Synthesis and biological evaluation of novel curcuminoid derivatives

Article abstract of DOI:10.3390/molecules191016349

Curcuminoids have been reported to possess multiple bioactivities, such as antioxidant, anticancer and anti-inflammatory properties. Three novel series of curcuminoid derivatives (11a-h, 15a-h and 19a-d) with enhanced bioactivity have been synthesized. Among the synthesized compounds, 11b exhibited the most significant activity with an MIC of 0.5 μ M /mL against selected medically important Gram-positive cocci (S aureus and S viridans) and Gram-negative bacilli (E. coli and E. cloacae). The derivatives exhibited remarkable results in an antioxidant test with an IC₅₀ 2.4- to 9.3-folder smaller than curcuminoids. With respect to antiproliferative activity against Hep-G2, LX-2, SMMC7221 and MDA-MB-231, the derivatives exhibited an effect stronger than curcuminoids with an IC₅₀ ranging from 0.18 to 4.25 μ M.

Full text of DOI:10.3390/molecules191016349

Molecules 2014, 19, 16349-16372; doi:10.3390/molecules191016349
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Novel
Curcuminoid Derivatives
1
,†
2,†
1
1
1,
Ya-Kun Cao , Hui-Jing Li , Zhi-Fang Song , Yang Li and Qi-Yong Huai *
1
Department of Applied Chemistry, Marine College, Shandong University, Weihai 264209, China;
E-Mails: yakun_cao@163.com (Y.-K.C.); zhifangdl@163.com (Z.-F.S.);
LYchemlh@hotmail.com (Y.L.)
2
Department of Environmental Engineering, School of Marine Science and Technology,
Harbin Institute of Technology, Weihai 264209, China; E-Mail: lihuijing@iccas.ac.cn
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: huaiqy06@sdu.edu.cn;
Tel./Fax: +86-631-568-8303.
External Editor: Derek J. McPhee
Received: 15 September 2014; in revised form: 26 September 2014 / Accepted: 29 September 2014 /
Published: 13 October 2014
Abstract: Curcuminoids have been reported to possess multiple bioactivities, such as
antioxidant, anticancer and anti-inflammatory properties. Three novel series of curcuminoid
derivatives (11a–h, 15a–h and 19a–d) with enhanced bioactivity have been synthesized.
Among the synthesized compounds, 11b exhibited the most significant activity with an
MIC of 0.5 µM /mL against selected medically important Gram-positive cocci (S. aureus
and S. viridans) and Gram-negative bacilli (E. coli and E. cloacae). The derivatives
exhibited remarkable results in an antioxidant test with an IC50 2.4- to 9.3-folder smaller
than curcuminoids. With respect to antiproliferative activity against Hep-G2, LX-2,
SMMC7221 and MDA-MB-231, the derivatives exhibited an effect stronger than
curcuminoids with an IC50 ranging from 0.18 to 4.25 µM.
Keywords: curcuminoids; antibacterial; antioxidant; antiproliferative

Molecules 2014, 19
. Introduction
Natural compounds derived from plants, animals and microorganisms proved to be an excellent
16350
1
source for therapeutic agents. Phytochemicals once served humankind as the source of new drug
development, and today, these natural products or their derivatives still represent preventive and
therapeutic agents against a wide range of human diseases [1,2]. Turmeric and ginger are spices that
belong to the Zingiberaceae family. Ginger, a common ingredient for various foods and beverages, is
also employed in medicine for the treatment of various ailments, like headaches, nausea, rheumatism and
colds [3]. Curcuminoids, viz., curcumin (CCM), demethoxycurcumin (DCM) and bisdemethoxycurcumin
(BCM), are the yellow pigments of turmeric [4]. (Figure 1) Recent studies have shown that curcuminoids
possess a wide range of biological activities, including antioxidant [5,6], anti-inflammatory [7,8],
anticancer [9,10] and anti-angiogenesis [11,12] properties, as well as medicinal applications,
including their use in HIV therapies [13] and Alzheimer’s disease [14]. The non-toxic food origin and
wide range of pharmaceutical properties of curcuminoids make them promising candidate molecules
for medicine. Efforts have been made to synthesize new curcuminoid derivatives with stronger
biological activities [15–17].
Figure 1. Curcumin, demethoxycurcumin and bisdemethoxycurcumin.
In addition to curcuminoids, some other natural products, such as methionine [18,19],
selenomethionine [19–22], caffeic acid [23–25], ferulic acid [26–28] and 18β-glycyrrhetinic acid [29–31],
have drawn our attention because of various pharmaceutical properties. Hence, we selected those
active ingredients as starting materials to design and synthesize three serials of curcuminoid
derivatives. The antioxidant ability, antibacterial activity, as well as cytotoxic potency of the
compounds against human tumor cells were evaluated.
2
. Results and Discussion
2
.1. Chemistry
Symmetrical curcuminoids, CCM and BCM were synthesized according to the procedure
previously reported (Scheme 1) [32]. Compounds 1a and 1b were obtained in an excellent yield. NMR
and other analytical data support the curcuminoids’ purity of >95% after recrystallization in methanol.

Molecules 2014, 19
Scheme 1. Syntheses of curcumin (1a) and bisdemethoxycurcumin (1b).
16351
The synthesis of curcuminoid derivatives, viz., 11a–h, 15a–h and 19a–d, were reported. In the first
series, selenomethionine (8) was prepared from methionine (2) through seven steps according to the
procedure previously reported (Scheme 2) [33]. The synthesis of 10a–h was carried out in dry CHCl
in the presence of dehydrating agents, 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride/
-Hydroxybenzotriazole (EDCI/HOBT) or Dicyclohexylcarbodiimide/4-dimethylaminopyridine
DCC/DMAP), as catalysts. Subsequently, deprotection of amino group were completed with HCl
3
1
(
in dry ethyl acetate (EA) at room temperature for 2–4 h to get methionine-substituted and
selenomethionine-substituted curcuminoids (11a–h).
Scheme
2.
Synthesis
of
selenomethionine-substituted
curcuminoids
and
methionine-substituted curcuminoids.
The synthesis of the second series depicted in Scheme 3, 13a or 13b, was reacted with
curcuminoids in the presence of EDCI/HOBT to obtain 14a–d in 45%–65% yield. Treatment of 13a or
1
3b with SOCl
acid-binding agent, using the esterification procedure furnished compounds 14e–h. Acetylated
compounds 14a–h resulted upon hydrolysis with CH ONa, resulting in their respective phenolic
2
was followed by the addition of curcuminoids and Triethylamine (TEA) as the
3
compounds, 15a–h. As shown in Scheme 4, we designed the synthesis of curcuminoid-glycyrrhetinic
acid conjugates (19a–d) using similar methods. All of the newly synthesized bioconjugates were
1
13
characterized by melting point, elemental analysis, ESI-MS, H-NMR and C-NMR.

Molecules 2014, 19
Scheme 3. Synthesis of curcuminoid-caffeic acid and curcuminoid-ferulic acid conjugates.
16352
Scheme 4. Synthesis of curcuminoid-glycyrrhetinic acid conjugates.
2
.2. Pharmacological Activities
.2.1. Antioxidant Activity
As one of their well-known mechanisms, antioxidants can inhibit lipid oxidation by scavenging free
2
radicals. Compared to other methods of measuring antioxidant activity, the DPPH radical method is
quite simple and rapid for screening specific compounds, as the DPPH radical is commercially
available and does not need to be generated prior to use. Thus, the antioxidant activity of all of the
synthesized new curcuminoid derivatives (11a–h, 15a–h and 19a–d) was assessed for their radical
scavenging ability using the stable DPPH radical method. The assay was conducted at six different
concentrations of antioxidants (2.5 µM, 5 µM, 10 µM, 20 µM, 40 µM and 80 µM) in a polar,
homogeneous medium. The antioxidant potency (IC50) is presented in µM concentrations of the
synthesized curcuminoid derivatives, as shown in Table 1. Besides, we tracked the change of absorbance
of the DPPH solution over time in the presence of curcuminoid derivatives at a concentration of
2
0 µM and, based on these data, determined the decay rates of the DPPH radicals (Table 1).

Molecules 2014, 19
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Table 1. In vitro DPPH free radical scavenging activity (FRSA) of the target compounds.
CCM, curcumin; BCM, bisdemethoxycurcumin.
DPPH FRSA%
Compound
IC50
(µM)
30
−
1
d[radical]/dt(µM·min )
2
.5 µM 5 µM 10 µM 20 µM 40 µM
80 µM
93.3
96.6
92.4
95.5
92.2
94.8
91.2
93.9
93.7
93.1
94.2
93.9
93.5
92.2
95.2
92.8
92.2
80.7
93.4
92.3
38.8
32.1
1
1a
1b
1c
1d
1e
1f
1g
1h
5a
5b
5c
5d
5e
5f
5g
5h
9a
9b
9c
9d
4.2
11.4
3.5
6.3
3.3
5.2
2.9
4.5
5.2
3.4
4.3
4.6
4.4
3.1
6.3
3.4
3.3
2.6
5.2
3.6
1.2
1.2
8.3
22.7
6.9
16.7
45.5
13.9
23.8
13.2
19.8
11.5
17.6
20.6
13.5
17.2
18.5
17.5
11.9
25.3
13.9
13.4
10.3
20.8
14.4
4.9
33.3
90.9
27.8
47.6
26.3
39.8
23.8
35.5
41.7
27.3
34.7
37.3
34.5
23.5
50.5
27.3
26.5
20.8
41.7
28.6
9.7
66.7
95.5
55.6
92.2
52.6
81.5
47.2
71.2
82.9
54.9
69.2
74.1
68.7
47.6
92.6
54.1
52.6
40.8
83.3
57.1
19.5
16.3
9.49
18.58
8.92
1
11
1
36
1
11.9
6.5
21
13.14
8.57
1
38
1
10.3
6.1
25
12.44
8.61
1
42
1
8.8
28
11.05
12.62
8.83
1
10.2
6.6
24
1
37
1
8.5
29
13.22
11.55
10.88
7.25
1
9.2
27
1
8.9
29
1
6.3
42
1
12.5
6.6
20
14.12
10.23
10.92
5.89
1
37
1
6.5
38
1
4.9
49
1
10.4
7.1
24
13.02
9.88
1
35
CCM
BCM
2.5
103
123
1.34
2.3
4.1
8.5
0.56
The radical-scavenging assay by the DPPH method clearly indicated that all curcuminoid
derivatives have lower IC50 values than curcuminoids. For instance, methionine-substituted curcumin
exhibited 2.5- to 3.4-fold lower IC50 values than did curcumin as the standard compound, which could
be attributed to the sulfur moiety present in it, acting as a good radical-scavenging agent.
However, selenomethionine-substituted curcuminoids showed 1.5- to 2.7-fold lower IC50 values
than did methionine-substituted curcuminoids, which revealed the important role of selenium in
scavenging radicals.
The results also showed that attachment of a caffeic acid and a ferulic acid moiety to the
curcuminoids significantly enhanced the antioxidant activity. For instance, curcumin-di-caffeic acid
(
15f) showed a potent DPPH free radical scavenging ability with IC50 of 29 µM , while the IC50 of
curcumin as the standard compound is 103 µM . In addition, curcuminoids with a caffeic acid moiety
showed a higher DPPH radical scavenging ability than those with ferulic acid, which revealed the great
influence of free hydroxyl groups on the DPPH radical scavenging activity of curcuminoid derivatives.
For instance, curcumin-mono-caffeic acid (15b) exhibited better antioxidant activity with an IC50 of
2
4 µM , while the corresponding curcumin-mono-ferulic acid (15a) showed an IC50 of 37 µM .
With respect to the third series of curcuminoid derivatives, the best radical scavenger is
curcumin-di-glycyrrhetinic acid (19c), whose scavenging ability is 4.3-folder higher than curcumin as

Molecules 2014, 19
16354
the standard compound. The curcuminoid-glycyrrhetinic conjugates also showed more efficiency than
curcuminoids. As the results revealed, the curcumin-di-glycyrrhetinic acid exhibited a 9.7-folder
higher scavenging radical rate than curcumin did.
2
.2.2. Antibacterial Activity
All curcuminoid derivatives were tested for their antimicrobial potential against Gram-positive
cocci (S. aureus and S. viridans) and Gram-negative bacilli (E. coli and E. cloacae). The lowest
concentration of curcuminoid derivatives in µmol/mL that prevented in vitro growth of microorganism
has been represented as being correlated with the zone of inhibition and MIC (minimum inhibitory
concentration), as shown in Table 2.
a
b
Table 2. Zone of inhibition and MIC correlation diagram of curcuminoid derivatives
against bacterial strains.
Name of Bacteria
Gram-positive
S. aureus S. viridans
Gram-negative
E. coli E. cloacae
Zone of Zone of
Compound
Zone of
Inhibition
19, 16, 14
26, 20, 18
19, 17, 15
24, 17, 14
18, 15, 13
21, 18, 16
18, 16, 14
20, 17, 15
19, 16, 14
22, 20, 18
18, 15, 13
20, 18, –
Zone of
MIC
MIC
MIC
MIC
Inhibition
19, 17, 14
24, 20, 18
18, 16, 14
22, 18, 15
18, 14, 10
21, 18, 16
22, 19, 17
23, 20, 18
19, 17, 14
21, 19, 16
19, 15, 13
20, 18, –
Inhibition
19, 17, 15
25, 20, 17
19, 16, 14
22, 17, 15
18, 15, 13
20, 17, 15
18, 16, 13
20, 15, 13
19, 17, 14
22, 20, 18
18, 16, 11
20, 18, –
Inhibition
18, 16, 14
23, 20, 18
19, 16, 14
24, 19, 16
18, 14, 13
21, 18, 15
20, 18, 16
22, 19, 17
19, 16, 15
22, 20, 18
18, 15, 12
21, 18, 17
19, 17, 13
21, 18, 16
19, 16, 14
26, 20, 18
19, 17, 16
20, 18, –
1
1a
1b
2.0
0.5
2.3
1.0
3.2
0.7
3.7
1.6
3.8
3.7
4.3
4.1
2.7
2.8
3.2
3.0
2.4
2.6
1.6
1.7
21.7
32.5
2.5
2.0
0.5
2.3
1.0
2.2
0.7
2.4
1.6
3.8
3.7
4.3
4.1
2.7
2.8
3.2
3.0
2.4
2.6
1.6
1.7
21.7
32.5
2.5
2.0
0.5
4.6
1.0
3.2
0.3
3.7
1.6
2.4
2.3
2.7
2.6
1.7
1.7
2.0
1.9
1.5
1.6
1.0
1.0
27.2
32.5
3.2
4.0
0.5
4.6
1.5
3.2
0.7
2.4
1.6
1.9
1.8
2.1
2.1
1.3
1.4
1.6
1.5
1.2
1.3
0.8
0.8
27.2
32.5
1
1
1c
1d
1e
1f
1
1
1
11g
11h
15a
15b
1
5c
5d
5e
5f
1
1
18, 15, 13
21, 17, 16
19, 17, 14
21, 19, 18
19, 17, 15
21, 19, 17
25, 20, 18
22, 20, 18
10, 8, 6
18, 15, –
16, 13, 10
21, 17, 16
19, 16, 14
26, 20, 18
20, 18, 17
21, 18, 17
24, 22, 20
22, 19, 17
10, 8, –
1
23, 17, 16
19, 16, 14
26, 20, 18
20, 18, 16
21, 18, 16
24, 21, 19
22, 20, 18
11, 9, –
15g
15h
19a
19b
1
9c
23, 21, 19
21, 19, 17
12, 8, –
1
9d
CCM
BCM
8, 5, –
8, 6, –
8, 5, –
9, 6, –
Ampicillin
21, 18, 15
20, 17, 15
20, 17,14
20, 18, 15
3.2
a
b
–
Resistant; the zone of inhibition was measured in mm at concentrations of 20, 10 and 5 µM/mL; MIC
(minimum inhibitory concentration) values were measured in µM/mL.

Molecules 2014, 19
16355
All curcuminoid derivatives exhibited better antibacterial activity than curcuminoids. The most
encouraging results were obtained in the case of curcumin-mono-selenomethionine (11b) having an
MIC of 0.5 µM/mL against S. aureus, while ampicillin, the best marketed antibiotic, shows an MIC of
2.5 µM/mL, showing that (11b) is five times more effective than the ampicillin at similar
concentrations. The zones of inhibition of 11b were 26 mm, 20 mm and 18 mm, while curcumin had
zones of 10 mm, 8 mm and 6 mm. The monoester 11b has shown highly satisfactory results as an
antibacterial agent, which may be due to the selenium and free hydroxyls. Curcuminoid-caffeic acid
and curcuminoid-ferulic acid conjugates showed an MIC ranging from 2.8 to 4.3 µM/mL. With respect
to the third series of curcuminoid derivatives, the best antibacterial agents are 19c and 19d, with an
MIC ranging between 1.6 µM/mL and 1.7 µM/mL, 1.5- to 1.6-fold lower than did ampicillin.
What is more, the second and third series of curcuminoid derivatives showed better antibacterial activity
against E. coli than ampicillin without exception. Curcuminoid-caffeic acid and curcuminoid-ferulic
acid conjugates showed an MIC ranging between 1.7 µM/mL and 2.7 µM/mL, 1.2- to 1.9-folder lower
than ampicillin did. Curcumin-glycyrrhetinic acid conjugates showed an MIC ranging between
0
.8 µM/mL and 1.3 µM/mL, 2.5- to 4.0-folder lower than ampicillin did.
The results of an antibacterial test against E. cloacae were also encouraging. The antibacterial
activity of the bioconjugates was 8.6–49.4-times higher than that of curcuminoids.
2
.2.3. Antiproliferative Activity
The inhibitory effects of curcuminoid derivatives on the growth of three lines of cultured human
hepatoma cells (Hep-G2, LX-2 and SMMC-7221) and one line of human breast cancer cells
MDA-MB-231) were determined using the MTT assay. For each experiment, CCM and BCM were
(
used as positive controls. The cytotoxic effects of curcuminoid derivatives were confirmed at
concentrations of 1 µM, 5 µM, 10 µM and 20 µM. Inhibitory activities (IC50) were presented in µM/L
concentrations of the synthesized curcuminoid derivatives, as shown in Table 3.
All curcuminoid derivatives showed remarkably stronger cancer growth inhibitory effects in
Hep-G2, LX-2, SMMC-7221 and MDA-MB-231 cells than did curcuminoids, as determined by the
MTT assay. The activity data suggests that the derivatives having methionine exhibited good
anticancer activity, and replacement of the selenium atom with the sulfur atom showed more
remarkable anticancer activity, which was effective against all cell lines. Compound 11b exhibited an
IC50 of 0.18–0.63 µM in Hep-G2, LX-2, SMMC-7221 and MDA-MB-231 cells, while the IC50 of 11a
was 4.9- to 9.8-fold higher (1.41–2.94 µM). We also determined that the monoesters demonstrated
improved anticancer properties compared to their corresponding diesters. The ester linkages to
selenomethionine and methionine could be hydrolyzed by esterase enzymes, releasing parent
curcuminoids at the target site. Thus, monoesters having one free phenolic hydroxyl and one ester
linkage to selenomethionine have great advantages and hold promise as prodrugs.
In the second series of curcuminoid derivatives, curcuminoid-caffeic acid conjugates showed a
stronger inhibition effect on the growth of tumor cells than curcuminoid-ferulic acid conjugates. For
instance, Compound 15a exhibited an IC50 of 2.75–3.96 μM in Hep-G2, LX-2, SMMC-7221 and
MDA-MB-231 cells, while the IC50 of 15b was between 1.11 and 2.16 μM.

Molecules 2014, 19
16356
Table 3. Inhibitory effects of curcuminoid derivatives on the growth of Hep-G2, LX-2,
SMMC7221 and MDA-MB-231.
IC50 (µM)
SMMC-7721
12.57
17.11
2.05
Compound
Hep-G2
LX-2
13.97
16.35
2.92
0.62
3.22
0.98
3.55
0.78
4.39
1.42
3.96
1.28
3.36
1.28
4.24
3.38
3.94
3.28
3.36
2.25
0.94
1.48
MDA-MB-231
11.45
14.10
2.64
CCM
BCM
24.95
32.65
2.54
0.31
3.01
0.75
3.54
0.95
4.01
0.96
3.75
1.11
4.25
3.11
5.15
2.16
3.75
3.11
4.25
3.16
0.75
1.52
1
1a
1b
1
0.18
0.52
1
1c
1d
1e
1f
2.35
1.84
1
0.55
0.85
1
3.41
3.59
1
1.35
1.22
11g
11h
15a
15b
4.17
4.62
1.21
1.82
2.75
3.23
2.16
1.72
1
5c
5d
5e
5f
3.32
2.75
1
1.16
2.05
1
3.23
3.05
1
2.98
2.80
15g
15h
19a
19b
2.75
3.23
2.16
2.72
3.32
2.75
2.28
2.15
1
9c
1.03
1.05
19d
1.65
1.78
IC50, the compound concentration required to inhibit cell proliferation by 50%. Hep-G2, LX-2, SMMC-7221
3
and MDA-MB-231 were seeded at a density of 2 × 10 Cells/well. The cells were then treated with various
concentrations (1 µM, 5 µM, 10 µM and 20 µM) of different curcuminoid derivatives for 72 h. The effects of
the different compounds on the growth of Hep-G2, LX-2, SMMC-7221 and MDA-MB-231 cells were
determined by the MTT assay.
Among the curcuminoid-glycyrrhetinic acid conjugates, 19c showed the best antiproliferative
activity with an IC50 of 0.75–1.05 µM . In the first series, we found that the monoesters exhibited better
anti-tumor activity than their corresponding diesters, but the third series is just the opposite. The IC50
of curcumin-mono-glycyrrhetinic acid (19a) ranged between 2.75 to 4.25 µM , while the IC50 of
curcumin-di-glycyrrhetinic acid (19c) was between 0.75 µM and 1.05 µM .
2
.3. Structure-Activity Relationship
Curcumin has two phenyl rings and substitutions at the 3 and 4 positions with methoxy and
hydroxyl groups, respectively. It has been reported to undergo extensive in vitro and in vivo phase I
and phase II metabolism through oxidation, reduction, glucuronidation and sulfation. The
glucuronidation and sulfation occur on the 4-OH groups present on both phenyl rings of curcumin. The
protection of the 4-OH groups improves its stability.

Molecules 2014, 19
16357
In the first series of curcuminoid derivatives, the assessment of their antioxidant, antimicrobial
and anticancer activities suggested that derivatives are relatively more active than curcuminoids,
which may be due to their increased solubility, slow metabolism and better cellular uptake.
Shiv K. Dubey et al. [34] enhanced the biological activities of curcumin by introducing glycine,
glutamic acid and valine on phenyl rings. They also found that monoesters of curcumin have even
better antimicrobial activity than their corresponding diesters, emphasizing the role of the free phenolic
group. That findings are consistent with our results. What is more, the CCM derivatives exhibited
better bioactivities than the corresponding BCM derivatives, which revealed the importance of
methoxy groups in the skeleton for biological activity.
Wisut et al. [35] synthesized a series of succinate prodrugs by esterification of curcuminoids with a
methyl or ethyl ester of succinyl chloride. These compounds showed better anticancer activity against
Caco-2 cells with IC50 values in the 1.8–9.6 µM range, Compared with succinylation, the conjugation
of caffeic acid and ferulic acid is in favor of the formation of hydrogen bond formation and the
extension of the conjugate structure. Zhang’s [36] research found that these factors greatly impact the
bioactivity of curcuminoid derivatives. In addition, the curcuminoids-caffeic acid conjugates showed
better activities than curcuminoids-ferulic acid conjugates, which may be attributed to different
numbers of hydroxyl groups in two substituents.
In the third series, 18β-glycyrrhetinic acid was used as the starting material to prepare prodrugs with
novel structures. To alter the lipophilicity of the molecule and to improve the bioavailability, the carboxylic
acid at position C-30 was transformed into esters by reacting with curcuminoids. The synthesized
derivatives retained the active groups of 18β-glycyrrhetinic acid, the C-3 hydroxyl group, which
proved to be essential for good biological activities. In addition, diesters with a two glycyrrhetinic acid
moiety showed better biological activities than monoesters, and the results agree with Fuchs’ findings [37].
Thus, we attempt to summarize the key issues:
1
2
3
. The absence of the methoxy group in curcuminoid derivatives leads to a decrease in the
biological activity.
. The difference in the biological activity between monoesters and diesters depends on the
substituent on the phenyl rings of curcuminoids.
. To some extent, but not as hard absolutes, the more hydroxyl groups the substituent contains,
the better biological activity the derivatives will have. In other word, the hydroxyl groups
incorporated are helpful for the improvement of biological activity.
3
. Experimental Section
3
.1. Chemicals and Reagents
All reagents and solvents were purchased from Aldrich Chemical Co. (Beijing, China) and used
without further purification. Se-Met was synthesized in-house. Melting points were determined on a
Fisher-Johns melting apparatus (Weihai, China). Electrospray ionization mass spectral (ESI-MS) data
1
were obtained on a Bruker Esquire HCT spectrometer (Waters, New York, NY, USA). The H-NMR
spectral data were recorded on a 400-MHz NMR spectrometer (Fällanden, Switzerland).

Molecules 2014, 19
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Human hepatoma cells (Hep-G2, LX-2 and SMMC-7221) and human breast cancer cells
MDA-MB-231) were obtained from Expression Systems (Beijing, China). Cultured cells were grown
(
at 37 °C in a humidified atmosphere of 5% CO
2
and were passaged twice a week.
3
.2. General Method for the Synthesis of CCM (1a) and BCM (1b)
Acetylacetone (10 mmol) was added to a solution of boric anhydride (5.0 mmol) in ethyl acetate
30 mL), followed by the addition of vanillin (20 mmol) or 4-hydroxybenzaldehyde (20 mmol) and
(
tributyl borate (40 mmol). The reaction mixture was stirred at 50 °C for 10 min. Subsequently,
n-butylamine (5.0 mmol) in ethyl acetate (5 mL) was added dropwise over 15 min at 50 °C and
additionally stirred for 4 hours at 80 °C. Hydrochloric acid (1 N, 30 mL) was added, and the mixture
was stirred for another 40 min. The organic layers were washed with water, dried with Na
2
SO . The
4
solvent was evaporated, and the residue was recrystallized from methanol to give the corresponding
curcuminoids (1a and 1b) as yellow solids.
1
CCM (1a). Yield 85%. m.p. 185–186 °C . H-NMR (CDCl
3
): δ 7.59 (d, J = 15.7 Hz, 2H), 7.13 (dd,
J = 8.1 and 1.8 Hz, 1H), 7.05 (d, J = 1.8 Hz, 2H), 6.94 (d, J = 8.1 Hz, 2H), 6.48 (d, J = 15.8 Hz, 2H),
−
5
5
.85 (s, 1H), 3.95 (s, 6H). ESI-MS (m/z): (M−H) = 367.03. Anal. Calcd. for C21
H
20
6
O : C, 68.47; H,
.47; O, 26.06%. Found: C, 68.40; H, 5.41; O, 26.12%.
1
BCM (1b). Yield 80%. m.p. 233–234 °C . H-NMR (CDCl
3
): δ 7.53 (d, J = 16.2 Hz, 2H).7.55 (d,
J = 8.6 Hz, 4H), 6.85 (d, J = 8.6 Hz, 4H), 6.65 (d, J = 15.9 Hz, 2H), 6.09 (s, 1H). ESI-MS (m/z):
−
(
M−H) = 307.03. Anal. Calcd. for C21
H
20
6
O : C, 74.01; H, 5.23; O, 20.76%. Found: C, 74.06; H, 5.21;
O, 20.82%.
3
.3. Procedure of the Preparation of Selenomethionine-Substituted Curcuminoids and
Methionine-Substituted Curcuminoids
According to the procedure previously reported [33], selenomethionine (8) was prepared.
Methionine (2) was used as the starting material in a three-pot, seven-step procedure to obtain
selenomethionine in 47% yield. In a 100-mL round-bottomed flask, selenomethionine or methionine
was dissolved in water and NaHCO
3
(3 eq.) was added. A solution of (BOC) O (1.5 eq.) in dioxane
2
was added to this mixture. The reaction mixture was stirred at 25 °C overnight. The reaction mixture
was washed with ethyl acetate. The resulting aqueous layer was acidified to pH 2 with concentrated
hydrochloric acid and extracted with ethyl acetate. The combined organic extracts were dried over
Na
2
SO , filtered and concentrated in vacuo to give 9a or 9b as colorless gums.
4
3
.3.1. General Procedure for the Synthesis of 10a–d
To a solution of 9a or 9b (10 mmol) in chloroform (50 mL) was added EDCI (12 mmol), HOBT
(
12 mmol) and DIEA (1 mL). After stirring at 0 °C for 1 h, a solution of CCM (10 mmol) or BCM
10 mmol) in chloroform (40 mL) was added dropwise to the reaction mixture, which was stirred
(
overnight at room temperature. After completion of the reaction, as indicated by TLC, the mixture was
washed with hydrochloric acid (1 M) and brine. The organic layer was dried over anhydrous Na SO
2
4

Molecules 2014, 19
16359
and concentrated in vacuo. Purification by column chromatography over polyamide gel (1:10,
CH OH/CCl ) gave 10a–d as yellow solids.
3
4
1
Curcumin-mono-N-(tert-butoxycarbonyl)-methionine (10a). Yield 48%. m.p. 165–166 °C. H-NMR
400 MHz, CDCl ): δ 7.63 (d, J = 5.6 Hz, 1H), 7.59 (d, J = 5.6 Hz, 1H), 7.21–7.07 (m, 4H), 7.06 (d,
J = 1.8 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 15.8 Hz, 1H), 6.49 (d, J = 15.8 Hz, 1H), 5.83 (s,
H), 5.21 (d, J = 8.0 Hz, 1H), 4.71 (s, 1H), 3.95 (s, 3H), 3.87 (s, 3H), 2.69 (t, J = 7.8 Hz, 2H), 2.34
(
3
1
1
3
(m, 2H), 2.15 (s, 3H), 1.47 (s, 9H). C-NMR (CDCl
3
): δ 198.9, 173.4, 155.2, 151.4, 149.5, 147.9,
1
42.8, 141.1, 130.6, 127.6, 122.2, 121.9, 116.8, 112.5, 110.9, 79.5, 56.5, 55.8, 51.9, 30.9, 29.7, 28.4.
−
ESI-MS (m/z): (M−H) = 598.31. Anal. Calcd. for C31
H
37NO S: C, 62.09; H, 6.22; N, 2.34%. Found:
9
C, 62.12; H, 6.24; N, 2.38%.
Curcumin-mono-N-(tert-butoxycarbonyl)-selenomethionine (10b). Yield 52%. m.p. 172–173 °C.
1H-NMR (400 MHz, CDCl
): δ 7.63 (d, J = 6.0 Hz, 1H), 7.59 (d, J = 6.0 Hz, 1H), 7.22–7.07 (m, 4H),
.06 (d, J = 1.6 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.56 (d, J = 15.8 Hz, 1H), 6.50 (d, J = 15.8 Hz,
H), 5.83 (s, 1H), 5.20 (d, J = 8.1 Hz, 1H), 4.71 (d, J = 5.0 Hz, 1H), 3.95 (s, 3H), 3.87 (s, 3H),
3
7
1
2
1
5
1
3
.75–2.67 (m, 2H), 2.41–2.21 (m, 2H), 2.05 (s, 3H), 1.47 (s, 9H). C-NMR (CDCl ): δ 198.5, 172.4,
3
55.9, 152.3, 149.5, 148.6, 142.6, 142.5, 133.8, 131.4, 125.6, 124.3, 122.9, 121.9, 116.8, 111.9, 110.9,
−
6.1, 55.8, 51.9, 28.4, 25.1. ESI-MS (m/z): (M−H) = 646.08. Anal. Calcd. for C31
H
37NO Se: C, 57.58;
9
H, 5.77; N, 2.17%. Found: C, 57.50; H, 5.71; N, 2.12%.
Bisdesmethoxycurcumin-mono-N-(tert-butoxycarbonyl)-methionine (10c). Yield 55%, m.p. 183–184 °C.
1H-NMR (400 MHz, CDCl
): δ 7.61 (d, J = 12.9 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.1 Hz,
H), 7.12 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 7.8 Hz, 2H), 6.56 (d, J = 15.8 Hz, 1H), 6.48 (d, J = 15.8
Hz, 1H), 5.80 (s, 1H), 5.24 (d, J = 7.8 Hz, 1H), 4.67 (s, 1H), 2.66 (t, J = 8.1 Hz, 2H),2.25 (m, 2H) 2.14
3
2
1
3
(
s, 3H), 1.48 (s, 9H). C-NMR (CDCl
3
): δ 198.5, 171.4, 158.8, 153.9, 150.2, 134.8, 131.9, 130.8,
−
1
30.2, 129.6, 121.6, 116.8, 79.2, 56.8, 51.9, 30.6, 28.6, 15.6. ESI-MS (m/z): (M−H) = 538.33. Anal.
Calcd. for C29
H
33NO S: C, 64.54; H, 6.16; N, 2.60%. Found: C, 64.50; H, 6.11; N, 2.65%.
7
Bisdemethoxycurcumin-mono-N-(tert-butoxycarbonyl)-selenomethionine (10d). Yield 60%. m.p.
1
1
92–193 °C. H-NMR (400 MHz, CDCl
3
): δ 7.61 (d, J = 14.9 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.43
(d, J = 8.1 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 6.85 (d, J = 5.5 Hz, 2H), 6.49 (d, J = 13.5 Hz, 2H), 5.80
(
s, 1H), 5.18 (d, J = 8.2 Hz, 1H), 4.66 (s, 1H), 2.66 (t, 2H), 2.41–2.31 (m, 2H), 2.03 (s, 3H), 1.48 (s,
1
3
9
7
H). C-NMR (CDCl
3
): δ 198.6, 170.5, 155.6, 150.4, 135.8, 131.6, 130.9, 130.4, 128.6, 122.5, 115.8,
−
9.4, 60.2, 51.8, 28.6, 25.1, 22.4, 9.5. ESI-MS (m/z): (M−H) = 586.22. Anal. Calcd. for
C
29
H
33NO Se: C, 59.38; H, 5.67; N, 2.39%. Found: C, 59.32; H, 5.61; N, 2.33.
7
3
.3.2. General Procedure for the Synthesis of 10e–h
To a stirred solution of 9a or 9b (10 mmol) in anhydrous chloroform (50 mL) was added a solution
of CCM or BCM (3 mmol) in chloroform (40 mL) dropwise. After 15 min, the reaction mixture was
basified with triethylamine (1 mL) and stirred for 20 min, followed by the addition of DCC (3 mmol).
The reaction mixture was stirred for 8 h and monitored by TLC. Upon completion of the reaction,

Molecules 2014, 19
16360
1
, 3-dicyclohexylurea (DCU) was removed by filtration, and the filtrate was evaporated. The resulting
residue was dissolved in chloroform (50 mL), washed consecutively with 1 M HCl (50 mL), 5%
NaHCO solution (50 mL), brine (50 mL) and H O (50 mL), dried over anhydrous Na SO , filtered
and concentrated in vacuo. Purification by column chromatography over polyamide gel (1:10,
CH OH/CCl ) gave 10e–h as yellow solids.
3
2
2
4
3
4
1
Curcumin-di-N-(tert-butoxycarbonyl)-methionine (10e). Yield 50%. m.p. 187–188 °C. H-NMR
400 MHz, CDCl ): δ 7.64 (d, J = 15.8 Hz, 2H), 7.21–7.09 (m, 6H), 6.60 (d, J = 15.8 Hz, 2H), 5.89 (s,
H), 5.25 (d, J = 7.7 Hz, 2H), 4.74 (d, J = 4.5 Hz, 2H), 3.89 (s, 6H), 2.71 (t, J = 7.6 Hz, 4H), 2.34 (m,
(
3
1
4
1
1
3
H), 2.18 (s, 6H), 1.49 (s, 18H). C-NMR (CDCl
3
): δ 198.5, 170.3, 155.6, 151.2, 142.5, 141.3, 131.8,
−
30.6, 124.4, 121.9, 110.9, 79.6, 56.7, 55.9, 51.3, 30.2, 29.4, 28.2, 15.1. ESI-MS (m/z): (M−H) = 829.33.
Anal. Calcd. for: C41
H N
54 2
O
12
S
2
: C, 59.26; H, 6.55; N, 3.37%. Found: C, 59.21; H, 6.51; N, 3.32%.
1
Curcumin-di-N-(tert-butoxycarbonyl)-selenomethionine (10f). Yield 58%. m.p. 200–201 °C. H-NMR
(400 MHz, CDCl
3
): δ 7.62 (d, J = 15.4 Hz, 2H), 7.24–6.81 (m, 6H), 6.58 (d, J = 14.6 Hz, 2H), 5.87 (s,
1
H), 5.19 (d, J = 5.7 Hz, 2H), 4.71 (s, 2H), 3.87 (s, 6H), 2.70 (t, 4H), 2.29 (m, 4H), 2.05 (s, 6H), 1.47
1
3
(s, 18H). C-NMR (CDCl
3
): δ 198.9, 170.7, 155.9, 151.8, 143.2, 141.1, 131.8, 124.4, 121.9, 110.9,
−
6
0.1, 55.8, 51.9, 28.4, 25.1, 9.4. ESI-MS (m/z): (M−H) = 926.08. Anal. Calcd. for: C41
H
54
N
2
O
12Se :
2
C, 51.58; H, 5.70; N, 3.17%. Found: C, 51.51; H, 5.73; N, 3.12%.
Bisdesmethoxycurcumin-di-N-(tert-butoxycarbonyl)-methionine (10g). Yield 65%. m.p. 207–208 °C.
1H-NMR (400 MHz, CDCl
): δ 7.64 (d, J = 15.8 Hz, 2H), 7.58 (d, J = 8.4 Hz, 4H), 7.16 (d, J = 8.4 Hz,
H), 6.59 (d, J = 15.8 Hz, 2H), 5.84 (s, 1H), 5.20 (d, J = 6.2 Hz, 2H), 4.66 (s, 2H), 2.66 (t, 4H), 2.31
3
4
1
3
(m, 2H), 2.15 (s, 6H), 2.10(m, 2H), 1.47 (s, 18H). C-NMR (CDCl
3
): δ198.9, 170.2, 159.8, 150.7,
−
1
35.2, 130.6, 129.4, 121.7, 79.7, 56.9, 52.3, 31.2, 30.4, 28.6, 15.5. ESI-MS (m/z): (M−H) = 769.5.
Anal. Calcd. for: C39
H N
50 2
O
10
2
S : C, 60.76; H, 6.54; N, 3.63%. Found: C, 60.71; H, 6.50; N, 3.67%.
Bisdesmethoxycurcumin-di-N-(tert-butoxycarbonyl)-selenomethionine (10h). Yield 62%. m.p. 214–215 °C .
1H-NMR (400 MHz, CDCl
): δ 7.65 (d, J = 15.8 Hz, 2H), 7.58 (d, J = 8.6 Hz, 4H), 7.16 (d, J = 8.6 Hz,
H), 6.59 (d, J = 15.8 Hz, 2H), 5.84 (s, 1H), 5.15 (d, J = 7.0 Hz, 2H), 4.65 (s, 2H), 2.67 (t, J = 7.4 Hz,
H), 2.44–2.29 (m, 4H), 2.34 (d, J = 18.5 Hz, 2H), 2.27–2.08 (m, 4H), 2.04 (s, 6H), 1.47 (s, 18H).
3
4
4
13C-NMR (CDCl
): δ 198.8, 170.2, 156.2, 150.7, 135.2, 130.5, 129.7, 122.2, 79.5, 60.3, 52.4, 28.7,
3
−
2
5.2, 22.3, 9.3. ESI-MS (m/z): (M−H) = 863.3. Anal. Calcd. for C39
H
50
N
2
O
10Se : C, 54.17; H, 5.83;
2
N, 3.24%. Found: C, 54.11; H, 5.89; N, 3.20%.
.3.3. General Procedure for the Synthesis of Compounds 11a–h
A solution of HCl/EA (4 mL, 4 M) in a 25-mL round-bottom flask equipped with a magnetic stir-bar
3
was cooled by an ice-water bath under nitrogen. Compound (10a–h, 1 mmol) was added in one portion
with stirring. The ice-bath was removed, and the mixture was stirred at 25 °C. After 30 min, TLC indicated
that the reaction was completed. The product separated as a precipitate, as it was insoluble in EA.
1
Curcumin-mono-methionine (11a). Yield 60%. m.p. 60–61 °C . H-NMR (400 MHz, DMSO): δ 7.63
(d, J = 7.6 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.40–7.24 (m, 4H), 7.18 (d, J = 8.2 Hz, 1H), 7.00 (d,

Molecules 2014, 19
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J = 16.0 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 15.9 Hz, 1H), 6.15 (s, 1H), 3.87 (s, 3H), 3.84
1
3
(
s, 3H), 2.78–2.71 (m, 2H), 2.28–2.23 (m, 2H), 2.12 (s, 3H). C-NMR (CDCl
3
): δ 198.7, 168.2, 151.7,
1
49.2, 147.5, 142.9, 141.4, 131.9, 130.5, 124.7, 122.9, 121.8, 117.2, 56.4, 52.3, 33.9, 29.7, 15.5.
−
ESI-MS (m/z): (M−H) = 498.17. Anal. Calcd. for C26
H29NO
7
S: C, 62.51; H, 5.85; N, 2.80%. Found:
C, 62.58; H, 5.80; N, 2.83%.
1
Curcumin-mono-selenomethionine (11b). Yield 65%. m.p. 69–70 °C . H-NMR (400 MHz, DMSO):
δ 7.63 (d, J = 7.7 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.41–7.23 (m, 4H), 7.18 (d, J = 8.3 Hz, 1H), 7.01
(
d, J = 16.0 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 15.8 Hz, 1H), 6.15 (s, 1H), 3.87 (s, 3H),
1
3
3
1
.84 (s, 3H), 2.78–2.69 (m, 2H), 2.33 (m, 2H), 2.02 (s, 3H). C-NMR (CDCl
3
): δ198.5, 167.8, 151.4,
49.2, 147.5, 142.9, 141.2, 131.7, 130.2, 123.1, 122.2, 120.8, 116.2, 55.8, 51.8, 32.8, 25.7, 9.4.
−
−
ESI-MS (m/z): (M−H) = 546.1. ESI-MS (m/z): (M−H) = 546.1. Anal. Calcd. for C26
2.51; H, 5.85; N, 2.80%. Found: C, 62.58; H, 5.80; N, 2.83%.
H
29NO Se: C,
7
6
1
Bisdesmethoxycurcumin-mono-methionine (11c). Yield 62%. m.p. 73–74 °C . H-NMR (400 MHz,
DMSO): δ 7.84 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 6.94 (d,
J = 16.0 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 6.74 (d, J = 15.9 Hz, 1H), 6.14 (s, 1H), 4.40 (s, 2H), 2.69
1
3
(
m, 2H), 2.37–2.21 (m, 2H), 2.11 (s, 3H). C-NMR (CDCl
3
): δ 198.9, 168.0, 157.4, 150.6, 135.1,
−
1
30.7, 130.5, 121.1, 115.2, 52.2, 34.1, 30.7, 15.4. ESI-MS (m/z): (M−H) = 438.17. Anal. Calcd. for
C
24
H
25NO S: C, 65.58; H, 5.73; N, 3.19%. Found: C, 65.53; H, 5.76; N, 3.21%.
5
1
Bisdesmethoxycurcumin-mono-selenomethionine (11d). Yield 65%. m.p. 77–78 °C . H-NMR (400 MHz,
DMSO): δ 7.84 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 6.95 (d,
J = 15.9 Hz, 1H), 6.85 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 15.9 Hz, 1H), 6.14 (s, 1H), 4.40 (s, 1H), 2.75
1
3
(
t, J = 8.8 Hz, 2H), 2.38–2.28 (m, 2H), 2.01 (s, 3H). C-NMR (CDCl
3
): δ198.7, 168.5, 157.6, 150.8,
−
1
36.3, 130.8, 130.4, 121.3, 115.2, 55.5, 51.9, 34.1, 25.7, 9.4. ESI-MS (m/z): (M−H) = 486.08. Anal.
Calcd. for C24
H25NO
5
Se: C, 59.26; H, 5.18; N, 2.88%. Found: C, 59.23; H, 5.16; N, 2.92%.
1
Curcumin-di-methionine (11e). Yield 70%. m.p. 82–83 °C . H-NMR (400 MHz, DMSO): δ 7.68 (d,
J = 16.0 Hz, 2H), 7.60 (s, 2H), 7.41 (d, J = 7.9 Hz, 2H), 7.06 (d, J = 15.9 Hz, 2H), 6.24 (s, 1H), 4.44
1
3
(
s, 2H), 3.87 (s, 6H), 2.82–2.69 (m, 4H), 2.32–2.24 (m, 4H), 2.12 (s, 6H). C-NMR (CDCl
3
): δ 197.7,
1
68.2, 151.6, 142.8, 141.3, 132.3, 130.6, 122.4, 120.3, 111.2, 56.5, 52.3, 33.9, 30.3, 15.7. ESI-MS
−
(
m/z): (M−H) = 629.25. Anal. Calcd. for C31
9.07; H, 6.11; N, 4.45%.
H
38
N
2
O
8
S
2
: C, 59.03; H, 6.07; N, 4.44%. Found: C,
5
1
Curcumin-di-selenomethionine (11f). Yield: 75%. m.p. 88–89 °C . H-NMR (400 MHz, DMSO):
δ 7.68 (d, J = 15.9 Hz, 2H), 7.60 (s, 2H), 7.40 (d, J = 7.8 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 7.06 (d,
J = 16.0 Hz, 2H), 6.23 (s, 1H), 4.44 (s, 2H), 3.87 (s, 6H), 2.80–2.69 (m, 4H), 2.35–2.26 (m, 4H), 2.02
1
3
(
s, 6H). C-NMR (CDCl
3
): δ 198.2, 168.1, 151.1, 143.1, 141.2, 131.8, 130.5, 124.4, 121.3, 110.2, 56.5,
−
5
3
2.3, 25.3, 9.2. ESI-MS (m/z): (M–H) = 725.17. Anal. Calcd. for C31
H
38
N
2
O Se: C, 51.39; H, 5.29; N,
8
.87%. Found: C, 51.37; H, 5.25; N, 3.84%.

Molecules 2014, 19
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1
Bisdesmethoxycurcumin-di-methionine (11g). Yield 77%. M.p. 92–93 °C . H-NMR (400 MHz, DMSO):
δ 7.86 (d, J = 8.5 Hz, 4H), 7.67 (d, J = 16.0 Hz, 2H), 7.35 (d, J = 8.5 Hz, 4H), 6.97 (d, J = 15.7 Hz,
1
3
2
H), 6.18 (s, 1H), 4.40 (s, 2H), 2.78–2.69 (m, 4H), 2.27 (d, 4H), 2.11 (s, 6H). C-NMR (CDCl
3
):
−
δ 198.8, 168.3, 150.5, 134.8, 130.8, 121.7, 121.8, 52.5, 33.8, 29.3, 15.4. ESI-MS (m/z): (M−H) = 570.25.
Anal. Calcd. for C29
H
34
N
2
O
6
S
2
: C, 61.03; H, 6.00; N, 4.91%. Found: C, 61.11; H, 6.07; N, 4.92%.
1
Bisdesmethoxycurcumin-di-selenomethionine (11h). Yield 70%. m.p. 97–98 °C . H-NMR (400 MHz,
DMSO): δ 7.86 (d, J = 8.6 Hz, 4H), 7.68 (d, J = 11.5 Hz, 2H), 7.35 (d, J = 8.6 Hz, 4H), 6.99 (d,
J = 16.0 Hz, 2H), 6.23 (s, 1H), 4.40 (t, J = 6.2 Hz, 2H), 2.81–2.68 (m, 4H), 2.41–2.24 (m, 4H), 2.01
1
3
(
s, 6H). C-NMR (CDCl
3
): δ 198.8, 168.2, 150.7, 135.0, 130.9, 129.8, 121.7, 55.8, 52.2, 25.3, 9.4.
−
ESI-MS (m/z): (M−H) = 665.00. Anal. Calcd. for C29
H N
34 2
O
6
Se : C, 52.42; H, 5.16; N, 4.20%.
2
Found: C, 52.49; H, 5.14; N, 4.23%.
3
.4. General Method for the Synthesis of Curcuminoid-Caffeic Acid and Curcuminoid-Ferulic
Acid Conjugates
3
.4.1. General Procedure for the Synthesis of 13a–b
1
2a or 12b (1 mmol) was heated under reflux with Ac
2
O (20 mmol) for 5 h. Then, acetic acid
solution was added to the still hot solution. The product, crystallized upon cooling, was filtered off and
washed with cold H
2
O and Et
2
O and dried in vacuo to give 13a or 13b as a white solid.
1
Ferulic acid acetate (13a). Yield 80%. H-NMR (400 MHz, CDCl
3
): δ 7.60 (d, J = 24 Hz, 1H), 7.01
−
(
m, 3H). 6.30 (d, J = 24 Hz, 1H), 3.90 (s, 3H), 2.33(s, 3H). ESI-MS (m/z): (M−H) = 236.08.
Anal. Calcd. for C12
H
12
O
5
: C, 61.01; H, 5.12; O, 33.87%. Found: C, 61.06; H, 5.16; O, 33.82%.
1
Caffeic acid acetate (13b). Yield 85%. H-NMR (400MHz, CDCl
3
): δ 7.47 (d, J = 15.8 Hz, 1H), 7.33
−
(
m, 3H), 6.33(d, J = 16.0 Hz, 1H), 2.30(s, 3H), 2.28(s, 3H). ESI-MS (m/z): (M−H) = 263.52.
Anal. Calcd. for C13
H
12
6
O : C, 59.09; H, 4.58; O, 36.33%. Found: C, 59.12; H, 4.52; O, 36.30%.
3
.4.2. General Procedure for the Synthesis of 9a–d
To a solution of 13a or 13b (1 mmol) in chloroform (50 mL), EDCI (1.2 mmol), HOBT
1.2 mmol) and DIEA (1 mL) were added. After stirring at 0 °C for 1 h, a chloroform solution (40 mL)
(
of CCM or BCM (1 mmol) was added dropwise to the mixtures, which was allowed to be stirred
overnight at room temperature. After completion of the reaction, as indicated by TLC, the mixture was
washed with hydrochloric acid (1 M) and then with brine. The organic layer was dried with anhydrous
Na
2
SO
4
and evaporated to dryness under vacuum. Purification by dry column flash chromatography on
:MeOH = 1:10) gave 14a–d as a yellow solid.
silica gel-G (CHCl
2
1
Curcumin-mono-ferulic acid acetate (14a). Yield 57%. 183–184 °C . HNMR (400 MHz, CDCl
.60 (d, J = 20.2 Hz, 1H),7.45 (d, J = 15.8 Hz, 1H), 7.10 (d, J = 2.5Hz, 1H), 7.05-6.92 (m, 6H), 7.01
m, 3H), 6.92–6.72 (m, 2H), 6.30 (d, J = 20.0 Hz,1H), 6.20(d, J = 16.0 Hz, 1H), 5.50 (s, 1H), 3.90 (s,
3
): δ
7
(
1
3
3
H), 3.91 ( s, 3H), 3.89 (s, 3H,), 3.87 (s, 3H), 2.32(s, 3H). C-NMR (CDCl ): δ 198.6, 169.0, 164.3,
3

Molecules 2014, 19
16363
1
5
51.2, 149.5, 147.6, 142.7, 137.5, 130.6, 128.3, 123.2, 122.5, 121.2, 116.8, 115.0, 111.2, 110.9, 55.9,
−
30
2.1, 20.1. ESI-MS (m/z): (M−H) = 553.5. Anal. Calcd. for C33H O10: C, 67.57; H, 5.15; O, 27.28%.
Found: C, 67.50; H, 5.18; O, 27.30%.
1
Curcumin-mono-caffeic acid acetate (14b). Yield 60%. m.p. 179–180 °C . H-NMR (400 MHz,
CDCl
.21–7.09 (m, 4H), 7.06 (d, J = 1.8 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.58 (d, J = 15.8 Hz, 1H), 6.50
d, J = 15.8 Hz, 1H), 6.33 (d, J = 16.0 Hz, 1H), 5.83 (s, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 2.30 (s, 3H),
3
): δ 7.65 (d, J = 6.0 Hz, 1H), 7.55 (d, J = 6.0 Hz, 1H), δ7.45 (d, J = 15.8 Hz, 1H), 7.33 (m, 3H),
7
(
1
3
2
1
.28 (s, 3H). C-NMR (CDCl
3
): δ 198.6, 169.2, 164.8, 151.7, 150.0, 148.1, 142.9, 137.8, 130.6, 128.2,
−
23.9, 122.8, 121.6, 117.2, 115.6, 111.8, 110.6, 55.9, 52.1, 20.5. ESI-MS (m/z): (M−H) = 613.18.
Anal. Calcd. for C34
H
30
O11: C, 66.44; H, 4.92; O, 28.64%. Found: C, 66.40; H, 4.88; O, 28.60%.
1
Bisdesmethoxycurcumin-mono-ferulic acid acetate (14c). Yield 55%. m.p. 197–198 °C . H-NMR
(
400 MHz, CDCl
3
): δ 7.67 (d, J = 15.5 Hz, 2H), 7.62 (d, J = 20.4 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H),
7
.46 (d, J = 8.6 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 7.01 (m, 3H), 6.85 (d, J = 7.5 Hz, 2H), 6.49 (d,
1
3
J = 13.5 Hz, 2H), 6.33 (d, J = 20.6 Hz, 1H), 5.80 (s, 1H), 3.90 (s, 3H), 2.31(s, 3H). C-NMR
(
CDCl
3
): δ 198.7, 169.0, 164.2, 157.6, 151.8, 150.6, 148.1, 141.3, 135.3, 132.0, 130.6, 124.8, 121.7,
−
1
7
15.8, 110.5, 110.5, 55.9, 52.2, 20.4. ESI-MS (m/z): (M−H) = 525.56. Anal. Calcd. for C31
H
26
8
O : C,
0.71; H, 4.98; O, 24.31%. Found: C, 70.65; H, 5.02; O, 24.36%.
1
Bisdesmethoxycurcumin-mono-caffeic acid acetate (14d). Yield 55%. m.p. 193–194 °C. H-NMR
(
400 MHz, CDCl
.43 (d, J = 8.1 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 7.36 (m, 3H), 6.85 (d, J = 7.8 Hz, 2H), 6.56 (d,
J = 15.8 Hz, 1H), 6.48 (d, J = 15.8 Hz, 1H), 6.38(d, J = 16.0 Hz, 1H), 5.80 (s, 1H), 2.32(s, 3H),
3
): δ 7.61 (d, J = 12.9 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), δ7.49 (d, J = 15.8 Hz, 1H),
7
1
3
2
1
.30(s, 3H). C-NMR (CDCl
3
): δ 198.7, 168.8, 164.2, 157.5, 150.7, 147.8, 142.6, 135.1, 130.6, 130.2,
−
26.3, 122.9, 121.5, 115.8, 115.2, 53.3, 20.7. ESI-MS (m/z): (M−H) = 553.10. Anal. Calcd. for
C
32
H
26
9
O : C, 69.31; H, 4.73; O, 25.97%. Found: C, 69.35; H, 4.76; O, 25.94%.
3
.4.3. General Procedure for the Synthesis of 14e–h
To a stirred, ice-cooled, solution of 13a or 13b (20 mL, 10 mmol), a solution of CCM or BCM
(
20 mmol) in dry CHCl (30 mL) was added dropwise, and the resulting solution was stirred for
3
1
1
0 min at 0 °C. Then, TEA (10 mmol) was added to the solution and stirred at room temperature for
0 h. After completion of the reaction, as indicated by TLC, the mixture was washed with sodium
bicarbonate (1 M) and then with brine. The organic layer was dried with anhydrous Na
2
SO
4
and
evaporated to dryness under vacuum. Purification by chromatography on silica (CHCl
3
:MeOH = 20:1)
gave 14e–h as a yellow solid.
1
Curcumin-di-ferulic acid acetate (14e). Yield 60%. m.p. 206–207 °C. HNMR (400 MHz, CDCl
3
):
δ 7.69 (d, J = 15.8 Hz, 2H), 7.64 (d, J = 20.8 Hz, 1H), 7.31–7.13 (m, 6H), 7.09 (m, 6H), 6.65 (d,
J = 15.8 Hz, 2H), 5.89 (s, 1H), 6.30 (d, J = 20.0 Hz, 2H), 3.90 (s, 6H), 2.33(s, 6H), 2.31(s, 6H).
13C-NMR (CDCl
): δ198.9, 169.0, 164.3, 151.5, 147.8, 142.5, 141.2, 137.4, 131.6, 130.4, 124.5, 121.9,
3

Molecules 2014, 19
16364
−
1
15.5, 110.8, 55.9, 51.9, 20.2. ESI-MS (m/z): (M−H) = 803.45. Anal. Calcd. for C45
H
40
O14: C, 67.16;
H, 5.01; O, 27.83%. Found: C, 67.20; H, 5.07; O, 27.80%.
1
Curcumin-di-caffeic acid acetate (14f). Yield 57%. m.p. 201–202 °C. HNMR (400 MHz, CDCl
1H-NMR (400 MHz, CDCl
) δ 7.66 (d, J = 16.2 Hz, 2H), δ7.47 (d, J = 15.8 Hz, 2H), 7.36–7.25 (m,
H), 7.21–7.10 (m, 6H), 6.60 (d, J = 16.0 Hz, 2H), 6.33(d, J = 16.0 Hz, 2H), 3.89 (s, 6H), 2.30 (s,
3
):
3
6
6
1
1
3
H), 2.28 (s, 6H). C-NMR (CDCl ): δ 198.9, 169, 164.3, 151.5, 147.9, 143.2, 142.6, 142.2, 137.4,
3
31.5, 131.0, 130.6, 126.8, 124.6, 123.8, 122.8, 121.2, 115.1, 110.7, 55.8, 51.8, 20.3. ESI-MS (m/z):
−
(
M−H) = 859.25. Anal. Calcd. for C47
H
40
O
16: C, 65.58; H, 4.68; O, 29.74%. Found: C, 65.50; H, 4.62;
O, 29.70%.
1
Bisdesmethoxycurcumin-di-ferulic acid acetate (14g). Yield 55%. m.p. 217–218 °C. H-NMR (400 MHz,
CDCl
3
): δ 7.68 (d, J = 15.8 Hz, 2H), 7.63 (d, J = 21.0 Hz, 2H), 7.60 (d, J = 8.5 Hz, 4H), 7.21 (d,
J = 8.6 Hz, 4H), 7.05 (m, 6H), 6.62 (d, J = 15.8 Hz, 2H), 6.30 (d, J = 20.8 Hz, 1H), 5.86 (s, 1H), 3.91
13
(
s, 6H), 2.33(s, 6H). C-NMR (CDCl
3
): δ 198.9, 169.3, 164.3, 151.5, 150.5, 147.9, 141.2, 134.8, 131.6,
1
30.7, 130.4, 129.6, 129.6, 124.4, 121.9, 121.5, 121.5, 115.5, 110.9, 55.8, 51.9, 20.4. ESI-MS (m/z):
−
(M−H) = 743.21. Anal. Calcd. for C43
H
36
O12: C, 69.35; H, 4.87; O, 25.78%. Found: C, 69.40; H, 4.80;
O, 25.82%.
1
Bisdesmethoxycurcumin-di-caffeic acid acetate (14h). Yield 65%. m.p. 213–214 °C. HNMR (400 MHz,
CDCl
3
): δ 7.68 (d, J = 15.8 Hz, 2H), 7.59 (d, J = 8.5 Hz, 4H), 7.50(d, J = 16.0 Hz, 2H), 7.35–7.25 (m,
6
H), 7.20 (d, J = 8.5 Hz, 4H), 6.61 (d, J = 16.2 Hz, 2H), 6.30 (d, J = 16.2 Hz, 2H), 2.34 (s, 6H), 2.32
13
(s, 6H). C-NMR (CDCl
3
): δ 198.9, 169.3, 164.3, 150.5, 147.9, 143.2, 142.5, 134.9, 131.2, 130.7,
−
1
30.4, 129.6, 126.4, 123.8, 122.9, 121.5, 121.5, 115.5, 51.9, 20.3. ESI-MS (m/z): (M−H) = 799.20.
Anal. Calcd. for C45
H
36
O14: C, 67.50; H, 4.53; O, 27.97%. Found: C, 67.55; H, 4.50; O, 27.92%.
3
.4.4. General Procedure for the Synthesis of 15a–h
A solution of 14a–h (1 mmol) and CH
temperature overnight. The solvent was evaporated and the residue diluted with water and HCl (1 M)
and extracted with CHCl . The organic layers were dried, and the solvent was evaporated in vacuo.
3
ONa (10 mmol) in MeOH (10 mL) was stirred at room
3
The residue was purified by flash chromatography on silica (CHCl
yellow powder.
3
:MeOH = 10:1) to give 15a–h as a
1
Curcumin-mono-ferulic acid (15a): Yield 65%. m.p 143–144 °C. H-NMR (400 MHz, CDCl
3
): δ 7.66
(d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.57 (d, J = 19.5 Hz, 1H), 7.38–7.26 (m, 4H), 7.19 (d,
J = 8.5 Hz, 1H), 7.03 (m, 3H), 7.07 (d, J = 16.0 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 15.8 Hz,
1
3
1
1
H), 6.36 (d, J = 20.0 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H). C-NMR (CDCl ): δ 198.8, 164.5, 151.2,
3
49.1, 147.5, 138.2, 132.2, 132.2, 130.4, 126.8, 123.8, 121.6, 116.8, 115.8, 112.2, 56.4, 52.6. ESI-MS
−
(
m/z): (M−H) = 543.10. Anal. Calcd. for C31
H O
28 9
: C, 68.37; H, 5.18; O, 26.44%. Found: C, 68.40; H,
5
.20; O, 26.50%.
1
Curcumin-mono-caffeic acid (15b): Yield 60%. m.p. 135–136 °C . H-NMR (400MHz, CDCl
3
): δ 7.66
(d, J = 7.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 15.8 Hz, 1H), 7.38–7.26 (m, 4H), 7.35 (m,

Molecules 2014, 19
16365
3
1
1
H), 7.20(d, J = 8.0 Hz, 1H), 7.04 (d, J = 16.0 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 15.9 Hz,
13
H), 6.38 (d, J = 16.0 Hz, 1H), 3.90 (s, 3H). C-NMR (CDCl
3
): δ 198.9, 164.3, 150.8, 147.9, 146.5,
45.9, 142.8, 139.2, 132.2, 131.2, 128.4, 125.8, 123.5, 122.6, 116.8, 115.5, 115.0, 114.2, 51.6. ESI-MS
−
(
m/z): (M−H) = 529.12. Anal. Calcd. for C30
H
26
O
9
: C, 67.92; H, 4.94; O, 27.14%. Found: C, 67.90; H,
4
.90; O, 27.10%.
1
Bisdesmethoxycurcumin-mono-ferulic acid (15c). Yield 65%. m.p. 155–156 °C . H-NMR (400 MHz,
3
CDCl ): δ 7.88 (d, J = 8.5 Hz, 2H), 7.63(d, J = 8.6Hz, 2H), 7.60 (d, J = 20.0 Hz, 1H), 7.38 (d, J = 8.6 Hz,
2
H), 7.01 (m, 3H), 6.98 (d, J = 15.9 Hz, 1H), 6.89 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 16.0 Hz, 1H), 6.30
1
3
(d, J = 20.6 Hz, 1H). C-NMR (CDCl
3
): δ198.5, 164.3, 157.3, 150.5, 149.4, 147.3, 130.8, 130.5,
−
1
21.5, 115.9, 111.6, 56.3, 52.5. ESI-MS (m/z): (M−H) = 483.19. Anal. Calcd. for C29
H
24
7
O : C, 71.89;
H, 4.99; O, 23.12%. Found: C, 71.80; H, 4.90; O, 23.14%.
1
Bisdesmethoxycurcumin-mono-caffeic acid (15d). Yield 65%. m.p 148–149 °C . H-NMR (400 MHz,
CDCl ): δ 7.86 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 15.8 Hz, 1H), 7.38 (d,
J = 8.5 Hz, 2H), 7.35-7.28 (m, 3H), 6.94 (d, J = 16.0 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 6.74 (d,
3
1
3
J = 15.9 Hz, 1H), 6.36 (d, J = 16.0 Hz, 1H). C-NMR (CDCl
3
): δ 198.9, 164.3, 157.9, 150.4, 147.98,
−
1
46.3, 145.8, 134.6, 130.4, 123.5, 121.3, 117.5, 115.9, 51.6. ESI-MS (m/z): (M−H) = 469.39.
Anal. Calcd. for C28
H
22
O
7
: C, 71.48; H, 4.71; O, 23.81%. Found: C, 71.40; H, 4.80; O, 23.94%.
1
Curcumin-di-ferulic acid (15e). Yield 60%. m.p. 166–167 °C . H-NMR (400 MHz, CDCl
3
): δ 7.66 (d,
J = 15.9 Hz, 2H), 7.60 (s, 2H), 7.56 (d, J = 23.8 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.2 Hz,
2
3
1
H), 7.05 (m, 6H), 7.01 (d, J = 16.0 Hz, 2H), 6.30 (d, J = 23.8 Hz, 2H), 3.90 (s, 6H), 3.88 (s, 3H),
1
3
.87 (s, 3H). C-NMR (CDCl
3
): δ 198.9, 164.5, 151.5, 149.2, 147.8, 142.5, 137.5, 131.6, 127.6, 124.4,
−
22.5, 121.4, 116.9, 109.6, 55.6, 52.1. ESI-MS (m/z): (M−H) = 719.20. Anal. Calcd. for C39
H
32
12
O :
C, 68.33; H, 5.03; O, 26.64%. Found: C, 68.40; H, 5.00; O, 26.70%.
1
Curcumin-di-caffeic acid (15f). Yield 62%. m.p. 162–163 °C . H-NMR (400 MHz, CDCl
3
): δ 7.69 (d,
J = 16.0 Hz, 2H), 7.49 (d, J = 15.8 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 7.9 Hz, 2H), 7.38
1
3
(
m, 6H), 6.38 (d, J = 16.0 Hz, 2H), 7.09 (d, J = 15.9 Hz, 2H), 3.90 (s, 6H). C-NMR (CDCl
3
): δ 198.9,
1
5
64.2, 151.7, 148.4, 146.4, 143.3, 137.9, 132.4, 130.6, 128.5, 124.6, 123.2, 121.9, 115.3, 110.5, 55.5,
−
1.5. ESI-MS (m/z): (M−H) = 743.25. Anal. Calcd. for C39
H
32
O12: C, 67.63; H, 4.66; O, 27.72%.
Found: C, 67.70; H, 4.70; O, 28.10%.
1
Bisdesmethoxycurcumin-di-ferulic acid (15g). Yield 57%. m.p. 191-192 °C . H-NMR (400 MHz,
CDCl
3
): δ 7.88 (d, J = 8.6 Hz, 4H), 7.70 (d, J = 11.5 Hz, 2H), 7.60 (d, J = 20.0 Hz, 2H), 7.38 (d,
J = 8.5 Hz, 4H), 7.25–7.13 (m, 6H), 6.96 (d, J = 16.0 Hz, 2H), 6.36 (d, J = 20.2 Hz, 1H), 3.90 (s, 6H).
13C-NMR (CDCl
): 198.9, 168.5, 148.7, 147.5, 134.6, 130.2, 129.5, 127.6, 123.3, 121.5, 116.8, 115.9,
3
−
1
49.2, 56.6, 52.2. ESI-MS (m/z): (M−H) = 659.10. Anal. Calcd. for C39
H
32
O10: C, 70.90; H, 4.88;
O, 24.22%. Found: C, 70.85; H, 4.90; O, 24.30%.
1
Bisdesmethoxycurcumin-di-caffeic acid (15h). Yield 55%. m.p. 181–182 °C . H-NMR (400 MHz,
CDCl ): δ 7.85 (d, J = 8.6 Hz, 4H), 7.69 (d, J = 16.0 Hz, 2H), 7.49 (d, J = 16.2 Hz, 2H), 7.35 (d,
3

Molecules 2014, 19
16366
1
3
J = 8.6 Hz, 4H), 7.30-7.18 (m, 6H), 6.97 (d, J = 15.7 Hz, 2H), 6.37 (d, J = 16.4 Hz, 2H). C-NMR
(
CDCl
3
): 198.9, 150.4, 146.5, 145.5, 139.5, 130.3, 129.8, 128.4, 123.5, 121.5, 117.5, 115.3, 51.5.
−
ESI-MS (m/z): (M−H) = 631.10. Anal. Calcd. for C37
28
H O10: C, 70.25; H, 4.46; O, 25.29%. Found:
C, 70.30; H, 4.50; O, 25.30%.
3
.5. General Procedure for the Synthesis of Curcuminoid-Glycyrrhetinic Acid Conjugates
Glycyrrhetinic acid (16, 1 mmol) was heated under reflux with Ac
2
O (50 mmol) for 2 h. Then,
acetic acid solution was added to the still hot solution. The product, crystallized upon cooling, was
filtered off and washed with cold H
2
O and Et
2
O and dried in vacuo to give 17 as a white solid.
1
Glycyrrhetinic acid acetate (17). Yield 79%. m.p. 317–318 °C. H-NMR (400 MHz, CDCl
3
): δ 5.76 (s,
1
2
3
H), 4.55 (dd, J = 11.8, 5.4 Hz, 1H), 2.85 (d, J = 13.9 Hz, 1H), 2.35 (s, 1H), 2.17 (d, J = 13.1 Hz, 1H),
.07(s, 3H), 2.09–1.57 (m, 12H), 1.45–1.01 (m, 7H), 1.35 (s, 3H), 1.25(s, 3H), 1.22(s, 3H), 1.15 (s,
−
H), 0.88 (s, 6H), 0.84 (s, 3H). ESI-MS (m/z): (M−H) = 511.65. Anal. Calcd. for C32
H
48
O : C, 74.96;
5
H, 9.44; O, 15.60%. Found: C, 74.95; H, 9.50; O, 15.55%.
3
.5.1. General Procedure for the Synthesis of Compounds 18a–b
To the solution of 17 (1 mmol) in chloroform (50 mL), EDCI (1.2 mmol), HOBT (1.2 mmol) and
DIEA (1 mL) were added. After stirring at 0 °C for 1 h, a chloroform solution (40 mL) of CCM
1 mmol) or BCM (1 mmol) was added dropwise to the mixtures, which was allowed to be stirred
(
overnight at room temperature. After completion of the reaction, as indicated by TLC, the mixture was
washed with hydrochloric acid (1 M) and then with brine. The organic layer was dried with anhydrous
Na
2
SO and evaporated to dryness under vacuum. Purification by dry column flash chromatography on
4
silica gel-G (EA:PE = 1:5) gave 18a–b as a yellow solid.
1
Curcumin-mono-glycyrrhetinic acid acetate (18a). Yield 55%. m.p. 225–226 °C . H-NMR (400 MHz,
CDCl ): δ 7.63 (d, J = 5.6 Hz, 1H), 7.59 (d, J = 5.6 Hz, 1H), 7.25–7.07 (m, 4H), 7.09 (d, J = 2.0 Hz,
H), 6.96 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 16.2 Hz, 1H), 6.49 (d, J = 15.8 Hz, 1H), 5.79 (s, 1H), 4.55
dd, J = 12.0, 5.8 Hz, 1H), 3.90(s, 3H), 3.88(s, 3H), 2.85 (d, J = 14.4 Hz, 1H), 2.38(s, 1H), 2.23(s, 3H),
3
1
(
2
3
1
6
2
.18 (d, J = 13.1 Hz, 1H), 2.09–1.57 (m, 12H), 1.46–1.03 (m,7H), 1.35 (s, 3H), 1.27(s, 3H), 1.24(s,
1
3
H), 1.17 (s, 3H), 0.89 (s, 6H), 0.85 (s, 3H). C-NMR (CDCl ): δ 200.9, 176.3, 170.6, 170.2, 151.5,
3
49.1, 147.9, 142.8, 141.5, 131.8, 130.6, 128.3, 127.8, 124.4, 122.5, 121.6, 116.8, 111.9, 110.9, 80.6,
1.3, 56.1, 55.9, 51.8, 48.2, 45.6, 43.6, 41.9, 41.1, 37.6, 37.1, 36.6, 35.7, 32.5, 32.1, 30.5, 28.3, 26.9,
−
6.7, 26.2, 25.7, 23.8, 23.6, 21.5, 18.4, 17.2, 16.8. ESI-MS (m/z): (M−H) = 861.40. Anal. Calcd. for
C
53
66
H O10: C, 73.75; H, 7.71; O, 18.54%. Found: C, 73.78; H, 7.80; O, 18.59%.
Bisdesmethoxycurcumin-mono-glycyrrhetinic acid acetate (18b). Yield 55%. m.p. 232–233 °C .
1H-NMR (400 MHz, CDCl
): δ 7.69 (d, J = 15.9 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.6
3
Hz, 2H), 7.17 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 7.8 Hz, 2H), 6.57 (d, J = 15.8 Hz, 1H), 6.45(d, J = 15.8
Hz, 1H), δ 5.79 (s, 1H), 4.58 (dd, J = 12.2, 6.0 Hz, 1H), 2.87 (d, J = 14.2 Hz, 1H), 2.36 (s, 1H), 2.25(s,
3
1
H), 2.19 (d, J = 13.8 Hz, 1H), 2.09–1.59 (m, 12H), 1.48–1.04 (m, 7H), 1.37 (s, 3H), 1.26 (s, 3H),
1
3
.23(s, 3H), 1.17 (s, 3H), 0.88 (s, 6H), 0.86 (s, 3H). C-NMR (CDCl ): δ 200.8, 176.3, 170.6, 170.2,
3

Molecules 2014, 19
16367
1
8
2
51.5, 149.1, 147.9, 142.8, 141.5, 131.8, 130.6, 128.3, 127.5, 124.3, 122.2, 121.4, 116.7, 111.6, 110.5,
0.3, 61.2, 55.5, 51.5, 48.1, 45.3, 43.2, 41.6, 41.0, 37.4, 37.0, 36.4, 35.5, 32.3, 32.0, 30.4, 28.1, 26.6,
−
6.5, 26.1, 26.0, 23.1, 23.3, 21.6, 18.4, 17.3, 16.9. ESI-MS (m/z): (M−H) = 819.40. Anal. Calcd. for
C
51
H
64
9
O : C, 74.61; H, 7.86; O, 17.54%. Found: C, 74.68; H, 17.60; O, 17.59%.
3
.5.2. General Procedure for the Synthesis of Compounds 18c–d
A stirred suspension of 17 (10 mmol) in acetyl chloride was heated up to 50 °C for 1 h resulting in a
clear solution. Excess acetyl chloride was removed under vacuum and the residue triturated using
diethyl ether. The residue was filtered and dried in vacuo to get glycyrrhetinic acid chloride. To a
stirred, ice-cooled, solution of glycyrrhetinic acid chloride (20 mL, 10 mmol), a solution of CCM or
BCM (20 mmol) in dry CHCl
3
(30 mL) was added dropwise, and the resulting solution was stirred for
0 min at 0 °C . Then, TEA (10 mmol) was added to the solution and stirred at room temperature for
0 h. After completion of the reaction, as indicated by TLC, the mixture was washed with sodium
SO and
:MeOH = 20:1)
1
1
bicarbonate (1M) and then with brine. The organic layer was dried with anhydrous Na
2
4
evaporated to dryness under vacuum. Purification by chromatography on silica (CHCl
3
gave 18c–d as a yellow solid.
1
Curcumin-di-glycyrrhetinic acid acetate (18c). Yield 65%. m.p. 238–239 °C . H-NMR (400 MHz,
CDCl
3
) δ 7.66 (d, J = 15.8 Hz, 2H), 7.21–7.13 (m, 6H), 6.65 (d, J = 15.8 Hz, 2H), 5.80 (s, 2H), 4.55
dd, J = 12.2, 5.6 Hz, 2H), 2.85 (d, J = 14.8 Hz, 2H), 2.38 (s, 2H), 2.25(s, 6H), 2.15 (d, J = 15.6 Hz,
H), 2.08–1.67 (m, 24H), 1.52–1.10 (m, 14H), 1.33 (s, 6H), 1.28(s, 6H), 1.26 (s, 6H), 1.15 (s, 3H),
(
2
0
1
4
1
1
1
3
.88 (s, 12H), 0.84 (s, 6H). C-NMR (CDCl ): δ 200.8, 198.9, 176.3, 171.6, 170.2, 151.7, 143.9, 141.3,
3
31.9, 130.6, 128.3, 125.4, 124.7, 121.9, 121.9, 110.9, 80.6, 61.3, 55.8, 54.8, 51.9, 48.2, 44.6, 43.6,
1.4, 41.1, 37.6, 37.1, 36.3, 35.8, 32.5, 32.1, 30.5, 28.5, 26.9, 26.7, 26.4, 25.7, 24.2, 23.7, 21.3, 18.7,
−
7.3, 16.8. ESI-MS (m/z): (M−H) = 1,355.80. Anal. Calcd. for C85
H
112
O14: C, 75.19; H, 8.31; O,
6.50%. Found: C, 75.20; H, 8.37; O, 16.59%.
1
Bisdesmethoxycurcumin-di-glycyrrhetinic acid acetate (18d). Yield 62%. m.p. 245–246 °C . H-NMR
(
400 MHz, CDCl
.63 (d, J = 15.8 Hz, 2H), 4.70 (dd, J = 16.2, 8.2 Hz, 2H), 2.86 (d, J = 14.8 Hz, 2H), 2.38 (s, 2H),
.28(s, 6H), 2.17 (d, J = 15.6 Hz, 2H), 2.11–1.78 (m, 20H), 1.58–1.19 (m, 14H), 1.37 (s, 6H), 1.30 (s,
3
): δ 7.69 (d, J = 16.4 Hz, 2H), 7.59 (d, J = 12.0 Hz, 4H), 7.18 (d, J =12.2 Hz, 4H),
6
2
6
1
5
2
1
3
H), 1.29 (s, 6H), 1.19 (s, 3H), 0.89 (s, 12H), 0.87 (s, 6H). C-NMR (CDCl ): δ 200.8, 198.9, 175.3,
3
70.6, 169.2, 168.7, 142.9, 140.3, 130.7, 130.2, 127.3, 124.4, 122.7, 121.9, 120.4, 111.5, 80.3, 60.2,
2.8, 50.9, 47.2, 44.2, 43.1, 41.0, 40.1, 36.6, 36.1, 35.3, 34.8, 32.5, 32.1, 30.5, 28.5, 26.5, 26.0, 25.7,
−
4.2, 23.4, 21.1, 18.2, 17.0, 16.8. ESI-MS (m/z): (M−H) = 1,355.80. Anal. Calcd. for C83
H
108
12
O :
C, 76.82; H, 8.39; O, 14.79%. Found: C, 76.80; H, 8.30; O, 14.69%.
3
.5.3. General Procedure for the Synthesis of Compounds 19a–d
A solution of 19a–d (1 mmol) and CH
temperature overnight. The solvent was evaporated and the residue diluted with water and HCl (1 M)
and extracted with CHCl . The organic layers were dried, and the solvent was evaporated in vacuo.
3
ONa (10 mmol) in MeOH (10 mL) was stirred at room
3

Molecules 2014, 19
16368
The residue was purified by flash chromatography on silica (CHCl
yellow powder.
3
:MeOH = 20:1) to give 19a–d as a
1
Curcumin-mono-glycyrrhetinic acid (19a). Yield 62%. 198–199 °C . H-NMR (400 MHz, CDCl
3
):
δ 7.69 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.45–7.28 (m, 4H), 7.17 (d, J = 8.0 Hz, 1H), 7.04
d, J = 16.0 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 16.2 Hz, 1H), 5.76 (s, 1H), 4.59 (dd,
(
J = 12.8, 5.4 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 2.85 (d, J = 14.0 Hz, 1H), 2.38 (s, 2H), 2.19 (d,
J = 14.0 Hz, 1H), 2.09(s, 3H), 2.15–1.76 (m, 12H), 1.47–1.15 (m, 7H), 1.38 (s, 3H), 1.28(s, 3H),
−
1
.25(s, 3H), 1.17 (s, 3H), 0.88 (s, 6H), 0.84 (s, 3H). ESI-MS (m/z): (M−H) = 819.40. Anal. Calcd. for
13
C
51
H
64
O
9
: C, 76.82; H, 8.39; O, 14.79%. C-NMR (CDCl ): δ 200.8, 198.9, 176.3, 170.6, 151.5, 149.1,
3
1
5
2
8
47.9, 142.8, 141.1, 131.8, 130.4, 128.3, 127.6, 124.4, 122.9, 121.9, 116.8, 111.9, 110.9, 78.6, 61.3,
6.1, 55.8, 54.6, 51.9, 48.2, 44.6, 43.6, 41.4, 41.1, 38.9, 36.7, 35.6, 33.2, 32.5, 30.6, 28.5, 27.4, 26.9,
6.5, 26.0, 25.4, 23.2, 18.8, 17.6, 17.0. Found: C, 76.80; H, 8.30; O, 14.69%. Found: C, 76.80; H,
.31; O, 14.71%.
1
Bisdesmethoxycurcumin-mono-glycyrrhetinic acid (19b). Yield 58%. 208–209 °C . H-NMR (400 MHz,
CDCl
3
): δ 7.88 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.6 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 6.99 (d,
J = 16.0 Hz, 1H), 6.89 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 16.2Hz, 1H), 2.88 (d, J = 14.0 Hz, 1H), 2.38
(
s, 1H), 2.19 (d, J = 13.9 Hz, 1H), 2.11(s, 3H), 2.11–1.69 (m, 12H), 1.40–1.19 (m, 7H), 1.39 (s, 3H),
1
3
1
1
1
2
.28 (s, 3H), 1.24 (s, 3H), 1.17 (s, 3H), 0.87 (s, 6H), 0.85 (s, 3H). C-NMR (CDCl ): δ 201.2, 198.8,
3
77.3, 171.6, 152.5, 149.3, 147.6, 142.4, 141.3, 131.5, 130.6, 128.5, 126.6, 125.2, 121.9, 121.2, 117.8,
11.9, 110.5, 78.6, 61.3, 55.8, 54.6, 51.9, 48.2, 44.6, 43.6, 42.4, 41.5, 38.5, 36.5, 35.6, 33.6, 32.2, 30.3,
−
8.5, 27.9, 26.8, 26.4, 26.2, 25.5, 23.3, 18.9, 17.9, 17.4. ESI-MS (m/z): (M−H) = 759.40. Anal. Calcd
for: C, 77.34; H, 7.95; O, 14.72%. Found: C, 77.40; H, 8.01; O, 14.68%.
1
Curcumin-di-glycyrrhetinic acid (19c). Yield 66%. 217–219 °C . H-NMR (400 MHz, CDCl
3
): δ 7.84
(
d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 16.2 Hz, 1H), 6.89
d, J = 8.5 Hz, 2H), 6.78 (d, J = 16.0Hz, 1H), 3.90 (s, 6H), 2.89 (d, J = 16.0 Hz, 2H), 2.38 (s, 2H),
(
2
6
1
1
2
.20 (d, J = 16.0 Hz, 2H), 2.07 (s, 3H), 2.09–1.57 (m, 24H), 1.55–1.19 (m, 7H), 1.35 (s, 6H), 1.28 (s,
1
3
H), 1.24 (s, 6H), 1.19 (s, 6H), 0.89 (s, 12H), 0.86 (s, 6H). C-NMR (CDCl ): δ 200.8, 198.5, 177.3,
3
71.6, 153.5, 150.1, 148.9, 143.8, 141.9, 132.2, 131.4, 129.3, 128.2, 125.4, 123.6, 122.3, 117.8, 113.9,
11.5, 79.5, 61.8, 57.2, 56.9, 55.6, 52.9, 49.2, 45.6, 44.1, 42.0, 41.2, 38.9, 36.9, 35.6, 33.2, 31.5, 30.6,
−
8.8, 27.3, 26.9, 26.5, 26.2, 25.8, 23.2, 19.8, 18.6, 17.6. ESI-MS (m/z): (M−H) = 1,243.70.
Anal. Calcd. for C79
104
H O12: C, 76.17; H, 8.42; O, 15.41%. Found: C, 76.10; H, 8.47; O, 15.46%.
1
Bisdesmethoxycurcumin-di-glycyrrhetinic acid (19d). Yield 66%. 225–226 °C . H-NMR (400 MHz,
CDCl
3
): δ 7.84 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 6.95 (d,
J = 16.4 Hz, 1H), 6.85 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 16.2 Hz, 1H), 2.88 (d, J = 13.9 Hz, 2H), 2.47
(
s, 2H), 2.17 (d, J = 14.0 Hz, 2H), 2.14–1.77 (m, 24H), 1.55–1.14 (m, 14H), 1.37 (s, 6H), 1.28(s, 6H),
1
3
1
1
7
.24(s, 6H), 1.18 (s, 6H), 0.88 (s, 12H), 0.85 (s, 6H). C-NMR (CDCl ): δ 202.2, 199.2, 179.3, 172.6,
3
53.6, 149.6, 147.6, 142.4, 141.3, 131.5, 130.6, 128.5, 126.6, 125.2, 121.9, 121.2, 117.8, 111.9, 110.5,
8.6, 61.3, 56.3, 54.8, 51.6, 48.5, 44.2, 43.8, 42.6, 41.6, 38.8, 36.2, 35.2, 33.2, 32.0, 30.5, 28.2, 27.9,

Molecules 2014, 19
16369
−
2
6.5, 26.0, 25.9, 25.5, 23.6, 18.5, 17.4, 17.1. ESI-MS (m/z): (M−H) = 1,211.75. Anal. Calcd. for
10: C, 78.18; H, 8.64; O, 13.18%. Found: C, 78.10; H, 8.67; O, 13.12%.
C
79
H
104
O
3
.6. Assay for DPPH-Scavenging Activity
Antioxidant activity was determined as radical-scavenging ability using the stable radical, DPPH.
Briefly, 100 µL of ethanol solutions of the synthesized curcumin or bisdesmethoxycurcumin
derivatives (5 µM, 10 µM, 20 µM, 40 µM, 80 µM and 160 µM) were added to 100 µL of an ethanol
solution of DPPH radicals (100 µM). After 30 min of incubation at 25 °C in a dark environment, the
absorbance was recorded at 517 nm, using ethanol as a blank for baseline correction. CCM and BCM
were used as references, and their concentrations were maintained to that of the synthesized
compounds. Sample concentrations providing 50% inhibition (IC50) were calculated from the graph of
inhibition percentage plotted against concentration. These experiments were carried out in triplicate,
and the resulting values were averaged. The radical-scavenging activity (%) was calculated using the
following formula:
Radical-scavenging activity (%) = [(Ac − As)/Ac] × 100]
(1)
where Ac is the absorbance of the control and As is the absorbance of the tested sample after 30 min.
3
.7. Antibacterial Assay
The in vitro antibacterial activity of each curcuminoid bioconjugate against Gram-positive cocci
(
S. aureus and S. viridans) and Gram-negative bacilli (E. coli and E. cloacae) was evaluated by the
microdilution broth susceptibility test method. The stock solutions of the conjugates along with
curcuminoids were prepared in DMSO. The tubes bore different concentrations (1 µg/mL, 1.25 µg/mL,
2
µg/mL, 2.5 µg/mL, 5 µg/mL and 10 µg/mL). All of the compounds and control tubes were incubated
at 37 °C for 24 h. After incubation, the antibacterial activity of molecules in the tube was detected by
the lack of turbidity, which indicated the inhibition of bacterial growth. The concentration in the tube
with the highest dilution showing no turbidity has been reported as the MIC. Each test was performed
in triplicate, and the MIC reported represents the result of at least three repetitions.
3
.8. Anticancer Assays
3
Hep-G2, LX-2, SMMC-7221 and MDA-MB-231 cells were seeded at a density of 2 × 10 cells/well.
These cells were incubated at 37 °C under a humidified atmosphere containing 5% CO for 24 h before
2
treatment with compounds. The cells were treated with various concentrations (1 µM, 5 µM, 10 µM
and 20 µM) of the curcumin and bisdesmethoxycurcumin derivatives for 72 h. After treatment, media
was removed, and the cells were washed with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide (MTT). Approximately 20 µL of the MTT solution were added to each well followed by
incubation for 6 h at 37 °C. The media was removed, followed by the addition of 200 µL of DMSO.
The absorbance was recorded on a microplate reader at 540 nm. The cell viability (%) was calculated
as the ratio of the number of surviving cells upon treatment with the test compounds compared to
the blank.

Molecules 2014, 19
. Conclusions
Three series of curcuminoid derivatives were synthesized through conjugation of bioactive
16370
4
compounds via ester bonds. The radical-scavenging assay by the DPPH method clearly indicated that
all curcuminoid derivatives have lower IC50 values than the curcuminoids. The results of the bioassay
showed that compounds containing one selenomethionine molecule (11b) attached on the hydroxy
group of curcuminoids showed the highest antimicrobial activity. As the starting materials showed broad
anti-tumor activities, we chose four different human cancer cell lines to evaluate the curcuminoid
derivatives. Three series of curcuminoid derivatives all inhibited the proliferation of all cancer cells.
The results suggest that the attempt to apply a structure combination to discover more efficient, less
toxic and more effective antibacterial, antioxidant and antiproliferative lead compounds is viable.
Acknowledgments
This study was supported by the Natural Science Foundation of Shandong province (No.
ZR2010BM021) and the National Natural Science Foundation of China (21202028, 21372054).
Author Contributions
Q.-Y.H. designed the research; Y.-K.C. performed the experimental work; H.-J.L., Z.-F.S., Y.L.
contributed ideas/bioassay; Y.-K.C. and Q.-Y.H. wrote the manuscript. All authors discussed, edited
and approved the final version.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 11a–h, 15a–h and 19a–d are available from the authors.
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