Newly synthesized curcumin derivatives: Crosstalk between chemico-physical properties and biological activity

Article abstract of DOI:10.1021/jm200872q

New curcumin analogues (ester and acid series) were synthesized with the aim to improve the chemical stability in physiological conditions and potential anticancer activity. Cytotoxicity against different tumorigenic cell lines (human ovarian carcinoma ce

Full text of DOI:10.1021/jm200872q

Article
pubs.acs.org/jmc
Newly Synthesized Curcumin Derivatives: Crosstalk between
Chemico-physical Properties and Biological Activity
Erika Ferrari,† Francesca Pignedoli,† Carol Imbriano,‡ Gaetano Marverti,§ Valentina Basile,‡
Ettore Venturi,† and Monica Saladini*,†
†Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41125 Modena, Italy
‡Department of Biology, University of Modena and Reggio Emilia, via Campi 213/D, 41125 Modena, Italy
§Department of Biomedical Science, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena, Italy
S
* Supporting Information
ABSTRACT: New curcumin analogues (ester and acid series)
were synthesized with the aim to improve the chemical stability
in physiological conditions and potential anticancer activity.
Cytotoxicity against different tumorigenic cell lines (human
ovarian carcinoma cells −2008, A2780, C13*, and A2780/CP,
and human colon carcinoma cells HCT116 and LoVo) was tested
to evaluate cellular specificity and activity. Physico-chemical
properties such as acidity, lipophilicity, kinetic stability, and free radical scavenging activity were investigated to shed light on the
structure−activity relationship and provide new attractive candidates for drug development. Most of ester derivatives show IC50
values lower than curcumin and exhibit selectivity against colon carcinoma cells. Especially they are extremely active after 24 h
exposure showing enhanced inhibitory effect on cell viability. The best performances of ester curcuminoids could be ascribed to
their high lipophilicity that favors a greater and faster cellular uptake overcoming their apparently higher instability in
physiological condition.
(p300/CBP, HDAC1), and genes regulating apoptosis (p53,
Bcl-2, Bcl-xL, Bax).4
1. INTRODUCTION
Medicinal plants have been an excellent source of pharmaceu-
tical agents for a long time. Curcuminoids are natural yellow
pigments and food-coloring agents present in the rhizomes of
the Asian tropical plant Curcuma longa L., which has been used
as a traditional medicinal herb for thousands of years. The
dried rhizome of C. longa has been widely used as an aromatic
stomachic, carminative, anthelmintic, laxative, as well as for liver
ailments and as condiment in foods. Curcumin, 1,7-bis(4-
hydroxy-3-methoxyphenyl)-1,6-heptadien-3,5-dione, is the pri-
mary bioactive compound isolated from this spice.
In the past decade, a large number of reports have been
published on the beneficial effects of curcumin, and it has
repeatedly been claimed that this natural product is efficient
and safe for the prevention and treatment of several diseases
including cancer.1,2
Curcumin inhibits cell proliferation of a variety of trans-
formed cell types, including T- and B-cell lymphomas, breast,
colon, gastric, ovarian, prostate, and oral epithelial carcinoma
cells.3 Moreover, its cytotoxic activity is confined to cancer
cells, thus promising low side effects of anticancer drugs based
on curcumin structure. Although numerous studies have been
performed, the origins of the curcumin anticancer effect have
not been elucidated yet. Curcumin acts by a multitude of dif-
ferent mechanisms and targets multiple proteins, both directly
and indirectly, including transcription factors (NF-kB, STAT3,
Egr-1, AP-1, PPAR-γ, β-catenin), coactivators and corepressors
A robust activity of curcumin was described in colon cancers,
showing the safety and tolerability of curcumin in patients with
colorectal carcinoma diseases.5 Curcumin inhibits proteasome
activity,6 exhibits changes in cell cycle progression,7 and induces
apoptosis in HCT116 cells, a human colon carcinoma cell line.8
Cells growth inhibition, cell cycle arrest in G2/M phase, and
apoptotic cell death were also observed in other human colon
cancer cells, LoVo, SW480, and HCT15.9
In addition, it has been shown that curcumin induces
apoptosis in ovarian carcinoma OVCA429 and SKOV3 cells10
and that it is also able to increase sensitivity of both wild type
and cisplatin (cDDP) resistant ovarian cancer cells.11 We have
previously reported that the cDDP-resistant human ovarian
carcinoma cell line, C13*, was also cross-resistant to curcumin
in comparison with the parental-sensitive cell line, 2008.12 In
fact, the observed resistant factor (RF = IC50 resistant/IC50
parent line)13 was 3.4 toward curcumin.
Unlike most chemotherapeutic agents, curcumin shows little
to no toxicity (no dose-limiting toxicity at doses up to 10 g/day
in humans),14 but no human studies were conducted to test the
dose levels which cause long-term toxicity so far.
However, clinical use of curcumin is severely limited by its
extremely low bioavailability, which is a consequence of poor
Received: July 4, 2011
Published: October 27, 2011
© 2011 American Chemical Society
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solubility and instability in aqueous solution, in particular at
alkaline pH.15,16
2. RESULTS AND DISCUSSION
2.1. Synthesis of Curcuminoids. In the present study, we
have synthesized new curcumin derivatives (Figure 1) in order
to investigate the effect of the insertion of an alkylic chain on
the β-keto-enolic moiety (C3 substitution) both on physico-
chemical properties and biological activity. We decided to add a
short chain to keep low the molecular weight. To understand
the role of polarity and acidity in relation to biological activity,
we investigated both ester and acid derivatives, here named E
and A series, respectively. We tested different ester protecting
groups (data not shown), but in many cases deprotection
conditions led to the breakage of curcumin backbone, and for
this reason we chose to protect the carboxyl with Boc function
which could be removed with no degradation of curcumin
structure.
As far as cancer is concerned, in vitro studies have
demonstrated that cancer cells do not die unless they are
exposed to curcumin concentrations of 5−50 μM for several
hours.17,18 Because of its poor bioavailability, these con-
centrations are not achieved outside the gastrointestinal tract
when curcumin is taken orally. Several reports have shown
that the concentrations of curcumin in plasma of people
taking relatively high oral doses are very low, typically in the
nanomolar range,17 hence its anticancer activity may be limited
to gastrointestinal tract.19 In addition, its limited bioavailability
and extensive metabolism suggest a possible disagreement
between anticancer effects observed in vitro and its in vivo
activity.17
To overcome the low oral bioavailability of curcumin, several
strategies have been proposed20 such as complexation with
cyclodextrin,16 conjugation with nucleosides,21 biopolymers,22
and composite nanoparticles.23 One of these strategies has
entered clinical trials and consists of using the black pepper
alkaloid piperine (bioperine) to increase the bioavailability of
curcumin.24 Besides, numerous analogues of curcumin have
been synthesized and tested to investigate their activity against
known biological targets to improve the pharmacological profile
of the natural product (i.e., improve their selectivity,
bioavailability, and stability).25,26 Recently, the introduction of
glycoside moiety at the phenyl ring of curcumin was found to
enhance its water solubility and chemical stability.12 In this case,
the substitution of the mobile phenolic hydrogens gave
compounds with no gain in antiproliferative activity with
respect to curcumin.
Four differently substituted benzaldehydes were chosen to
investigate the role of electronic effects and intramolecular
hydrogen bond on chemical and biological properties.
E and A series, except of compound 8, were synthesized
from 1 (route a, Scheme 1) employing the renowned Pabon
reaction27 under modified conditions as previously reported by
the authors for curcumin.7 Acetylacetonate−boron complex
is formed with the aim of activating the methyl positions of
2,4-pentanedione for aldol condensation and preventing
Knoevenagel condensation the on methylene group. The
required condensation product is set free from boron by
addition of an acid aqueous solution. We have optimized the
reaction conditions of route a with respect to the previously
investigated substituted curcuminoids.26,28,29
In route a, compounds 3−6 were obtained using DMF
instead of EtOAc, the commonly used solvent for Pabon
reaction, in view of its ability to provide: (i) high solubility of
reactants and intermediates, (ii) suitable polarity for the
process, and (iii) easy reaction workup, particularly the
isolation and separation of curcuminoids from byproduct. To
maintain satisfactory reaction yields, the aldol condensation
reaction is carried out under anhydrous conditions, and
tributylborate (n-BuO)3B is added as drying agent. On the
whole, this one-pot reaction is characterized by quite good
yields (∼65%), short time (6 h), and small volumes of solvents
(1 mL DMF/0.5 mmol product). The following deprotection
of Boc group affords the acid derivatives 7, 9, and 10. Com-
pound 8 is unstable under the reaction conditions employed for
Boc deprotection (50% TFA in CH2Cl2 at 0 °C); for this
reason a new strategic synthetic pathway (route b, Scheme 1)
was used. Defferently from route a, Boc group deprotection
was performed before aldol condensation.
In this scenario, we have here designed and synthesized new
curcumin analogues (Figure 1) in which we have modified the
Figure 1. Structures of curcumin and newly synthesized curcuminoids.
β-keto-enolic moiety, inserting an alkylic group with the aim to
improve the chemical stability in physiological conditions and
the potential anticancer activity with respect to the parent
compound. Cytotoxicity against different tumorigenic cell lines
(cisplatin-sensitive human ovarian carcinoma cell lines 2008
and A2780, compared with their respective -resistant counter-
parts, C13* and A2780/CP cells, and human colon carcinoma
cell lines, HCT116 and LoVo) is tested with the purpose of
evaluating possible specificity and selectivity of the new
compounds. Furthermore, results from in vitro biological
assays are here related to chemico-physical properties such as
acidity, lipophylicity, kinetic stability, and free radical
scavenging activity in order to shed light on structure−activity
relationship and supply new attractive candidates for drug
development.
According to route b, curcumin derivative 8 was prepared
directly from 2 in reaction condition similar to those reported
by Babu.30
The choice of Boc deprotection as the first step in route b
allows obtaining compound 8 in milder acidic conditions,
overcoming its instability. The synthetic pathway b was also
tested to synthesize compound 7, 9, and 10 but reaction yields
were significantly lower than those achieved by route a.
2.2. X-ray Crystallography. ORTEP31 view of 5 structure
is reported in Figure 2, geometric parameters are reported in
Table 1. The compound is in the keto-enolic form with a strong
intramolecular hydrogen bond [O1−H1 1.05(6) Å, O1···O2
2.428(5) Å, H1···O2 1.4386) Å, O1−H1···O2 158(4)°].
Although the presence of a bulky electron-withdrawing group
on the central carbon in β-diketones was found stabilizing the
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a
Scheme 1. General Synthetic Strategies for Symmetric Curcumin Analogues
a
The two different investigated routes, a and b, are represented with dotted and solid arrows respectively.
Table 1. Bond Lengths [Å] and Angles [deg] for Compound 5
O(1)−C(18)
O(2)−C(2)
O(3)−C(13)
O(4)−C(13)
O(4)−C(14)
O(5)−C(7)
O(5)−C(11)
O(6)−C(25)
O(6)−C(27)
C(1)−C(2)
C(1)−C(18)
C(1)−C(12)
C(2)−C(3)
C(3)−C(4)
C(4)−C(5)
C(5)−C(6)
C(5)−C(10)
1.293(4) C(6)−C(7)
1.299(4) C(7)−C(8)
1.194(4) C(8)−C(9)
1.335(5) C(9)−C(10)
1.478(5) C(12)−C(13)
1.371(5) C(14)−C(15)
1.424(6) C(14)−C(17)
1.368(5) C(14)−C(16)
1.430(6) C(18)−C(19)
1.398(5) C(19)−C(20)
1.403(5) C(20)−C(21)
1.525(5) C(21)−C(26)
1.452(6) C(21)−C(22)
1.318(5) C(22)−C(23)
1.468(6) C(23)−C(24)
1.381(5) C(24)−C(25)
1.385(6) C(25)−C(26)
1.377(6)
1.369(6)
1.378(6)
1.375(6)
1.506(6)
1.500(7)
1.503(6)
1.514(6)
1.464(6)
1.311(5)
1.464(6)
1.381(6)
1.386(6)
1.379(7)
1.361(7)
1.366(6)
1.376(6)
Figure 2. ORTEP view of compound 5.
diketo form,32 the considerable charge delocalization over the
whole framework contribute to stabilize the keto-enolic tautomer.
This behavior was previously found for curcumin in the solid
state33 and for its derivatives.34 The geometry around C1 indicates
an sp2 coordination, as expected for the keto-enol tautomer.
2.3. Acidity. pH-Metric titrations were performed by
means of UV−vis spectroscopy on A compounds in order to
assess their acidity and predict the most abundant species in
physiological pH range. All the compounds undergo a
tautomeric equilibrium (Scheme 2) which is highly solvent
dependent. Anyway, the prevailing tautomer for both E and A
families in physiological condition is the diketo form, as
suggested by the value of λmax around 320 nm in UV−vis
spectra;35 the opposite takes place in aprotic/apolar solvents, in
which the keto-enolic tautomer is the most abundant, as shown
by NMR data (Experimental Section).
Increasing pH, simultaneous equilibria, involving keto-enolic
tautomerism and acid dissociations, take place, giving rise to a
intricate titolative spectral pattern (Figure 3). Despite this
apparent complex behavior, for all A molecules, the plot of
absorbance at λmax vs pH shows titolative trend characterized
by two equivalent points: the first (pH ∼ 4.5) corresponds to
the dissociation of carboxyl group and the second one (pH ∼ 9)
is due to the keto-enol deprotonation. Overall protonation
constants were calculated from spectrophotometric data and
optimized by means of pHab software,36 and log β and pKa
values are summarized in Table 2. The carboxylic pKa value is
similar for all the compounds, while the enolic dissociation is
strongly influenced by the electron withdrawing effect of the
aromatic substituents, in the order of increasing acidity 7 < 9 <
10 < 8, a similar behavior was previously observed for other
curcuminoids substituted on the aromatic ring as reported by
C(13)−O(4)−C(14) 121.9(3)
C(7)−O(5)−C(11) 118.0(4)
C(25)−O(6)−C(27) 118.5(5)
C(2)−C(1)−C(18)
C(2)−C(1)−C(12)
O(3)−C(13)−C(12)
O(4)−C(13)−C(12)
O(4)−C(14)−C(15)
O(4)−C(14)−C(17)
124.0(4)
110.9(4)
109.9(3)
101.9(4)
119.2(4)
120.0(4)
C(15)−C(14)−C(17) 110.7(5)
O(4)−C(14)−C(16) 109.5(4)
C(18)−C(1)−C(12) 120.8(4)
O(2)−C(2)−C(1)
O(2)−C(2)−C(3)
C(1)−C(2)−C(3)
C(4)−C(3)−C(2)
C(3)−C(4)−C(5)
C(6)−C(5)−C(10)
C(6)−C(5)−C(4)
C(10)−C(5)−C(4)
C (7)-C(6)−C(5)
C(8)−C(7)−O(5)
C(8)−C(7)−C(6)
O(5)−C(7)−C(6)
C(7)−C(8)−C(9)
C(10)−C(9)−C(8)
C(9)−C(10)−C(5)
120.8(4)
115.4(4)
123.8(4)
123.8(4)
127.5(4)
118.1(4)
119.4(4)
122.5(4)
121.8(4)
123.9(5)
120.1(4)
116.0(5)
118.3(5)
122.2(5)
119.5(4)
C(15)−C(14)−C(16) 112.5(4)
C(17)−C(14)−C(16) 111.8(4)
O(1)−C(18)−C(1)
O(1)−C(18)−C(19)
C(1)−C(18)−C(19)
C(20)−C(19)−C(18) 123.3(5)
C(19)−C(20)−C(21) 127.8(5)
C(26)−C(21)−C(22) 117.9(5)
C(26)−C(21)−C(20) 122.5(5)
C(22)−C(21)−C(20) 119.6(5)
C(23)−C(22)−C(21) 120.2(5)
C(24)−C(23)−C(22) 121.1(5)
C(23)−C(24)−C(25) 119.3(5)
C(24)−C(25)−O(6)
C(24)−C(25)−C(26) 120.3(5)
O(6)−C(25)−C(26) 115.9(5)
C(21)−C(26)−C(25) 121.2(5)
120.5(4)
114.8(5)
124.7(5)
123.8(6)
C(13)−C(12)−C(1) 113.8(4)
O(3)−C(13)−O(4) 125.1(4)
Caselli et al.,35 Ferrari et al.,12 and references cited therein. As
shown by the species distribution curves (Figure 4), the pre-
vailing species at physiological pH (7.4) is the monodissociated
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The investigation on the acidity of E compounds was
precluded by the low stability in basic condition of Boc group,
therefore it is not possible to determine enolic pKa, which is the
only acid moiety. Assuming that this value is ∼9, as found in A
compounds, it is reasonable to assess that the most abundant
species is the neutrally charged one in physiological condition.
2.4. Kinetic Stability. In the present investigation, we want
to test if the introduction of an alkylic chain on C-3 position
of curcumin backbone can improve stability in physiological
conditions. In fact, several pharmacokinetics studies on
curcumin showed that its main drawbacks are the extremely
fast degradation and its extensive metabolism that both induce
particularly low plasma concentration, typically in the nano-
molar range.19 Figure 5 reports the decomposition kinetics
profiles of A and E compounds in simulated physiological
conditions (parts A and B) and their linear fittings37 (parts C
and D). Table 3 summarizes hyperbolic function parameters (a
and b) together with their reciprocal values and statistical
parameter R2. The reciprocal of a (1/a) corresponds to the
minimum percentage of residual compound achieved in
physiological condition. The value (−1/b) is related to the
degradation rate: the higher the value the faster the degradation
process. Compounds 7 and 9 show the highest values for 1/a
corresponding to the more stable compounds. 10 is
characterized by the lowest values of 1/a and −1/b in fact
decomposes up to ∼25% in 8 h, while 8 decomposes quite
rapidly up to ∼20% during the first 2 h, showing the highest
value of kinetic rate (−1/b). These data point out a strong
relationship between the aromatic substituents and kinetics
profiles, in particular, the absence of para substitution seems to
be fundamental in order to achieve stability, while the meta
group looks like of minor importance. Moreover, the possibility
to form an intramolecular hydrogen bond, such as for 8,
appears the driving force in the decomposition process, as
observed for curcumin in comparison with derivatives
substituted on the aromatic ring.7 Anyway, compound 8 is
more stable than curcumin, as after 2 h it is only 20% degraded
vs 40% of curcumin,12 therefore C-3 substitution on keto-enolic
moiety seems to be a key factor in slowing the degradation
process of curcuminoids.
Scheme 2. General Scheme of Tautomeric Equilibrium
between Keto-enolic (KE) and Di-ketonic (DK) Forms of
New Curcumin Analogues E and A
Figure 3. pH-Metric spectrophotometric titration of 10; the insert
shows the plot of absorbance (A) vs pH at λmax = 417 nm.
Table 2. Logarithm of Protonation Constants (logβ LH) and
pKa Values Calculated with pHab36 from Spectrophoto-
metric Titrations Performed at 25 °C, I = 0.1 M (NaNO3)
7
8
9
10
logβ11
logβ12
logβ13
logβ14
pKa1
10.46(1)
15.10(4)
13.35(1)
22.98(2)
31.40(1)
35.84(1)
4.44(5)
9.44 (4)
9.04(1)
14.52 (2)
13.47(1)
4.54(5)
5.08(6)
9.44(4)
4.43(2)
9.04(1)
pKa2
10.46(1)
8.42(4)
E compounds decompose more than their analogues A,
reaching a residual percentage beneath 50% even though with
slower rates as shown by −1/b values, which are on average
minor than their carboxylic counterparts.
This apparent instability of E series may be due to the
presence of Boc group, which can hydrolyze in slightly basic
pKa3
9.63(3)
pKa4
13.35(1)
one for all A compounds. Anyway for 8 about 15% of the
dianionic species is present as well.
Figure 4. Species distribution curves for 7 (left) and 8 (right), [L]total = 5 × 10−3 M.
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Figure 5. (A,B) Decomposition kinetics profiles of E and A compounds in buffered aqueous solution (TRIS-HCl pH = 7.4) at 37 °C in darkness
over a period of 8 h. Residual concentration is expressed as percentage with respect to concentration at time zero. Concentrations were determined
by reading absorbance at 300 nm. (C,D) Linearization of kinetics profiles of A and E compounds by the hyperbolic function t/f% = at + b, where t is
time (min) and f% represents concentration as residual percentage.
Lipinski’s “rule of 5”: there are less than 5 H-bond donors, MW
is lower than 500 Da, log P is <5, and there are less than 10 H-
bond acceptors.38
Table 3. Linear Fittings for Kinetics Profiles Obtained Using
the Function: t/f% = at + b
a
compd
a
(1/a)
b
(−1/b)
R2
Table 4 reports log Poct and water-solubility values, which
show inverse proportionality. 8 and 10 exhibit the lower log
3
0.0236
0.022
42.37
45.45
13.72
70.92
92.59
81.30
90.09
76.34
−0.3353
−0.3257
−2.6655
−0.1195
−0.0501
−0.0143
−0.0562
−0.1934
2.98
3.07
0.9958
0.9851
0.9679
0.9975
0.9995
0.9999
0.9993
0.9959
4
5
0.0729
0.0141
0.0108
0.0123
0.0111
0.0131
0.38
6
8.37
Table 4. Summary of Experimental Physico-Chemical
Properties of New Curcuminoids: log P (Logarithm of
Partition Coefficient Defined As the Concentration Ratio of
Each Compound in n-Octanol and Aqueous Buffer Solution,
7
19.96
69.93
17.79
5,17
8
9
a
10
pH = 7.4 and Solubility (mM)
a
Equation parameters (a and b) and statistical parameter R2 were
extracted by a least-squares fitting on the experimental UV-Vis results.
compd
log P
solubility
7
8
1.14 ± 0.08
−0.7 ± 0.1
1.25 ± 0.05
0.25 ± 0.02
0.92 ± 0.02
>100
condition. Anyway, at physiological pH, they have a kinetic
stability comparable with curcumin.12
9
0.55 ± 0.02
1.60 ± 0.01
10
2.5. Lipophilicity. The primary property for orally active
drugs is the ability to be efficiently absorbed from the
gastrointestinal tract (GIT) and to cross biological membranes.
The molecular size is a critical factor which influences the rate
of drug absorption. The “cutoff” molecular weight for the
paracellular route in the human small intestine is 500 Da,38 and
A and E molecules are talented with this prerequisite. In addi-
tion, lipophilicity, which is usually quantified by the logarithm
of the 1-octanol/water partition coefficient (log Poct), is a
fundamental chemico-physical factor, because it is correlated to
permeability and drug penetration into cells and into the
central nervous system.39 To achieve efficient oral absorption,
log Poct should be greater than −0.740 and lower than 5.38 For
all the synthesized curcuminods we can potentially predict
good absorption and permeation because they satisfy the
a
The mean value of three independent experiments ± SD is reported.
Poct; this seems to be related to their higher acidity with respect
to 9 and 7. In particular, 8, in which the enolic function is
partially dissociated at physiological condition, displays an
extremely high water solubility and the lowest log Poct. As a
consequence, with the exception of 8, we can expect effective
penetration of the drug into cells for all A derivatives. E
compounds demonstrated a great lipophilicity, which prevented
log Poct measurement; therefore also for these molecules we can
suggest a good cell permeability.
2.6. Free Radical Scavenging Ability. The free radical
scavenging ability of synthesized curcuminoids was evaluated by
the DPPH radical assay, which is one of the most widely used
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method to determine antioxidant activity. Results, summarized
in Figure 6, point out that curcuminoids can be divided into
two main groups according to their DPPH inhibition, expressed
similar free radical scavenging ability for all our compounds; on
the contrary, our findings evidenced a significant inhibition of
DPPH only for those compounds characterized by the presence
of phenolic groups (4 and 8), therefore we suggest that the
mechanism of H-transfer originates mainly from phenolic
groups rather than from keto-enolic moiety; in this way
phenolic OH groups are mainly responsible for antioxidant
activity of curcumin confirming the mechanism suggested by
Priyadarsini et al.42
2.7. Biological Assays. The effectiveness of curcumin and
curcuminoids against human ovarian and colon carcinoma cell
lines, expressed as IC50 values, are reported in Table 5.
With the exception of 8 and 10, all compounds show IC50
values after 72 h treatment equal or lower than curcumin on
cDDP-sensitive human ovarian carcinoma cells. As already
reported, the cDDP-resistant counterpart C13* is cross-
resistant to curcumin.12 In addition, we display now that this
effect occurs in a time-dependent manner because it increases
with the exposure time. Our compounds do not show such
cross-resistance, giving similar cell growth inhibition in both
2008 and C13*cell lines; in addition they are all more effective
even than curcumin. Of note, 9 is the most potent cell growth
inhibitor toward this cDDP-sensitive and -resistant cell model
after 48 h treatment. Drug accumulation is the result of uptake
and efflux from cells, and many resistant cells expel the drug
through the multidrug resistance (MDR) pump,43 and even
cDDP-resistant cells exploit it to extrude most of the drugs.44
The expression of mdr-1 is enhanced in a dose-dependent
manner as a series of bladder transitional carcinoma cells
acquires progressive cDDP-resistance, indicating a possible role
of multidrug resistance-associated protein (MRP).45 Physical
characteristics commonly seen in drugs showing MDR46 may
suggest that most of the compounds could become substrates
of P170, the product of mdr-1 gene, and so be expelled from
Figure 6. The percentage inhibition of free DPPH radical in presence
of different curcuminoids; curcuminoid concentration is expressed as
molar ratio (mol of curcuminoid/mol of DPPH).
as EC50 (the antioxidant concentration necessary to decrease
the initial amount of DPPH by 50%). 4 and 8 show EC50 values
of 16 ± 1 μM and 11 ± 1 μM, respectively, which are close to
that of curcumin (13 ± 1 μM). For all the other compounds,
EC50 is not reached within 1/1 molar ratio.
Some studies on antioxidant properties of curcumin41
highlighted a mechanism involving H-atom transfer from the
keto-enolic moiety, excluding the extraction of phenolic H-
atoms due to their participation in intramolecular H-bonds with
adjacent methoxyl groups. On this basis, we should predict
Table 5. IC50 Values (μM) for Curcumin and Its Eight Derivatives against Two cDDP-Sensitive Human Ovarian Carcinoma Cell
Lines (2008, A2780) and Their Resistant Counterparts (C13*, A2780/CP) and Two Human Colon Carcinoma Cell Lines
a
(HCT116, LoVo)
cell line
2008
t (h)
CURC
3
4
5
6
7
8
9
10
24
48
72
18 ± 1
7 ± 1
4.7 ± 0.5
18 ± 2
15 ± 4
3.1 ± 0.6
55 ± 7
35 ± 4
9.6 ± 0.2
23 ± 4
6.9 ± 0.3
2.1 ± 0.4
34 ± 5
18 ± 3
1.1 ± 0.2
32 ± 4
17 ± 3
2.5 ± 0.3
>100
14 ± 1
6.8 ± 0.4
2.0 ± 0.4
72 ± 8
66 ± 8
24 ± 3
98 ± 9
70 ± 8
C13*
A2780
24
48
72
26 ± 3
18 ± 2
16 ± 2
25 ± 4
17 ± 4
2.5 ± 0.4
49 ± 6
50 ± 6
10 ± 1
16 ± 2
11 ± 1
2.6 ± 0.3
32 ± 4
16 ± 2
3.1 ± 0.4
25 ± 2
16 ± 3
3.5 ± 0.3
95 ± 11
99 ± 9
100 ± 11
11 ± 1
8 ± 1
3.5 ± 0.2
>100
80 ± 11
60 ± 5
24
48
72
38 ± 5
12 ± 1
8 ± 1
40 ± 6
41 ± 2
9 ± 1
9.4 ± 0.4
7 ± 1
6.4 ± 0.4
9 ± 1
5.8 ± 0.4
8 ± 1
76 ± 8
28 ± 5
4 ± 1
40 ± 3
36 ± 8
10 ± 1
>100
>100
17 ± 2
9 ± 1
7 ± 1
>100
>100
54 ± 7
46 ± 8
A2780/CP
24
48
72
28 ± 3
18 ± 2
11 ± 2
>100
9 ± 1
6.3 ± 0.5
7 ± 1
8 ± 1
6 ± 1
5.3 ± 0.1
>100
35 ± 6
5 ± 1
>100
24 ± 6
9 ± 1
>100
>100
21 ± 2
10 ± 1
7 ± 1
>100
>100
39 ± 7
13 ± 1
91 ± 3
47 ± 6
HCT116
LoVo
24
48
13 ± 1
4 ± 2
5 ± 1
3.6 ± 0.4
3.6 ± 0.6
2.3 ± 0.6
3.5 ± 0.4
2.7 ± 0.8
3 ± 1
2.7 ± 0.3
17 ± 3
13 ± 2
>100
14 ± 1
8 ± 1
56 ± 9
60 ± 10
93 ± 17
24
48
16 ± 2
10 ± 1
8 ± 2
3.9 ± 0.3
4.1 ± 0.4
7 ± 1
4.6 ± 0.4
4.0 ± 0.2
4.2 ± 0.5
3.5 ± 0.3
27 ± 6
26 ± 4
>100
>100
20 ± 2
17 ± 1
>100
71 ± 17
a
The IC50 is defined as the concentration causing 50% growth inhibition in treated cells when compared to control cells after 24, 48 or 72 h drug
exposure. Values are means ± SD of five separate experiments performed in duplicate.
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Figure 7. Dose−response curves of curcumin and its eight derivatives against 2008, A2780, HCT116, and LoVo cell lines. Twenty-four h after
seeding, ovarian cancer cells (upper panels) and colon cancer cells (lower panels) were exposed to the indicated concentrations of the drugs (open
symbols, A series; closed symbols, E series) for 48 h and then stained with crystal violet solution. Results represent the mean of three separate
experiments performed in duplicate. Error bars, SEM.
resistant cells, accounting for the impairment of drug
accumulation and cross-resistance.
its derivatives with respect to A2780 and A2780/CP. On the
contrary, compounds 4 and 5 result more cytotoxic against the
latter cells than the former ones.
On the contrary, in the other couple of ovarian cancer cell
lines, A2780 and A2780/CP, no cross-resistance was observed
toward both curcumin and its derivatives; probably, these cell
lines accumulate equal amounts of each compound that could
also undergo the same biotransformation. In addition, because
cDDP-resistance in C13* cells has been partly related to
mitochondria alterations raised during resistance selection,47
and never reported in A2780/CP cells, it could be suggested
that these mitochondrial defects in C13* cells may impair
curcumin and its derivatives effects on these organelles.48
A time-dependent behavior is observed only for curcumin
and the less active compounds on ovarian carcinoma cell lines
A2780 and A2780/CP, while the more cytotoxic ones (4, 5, 9)
manifest a cell growth inhibition at 24 h exposure similar or
lower than the one obtained with curcumin after 72 h.
In addition, from Table 5, it is also evident that 2008 and
C13* cells are more responsive to both curcumin and most of
Curcumin derivatives were also tested on human colon
carcinoma cell lines (HCT116 and LoVo) (Table 5), displaying
in general lower IC50 values than those gained against human
ovarian carcinoma cells previously discussed. Differently from
curcumin, which showed a time-dependent inhibitory activity
on cells doubling, the synthesized derivatives did not lower
their IC50 values with prolonged exposure, as indicated by the
comparison between 24 h and 48 h treatments. This
phenomenon could be the consequence of a rapid cellular
uptake along with a fast metabolic degradation.
Figure 7 depicts the dose−response curve of curcumin and
its eight derivatives, in two cDDP-sensitive human ovarian
carcinoma cell lines (parts A and B) and two colon cancer cell
lines (parts C and D) after 48 h exposure, corresponding to
about two cell cycle span. As it appears, most of the drugs
killed more than 70% of 2008 cells within 25 μM, or at
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Figure 8. Correlations between IC50 values experimentally obtained after 48 h exposure with synthetic A series curcuminoids and log Poct values
together with linear regressions on human ovarian carcinoma cells (A) and human colon carcinoma cells (B).
concentrations slightly higher in A2780 cells, similarly to
curcumin. Compounds 8 and 10 were poorly cytotoxic even at
high concentrations (>100 μM).
(v) protic groups (phenols) are essential for the curcumi-
noids to exert a radical scavenging ability comparable to
the parent compound.
As it concerns colon cancer cells (parts C and D of Figure 7),
the E series is able to inhibit cell proliferation up to 70% in a
lower and closer concentration range (4−5 μM in HCT116
and 6−9 μM in LoVo cells) than the A series compounds
(>100 μM both in HCT116 and in LoVo cells), suggesting that
Boc group positively affects the pharmacokinetics of these
drugs, making them more lipophilic and thus facilitating their
intracellular accumulation. E compounds are more potent
inhibitors than curcumin, which requires a concentration of
4.4 μM and 20 μM on HCT116 and LoVo cell lines, respectively.
Figure 8 shows the correlation between lipophilicity, expressed
by log Poct, and biological activity defined as IC50. For all cell
lines, a linear regression is evident, showing a decrease in IC50
value as log Poct is increased, suggesting the importance of this
chemico-physical property as the driving force in cellular activity
despite acidity and chemical stability, which do not show any
direct relationship with cell growth inhibition.
Furthermore, our data clearly highlight an interesting
antiproliferative effect for most of the synthesized com-
pounds, suggesting that the alkyl substitution in α position
of β-diketo moiety does not affect the cytotoxic activity
associated with curcumin backbone. In particular, the E
series shows IC50 values similar or in many cases better than
curcumin in all tested cell lines and exhibits selectivity
against colon carcinoma cells. Indeed, they are extremely
active in HCT116 and Lovo cells after 24 h exposure at μM
concentrations ca. 1/3 of curcumin: 4, 5, and 6 are the most
effective ones. The best performances of E compounds with
respect to A could be ascribed to their high lipophilicity that
favors a greater and faster cellular uptake overcoming their
apparently higher instability in physiological condition,
however the E series maintains kinetic stability better than
curcumin.
Interestingly, the compound 4 behaves similarly to curcumin
as it concerns its free radical scavenging ability, principally due
to the presence of phenolic groups on aromatic rings, and it has
an increased antiproliferative activity particularly on human
colon carcinoma cell lines. Even though further experiments
will be needed, it is appealing to speculate that this similarity to
curcumin in radical scavenging ability may lead on one side to
antioxidant/protective properties in normal cells and, on the
other side, may trigger apoptosis in transformed cells via
enhanced ROS production.49 Therefore the compound 4
should be considered a good candidate for future anticancer
drug developments.
3. CONCLUSION
The investigation here was performed to elucidate the effect on
physicochemical properties of C-3 substitution in curcumin
analogues highlighted interesting remarks:
(i) the insertion of alkylic chain, both in the ester and acid
form, increases acidity of keto-enolic moiety and
pharmacokinetic stability in physiological condition
with respect to parent curcuminoid;
(ii) the alkylic chain increases molecular polarity and
consequently diminishes lipophilicity with respect to
curcumin, particularly for the acid series;
(iii) the C-3 substitution has no effect on radical scavenging
ability with respect to the parent compounds.
4. EXPERIMENTAL SECTION
Besides these general observations on C-3 substitution,
which are strongly related to molecular conformations
and tautomeric equililibria, the effect of aromatic
substituents is instead principally connected to electronic
density and resonance/inductive effects, as observed for
previously investigated aromatic ring substituted curcu-
minoids:
4.1. General Procedures and Chemicals. Elemental analysis
was performed on a CE Instruments EA 1110.
LC-MS spectra were recorded on 6310A ion trap LC-MS (Agilent
Technologies) in isocratic condition (20% H2O (0.1% HCOOH),
80% ACN) and in positive mode detection.
All chemicals were reagent grade and used without further
purification unless otherwise specified. They were purchased form
Sigma-Aldrich. The purity of all final compounds was determined to be
at least 95% pure by a combination of HPLC, LCMS, NMR, and
combustion analysis.
(iv) among the same series, electro-withdrawing groups
increase acidity of keto-enolic moiety favoring hydro-
philicity and instability in physiological conditions;
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(6). Yellow powder, 47% yield; mp 152−153 °C. LC-MS-IT m/z
567.3 (M + Na)+; 589.3 (M + H)+.
tert-Butyl 3-acetyl-4-oxopentanoate (1) and 3-acetyl-4-oxopentanoic
acid (2) were synthesized as previously reported by Ferrari et al.50
Preparation of E Series (3−6). A suspension of B2O3 (1 mmol)
and 1 (1 mmol) in DMF (1.5 mL) was stirred for 30 min at 80 °C, and
then tributylborate (4 mmol) was added. After 30 min, the appropriate
benzaldehyde (1.8 mmol) was added and followed by slow addition of
n-butylamine (0.4 mmol in 0.5 mL of DMF). After stirring at 80 °C for
4 h, the solution was acidified with 0.5 M HCl (8 mL) and cooled
down to room temperature. The yellow−orange solid was suspended
in water, filtered, and dried under vacuum. All crude compounds were
recrystallized in EtOH to give the pure products.
KE 43%: 1H NMR (CDCl3) δ 3.50 (s, 2H; H-2), 7.16 (d, 2H; H-5,
J = 15.2 Hz), 7.77 (d, 2H; H-6, J = 15.2 Hz), 7.19 (d, 2H; H-8, J = 1.7
Hz), 7.25 (d, 2H; H-11, J = 8.1 Hz), 7.11 (dd, 2H; H-12, J = 1.7 Hz,
J = 8.1 Hz), 3.89 (s, 6H; Ar-OCH3), 2.36 (s, 6H; Ar−O(CO)CH3),
1.47 (s, 9H, −COOC(CH3)3); 13C NMR (CDCl3) δ 170.7 (C-1),
33.5 (C-2), 105.6 (C-3), 183.4 (C-4), 120.9 (C-5), 141.5 (C-6), 134.2
(C-7), 111.9 (C-8), 151.4 (C-9), 141.9 (C-10), 121.0 (C-11), 123.21
(C-12), 56.0 (Ar−OCH3), 20.6 (Ar−O(CO)CH3), 81.4 (−COOC-
(CH3)3), 28.1 (−COOC(CH3)3), 168.7 (Ar−O(CO)CH3). DK 57%:
1H NMR (CDCl3) δ 3.01 (d, 2H; H-2, J = 7.1 Hz), 4.67 (t, 1H; H.3,
(3Z-5E)-tert-Butyl-3-cinnamoyl-4-hydroxy-6-phenylhexa-3,5-di-
enoate (3). Yellow powder, 80% yield; mp 149−150 °C. LC-MS-IT
m/z 413.3 (M + Na)+; 391.3 (M + H)+.
J = 7.1 Hz), 6.83 (d, 2H; H-5, J = 15.8 Hz), 7.69 (d, 2H; H-6, J = 15.8
Hz), 7.15 (d, 2H; H-8, J = 1.7 Hz), 7.25 (d, 2H; H-11, J = 8.1 Hz),
7.11 (dd, 2H; H-12, J = 1.7 Hz, J = 8.1 Hz), 3.93 (s, 6H; Ar-OCH3),
2.37 (s, 6H; Ar−O(CO)CH3), 1.47 (s, 9H; COOC(CH3)3); 13C
NMR (CDCl3) δ 170.3 (C-1), 33.9 (C-2), 60.1 (C-3), 193.7 (C-4),
124.0 (C-5), 144.2 (C-6), 133.0 (C-7), 111.6 (C-8), 151.4 (C-9),
141.9(C-10), 120.9 (C-11), 122.0 (C-12), 56.0 (Ar−OCH3), 20.6
(Ar−O(CO)CH3), 81.4 (−COOC(CH3)3), 28.1 (−COOC(CH3)3),
168.7 (Ar−O(CO)CH3).
Preparation of A Series (7−10). Route a. The removal of the
Boc group from E compounds gives A series. The reaction was
accomplished by addition TFA (50% in CH2Cl2) under continuous
stirring for 1 h at 0 °C.51 The organic phase was washed three times
with distilled water, and then dried under Na2SO4, concentrated, and
purified through flash column chromatography (silica gel, mesh
0.035−0.070 mm, mobile phase: n-hexane/EtOAc 5/5).
KE 60%: 1H NMR (CDCl3) δ 3.52 (s, 2H; H-2), 7.26 (d, 2H; H-5,
J = 15.2 Hz), 7.83 (d, 2H; H-6, J = 15.2 Hz), 7.64 (dd, 4H; H-8, J =
8.1 Hz, J = 1.7 Hz), 7.64 (m, 4H; H-9), 7.44 (m, 2H; H-10), 1.49 (s,
9H; COOC(CH3)3); 13C NMR (CDCl3) δ 170.9 (C-1), 33.5 (C-2),
105.7 (C-3), 183.6 (C-4), 120.7 (C-5), 142.0 (C-6), 135.2 (C-7),
128.2 (C-8), 128.4 (C-9), 130.1 (C-10), 81.9 (−COOC(CH3)3), 28.3
1
(−COOC(CH3)3). DK 40%: H NMR (CDCl3) δ 3.01 (d, 2H; H-2,
J = 7.3 Hz), 4.69 (t, 1H; H-3, J = 7.3 Hz), 6.91 (d, 2H; H-5, J = 15.8
Hz), 7.75 (d, 2H; H-6, J = 15.8 Hz), 7.64 (dd, 4H; H-8, J = 8.1 Hz,
J = 1.7 Hz), 7.60 (m, 4H; H-9), 7.44 (m, 2H; H-10), 1.48 (s, 9H;
−COOC(CH3)3); 13C NMR (CDCl3) δ 170.4 (C-1), 34.0 (C-2), 60.1
(C-3), 194.0 (C-4), 124.0 (C-5), 144.9 (C-6), 134.1 (C-7), 128.7
(C-8), 128.9 (C-9), 130.9 (C-10), 81.9 (−COOC(CH3)3), 28.3
(−COOC(CH3)3).
Route b. A suspension of B2O3 (2.25 mmol) and 2 (3.0 mmol) in
DMF (4 mL) was stirred for 30 min at 70 °C. A solution of the
appropriate benzaldehyde (6.0 mmol), AcOH (1,1 mL), and
morpholine (0.60 mmol) in DMF (4 mL) was added. The stirring
continued for 5 h at 70 °C. The mixture was then hydrolyzed at room
temperature by adding an aqueous solution 20% AcOH (36 mL) and
stirring 1 h. The mixture was then extracted twice with EtOAc. The
combined organic layers were washed with brine and water until
neutral pH, dried over Na2SO4, and evaporated under reduced pres-
sure to afford an oil, which was purified by flash column chroma-
tography. Silicagel, mesh 0.035−0.070 mm, mobile phase referred to
compound 8: petroleum ether/EtOAc/AcOH 70/30/0.3.
(3Z-5E)-tert-Butyl-4-hydroxy-6-(3-methoxy-4-hydroxyphenyl)-
acrolyl)hexa3,5-dienoate (4). Orange−red powder, 60% yield; mp
116.5 °C. LC-MS-IT m/z 505.2 (M + Na)+; 483.3 (M + H)+.
1
KE 50%: H NMR (MeOD-d4) δ 3.65 (s, 2H; H-2), 7.12 (d, 2H;
H-5, J = 15.2 Hz), 7.67 (d, 2H; H-6, J = 15.2 Hz), 6.84 (d, 2H; H-8,
J = 1.7 Hz), 7.17 (d, 2H; H-11, J = 8.1 Hz), 7.26 (dd, 2H; H-12,
J = 1.7 Hz, J = 8.1 Hz) 3.94 (s, 6H; Ar-OCH3), 1.45 (s, 9H;
−COO(CH3)3); 13C NMR (MeOD-d4) δ 172.2 (C-1), 32.6 (C-2),
105.1 (C-3), 183.2 (C-4), 117.4 (C-5), 142.2 (C-6), 127.2 (C-7),
115.2 (C-8), 148.0 (C-9), 149.8 (C-10), 123.7 (C-11), 110.6 (C-12),
55.1 (Ar−OCH3), 80.9 (−COOC(CH3)3), 26.8 (−C(CH3)3). DK
50%: 1H NMR (MeOD-d4) δ 2.90 (s, 2H; H-2), 6.93 (d, 2H; H-5, J =
15.9 Hz), 7.70 (d, 2H; H-6, J = 15.9 Hz), 6.86 (d, 2H; H-8, J = 1.7
Hz), 7.17 (d, 2H; H-11, J = 8.1 Hz), 7.26 (dd, 2H; H-12, J = 1.7 Hz,
(3Z-5E)-3-Cinnamoyl-4-hydroxy-6-phenylhexa-3,5-dienoic Acid
(7). Pale-yellow powder, yield: 67% (route a); mp 163−164 °C.
1
LC-MS-IT m/z 357.2 (M + Na)+; 335.3 (M + H)+. KE 48%: H
J = 8.1 Hz), 3.87 (s, 6H; Ar-OCH3), 1.44 (s, 9H; −COO(CH3)3); 13
C
NMR (MeOD-d4) δ 3.72 (s, 2H; H-2), 7.29 (d, 2H; H-5, J = 15.2 Hz),
7.76 (d, 2H; H-6, J = 15.2 Hz), 7.67 (dd, 4H; H-8, J = 1.8 Hz,, J = 8.2
Hz), 7.67 (m, 4H; H-9), 7.43 (m, 2H; H-10); 13C NMR (MeOD-d4) δ
174.4 (C-1), 30.8 (C-2), 105.5 (C-3), 183.0 (C-4), 120.3 (C-5), 141.9
(C-6), 134.1 (C-7), 130.9 (C-10), 128.4 (C-11), 127.9 (C-12). DK
52%: 1H NMR (MeOD-d4) δ 3.00 (s, 2H; H-2), 7.08 (d, 2H; H-5, J =
15.8 Hz), 7.77 (d, 2H; H-6, J = 15.8 Hz), 7.67 (dd, 4H; H-8, J = 1.8
Hz, J = 8.2 Hz), 7.67 (m, 4H; H-9), 7.43 (m, 2H; H-10); 13C NMR
(MeOD-d4) δ 173.3 (C-1), 32.0 (C-2), 57.9 (C-3), 194.7 (C-4), 124.5
(C-5), 144.6 (C-6), 135.2 (C-7), 128.1 (C-8), 128.4 (C-9), 130.9
(C-10).
NMR (MeOD-d4) δ 171.0 (C-1), 33.7 (C-2), 58.0 (C-3), 194.8 (C-4),
121.5 (C-5), 145.3 (C-6), 126.2 (C-7), 115.2 (C-8), 148.0 (C-9),
149.8 (C-10), 123.7 (C-11), 110.6 (C-12), 55.0 (Ar−OCH3), 80.9
(−C(CH3)3), 26.8 (−C(CH3)3).
(3Z-5E)-tert-Butyl-4-hydroxy-6-(3-methoxyphenyl)acrolyl)-
hexa3,5-dienoate (5). Dark-yellow powder, 60% yield; mp 152−
153 °C. LC-MS-IT m/z 473.2 (M + Na)+; 451.3 (M + H)+.
KE 52%: 1H NMR (CDCl3) δ 3.51 (s, 2H; H-2), 7.22 (d, 2H; H-5,
J = 15.3 Hz), 7.78 (d, 2H; H-6, J = 15.3 Hz), 7.15 (d, 2H; H-8, J = 1.9
Hz), 6.98 (m, 2H; H-10), 7.36 (t, 2H; H-11, J = 8.2 Hz), 7.25 (dd, 2H;
H-12, J = 1.9 Hz, J = 8.2 H), 3.90 (s, 6H; Ar−OCH3), 1.48 (s, 9H;
−C(CH3)3); 13C NMR (CDCl3) δ 170.87 (C-1), 33.5 (C-2), 105.8
(C-3), 183.5 (C-4), 121.0 (C-5), 141.9 (C-6), 136.9 (C-7), 113.4
(C-8), 159.8 (C-9), 115.7 (C-10), 129.8 (C-11), 120.7 (C-12), 55.4
(Ar−OCH3), 81.5 (−C(CH3)3), 28.0 (−C(CH3)3). DK 48%: 1H
NMR (CDCl3) δ 3.01 (d, 2H; H-2, J = 7.2 Hz), 4.69 (t, 1H; H-3, J =
7.2 Hz), 6.88 (d, 2H; H-5, J = 15.9 Hz), 7.71 (d, 2H; H-6, J = 15.9
Hz), 7.10 (d, 2H; H-8, J = 1.9 Hz), 6.98 (m, 2H; H-10), 7.36 (t, 2H;
H-11, J = 8.2 Hz), 7.25 (dd, 2H; H-12, J = 1.9 Hz, J = 8.2 H), 3.86 (s,
6H; Ar-OCH3), 1.48 (s, 9H; COOC(CH3)3); 13C NMR (CDCl3) δ
170.3 (C-1), 34.0 (C-2), 60.0 (C-3), 193.8 (C-4), 124.2 (C-5), 144.8
(C-6), 136.9 (C-7), 113.2 (C-8), 159.9 (C-9), 115.7 (C-10), 129.8
(C-11), 120.7 (C-12), 55.4 (Ar−OCH3), 81.4 (−COOC(CH3)3), 27.9
(−COOC(CH3)3).
(3Z,5E)-6-(3-Methoxy-4-hydroxyphenyl)-3-((E)-3-(3-methoxy-4-
hydroxyphenyl)acryloyl)-4-hydroxyhexa-3,5-dienoic Acid (8). Orange−
red powder, yield: 21% (route b); mp 158−159 °C. LC-MS-IT m/z
449.1 (M + Na)+; 427.1 (M + H)+.
1
KE 25%: H NMR (MeOD-d4) δ 3.68 (s, 2H, H-2), 7.08 (d, 2H,
H-5, J = 15.1 Hz), 7.66 (d,2H, H-6, J = 15.1 Hz), 7.24 (d, 2H, H-8, J =
1.6 Hz), 6.82 (d, 2H, H-11, J = 8.2 Hz), 7.13 (dd, 2H, H-12, J = 1.6
Hz, J = 8.2 Hz), 3.91 (s, 6H, Ar-OCH3); 13C NMR (MeOD-d4) δ
176.4 (C-1), 32.7 (C-2), 106.5 (C-3), 184.8 (C-4), 118.8 (C-5), 143.6
(C-6), 128.9 (C-7), 112.0 (C-8), 149.4 (C-9), 150.5 (C-10), 116.6
(C-11), 124.3 (C-12), 56.5 (Ar−OCH3). DK 75%: 1H NMR (MeOD-d4)
δ 2.96 (s, 2H, H-2), 6.91 (d, 2H, H-5, J = 15.9 Hz), 7.68 (d, 2H, H-6,
J = 15.9 Hz), 7.22 (d, 2H, H-8 J = 1.6 Hz), 6.81 (d, 2H, H-11, J = 8.2
Hz), 7.14 (dd, 2H, H-12, J = 1.6 Hz, J = 8.2 Hz), 3.85 (s, 6H, Ar-
OCH3); 13C NMR (MeOD-d4) δ 175.1 (C-1), 33.7 (C-2), 59.4 (C-3),
196.2 (C-4), 123.1 (C-5), 146.8 (C-6), 127.6 (C-7), 112.0 (C-8),
4,4′-((1E,3Z,6E)-4-(2-tert-Butoxy-2-oxoethyl)-3-hydroxy-5-oxo-
hepta-1,3,6-triene-1,7-diyl)bis(2-methoxy-4,1-phenylene)diacetate
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149.5 (C-9), 151.4 (C-10), 116.6 (C-11), 125.2 (C-12), 56.4 (Ar−
OCH3).
Table 6. Crystal Data and Structure Refinement for
Compound 5
(3Z-5E)-4-Hydroxy-6-(3-methoxyphenyl)acrolyl)hexa3,5-dienoic
Acid (9). Orange−yellow powder, yield: 62% (route a); mp 191−
192 °C. LC-MS-IT m/z 417.2 (M + Na)+; 395.2 (M + H)+. KE 43%: 1H
NMR (MeOD-d4) δ 3.70 (s, 2H; H-2), 7.28 (d, 2H; H-5, J = 15.3 Hz),
7.72 (d, 2H; H-6, J = 15.3 Hz), 7.22 (d, 2H, H-8, J = 1.9 Hz), 7.00 (dd,
2H, H-10, J = 1.9 Hz, J = 8.2 Hz), 7.34 (t, 2H, H-11, J = 8.2 Hz), 7.24
(dd, 2H, H-12, J = 1.9 Hz, J = 8.2 Hz), 3.86 (Ar-OCH3); 13C NMR
(MeOD-d4) δ 174.9 (C-1), 31.6 (C-2), 106.1 (C-3), 183.1 (C-4),
120.8 (C-5), 141.6 (C-6), 160.1 (C-9), 54.5 (Ar−OCH3). DK 57%:
1H NMR (MeOD-d4) δ 2.98 (s, 2H, H-2), 7.08 (d, 2H, H-5, J = 15.9
empirical formula
formula wt
C27 H30 O6
450.51
temp
293(2) K
wavelength
0.71073 Å
crystal system, space group
unit cell dimensions
triclinic P1
a = 10.3080(10) Å α = 63.957(4)°
b = 10.9062(9) Å β = 82.789(3)°
c = 12.1041(9) Å γ = 86.318(3)°
1212.86(18) Å3
2, 1.234 g/cm3
0.086 mm−1
vol
Hz), 7.73 (d, 2H, H-6, J = 15.9 Hz), 7.22 (d, 2H, H-8, J = 1.9 Hz),
7.00 (dd, 2H, H-10, J = 1.9 Hz, J = 8.2 Hz), 7.34 (t, 2H, H-11, J = 8.2
Z, calcd density
Hz), 7.24 (dd, 2H, H-12, J = 1.9 Hz, J = 8.2 Hz), 3.82 (Ar-OCH3); 13
C
absorption coefficient
F(000)
NMR (MeOD-d4) δ 173.8 (C-1), 32.4 (C-2), 57.6 (C-3), 194.8 (C-4),
124.9 (C-5), 144.5 (C-6), 136.5 (C-7), 112.6 (C-8), 160.1 (C-9),
116.7 (C-10), 129.8 (C-11), 120.2 (C-12), 54.1 (Ar−OCH3).
480
crystal size
0.3 × 0.2 × 0.3 mm
3.77−19.19°
−9≤h ≤ 9, −9≤k ≤ 10, −11≤l ≤ 10
5076/1946 [R(int) = 0.0449]
96.6%
θ range for data collection
limiting indices
(3Z,5E)-6-(4-Acetoxy-3-methoxyphenyl)-3-((E)-3-(4-acetoxy-3-
methoxyphenyl)acryloyl)-4-hydroxyhexa-3,5-dienoic Acid (10). Orange−
yellow powder, yield: 60% (route a); mp 130−131 °C. LC-MS-IT
m/z 533.2 (M + Na)+; 511.2 (M + H)+. KE 30%: 1H NMR (MeOD-d4)
δ 3.76 (s, 2H, H-2), 6.96 (d, 2H, H-5, J = 15.2 Hz), 7.73 (d, 2H, H-6,
J = 15.2 Hz), 7.39 (d, 2H, H-8, J = 1.7 Hz), 7.28 (d, 2H, H-11, J = 8.1
Hz), 7.10 (dd, 2H, H-12, J = 1.7 Hz, J = 8.1 Hz), 2.29 (Ar−
O(CO)CH3), 3.90 (Ar−OCH3); 13C NMR (MeOD-d4) δ 174.6 (C-
1), 31.2 (C-2), 106.7 (C-3), 183.2 (C-4), 115.2 (C-5), 141.1 (C-6),
134.3 (C-7), 111.4 (C-8), 151.6 (C-9), 141.9 (C-10), 121.1 (C-11),
122.9 (C-12), 55.1 (Ar−OCH3), 19.0 (Ar−O(CO)CH3), 169.0 (Ar−
reflns collected/unique
completeness to θ = 19.19
absorption correction
refinement method
data/restraints/parameters
goodness-of-fit on F2
final R indices [I > 2σ (I)]
R indices (all data)
extinction coefficient
largest diff. peak and hole
none
full-matrix least-squares on F2
1946/0/389
0.800
R1 = 0.0399, wR2 = 0.0943
R1 = 0.0761, wR2 = 0.1167
0.0000(12)
0.106 and −0.103 e·Å−1
1
O(CO)CH3). DK 70%: H NMR (MeOD-d4) δ 3.01 (s, 2H, H-2),
7.09 (d, 2H, H-5, J = 15.8 Hz), 7.75 (d, 2H, H-6, J = 15.8 Hz), 7.37 (d,
2H, H-8, J = 1.7 Hz), 7.28 (d, 2H, H-11, J = 8.1 Hz), 7.10 (dd, 2H, H-
12, J = 1.7 Hz, J = 8.1 Hz), 2.28 (Ar−O(CO)CH3), 3.84 (Ar−OCH3).
13C NMR (MeOD-d4) δ 173.5 (C-1), 32.2 (C-2), 58.1 (C-3), 194.7
(C-4), 124.9 (C-5), 143.7 (C-6), 133.3 (C-7), 111.4 (C-8), 151.6
(C-9), 141.9 (C-10), 121.1 (C-11), 122.9 (C-12),55.1 (Ar−OCH3),
19.0 (Ar−O(CO)CH3), 169.0 (Ar−O(CO)CH3).
change in absorbance in the 200−600 nm range over an overall period
of 8 h. Then 50 μM solutions of the ligands were prepared in 0.1 M
TRIS-HCl buffer (pH 7.4). A constant ionic strength of 0.1 M
(NaNO3) was maintained in all experiments. Spectra were recorded
every 30 min. All profiles were linearized by an hyperbolic function
(eq 1), which represents an empirical model that well describes drug
decomposition or release.37
4.2. Crystal Data. The diffraction data were collected at room
temperature with Bruker-Nonius X8APEX single-crystal diffractometer
controlled by Bruker-Nonius X8APEX software using Mo Kα radiation
(λ = 0.71073 Å). The structure was solved by direct method and
(1)
2
refined by full-matrix, lest-squares procedures (based on Fo ) using
WINGX system of crystallographic computer programs.52 All non-
hydrogen atoms were refined anisotropically, and hydrogen atoms
were located in a Fourier map and refined isotropically. The crystal
data and refinement parameters are summarized in Table 6.
4.3. Spectroscopy. Spectrophotometric measurements were
performed using Jasco V-570 spectrophotometer at 25 ± 0.1 °C in
the 200−600 nm spectral range employing 1 cm quartz cells. pH-
Metric titrations were obtained varying the pH value by adding small
amounts of concentrated NaOH or HCl in the pH range 1−11. A
constant ionic strength of 0.1 M (NaNO3) was maintained in all
experiments. The overall stability constants (expressed as log βLH) and
pKa values were evaluated from spectrophotometric data using the
software pHab.36
where f% is the fraction of residual compound at time t (min)
expressed as percentage referred to starting concentration at time zero.
4.5. Solubility. The appropriate amount of each A compound was
shaken with a known volume of buffered aqueous solution (0.1 M
TRIS-HCl, pH = 7.4) at room temperature for 30 min in order to
provide a saturated solution with a precipitate. The supernatant was
filtered through a 0.2 μm disposable membrane filter (OlimPeak,
Tekno Kroma), and the concentration of ligand in the resultant
solution was determined (after an appropriate dilution) by UV−vis
spectroscopy.
4.6. Partition Coefficient. The shaken-flask method was
performed in buffered condition (TRIS-HCl pH 7.4). Ten mL stock
solutions of each compound (50 μM) were mixed in a vessel with
known volumes of high purity analytical grade n-octanol (2, 4, 6, 8,
10 mL) and shaken at 37 °C for 2 h. After separating the two phases,
the ligand concentration in the aqueous solution was determined by
UV−vis spectroscopy by reading absorbance at λ = 300 nm.
4.7. Antioxidant Activity (DPPH Radical Scavenging
Method). The antioxidant activity of curcuminoids was determined
in terms of hydrogen donating or radical scavenging ability, using the
stable radical DPPH. A variable amount (15, 30, 45, 75, 105, and
150 μL) of a methanolic solution (1.2 mM) of each compound,
including curcumin as benchmark, was placed in a cuvette, and 3 mL
of a 6 × 10−5 M methanolic solution of DPPH was added. Absorbance
measurements initiated immediately. The decrease in absorbance at
517 nm was determined continuously every minute up to 5 min, then
every 5 min up to 30 min and every 30 min until reaction reaches
completeness and absorbance stabilizes attaining a plateau after
NMR spectra were recorded on a Bruker Avance AMX-400
spectrometer with a Broad Band 5 mm probe (inverse detection).
Nominal frequencies were 100.13 MHz for 13C and 400.13 MHz for
1H. The typical acquisition parameters for 1H were as follows: 20 ppm
1
spectral bandwidth (SW), 6.1 μs pulse width (90° hard pulse on H),
0.5−1 s pulse delay, 216−512 number of scans. For 2D H,H-homo-
nuclear correlated spectroscopy (COSY), typical parameters were
used. For 2D H,X-hetero correlated spectroscopy, HMBC and HMQC
opportune parameters were used (50−90° pulses; 32 k data points; 1 s
1
3
relaxation delay; 8−64 k transients; JH−C 125−145 Hz; JH−C 5−15
Hz). CD3OD-d4, DMSO-d6, and CDCl3 were used as NMR solvent.
All reported chemical shifts (δ) are expressed in ppm and are referred
to TMS.
4.4. Kinetic Stability. The chemical stability at 37 °C in darkness
of both T and A series was evaluated by UV−vis spectroscopy as a
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Journal of Medicinal Chemistry
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120 min. Methanol was used to zero the spectrophotometer. The
absorbance of the DPPH radical without antioxidant, i.e. the control,
was measured daily, and concentration was calculated applying eq 2:53
ACKNOWLEDGMENTS
■
We are grateful to the “Centro Interdipartimentale Grandi
Strumenti−CIGS” of the University of Modena and Reggio
Emilia for supplying advanced instrumentations and skilled
technicians. This work was supported by Associazione Italiana
per la Ricerca sul Cancro-MFAG to CI (grant no. 6192).
(2)
The percentage of inhibition (%In) of the DPPH radical by each
sample was calculated according to the formula:
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ASSOCIATED CONTENT
* Supporting Information
■
S
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Crystallographic data (excluding structure factors) have been
deposited with the Cambridge Crystallographic Data Center as
Supplementary Publication no. CCDC 838903. Copies of the
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AUTHOR INFORMATION
Corresponding Author
*Phone: +39 059 2055040. Fax: +39 059373543. E-mail:
monica.saladini@unimore.it.
■
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