-H18•••O2
C19-H19•••O2
0.930(3)
0.960(3)
0.929(2)
0.930(4)
2.417(2)
2.825(2)
2.718(2)
2.846(2)
3.328(4)
3.475(4)
3.332(3)
3.397(4)
166.0(2)
125.8(2)
124.3(1)
119.1(2)
−x
x − 1
−x
−y + 1
−z
+y
+z
−y
−z + 1
−z + 1
−x
−y
C13-H13•••O11
0.929(3)
0.930(3)
0.931(3)
0.930(3)
0.960(4)
2.496(3)
2.581(3)
2.600(2)
2.672(2)
2.550(2)
3.413(4)
3.504(4)
3.499(4)
3.485(4)
3.475(4)
169.2(2) 1
172.0(2) 2
162.6(2) 2
146.3(2) 4
161.9(2) 5
−x + 1
−x + 1
−x + 1
−x
−y + 1
−y + 1
−y + 1
−y + 1
+y
−z
−z + 1
−z + 1
−z
C5-H5•••O20
C25-H25•••O20
C2-H2•••O22
C23-H23C•••O18
−x + 1
+z
In the molecular structures of
7, 8, 9, 10, 12, 13, and 14, the least-squares plane calculation shows
◦
◦
◦
◦
◦
that the dihedral angles between the two benzene rings are 80.7(1) , 11.9(7) , 61.6(5) , 79.7(6) , 38.7(1) ,
◦ ◦
8
7.2(1) , and 75.2(2) , respectively. The dihedral angles between the C-phenyl-ring and –O-phenyl
◦
◦
◦
◦
◦
ring and the central moiety for structures
7,
8,
9
,
10,
12
,
13, and 14 are 3.2(1) and 77.6(1) , 37.8(1) and
◦
◦
◦
◦
◦
◦
◦
◦
4
8.4(1) , 2.2(1) and 63.3(1) , 2.9(1) and 81.0(1) , 13.2(1) and 25.8(2) , 3.2(1) and 88.2(1) , and 6.4(1) ,
◦
and 77.5(1) , respectively. This clearly shows that in structures
7
,
9,
10,
13, and 14, the –C-phenyl is
coplanar with the central moiety.
This result agrees with the dihedral angle of 8.2 between the two benzene rings in the structure
of 1,7-bis(4-ethyloxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione [17]. In contrast, the dihedral angle
◦
◦
◦
between the –O-phenyl ring and the central moiety varies from 63.3 to 88.2 . These features are shown
in previous reports with -unsaturated aromatic ester compounds [18 22], where aromatic, double
α
,β
–
bond, and ester groups are in a planar configuration and the substituents bonded to ester groups show
some deviation. For structures 8 and 12 the dihedral angles show no significant difference.
2
.3. Cytotoxicity and Antioxidant Activity
Cytotoxic activity of all synthesized compounds was first measured in healthy and cancerous
cell lines. None of the compounds injured healthy cells; however, only compounds
showed potential cytotoxic activity. Compounds , and 10 (ortho, meta, and ortho substituents,
respectively) had IC50 values similar to curcumin and displayed better anticancer activity against
K562, MCF-7, and SKLU-1 cells than compound (Table 3). These results agree with a previous
7, 8, 9, and 10
7, 8
9
study of the cytotoxicity of dehydrozingerone derivatives, which reported that compounds with
hydroxyl at the ortho position, and ketone and phenyl at the C-3 and C-4 positions, respectively, are
necessary for optimal cytotoxic activity [8]. The presence of α,β unsaturated systems, which has
previously been described as an enhancer of cytotoxic activity, may also account for the biological
effect of retro-curcuminoids. The ketone form in particular enhances toxicity [23], and the molecules we

Molecules 2017, 22, 33
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synthesized have double
α
,β
unsaturated systems. The cytotoxic specificity of these new derivatives
of curcumin and dehydrozingerone is related to precise differences in chemical structure, which could
form the basis of future studies of the anticancer properties of retro-curcuminoids.
Table 3. Cytotoxicity screening of retro-curcuminoids 7, 8, 9, and 10.
IC50 (µM) on Cell Line
Retro-Curcuminoid
K562
MCF-7
SKLU-1
Curcumin
9.75 ± 0.6
10.74 ± 0.5
12.8 ± 1.9
10.43 ± 1.8
13.19 ± 1.4
18.68 ± 2.5
63.46 ± 10.8 *
12.01 ± 0.62
7.78 ± 0.64
11.59 ± 1.2
12.85 ± 0.9
63.81 ± 2.7 *
8.07 ± 0.55
7
8
9
28.15 ± 2.5 *
1
0
8.60 ± 0.44
Data are represented as mean
adenocarcinoma cells, HCT-15: human colon cancer cells, MCF-7: human mammary adenocarcinoma cells, and
SKLU-1: human lung adenocarcinoma cells.
±
standard error of mean (SEM). * p < 0.01 compared to curcumin. K562: leukemia
The effect on lipid peroxidation by compounds
only retro-curcuminoid showed a high degree of inhibition. The unique retro-curcuminoid with
antioxidant activity in vitro was compound 10, as shown in Table 4. In contrast with the reported high
antioxidant activity of curcumin and derivatives [ ], the retro-curcuminoids described here had no
7, 8, 9, and 10 was tested on rat brain, but
7
1
important prophylactic effect on the oxidative process, probably because of the structural occupation
of phenyl groups and the loss of free hydroxyls, chemical groups related to activity against free
radicals. Preliminary analysis of antioxidant activity in vitro by DPPH assay did not show antioxidant
properties of any of the retro-curcuminoids. These data suggest that changes in the chemical structure
of these compounds decrease antioxidant activity, but enhance their anticancer properties.
Table 4. Inhibition of lipid peroxidation of retro-curcuminoids 7, 8, 9, and 10 in rat brain.
Retro-Curcuminoid
Concentration (µg/mL)
% Inhibition
5
5
0
4.33 ± 1.06 *
7
1.97 ± 0.77 *
5
5
0
5
1.77 ± 0.22 *
0.27 ± 0.06 *
8
9
0
3.30 ± 0.78 *
5
3.19 ± 0.53 *
5
5
0
96.18 ± 6.53
14.81 ± 2.33
1
0
Data are represented as mean ± standard error of mean (SEM). * p < 0.01 compared to α-tocopherol.
3
. Experimental Section
3
.1. General Information
All chemicals were reagent grade and were used as received. Solvents were purified by standard
methods [24].
3
.2. Physical Measurements
Melting points were determined on an Electrothermal IA9100 digital melting point apparatus (Bibby
Scientific Limited, Staffordshire, UK) and are uncorrected. IR absorption spectra were recorded in the
−
1
1
4
000–400 cm range as KBr pellets on a Perkin Elmer 283-B spectrophotometer (PerkinElmer, Waltham,
13
MA, USA). H- and C-NMR spectra were recorded in CDCl on a Bruker 500 MHz (Billerica, MA,
3
USA) or a 600 MHz NMR Agilent technologies spectrometer equipped with a DD2 Oneprobe (Agilent

Molecules 2017, 22, 33
8 of 14
Technology, Santa Clara, CA, USA), using tetramethylasilane (TMS) as internal reference. NMR spectra
were processed with MESTRENOVA version 10.0.2 and can be found in the Supplementary Materials.
3
.3. Synthetic Procedures
Compounds and
thionyl chloride in CHCl containing a catalytic amount of dimethylformamide (DMF), and refluxed
1
2 were prepared in high yield from acetylated ferulic or cinnamic acid with
3
for 4 h according to the synthetic route reported [25,26]. The precursors 3, 4, 5, and 6 were obtained
using a Claisen-Schmidt reaction with acetone and corresponding aldehyde in equimolar amounts,
and sodium hydroxide as a powdered base, in accordance with a published synthetic method [27].
Retro-Curcuminoids 7–14
A solution of the corresponding acid chloride (
1
or
2
) (1.0 eq in anhydrous THF) was added to a
◦
solution of DHZ analog (
3–
5) or DHZ (
6
) (1.0 eq in anhydrous THF) at 0 C containing triethylamine
◦
(
1.2 eq). The reaction mixture was stirred at 30 C until the disappearance of the starting material was
observed by TLC. The reaction was quenched by the addition of aqueous NH Cl, and the mixture was
4
extracted with EtOAc (3
×
15 mL). The combined extracts were dried over anhydrous Na SO and
2 4
filtered, and the solvent was removed in vacuo. The resulting precipitate was treated with adequate
solvents to give the corresponding crystalline product.
2
-((E)-3-oxobut-1-en-1-yl)phenyl cinnamate (
7
). Obtained as light green needles From ethyl ether/n-hexane
90%), m.p. 138.9 C. H-NMR (500 MHz, Chloroform-d) (ppm) 7.93 (dd, J = 16.1, 0.5 Hz, 1H), 7.72–7.58
m, 4H), 7.49–7.41 (m, 4H), 7.30 (dddd, J = 7.9, 7.4, 1.2, 0.6 Hz, 1H), 7.22 (ddd, J = 8.2, 1.3, 0.4 Hz, 1H), 6.72
dd, J = 18.8, 16.2 Hz, 2H), 2.34 (s, 3H). 13C-NMR (125 MHz, CDCl3)
(ppm) 198.03, 165.02, 151.61, 149.65,
47.59, 136.58, 133.92, 131.31, 130.99, 129.04, 128.87, 128.45, 127.53, 127.34, 126.35, 123.19, 116.38, 27.62.
◦
1
(
(
(
δ
δ
1
−
1
−1
−1
−1
−1
−1
−1
IR; 3060.72 cm , 3030.22 cm , 1728.16 cm , 1634.69 cm , 1600.59 cm , 1133.75 cm , 984.56 cm
m/z [293 ].
.
+
3
-((E)-3-oxobut-1-en-1-yl)phenyl cinnamate (8). Light yellow needless from CH Cl /n-hexane (90%), m.p.
2 2
◦
1
1
2
2
1
3
00 C. H-NMR (500 MHz, Chloroform-d)
δ
(ppm) 7.89 (d, J = 16.0 Hz, 1H), 7.60 (dd, J = 16.0, 3.0 Hz,
H), 7.53–7.35 (m, 7H), 7.22 (dt, J = 6.9, 2.3 Hz, 1H), 6.72 (d, J = 16.0 Hz, 1H), 6.64 (d, J = 16.0 Hz, 1H),
.38 (s, 3H). 13C-NMR (125 MHz, CDCl3)
(ppm) 198.10, 165.16, 151.25, 147.04, 142.25, 136.05, 134.02,
30.84, 130.59, 129.95, 129.06, 129.02, 128.34, 128.26, 127.92, 125.75, 123.66, 121.06, 116.90, 27.67. IR;
δ
−
1
1
−1
−1
−1
−1
−1
−1
052.19 cm , 2923.64 cm , 1717.09 cm , 1663.80 cm , 1631.63 cm , 1310.01 cm , 1257.60 cm ,
−1
+
1
150.97 cm , 977.46 cm . m/z [293 ].
4
1
-((E)-3-oxobut-1-en-1-yl)phenyl cinnamate (
9
). Colorless needles from EtOAc/n-hexane (90%), m.p.
39.15 C. H-NMR (500 MHz, Chloroform-d) (ppm) 7.87 (dd, J = 16.0, 15.9 Hz, 2H), 7.63–7.47 (m, 4H),
.48–7.37 (m, 3H), 7.29–7.20 (m, 2H), 6.66 (d, J = 16.0 Hz, 1H), 6.53 (d, J = 15.9 Hz, 1H), 2.38 (s, 3H).
◦
1
δ
7
13
13
C-NMR (CDCl3). C-NMR (125 MHz, CDCl3)
δ (ppm) 198.17, 164.96, 162.43, 152.44, 148.60, 147.08,
1
1
1
42.29, 133.98, 133.69, 132.03, 131.23, 130.84, 129.37, 129.03, 128.99, 128.54, 128.31, 127.13, 122.25, 116.84,
−
1
−1
−1
−1
−1
−1
16.73, 27.56. IR; 3062.07 cm , 3027.85 cm , 2926.10 cm , 1726.81 cm , 1662.5 cm , 1629.55 cm ,
−
1
−1
−1
+
214.85 cm , 1140.63 cm , 978.60 cm . m/z [293 ].
2
-((E)-3-oxobut-1-en-1-yl)phenyl(E)-3-(4-acetoxy-3-methoxyphenyl) acrylate (10). Light green needles from ethyl
◦
1
ether/n-hexane (90%), m.p. 160 C. H-NMR (500 MHz, Chloroform-d)
.66 (dd, 2H), 7.49–7.41 (m, 1H), 7.33–7.17 (m, 4H), 7.10 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 16.3 Hz, 1H), 6.64
d, J = 16 Hz, 1H), 3.90 (s, 3H), 2.34 (s, 6H). 13C-NMR (125 MHz, Chloroform-d)
(ppm) 198.02, 168.65,
64.89, 151.54, 149.58, 146.81, 142.04, 136.49, 132.80, 131.32, 128.82, 127.52, 127.30, 126.39, 123.41, 123.15,
δ (ppm) 7.88 (d, J = 16.0 Hz, 1H),
7
(
δ
1
1
1
−1
−1
−1
−1
21.69, 116.55, 111.53, 55.97, 27.69, 20.61. IR; 3072.19 cm , 3011.55 cm , 2956.45 cm , 1759.78 cm ,
− −1 −1 −1 −1 −1
733.54 cm , 1604.83 cm , 1258.74 cm , 1201.97 cm , 1120.02 cm , 984.99 cm . m/z [381+].
1
3
-((E)-3-oxobut-1-en-1-yl)phenyl(E)-3-(4-acetoxy-3-methoxyphenyl) acrylate (11). Colorless needles from
◦
1
CH Cl /ethyl ether/n-hexane (90%), m.p. 101.6 C. H-NMR (500 MHz, Chloroform-d)
δ 7.84
2
2

Molecules 2017, 22, 33
9 of 14
(
6
d, J = 16.0 Hz, 1H), 7.50 (d, J = 16.3 Hz, 1H), 7.47–7.36 (m, 3H), 7.25–7.15 (m, 3H), 7.10 (d, J = 8.1 Hz, 1H),
.72 (d, J = 16.3 Hz, 1H), 6.58 (d, J = 15.9 Hz, 1H), 3.89 (s, 3H), 2.38 (s, 3H), 2.34 (s, 3H). 13C-NMR
(
126 MHz, CDCl3)
δ
(ppm) 198.07, 168.67, 164.98, 151.50, 151.17, 146.24, 142.18, 141.89, 136.06, 132.93,
−
1
1
2
9
29.94, 127.91, 125.77, 123.60, 123.38, 121.50, 121.00, 117.07, 111.48, 55.92, 27.64, 20.60. IR; 3070.16 cm
,
,
−
1
−1
−1
−1
−1
−1
−1
945.64 cm , 1762.35 cm , 1720.60 cm , 1611.18 cm , 1237.04 cm , 1191.03 cm , 1150.22 cm
−
1
+
75.91 cm . m/z [381 ].
4
-((E)-3-oxobut-1-en-1-yl)phenyl(E)-3-(4-acetoxy-3-methoxyphenyl) acrylate (12). Colorless needles from
◦
1
EtOAc/n-hexane (88%), m.p. 172 C. H-NMR (500 MHz, Chloroform-d)
.60 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 16.3 Hz, 1H), 7.26–7.15 (m, 4H), 7.10 (d, J = 8.1 Hz, 1H), 6.70
d, J = 16.2 Hz, 1H), 6.58 (d, J = 15.9 Hz, 1H), 3.89 (s, 3H), 2.39 (s, 3H), 2.34 (s, 3H). 13C-NMR (125 MHz,
δ (ppm) 7.84 (d, J = 15.9 Hz, 1H),
7
(
CDCl ) δ (ppm) 198.11, 168.64, 164.78, 152.34, 151.47, 146.29, 142.21, 141.89, 132.89, 132.08, 129.37, 127.16,
3
−
−1
1
−1
−1
−1
−1
1
1
1
23.38, 122.21, 121.50, 117.02, 111.46, 55.93, 27.58, 20.61. IR; 3066.07 cm , 2982.16 cm , 1757.33 cm
725.80 cm , 1632.44 cm , 1585.39 cm , 1256.04 cm , 1203.46 cm , 1166.33 cm , 1119.10 cm
012.64 cm , 974.26 cm . m/z [381 ].
,
,
−
1
−1
−1
−1
−1
−1
+
2
-Methoxy-4-((E)-3-oxobut-1-en-1-yl)phenyl cinnamate (13). Obtained as yellow needles from EtOAc/n-
◦
1
hexane (80%), m.p. 132 C. H-NMR (600 MHz, Chloroform-d)
J = 6.6, 2.9 Hz, 2H), 7.49 (d, J = 16.2 Hz, 1H), 7.42 (dd, J = 4.9, 1.9 Hz, 3H), 7.16 (d, J = 3.7 Hz, 3H), 6.67
dd, J = 16.1, 9.1 Hz, 2H), 3.87 (s, 3H), 2.39 (s, 3H). 13C-NMR (125 MHz, CDCl3)
(ppm) 198.10, 164.59,
51.59, 146.95, 145.68, 143.71, 142.67, 141.69, 134.06, 133.29, 130.71, 128.92, 128.29, 127.23, 123.40, 121.49,
δ (ppm) 7.88 (d, J = 16.0 Hz, 1H), 7.59 (dd,
(
δ
1
1
1
−1
−1
−1
−1
−1
16.57, 111.30, 55.92, 27.46. IR; 3069.35 cm , 3012.15 cm , 2933.29 cm , 1733.69 cm , 1254.52 cm ,
122.37 cm , 1030.06 cm , 971.39 cm . Calculated mass 322 (not observed).
−1
−1
−1
2
-Methoxy-4-((E)-3-oxobut-1-en-1-yl)phenyl(E)-3-(4-acetoxy-3-methoxyphenyl) acrylate (14). Colorless
◦
1
needles from ethyl ether/n-hexane (80%), m.p. 181 C. H-NMR (600 MHz, Chloroform-d)
.84 (d, J = 15.9 Hz, 1H), 7.50 (d, J = 16.3 Hz, 1H), 7.17 (d, J = 7.3 Hz, 5H), 7.09 (d, J = 8.0 Hz, 1H), 6.68
d, J = 16.3 Hz, 1H), 6.61 (d, J = 15.9 Hz, 1H), 3.89 (s, 6H), 2.40 (s, 3H), 2.34 (s, 3H); 13C-NMR (CDCl3)
δ = 20.63, 27.30, 27.60, 55.90, 111.35, 116.70, 121.50, 123.40, 133.4, 141.70, 142.76, 146.29, 151.56, 164.60,
δ (ppm)
7
(
−
−1
1
−1
−1
−1
−1
−1
1
1
68.6, 198.3. IR; 3079.23 cm , 3010.78 cm , 1758.89 cm , 1722.39 cm , 1196.83 cm , 1152.87 cm ,
120.88 cm , 1027.43 cm 978.07 cm . m/z [411 ].
−
1
−1
+
3
.4. Crystallography
The X-ray data for 14 was collected by using an Agilent-Gemini-Atlas area-detector diffractometer
with CuK (1.54184 Å) radiation at room temperature (293 K). The data reduction was carried out using
α
CrysAlisPro (v38.43), and the absorption corrections were applied using the CrysAlisPro analytical
method [28]. Crystallographic data, together with refinement details for compound 14, are deposited at
the Cambridge Crystallographic Data Center (CCDC) (No. 1056387). The X-ray data for compounds
10 12, and 13 were collected using a Bruker Smart APEX AXS CCD area detector with a graphite
monochromator and Mo K radiation ( = 0.71073 Å) at room temperature in scan mode. Collected data
7,
8,
9,
,
α
λ
were reduced using SAINT [29] and empirical absorption corrections were performed using SADABS
software [29,30]. Crystallographic data together with refinement details are summarized in Table 1.
2
All reflections were defined based on F . The weighted R-factor wR and goodness of fit S are based
2
2
on F , and conventional R-factors R are based on F, with F set to zero for negative F . The threshold
2
2
expression of F >
σ
(F ) is used only for calculating R-factors (gt), etc., and is not relevant to the choice
2
of reflections for refinement. R-factors based on F are statistically about twice as large as those based
on F, and R-factors based on all data will be even larger. The crystallographic formulas for R , R ,
int
1
wR and S were used for refinement of structures.
2
ꢀ
ꢁ
ꢂꢀ
ꢀ
2
2 ꢀ
∑
F − F
∑ k Fo| − |Fc k
∑|Fo|
o
o
Rint
=
, R =
,
1
2
∑
F
o

Molecules 2017, 22, 33
10 of 14
v
u
u
t
s
2
2
2
2
2
2
∑
w(F − F )
∑ w(F − F )
o
c
o
c
wR =
, S =
2
2
2
m − n
∑
w(F )
o
The hydrogen atoms attached to the carbon atoms were placed in their calculated positions and
included in the isotropic refinement using the riding model with C–H = 0.93 Å (
Å with Uiso (H) = 1.2 Ueq (C-H) and 1.5 Ueq (C-H3).
−
CH) or 0.96 Å (
−
CH3)
Figure 1 shows the molecular structures and atom-labeling scheme for compounds
7,
8,
9,
10
,
1
2
,
13, and 14. All structures were drawn with 50% displacements using Mercury 3.7 software for
Windows [31]. The geometry of the molecules was calculated using PARST software [32].
Atomic coordinates and displacement parameters, and bond distances and bond angles for all
seven retro-curcuminoids are given as Supplementary Materials.
3
.5. Cell Lines, Culture Medium, and Cytotoxicity Assay
All synthetized compounds and precursors DHZ and ferulic acid, were initially screened in vitro
at doses of 10
(
(
µ
g/mL and 50
human chronic myelogenous leukemia), MCF-7 (human mammary adenocarcinoma), and SKLU-1
human lung adenocarcinoma). No cytotoxic activity was detected for DHZ and ferulic acid, although
16]. After selection
, and 10 were calculated in triplicate
µg/mL against human polymorphonuclear and cancer cell lines: K562
some activity has been reported for these compounds with other cell lines. [12
by preliminary screening, the IC50 values of compounds
–
7, 8, 9
following standard protocols. Clearly, the ester groups in the retro-curcuminoids play an important role
in the exertion of cytotoxic activity in vitro. Cell lines were supplied by U.S. National Cancer Institute
(
NCI). The human tumor cytotoxicity was determined using the protein-binding dye sulforhodamine
B (SRB) in microculture assay to measure cell growth, as described in the protocols established by the
NCI [33]. The cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine
serum, 2 mM L-glutamine, 10,000 units/mL penicillin G sodium, 10,000 µg/mL streptomycin sulfate,
2
5
µ
g/mL amphotericin B (Invitrogen/Gibco
™
, Thermo Fisher Scientific, Waltham, MA, USA), and
◦
1% non-essential amino acids (Gibco). They were maintained at 37 C in a humidified atmosphere
with 5% CO . The viability of the cells used in the experiments exceeded 95% as determined with
2
trypan blue.
Cytotoxicity after treatment with the test compounds of the normal and tumor cells was
determined using the protein-binding dye sulforhodamine B (SRB) in microculture assay to measure
cell growth, as described in a previous study [34]. The cells were removed from the tissue culture flasks
by treatment with trypsin and diluted with fresh media. From these cell suspensions, 100
µL, containing
5
000–10,000 cells per well, was pipetted into 96-well microtiter plates (Costar, Cambridge, MA, USA)
◦
and the material was incubated at 37 C for 24 h in a 5% CO atmosphere. Subsequently, 100
µ
L of
a solution of the compound obtained by diluting the stocks was added to each well. The cultures
were exposed for 48 h to the compound at concentrations of 50 g/mL and 10 g/mL. After the
L of cold 50%
2
µ
µ
incubation period, cells were fixed to the plastic substratum by the addition of 50
µ
◦
aqueous trichloroacetic acid. The plates were incubated at 4 C for 1 h, washed with tap H O, and
2
air-dried. The trichloroacetic-acid-fixed cells were stained by the addition of 0.4% SRB. Free SRB
solution was removed by washing with 1% aqueous acetic acid. The plates were air-dried, and the
bound dye was solubilized by the addition of 10 mM unbuffered Tris base (100 µL). The plates were
placed on a shaker for 10 min, and the absorption was determined at 515 nm using an enzyme-linked
immunosorbent assay (ELISA) plate reader (Bio-Tek Instruments, Winooski, VT, USA).
3
.6. Inhibition of Lipid Peroxidation on Rat Brain
Animals. Adult male Wistar rats (200–250 g) were provided by the Instituto de Fisiología Celular,
Universidad Nacional Autónoma de México (UNAM). Procedures and care of animals were conducted

Molecules 2017, 22, 33
11 of 14
in conformity with the Mexican Official Norm for Animal Care and Handling NOM-062-ZOO-1999.
◦
They were maintained at 23 ± 2 C on a 12/12 h light-dark cycle with free access to food and water.
Rat Brain Homogenate Preparation. Animal sacrifice was carried out avoiding unnecessary
pain. Rats were sacrificed with CO . The cerebral tissue (whole brain), was rapidly dissected and
2
homogenized in phosphate-buffered saline (PBS) solution (0.2 g of KCl, 0.2 g of KH PO , 8 g of
2
4
NaCl, and 2.16 g of NaHPO4 7H O/L, pH adjusted to 7.4) as described elsewhere [35,36] to produce
·
2
a 1/10 (w/v) homogenate. The homogenate was then centrifuged for 10 min at 800 rcf (relative
centrifugal field) to yield a pellet that was discarded. The supernatant protein content was measured
using Folin and Ciocalteu’s phenol reagent [37] and adjusted with PBS at 2.666 mg of protein/mL.
Induction of lipid peroxidation and thiobarbituric acid reactive substances (TBARS) quantification.
As an index of lipid peroxidation, TBARS levels were measured using rat brain homogenates according
to the method described by Ng and co-workers [38], with some modifications. Supernatant (375
was added with 50 L of 20 M EDTA and 50 L of each sample concentration dissolved in DMSO
50 L of DMSO for control group) and incubated at 37 C for 30 min. Lipid peroxidation was started
µL)
µ
µ
µ
◦
(
µ
adding 50
µ
L of freshly prepared 100
µ
M FeSO solution (final concentration 10
µ
M), and incubated
4
◦
at 37 C for 1 h. The TBARS content was determined as described by Ohkawa and co-workers [39],
with some modifications. 500 µL of TBA reagent (1% 2-thiobarbituric acid in 0.05 N NaOH and 30%
trichloroacetic acid, in 1:1 proportion) was added to each tube and the final suspension was cooled
◦
on ice for 10 min, centrifuged at 13,400 rcf for 5 min and heated at 80 C in a water bath for 30 min.
After cooling at room temperature, the absorbance of 200 µL of supernatant was measured at λ = 540 nm
in a UltraMicroplate (Synergy/HT BIOTEK Instrument, Inc., Winooski, VT, USA). The concentration
of TBARS was calculated by interpolation on a standard curve of tetra-methoxypropane (TMP) as a
precursor of MDA [40]. Results were expressed as nmoles of TBARS per mg of protein. The inhibition
ratio (IR [%]) was calculated using the formula IR = (C – E)
and E is the sample absorbance. Butylated hydroxytoluene (BHT) and
positive standards. All data were represented as mean standard error (SEM). Data were analyzed by
×
100/C, where C is the control absorbance
α
-tocopherol were used as
±
one-way analysis of variance (ANOVA) followed by Dunnett’s test for comparison against control.
Values of p ≤ 0.05 (*) and p ≤ 0.01 (**) were considered statistically significant.
4
. Conclusions
Seven new retro-curcuminoids were synthesized with a chemical modification strategy in good
yield, and NMR data confirmed the formation of the ester in ortho, meta, and para phenol positions.
The crystalline descriptions indicate that the compounds are formed by two aromatic rings (phenyl-C–;
phenyl-O–) connected by a moiety C(sp2)-C(sp2)-C(sp2)-C(sp2)-O(sp3)-C(sp2) chain of atoms, and
–C-phenyl is coplanar with the central moiety. Retro-curcuminoids with ortho substituents had the
highest cytotoxic activity. Only 2-((E)-3-oxobut-1-en-1-yl)phenyl(E)-3-(4-acetoxy-3-methoxyphenyl)
acrylate showed relevant antioxidant activity. Comparison of the cytotoxicity of retro-curcuminoids
with their synthetic precursors allows us to postulate that the ester function plays an important role
in biological activity in vitro. The results of this study suggest DHZ-based derivatives in the ortho
position as a new template for future anticancer and antioxidant studies of this family of compounds.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/22/
1
/33/s1.
Acknowledgments: Raúl G. Enríquez acknowledges support from DGAPA, UNAM (IN208516) and CONACyT
(
CB-252524). Marco A. Obregón-Mendoza acknowledges CONACyT CVU 599367. Dino Gnecco acknowledges
support from DGI-VIEP, BUAP (00139).
Author Contributions: Raúl G. Enríquez and Dino Gnecco designed and planned the research; Marco A.
Obregón-Mendoza, María Mirian Estévez-Carmona, Laura Orea and Hugo Pilotzi assisted in synthetic work.
Julia Cassani carried out special NMR determinations. Simón Hernández-Ortega performed X-ray determinations,
Manuel Soriano-García contributed with crystallographic discussions, and María Teresa Ramírez-Apan performed
biological evaluations. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.

Molecules 2017, 22, 33
12 of 14
Abbreviations
The following abbreviations are used in this manuscript:
DHZ
TPA
IC50
Dehydrozingerone
12-O-tetradecanoylphorbol-13-acetate
Inhibitory Concentration 50
Appendix A
CCDC 1451617, 1451555, 1451556, 1451621, 1451557, and 1452007 contain the supplementary crystallographic
data for C19H16O3 ( ), C19H16O3 ( ), C19H16O3 ( ), C22H20O6 (10), C22H20O6 (12), and C20H18O4 (13). These data
7
8
9
can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336-033; or E-Mail:
deposit@ccdc.cam.ac.uk.
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Sample Availability: Some samples of the compounds are available from the authors.
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